Geoffrey Hale

REMISSION INDUCTION IN NON-HODGKIN LYMPHOMA WITH RESHAPED HUMAN MONOCLONAL ANTIBODY CAMPATH-1H

A genetically reshaped human IgG1 monoclonal antibody (CAMPATH-1H) was used to treat two patients with non-Hodgkin lymphoma. Doses of 1-20 mg daily were given intravenously for up to 43 days. In both patients lymphoma cells were cleared from the blood and bone marrow and splenomegaly resolved. One patient had lymphadenopathy which also resolved. These effects were achieved without myelosuppression, and normal haemopoeisis was restored during the course of treatment, partially in one patient and completely in the other. No antiglobulin response was detected in either patient. CAMPATH-1H is a potent lympholytic antibody which might have an important use in the treatment of lymphoproliferative disorders and additionally as an immunosuppressive agent.

T cell depletion with CAMPATH-1 in allogeneic bone marrow transplantation.

A total of 282 patients with leukemia have been treated by transplantation from HLA-matched siblings using marrow depleted of T cells with CAMPATH-1 and autologous complement. The incidence of graft-versushost disease (GVHD) of grades 2–4 was only 12% but the maximum incidence of graft failure was 15%. A significant increase in relapse cannot yet be detected in acute leukemia but relapse in chronic granulocytic leukemia (CGL) was substantially above that reported before T cell depletion. The most important predictive factor for relapse in CGL appeared to be slow engraftment. This finding suggests an alternative explanation for the graft-versus-leukemia effect other than a direct attack on leukemia cells. This is that donor T cells may affect the balance of competition between donor and recipient haemopoesis by preventing a rejection reaction to donor stem cells. Recipient leukemic cells would benefit (i.e. relapse) if recipient hemopoiesis gained an advantage. If this explanation were true we would expect extra immunosuppressive preconditioning of recipients to reduce the incidence of relapse, as well as preventing graft rejection.

Efficient complement-mediated lysis of cells containing the CAMPATH-1 (CDw52) antigen.

CAMPATH-1 antibodies recognise a unique molecule on human lymphocytes and are unusually efficient at causing cell lysis with homologous complement. They have been successfully used for lymphocyte depletion in vivo in a variety of diseases. The antigen is a small glycosylphosphatidylinositol (GPI)-anchored glycoprotein with a mature peptide comprising only 12 amino acids and one N-linked glycosylation site at Asn3. The antigenic epitope is found in a proteolytic fragment containing the C-terminal tripeptide and the GPI anchor. Both the native and deglycosylated antigen as well as the proteolytic fragment can be reincorporated into various target cells, conferring sensitivity to lysis by CAMPATH-1 antibodies. These results imply that the special feature of the antigen which makes it a good target does not reside in the N-linked sugar or the first nine amino acids: instead they support the hypothesis that the proximity of the antigenic epitope to the cell membrane is more important.

The glycosylphosphatidylinositol-anchored lymphocyte antigen CDw52 is associated with the epididymal maturation of human spermatozoa.

The CAMPATH-1 (CDw52) antigen is a small glycosylphosphatidylinositol (GPI) anchored glycoprotein with a mature peptide comprising only 12 amino acids. It is abundantly expressed on human lymphocytes and is an unusually good target for complement-mediated cell lysis. The immunosuppressive and lymphocyte-depleting effects of CAMPATH-1 antibodies are being tested in a variety of diseases. Here we show that the antigen is also expressed at a high level in the male reproductive system, being found in the epididymis, seminal vesicle, seminal plasma and on the surface of mature (but not testicular) spermatozoa. Its possible transfer from epithelial cells in the epididymis to maturing sperm may represent a novel method of acquisition of cell surface antigens. In the presence of human complement, CAMPATH-1 antibodies inhibited the motility of washed sperm. However, seminal plasma blocks antibody binding and can protect sperm from this cytotoxic effect.

A therapeutic human IgG4 monoclonal antibody that depletes target cells in humans

It is traditionally held that human IgG4 MoAbs should not deplete target cells in vivo, as this isotype is inactive in a number of in vitro assays that measure effector function. We have previously challenged this dogma, and the current study was designed to investigate the in vivo biological effects in humans of a MoAb of human IgG4 isotype. Nine patients with refractory rheumatoid arthritis (RA) fulfilling ARA criteria, and one with ankylosing spondylitis (AS) received a human IgG4 Campath‐1 MoAb (with specificity against the pan‐lymphocyte antigen CD52) as part of a two‐stage therapeutic protocol. In stage 1, patients received a single dose of this MoAb. Stage 2, starting 48 h later, comprised a 5‐day course of a human IgG1 Campath‐1 MoAb with identical V‐region (CAMPATH‐1H), as previously used in the management of RA patients. The intervening 48 h provided a window of opportunity to monitor the biological effects of the IgG4 MoAb for comparison with the IgG1. The two MoAbs were also compared for in vitro biological activity. IgG4 depleted peripheral blood lymphocytes (PBL), albeit less efficiently than IgG1. It produced a first‐dose reaction of similar intensity, although associated circulating tumour necrosis factor‐alpha (TNF‐α) levels were lower. TNF‐α release from whole blood in vitro was also greater with the IgG1 MoAb. The study design did not permit conclusions to be drawn regarding therapeutic efficacy of the IgG4 MoAb. In summary, a human IgG4 Campath‐1 MoAb depletes target cells in vivo. Importantly, this study demonstrates for the first time in humans that in vitro assays may not predict the in vivo effector function of therapeutic MoAbs.

Immune reconstitution after allogeneic bone marrow transplantation depleted of T cells.

BACKGROUND Immune reconstitution following transplantation in individuals who had received T-cell-depleted marrow from HLA identical siblings was serially documented and correlated with the clinical recovery. METHODS Patients were preconditioned with radiation containing programs. GvHD prophylaxis was by T-cell depletion with CAMPATH 1G (ex vivo; median dose 20 mg). After transplantation lymphoid development was studied by flow cytometry and serum Ig concentrations were determined. Charts were reviewed to determine the effects of the immune reconstitution on the clinical performance. RESULTS The mean donor mononuclear cell number infused was 0.89x10(8)/kg. Within 6 months all the patients recovered their blood parameters and only one required therapy for GvHD. However, despite normal blood counts, 15 suffered life-threatening opportunistic infections, developing at a median of 24 weeks post grafting, but occurring even after 11 months. At 8 weeks from marrow infusion when leukocyte values had normalized in 15/20, compared to normal, immunophenotyping of blood cells from BMT revealed a significantly reduced mean lymphocyte count (1.06, SD 0.83x10(9)/l; P = 0.01), cells expressing CD3 (0.7x10(9)/l, SD 0.68; P = 0.05), CD4 (0.13x10(9)/l, SD 0.21; P = 0.0001) and CD19 (0.04x10(9)/l, SD 0.05; P = 0.001). Populations expressing CD8 and CD56 remained within normal range throughout the study. Normalization of cell numbers displaying CD2, CD3 and CD19 was delayed until 52, 52 and 24 weeks respectively, while CD4 counts persisted subnormal even at 72 weeks. Serum IgA levels were significantly decreased for the entire study period. CONCLUSIONS T-cell depletion with CAMPATH 1G while effectively preventing GvHD, also causes clinically significant and prolonged immunosuppression with apparently important clinical implications.

Molecular weight and symmetry of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (Do20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10−11m2/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (so20,w = 60.2 (±0.4) S) and partial specific volume (v = 0.735 (±0.01) ml/g), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 106. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78. From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core. The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (so20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.

Repeated cleaning of protein A affinity column with sodium hydroxide

Protein A attached to an affinity adsorbent was shown to be remarkably resistant to 0.5 M NaOH. Even repeated treatments gave only a small decrease in functional capacity and no adverse effect on leakage of the protein A into the eluate. This simple cleaning procedure should be useful in applications where antibodies need to be purified free from the risk of contamination with endotoxins or micro-organisms, e.g., for in vivo therapy, either in clinical trials or experimental animals. It can also prevent cross-contamination when the same protein A adsorbent is used for different batches of antibody.

Synthetic peptide mimotope of the CAMPATH-1 (CD52) antigen, a small glycosylphosphatidylinositol-anchored glycoprotein

BACKGROUND CAMPATH-1 (CD52) antibodies are among the most powerful and specific lympholytic agents in humans and have numerous potential applications for human therapy. The CD52 antigen is a GPI-anchored glycoprotein with an exceptionally short peptide sequence of only 12 amino acids and a single, complex, N-linked oligosaccharide. Antibodies bind to the deglycosylated antigen and to a proteolytic fragment, but not to the synthetic peptide alone. OBJECTIVES To characterise the antigenic epitope more precisely and to construct a synthetic analogue. Such an analogue would be useful for assay and purification of the therapeutic CAMPATH-1 antibodies as well as for studies of the antibody-antigen binding site. STUDY DESIGN Collections of synthetic peptides based on the natural sequence were screened with a panel of CD52 antibodies. RESULTS AND CONCLUSION A synthetic peptide composed of the natural C-terminal amino acids plus two additional residues was found to mimic the antigen with sufficient affinity to be useful for a variety of assays and for construction of an affinity matrix for antibody purification. Systematic mutation of this peptide enabled the definition of the critical residues for antibody binding, which will be of great help in building a model of the antibody-antigen interaction. Peptide mimotopes synthesised using a natural sequence as a starting point, rather than a completely random library, may be useful in many other similar circumstances.

Humanized anti-CD4 monoclonal antibody therapy of autoimmune and inflammatory disease

We have investigated the biological and therapeutic properties of a humanized anti‐CD4 MoAb, hlgGl‐CD4, in patients with refractory psoriasis and rheumatoid arthritis (RA). hIgGl‐CD4 is a modulating, non‐depleting MoAb, which induced a first‐dose reaction in most patients treated. It provided brief symptomatic relief in both conditions, and psoriasis appeared easier to control with conventional agents after MoAb therapy. At the doses used, hIgGl‐CD4 did not synergize therapeutically with the pan‐lymphocyte MoAb CAMPATH‐1H (C1H) in patients with RA treated sequentially with both agents. There were no serious adverse effects definitely attributable to therapy. Our results are compared with those of other CD4 MoAb studies, and factors influencing the outcome of therapy are discussed.