G T Stevenson

Preliminary experience in treating lymphocytic leukaemia with antibody to immunoglobulin idiotypes on the cell surfaces.

Tumour-specific antiserum was raised in sheep against idiotypic determinants on the surface immunoglobulin of neoplastic lymphocytes from a patient with chronic lymphocytic leukaemia (prolymphocytic variant). The complement-activating IgG1 subclass of the anti-idiotype was prepared from the serum in monodisperse form for infusion. Two treatments of 480 and 1200 mg caused the white-cell count to fall by one-third and one-half respectively. However, there was a rapid resurgence, so that by 8 days after each treatment the counts were restored to approximately 85% of their former levels. No change was noted in the size of spleen or lymph nodes. Each treatment probably destroyed 4-8 X 10(11) cells, some 10% of the total tumour load. The antibody was rapidly consumed, and there was evidence of heavy utilization of complement.

Follicular lymphoma and the immune system: from pathogenesis to antibody therapy

Follicular lymphoma (FL) is a B-cell tumor arising in germinal centers and retaining features of its normal B-cell counterpart. Lymphomagenesis appears stepwise from the t(14;18) translocation, through FL-like cells, to FL in situ, then to overt FL. Surface Ig is mandatory and carries a striking V-region modification because of introduction of glycan addition sites during somatic mutation. These are positively selected and acquire unusual high mannoses, which interact with lectins. The Ig-associated mannoses appear essential for FL, providing a disease- specific target for antibody attack. Antibody therapy is currently focused on anti-CD20 (rituximab), which appears to rely predominantly on the Fcγ module recruiting suitably activated macrophages. Immunogloblulin and, to some extent, CD20, can each escape antibody attack in vitro by modulation, but this is difficult to demonstrate clinically. Instead, studies of anti-CD20 therapy of FL suggest that effector modulation, similar to that seen in the suppression of autoimmune inflammation by infusions of normal human IgG, may be important. Both antigenic and effector modulations might be minimized by repeated small doses of more potent antibodies. Clearly, mechanisms of attack vary with the malignancy, the target molecule, and the antibody design, offering opportunities for optimizing this promising strategy.

Surface immunoglobulins on Xenopus laevis lymphocytes

Abstract o 1. The surface immunoglobulins (Ig) on Xenopus laevis lymphocytes have been studied by immunofluorescence. 2. 60–85% of the lymphocytes in adult peripheral blood possess surface Ig. In post-Stage 48 tadpoles the proportion of such cells varies between 35 and 45%, the proportion rising to the adult level in the immediate post-metamorphosis period. 3. The proportions of surface Ig-carrying lymphocytes in the spleen and other lymphomyeloid organs are similar to those in the blood at any given developmental stage. The thymus is an exception: in tadpoles some 47–55%, and in metamorphs 70–75% of the thymocytes have surface Ig; this figure declines to between 8 and 12% in the adult. 4. In all situations the surface Ig appears to be almost exclusively IgM; IgG only presents on 5% or less of the cells. On no cells are both IgM and IgG present together. 5. The surface Ig can be capped, and can be removed by limited papain proteolysis of the cell surface: subsequent to capping or proteolysis the Ig is regenerated. 6. The phylogenetic significance of these results is discussed in terms of B and T cell delineation and differentiation.

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

SummaryThe three forms of Fcγ receptor carried by monocytes (FcγRI, II) and natural killer (NK) cells (FcγRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab′γ)2 bispecific antibodies, specifically targetting individual FcγR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcγR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab′ from an anti-FcγR mAb linked to Fab′ from a common anti-target mAb (BsAb), or Fab′ from the common anti-target mouse antibody linked to human Fcγ (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L2C, regularly resulted only via the anti-FcγRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcγRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (≈50%) lysis occurred with effector cell populations magnetically depleted through either FcγRII or FcγRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fcγ. The lysis mediated by BsAb reactive with FcγRI or II was unaffected by the presence of human Fcγ at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcγRIII and all the Fc-containing derivatives were completely inhibited.

Secretion of immunoglobulin by neoplastic B lymphocytes from lymph nodes of patients with lymphoma

An investigation has been made into the ability of neoplastic B lymphocytes obtained from lymphoid tissue of patients with non-Hodgkins lymphoma (NHL) to secrete immunoglobulin (Ig) in vitro. The majority of the cell populations secreted IgM (17/24 patients), identified as pentameric in three cases examined, and free monotypic light chains (23/24 patients) of the same type as the surface Ig. Secretion of IgD (6/21 patients) and IgG (3/21 patients) was found less frequently. The amounts of Ig secreted were variable and there was no significant difference in the patterns of secretion of cells from NHL patients when compared to previous studies of chronic lymphocytic leukaemia (CLL), nor was there any clear correlation with the histological type. For four of the patients, anti-idiotypic antibody was produced and was used to demonstrate the idiotypic nature of the secreted Ig, and also to show its presence in the serum. The level of idiotypic IgM was measured in one patient during chemotherapy and appeared to correlate well with disease. Such idiotypic Ig must be taken into account when planning treatment of B cell neoplasms with antiidiotypic antibody since it could act as a block to antibody attack. Assessment of the ability of tumour cells to secrete Ig in vitro provides a useful preliminary screen when choosing such patients since a high secretion rate together with extensive disease could lead to unacceptable levels of serum idiotypic Ig.

Consumption of monoclonal anti-idiotypic antibody by neoplastic B lymphocytes: a guide for immunotherapy.

A quantitative analysis in vitro of events which might occur on administration of mouse monoclonal anti-idiotypic antibody to a recipient with a B cell neoplasm has been made. The L2C leukaemic cells of guinea pigs, which closely resemble those of human lymphoma in expression and metabolism of immunoglobulin have been used as a model. Exposure of neoplastic B cells to antibody results in rapid binding of approximately 420,000 molecules of antibody per cell at saturation, and the amount consumed does not increase markedly over the next 4 h of exposure at 37 degrees C. This is in spite of the fact that secretion of idiotypic IgM continues unaffected by the presence of antibody, and reflects the fact that the amount of IgM secreted during this period is low compared to the amount displayed on the cell surface. If cells undergo lysis, however, the antibody consumed is approximately doubled: thus a recipient with an estimated tumour load of 10(12) cells would require 200 mg of monoclonal anti-idiotype for binding to surface and intracellular antigen. The effect of the soluble idiotypic IgM found in serum on the ability of antibody to bind target cells has been examined by means of the fluorescence activated cell sorter. Access of antibody to the cells is efficiently blocked by competing idiotypic IgM in the fluid phase, with no indication of preferential binding to cell surface idiotype. Immunotherapeutic doses should be designed therefore to overcome this additional antigenic load in secreting tumours, which form the majority of B cell neoplasms.

Three major uncertainties in the antibody therapy of cancer

Antibodies against surface molecules of human tumors are now frequently administered in combination with strong chemotherapy, increasing therapeutic efficacy but making the task of elucidating immunological events more difficult. Experiments on genetically manipulated mice indicate that antibody efficacy is greatest when IgG antibody coating tumor cells is engaged by the Fcγ-receptors of effector cells, chiefly the monocyte/macrophage lineage. Evidence suggests lesser roles for NK cells, neutrophils, receptor-mediated cytotoxicity and complement-mediated cytotoxicity. The classical mode of killing employed by macrophages is phagocytosis, but much has to be learned about optimally activating macrophages for this task, and about any other modes of cytotoxicity used. There is renewed interest in antigenic modulation, which implies removal of therapeutic antibody linked with antigen from target-cell surfaces. It is now apparent that this removal of immune complexes can be achieved either by internalization by the target cell, or by transfer of the complexes to another cell by trogocytosis. In trials, anti-idiotype antibodies surprisingly proved therapeutically more effective than anti-CD20, despite anti-idiotype being more effectively removed from target-cell surfaces by antigenic modulation. This anomalous result might reflect the fact that persistence of anti-CD20 immune complexes in large amounts induces serious effector modulation, which paralyzes macrophage attacks on antibody-coated cells. The case for effector modulation is argued by analogy with the therapeutic suppression of autoimmune inflammation by effector modulation, achieved by infusion either of normal IgG in large amounts, or of anti-red cell IgG in relatively small amounts.

Humoral antibodies to soluble antigens in larvae of Xenopous laevis

Abstract 1. 1. Larvae (tadpoles) of Xenopus laevis produce precipitating humoral antibodies to both “alum-precipitated” human immunoglobulin light chains and to Limulus haemocyanin. 2. 2. Most of the larval antibody is in the 19 S serum protein fraction, but a significant proportion is in the 7 S protein fraction. 3. 3. Immuno of the larval antibody shows that Xenopus tadpoles are capable of synthesizing both IgM and IgG antibodies. 4. 4. The phylogenetic significance of these findings is discussed.

Immunoglobulin classes in Xenopus laevis.

1. 1. Electrophoretically pure 19 S and 7 S immunoglobulins, designated IgMand IgG, were prepared from Xenopus laevis serum. Both immunoglobulins possessed antibody activity. 2. 2. The sedimentation coefficient for IgM is 19·5 S, for IgG 6·1 S. Reduction and alkylation of IgM yielded subunits with a sedimentation coefficient of 4·1 S. 3. 3. The immunoglobulins were dissociated into heavy and light chains. IgM and IgG have identical light chains; however, the respective heavy chains share no antigenic determinants. 4. 4. The phylogenetic significance of this finding is discussed.

Antibody treatment of lymphoma: experience and prospects

Lymphomas which appear after transplantation fare poorly with standard chemotherapeutic regimes, so a report on the therapeutic use of antibody in these cases has aroused considerable interest [1]. We present here our experience with antibody treatment of lymphomas in general, and discuss the prospects for applying some of our newer antibody derivatives to treating the post-transplantation tumours.