CD40 antibody evokes a cytotoxic T-cell response that eradicates lymphoma and bypasses T-cell help.

CD40 is essential in enabling antigen-presenting cells to process and present antigen effectively to T cells. We demonstrate here that when antibody against CD40 is used to treat mice with syngeneic lymphoma, a rapid cytotoxic T-cell response independent of T-helper cells occurs, with tenfold expansion of CD8+ T cells over a period of 5 days. This response eradicates the lymphoma and provides protection against tumor rechallenge without further antibody treatment. Thus, it seems that by treating mice with monoclonal antibody against CD40, we are immunizing against syngeneic tumors. The phenomenon proved reproducible with two antibodies against CD40 (3/23 and FGK-45) in three CD40+ lymphomas (A20, A31 and BCL1) and gave partial protection in one of two CD40- lymphomas (EL4 and Ten1). Although the nature of the target antigens on these lymphomas is unknown, CD8+ T cells recovered from responding mice showed powerful cytotoxic activity against the target B-cell lymphoma in vitro.

Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16).

Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.

Emergence of immunoglobulin variants following treatment of a B cell leukemia with an immunotoxin composed of antiidiotypic antibody and saporin.

The potency and specificity of immunotoxins consisting of monoclonal antiidiotype conjugated to the ribosome-inactivating protein, saporin, have been evaluated in the treatment of guinea pig L2C B lymphocytic leukemia. The immunotoxins were therapeutically much more effective than their parent antibodies. Their specificity reflected that of their antiidiotype component. Although the leukemia emerged eventually in most animals treated with these conjugates, most of the cells showed altered Ig expression, which rendered them resistant to the therapy. Commonly, the emerging cells had lost mu heavy chain production, leaving them negative for intracellular, surface, and secreted IgM, but still positive for lambda light chain production. In addition, a minor group of L2C variants was identified in a protocol designed to detect mutants at very low frequency: here the cells were exposed in vitro to immunotoxin and, while still viable as judged by dye-exclusion, inoculated in large numbers into animals. In tumor that emerged under these circumstances, the majority of cells were again immunoglobulin-negative; however a minority exhibited IgM with an altered idiotype (Idiotope-loss variants), rendering them unreactive with immunotoxin. Immunotherapy with unmodified anti-Id antibody alone does not reveal these variants, and we suggest it is the increased selective force exerted by the highly potent immunotoxins that allow these minor nonreactive populations to emerge.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

Signaling antibodies in cancer therapy.

Over the past 10-15 years, genetic engineering of monoclonal antibodies has greatly improved their utility in humans and in particular their ability to recruit immunological effectors such as natural killer cells and macrophages. Clinical results now confirm that these new reagents, when directed at the appropriate tumor markers (e.g. CD20 or Her-2), can control disease without untoward side effects. However, despite such success it is still unclear exactly how monoclonal antibodies (mAbs) destroy tumors in vivo. The ability of mAbs to crosslink membrane receptors and generate intracellular signals is part of the mechanism by which they control tumor growth. New data show that such 'signaling' mAbs can be used to sensitize tumors to the action of conventional DNA-damaging drugs.

Delivery of saporin to human B-cell lymphoma using bispecific antibody: targeting via CD22 but not CD19, CD37, or immunoglobulin results in efficient killing.

A panel of bispecific F(ab')2 antibodies (BsAb) have been constructed for delivering the ribosome-inactivating protein saporin to human B cell lymphoma. Each derivative was prepared with specificity for saporin and CD19, CD22, CD37, or immunoglobulin. In vitro studies measuring inhibition of [3H]leucine uptake by cultured Daudi and Raji cells demonstrated that, despite all BsAb capturing saporin on the cell surface, BsAb targeting through CD22 were far more cytotoxic than those functioning via CD19, CD37, or surface immunoglobulin. This exceptional activity of the CD22-specific BsAb appears to derive from its ability to deliver and accumulate saporin inside the target cells. Further studies showed that four CD22-specific BsAb all performed with equal potency and were able to increase saporin toxicity (50% inhibitory concentration) up to 1000-fold, from 2 x 10(-7) M to 2 x 10(-10) M. Pairs of anti-CD22 BsAb which recognized different nonblocking epitopes on the saporin molecule were able to bind saporin more avidly to the target cell and, as a consequence, increased cytotoxicity by at least an additional 10-fold, resulting in 50% inhibitory concentration for protein synthesis of 2 x 10(-11) M. These results suggest that selected combinations of BsAb which bind cooperatively to a toxin and the cell surface may provide an efficient way of delivering toxins to unwanted cells in patients.

Cooperative mixtures of bispecific F(ab')2 antibodies for delivering saporin to lymphoma in vitro and in vivo.

We report that selected combinations of two or more monoclonal bispecific F(ab')2 antibodies (BsAbs) far outperform single derivatives in the delivery of the ribosome-inactivating protein, saporin, to guinea pig L2C leukemic cells. Throughout the work, BsAbs were constructed by thioether-linking the hinges of two Fab'gamma, one from monoclonal anti-L2C-idiotype antibody and the other from anti-saporin antibody. The latter was either affinity-purified rabbit polyclonal or one of a panel of five mouse monoclonal antibodies. In vitro cytotoxicity studies showed that, though all derivatives were effective, the BsAb made with the polyclonal antibody was always 10 to 20 times more potent than those made with a monoclonal antibody in yielding 50% inhibition of [3H]leucine uptake. This superior activity could be matched by selective mixtures of two or more of the monoclonal derivatives. Furthermore, in immunotherapeutic delivery of saporin to tumor, a pair of BsAbs performed significantly better than did either individually. Binding and uptake studies with radiolabeled saporin demonstrated a 20-fold increase in functional affinity when saporin was held at the cell surface by an appropriate BsAb mixture rather than by a single BsAb. In contrast, only small differences were recorded in the rate at which saporin was internalized as a result of the same maneuver. We conclude that the improved performance of combinations of BsAbs arises from their ability to provide multiple linkages between saporin molecules and cell surfaces, significantly increasing the functional affinity with which saporin is tethered to the cell, but, in this system at least, having only a minor effect on the rate at which it is internalized. Cocktails of two or more BsAbs, selected to bind to multiple epitopes on ribosome-inactivating proteins and perhaps also on unwanted cells, could provide an important new strategy in immunotherapy.

Evaluating antibodies for their capacity to induce cell-mediated lysis of malignant B cells.

Promising results from clinical trials have led to renewed interest in effector mechanisms operating in antibody-based therapy of leukemia and lymphoma. We tested a panel of B-cell antibodies from the Sixth Human Leukocyte Differentiation Antigen workshop for their capacity to mediate antibody-dependent cellular cytotoxicity, often considered to be one of the most potent effector mechanisms in vivo. As effector cells, mononuclear cells and polymorphonuclear (PMN) cells from healthy donors were compared with Fc gammaRI (CD64)-expressing PMN cells from patients receiving granulocyte colony-stimulating factor (G-CSF) treatment. Of the 29 IgG workshop antibodies binding most strongly to the tested malignant human B-cell lines, only 3 consistently induced target cell lysis. These three antibodies were determined to be HLA DR reactive. Experiments with a panel of HLA class II antibodies showed the involvement of individual Fc gamma receptors on effector cells to be strongly dependent on the antibody isotype. We then compared killing mediated by chimeric IgG1 antibodies with that from Fc gammaRI-directed bispecific antibodies, targeting classical HLA class II, or the Lym-1 and Lym-2 antigens. The latter two are variant forms of HLA class II, which are highly expressed on the surface of malignant B cells but which are found only at low levels in normal cells. With blood from G-CSF-treated donors, bispecific antibodies showed enhanced killing compared to their chimeric IgG1 derivatives, because they were more effective in recruiting Fc gammaRI-expressing PMN cells. G-CSF- and Fc gammaRI-directed bispecific antibodies to HLA class II, therefore, seem to be an attractive combination for lymphoma therapy.

Complement mediated cell death is associated with DNA fragmentation.

In this study, we demonstrate for the first time that complement attack of target cells, in the presence of suitably high levels of serum, can induce the oligonucleosomal DNA fragmentation characteristic of apoptosis. This phenomenon requires membrane permeabilisation induced by formation of the complete membrane attack complex and relies on physiologically relevant levels of serum. TUNEL analysis detected complement mediated DNA fragmentation as early as 30 min after the addition of serum and electron microscopy confirmed that chromatin became condensed after complement attack. Various experiments implicate serum DNase I as the mediator of this DNA fragmentation. Intriguingly, membrane permeability induced by melittin gave rise to similar serum dependent DNA fragmentation. The implications of these results for the study of apoptosis in vitro and in vivo are discussed.

Characterization of a new monoclonal anti-Fc gamma RII antibody, AT10, and its incorporation into a bispecific F(ab')2 derivative for recruitment of cytotoxic effectors.

Fc gamma RII (CDw32) on monocytes is capable of triggering both phagocytosis and lysis of chick red blood cells (CRBC) coated with antibody of the appropriate isotype. In this report we describe the production and characterization of a mouse monoclonal IgG1 antibody specific for Fc gamma RII and compare its activity in binding studies, tissue distribution and redirected cellular cytotoxicity (RCC), with the previously identified anti-Fc gamma RII antibodies KB61 and IV.3. Immunohistochemical and flow cytometry analyses demonstrated that AT10 binds very strongly to Fc gamma RII on normal monocytes, but only weakly to that expressed on lymphocytes. This pattern does not correspond to the staining seen with either KB61 or IV.3, and appears to give an intermediate profile. The binding constant (Ka) for the Fab' fragment of AT10 was calculated at 5.3 x 10(8) M-1, four times higher than that for KB61 (1.4 x 10(8) M-1). Bispecific F(ab')2 antibodies were constructed from Fab' fragments of AT10 or KB61 thioether-linked to Fab' from an anti-CRBC monoclonal antibody. These bispecific derivatives directed monocyte cytotoxicity against CRBC as efficiently as either a monoclonal or polyclonal anti-chick erythrocyte antibody. The bispecific F(ab')2 antibodies had a distinct advantage over the conventional reagents, in that they were not blocked in the presence of human Fc gamma at 3.5 mg/ml (a concentration comparable with that provided by IgG in serum). Therefore, bispecific derivatives constructed with the high affinity anti-Fc gamma RII antibody, AT10, may be used as therapeutic reagents for targeting tumour cell lysis in vivo.

Measurement of kinetic binding constants of a panel of anti-saporin antibodies using a resonant mirror biosensor.

We have used a resonant mirror biosensor to determine the kinetics of binding of four antibodies, and their Fab' fragments, to their antigen, the plant-derived ribosome-inactivating protein (RIP) saporin. The analysis of the affinity of the antibodies was in reasonable agreement with values obtained by conventional techniques. However, the kinetic data showed that all four antibodies have a high dissociation rate constant (kdiss). These antibodies have been used in the construction of bispecific antibodies used to deliver saporin to tumour cells, and it is highly probable that the in vivo efficacy of the bispecific antibodies is limited by the high rate of dissociation of antibody-toxin complexes.

Mechanisms of G-CSF- or GM-CSF-stimulated tumor cell killing by Fc receptor-directed bispecific antibodies.

Studies with gene-modified mice have recently reinforced the importance of Fc receptor-mediated effector mechanisms for the therapeutic efficacy of rituxan and herceptin - two clinically approved antibodies for the treatment of tumor patients. We investigated Fc receptor-dependent tumor cell killing by mononuclear and granulocytic effector cells - comparing human IgG1 antibodies against CD20 or HER-2/neu with their respective FcgammaRI (CD64)-, FcgammaRIII (CD16)-, or FcalphaRI (CD89)-directed bispecific derivatives. With blood from healthy donors as effector source, human IgG1 and FcgammaRIII (CD16)-directed bispecific antibodies proved most effective in recruiting mononuclear effector cells, whereas tumor cell killing by granulocytes was most potently triggered by FcalphaRI-directed bispecific constructs. Granulocyte-mediated tumor cell lysis was significantly enhanced when blood from G-CSF- or GM-CSF-treated patients was investigated. Interestingly, however, both myeloid growth factors improved effector cell recruitment by different mechanisms, which were furthermore dependent on the tumor target antigen, and on the selected cytotoxic Fc receptor.

CD40 ligation for immunotherapy of solid tumours.

Tumour vaccines provide an important focus of current cancer research and are often based on the premise that although T-cells do respond naturally to certain tumours, this is usually weak and therefore ineffective at controlling disease. An integral and necessary part of a T-cell immune response involves triggering of CD40 on antigen-presenting cells (APC) by its ligand, CD154, on responding T helper (Th) cells. Furthermore, cytotoxic responses to tumours may fail because the Th-cell response is inadequate and unable to provide CD40 stimulation of APC. Growing evidence shows that stimulating APC with soluble CD40L or an agonistic anti-CD40 mAb can, at least in part, replace the need for Th cells and generate APC that are capable of priming cytotoxic T lymphocytes (CTL). The aim of this study was to investigate whether a range of solid tumours (CD40(-)) could be treated with anti-CD40 mAb. It was found that this treatment was effective, and correlated with the intrinsic immunogenicity and aggressiveness of the tumours. The mAb could be delivered locally or at a distal site, but increased antigen load provided by irradiated tumour cells added little to the effectiveness of the treatment. T-cells were required since cytokine (interferon-gamma) and CTL activity were demonstrated following treatment and the therapeutic efficacy was lost in nude mice. In addition, depletion of CD8(+) cells abrogated protection whilst depletion of CD4(+) cells had no effect. This study demonstrates that solid CD40(-) tumours are sensitive to anti-CD40 mAb therapy and that the response bypasses the need for Th cells.

Development of a novel flow cytometric cell-mediated cytotoxicity assay using the fluorophores PKH-26 and TO-PRO-3 iodide.

A flow cytometric (FCM) assay has been developed for the determination of cell-mediated cytotoxicity (CMC). In the assay, the target tumour cell population was labelled with a membrane dye, PKH-26, prior to incubation with splenocyte effector cells. Cell death within the target population was assessed by the addition of the viability probe TO-PRO-3 iodide (TP3) and analysed by flow cytometry. The extent of cytotoxicity was determined by the relative number of live target cells labelled with PKH-26 only and dead, permeabilised cells labelled with both PKH-26 and TP3. This CMC method allows the analysis to be conducted on a single cell basis and overcomes the need for radiochemicals. This communication indicates that the FCM assay is an accurate and reproducible experimental system capable of analysing natural killer (NK) cell and antibody-dependent cell-mediated cytotoxicity. The procedure is comparable to the chromium release assay. We believe that this is one of the first demonstrations of an FCM-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay.

Synthesis, conformational properties, and antibody recognition of peptides containing beta-turn mimetics based on alpha-alkylproline derivatives.

Peptide recognition by monoclonal antibodies may provide a useful model for drug development, in particular to test the effects of conformational restriction on ligand binding. We have tested the influence of novel peptide mimetics upon conformation and binding affinity for the case of monoclonal antibodies raised to a peptide antigen which displays a preference for a beta-turn conformation in aqueous solution. Two monoclonals were isolated that recognized the peptide Ac-Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala specifically at the beta-turn formed by Tyr-Pro-Tyr-Asp. Peptide analogues were then synthesized containing mimetics designed to stabilize this conformation. One, analogue (3), contained a spirocyclic gamma-lactam bridge between the alpha-position of proline-2 and the N atom of tyrosine-3, while another (2) contained (S)-alpha-methylproline at position 2. NMR spectroscopy and molecular modeling suggest that both analogues adopt reverse-turn conformations stabilized relative to that in the native sequence. For the (S)-alpha-methylproline analogue binding to both monoclonal antibodies was substantially improved, compared with the native antigen, whereas the gamma-lactam analogue (3) was not recognized by either antibody. Quantitative equilibrium ultrafiltration binding assays showed that the affinities of the (S)-alpha-methylproline analogue (2) for the two antibodies were improved over those measured with the native antigen by -2.3 and -0.65 kcal/mol. The origins of these free energy differences cannot be explained wholly on the basis of presumed extra hydrophobic contacts between the new methyl substituent and the antigen binding sites. We propose that the increased conformational stability of the analogue plays a decisive role, implying that the reverse turn detected in the native antigen, possibly a type-I turn, is important for recognition by the two antibodies.

Response of B-cell lymphoma to a combination of bispecific antibodies and saporin.

Observations are described using a combination of two bispecific F(ab')2 antibodies (BsAb) to deliver the ribosome-inactivating protein, saporin, in the treatment of low-grade, end-stage, B-cell lymphoma. Two BsAb were used, each having one arm directed at saporin and one at the CD22 on target B cells. The BsAb, however, recognized different, non-overlapping epitopes on each molecule, a strategy which permits high-avidity double attachment of saporin to the target. The BsAb and saporin were pre-mixed at a molar ratio of 3:1 24 h before treatment and infused intravenously over a period of 1 h. Five patients have been treated, mostly with weekly doses of between 2 and 4 mg of saporin for a period of up to 6 weeks. Toxicity was minimal. Three complained of weakness and myalgia for 1 to 2 days after treatment, without objective neurological deficit or rise in serum creatine kinase. One patient produced an anti-mouse Fab' and an anti-saporin response. All patients showed a rapid and beneficial response to treatment. When present, circulating tumor cells were cleared (4/4 patients), ascitic and pleural effusions were eliminated (2/2 patients) and one patient with splenomegaly showed a marked reduction in tumor bulk. Malignant lymph nodes showed significant, but partial, shrinkage in all patients and finally marrow responded well with tumor clearance in biopsy material and impressive resolution of pancytopenia in some patients. While these responses were mainly short-lived, with tumor progression once the treatment was stopped, their speed and magnitude, and the relative lack of associated toxicity warrants further study of this treatment to determine maximum tolerated doses and therapeutic utility.

HLA class II antibodies recruit G-CSF activated neutrophils for treatment of B cell malignancies.

Monoclonal antibodies offer the potential to improve specificity of oncological therapy. However, future success in the clinic depends on enhancing antibody effector functions. Here, we suggest that target antigen selection may influence recruitment of effector cells for antibody therapy, and may improve the outcome of antibody treatment in patients. Comparing a wide range of antibodies to different B cell antigens, we found most were able to mediate antibody dependent cellular cytotoxicity (ADCC) with blood mononuclear cells (MNC). In direct contrast, however, polymorphonuclear granulocytes (PMN) from the same donors showed ADCC only with HLA class II antibodies. Based on this observation, we propose a therapeutic strategy with a combination of HLA class antibodies and G-CSF, the latter being required to increase number and activational state of neutrophils. In particular, we suggest using bispecific antibodies (BsAb) in which one arm binds to HLA class II on tumor cells, and the second to FcgammaRI on activated effector cells. The clinical potential of this approach for the treatment of B cell malignancies looks most attractive.

Preparation and properties of FabIgG, a chimeric univalent antibody designed to attack tumour cells.

In order to promote the killing of tumour cells by antibody a derivative has been synthesized in which Fab'gamma from xenogeneic antibody is thioether-bonded to half-cystine on normal IgG of the species to be treated. The resulting entity, FabIgG, is obtained with about a 40% yield of the starting Fab'gamma. Being univalent it evades antigenic modulation. It activates complement efficiently, is minimally immunogenic, and appears to be catabolized at the slow rate characteristic of autologous IgG.

The application of monoclonal antibodies in the treatment of lymphoma.

Monoclonal antibodies (mAbs) appear to offer many benefits for the treatment of cancer and in particular lymphoma (1). They are natural products that can be made with precise specificity and in almost unlimited amounts. In addition, mAbs can be selected or engineered to efficiently recruit the body's effector systems, such as complement and natural killer cells, against the unwanted cells in much the same way as they might destroy an invading pathogen. Unfortunately, progress in the clinic has been slow, and the cytotoxic activity achieved with mAb in vitro has failed to be transferred into patients. Despite this rather disappointing outcome, recent results in treating non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL) with anti-CD20 and anti-CD52 (CAMPATH 1) mAb suggest, at least for certain neoplasms, that the situation may be changing (2,3). Stevenson and colleagues (personal communication) have recently achieved more than 70% complete responses in posttransplant lymphoma treated with a chimeric anti-CD20 mAb, and Maloney and co-workers (2) recently reported a 50% response rate in relapsed, low-grade, NHL, with a 10---11 mo duration. Encouragingly, patients in these studies did not raise antibody responses to the treatment anti-CD20 mAb and, unlike the situation following therapy in many lymphomas with anti-idiotype (Id) mAb, the emergence of antigen-negative tumors has not been seen (4). To underline its clinical success, anti-CD20 mAb (Rituximab) has now become the first anticancer mAb to become licensed by the FDA for lymphoma treatment. One of the most encouraging aspects of Rituximab treatment is that, in addition to its therapeutic activity, which appears to match that of more conventional chemotherapy in a similar setting, it has very few adverse effects and can be given to patients who are in poor condition with advanced disease. Early experience suggests that it will be this lack of adverse effects that will be its most attractive feature.

Monoclonal antibodies raised against the idiotype of the murine B cell lymphoma, BCL1 act primarily with heavy chain determinants.

Anti-idiotypic antibodies can be used as probes to distinguish neoplastic cells from their normal counterparts. In addition they have been used in the passive therapy of B cell tumors. In this report we describe a panel of 7 rat monoclonal antibodies raised against idiotypic determinants carried by the IgM molecule of the BCL1 lymphoma. The majority (6/7) of these antibodies recognize private idiotypic determinants that are carried on the isolated mu heavy chain of the molecule, and do not require the lambda chain for reactivity. This is unusual for antibodies raised against the idiotype of the whole immunoglobulin molecule, which normally require both chains for reactivity. The antibodies do not, however, bind peptides corresponding to the complementarity determining regions of the mu heavy chain of BCL1. The antibodies perform well in complement mediated cytotoxicity, and, in at least one case, are effective in the passive immunotherapy of BCL1 lymphoma.