Immunological Methods to Study Monoclonal Antibody Activity in Chronic Lymphocytic Leukaemia.

Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous "normal" cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities.

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.

How to make bispecific antibodies.

This protocol describes the production of bispecific F(ab')2 antibody derivatives (BsAbs) by the linking of two Fab' fragments via their hinge region SH groups using the bifunctional crosslinker o-phenylenedimaleimide (o-PDM) as described by Glennie et al. (1,2). The procedure is illustrated in Fig. 1. The first step is to obtain F(ab')2 from the two parent IgG antibodies. Methods for digestion of IgG to F(ab')2 are described in Chapter 25. Fab' fragments are then prepared from the two F(ab')(2) species by reduction with thiol, thus exposing free SH groups at the hinge region (three SH-groups for mouse IgG1 and IgG2a antibodies) (see Note 1 ). One of the Fab' species (Fab'-A) is selected for alkylation with o-PDM. Because o-PDM has a strong tendency to crosslink adjacent intramolecular SH-groups, two of the three hinge SH-groups will probably be linked together, leaving a single reactive maleimide group available for conjugation (Fig. 1; see Note 2 ). Excess o-PDM is then removed by column chromatography, and the Fab'-A(mal) is mixed with the second reduced Fab' (Fab'-B) under conditions favoring the crosslinking of the maleimide and SH groups. When equal amounts of the two parent Fab' species are used, the major product is bispecific F(ab')(2), resulting from the reaction of one Fab'-A(mal) with one of the SH groups at the hinge of Fab'-B. Increasing the proportion of Fab'-A(mal) in the reaction mixture results in a significant amount of F(ab')(3) product by the reaction of two molecules of Fab'-A(mal) with two free SH-groups at the hinge of a single Fab'-B molecule (see Note 3). The remaining free SH groups on Fab'-B are alkylated, and the F(ab')(2) bispecific antibody product (Fab'-A x Fab'-B) is separated by gel filtration chromatography. Each stage of the procedure is checked by HPLC.

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.

The importance of antibody-specificity in determining successful radioimmunotherapy of B-cell lymphoma.

We report the radioimmunotherapy of mouse B-cell lymphoma, BCL1, using a panel of anti-B-cell monoclonal antibodies (MoAb) (anti-CD19, anti-CD22, anti-major histocompatibility complex (MHC) II, and anti-idiotype (Id) radiolabeled with 131-iodine. When administered early in disease (day 4), the 131I-anti-MHCII MoAb cured tumors as a result of targeted irradiation alone, the unlabeled MoAb being nontherapeutic. In contrast, 131I-anti-Id, despite targeting irradiation and having therapeutic activity as an unconjugated antibody, protected mice for only 30 days; 131I-anti-CD19 and anti-CD22 were therapeutically inactive. Binding and biodistribution studies showed that the anti-Id, unlike anti-MHCII, MoAb was cleared from target cells in vivo and delivered 4 times less irradiation to splenic tumor. Treating later in the disease (day 14) increased tumor load and produced the expected reduction in therapeutic activity with the anti-MHCII, but surprisingly, allowed 131I-anti-Id to cure most mice. This unexpected potency of 131I-anti-Id late in the disease appeared to result from the direct cytotoxicity of the anti-Id MoAb, which was more active in established disease, in combination with targeted irradiation. We believe the ability of targeted irradiation and certain cytotoxic MoAb to work cooperatively against tumor in this way has important implications for the selection of reagents in radioimmunotherapy of B-cell lymphoma.

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.

Analysis of the interaction of monoclonal antibodies with surface IgM on neoplastic B-cells.

In vitro studies identified three Burkitts lymphoma cell lines, Ramos, MUTU-I and Daudi, that were growth inhibited by anti-IgM antibody. However, only Ramos and MUTU-I were sensitive to monoclonal antibodies (mAb) recognizing the Fc region of surface IgM (anti-Fc mu). Experiments using anti-Fc mu mAb (single or non-crossblocking pairs), polyclonal anti-mu Ab, and hyper-crosslinking with a secondary layer of Ab, showed that growth inhibition of B-cell lines was highly dependent on the extent of IgM crosslinking. This was confirmed by using Fab', F(ab')2 and F(ab')3 derivatives from anti-Fc mu mAb, where increasing valency caused corresponding increases in growth arrest and apoptosis, presumably as a result of more efficient BCR-crosslinking on the cell surface. The ability of a single mAb to induce growth arrest was highly dependent on epitope specificity, with mAb specific for the Fc region (C mu2-C mu4 domains) being much more effective than those recognizing the Fab region (anti-L chain, anti-Id and anti-Fd mu, or C mu1). Only when hyper-crosslinked with polyclonal anti-mouse IgG did the latter result in appreciable growth inhibition. Binding studies showed that these differences in function were not related to differences in the affinity, but probably related to intrinsic crosslinking capacity of mAb.

Internalization of the lymphocytic surface protein CD22 is controlled by a novel membrane proximal cytoplasmic motif.

CD22 is a key receptor on B-lymphocytes that modulates signaling during antigenic stimulation. We have defined a novel cytoplasmic motif in human CD22 that controls its unusually rapid turnover at the plasma membrane. Chimeric and mutated CD22alpha cDNA vectors were constructed and stably transfected in CD22-negative Jurkat T-lymphocytic cells. Two assays were employed to measure CD22alpha internalization: first, cytoplasmic uptake of radioiodinated anti-CD22 monoclonal antibody; and second, lethal targeting of a toxin, saporin, into cells via CD22 using bispecific F(ab')2 ([anti-CD22 x anti-saporin]) antibody. Results showed that CD22alpha lacking a cytoplasmic tail was not internalized and that replacement of the cytoplasmic tail of CD19 with that of CD22alpha resulted in a chimeric molecule that behaved like CD22alpha and internalized rapidly. Step-wise deletion of the cytoplasmic tail of CD22alpha located the internalization motif to a polar region of 11 residues (QRRWKRTQSQQ) proximal to the plasma membrane, a part of the molecule predicted to form a coil or turn structure. Interestingly, additional CD22 mutants showed that the two glutamine residues sandwiching the serine are critical to internalization but that the serine itself is not.

Monoclonal antibody therapy of B cell lymphoma: signaling activity on tumor cells appears more important than recruitment of effectors.

Despite the recent success of mAb in the treatment of certain malignancies, there is still considerable uncertainty about the mechanism of action of anti-cancer Abs. Here, a panel of rat anti-mouse B cell mAb, including Ab directed at surface IgM Id, CD19, CD22, CD40, CD74, and MHC class II, has been investigated in the treatment of two syngeneic mouse B cell lymphomas, BCL1 and A31. Only three mAb were therapeutically active in vivo, anti-Id, anti-CD19, and anti-CD40. mAb to the other Ags showed little or no therapeutic activity in either model despite giving good levels of surface binding and activity in Ag-dependent cellular cytotoxicity and complement assays, and in some cases inhibiting cell growth in vitro. We conclude that the activity of mAb in vitro does not predict therapeutic performance in vivo. Furthermore, in vivo tracking experiments using fluorescently tagged cells showed that anti-Id and anti-CD40 mAb probably operate via different mechanisms: the anti-Id mAb cause growth arrest that is almost immediate and does not eliminate cells over a period of 5 or 6 days, and the anti-CD40 mAb have a delayed effect that allows tumor to grow normally for 3 days, but then abruptly eradicates lymphoma cells. This work supports the belief that mAb specificity is critical to therapeutic success in lymphoma and that, in addition to any effector-recruiting activity they may possess, in vivo mAb operate via mechanisms that involve cross-linking and signaling of key cellular receptors.

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.

The development of anti-CD79 monoclonal antibodies for treatment of B-cell neoplastic disease.

The B-cell antigen receptor (BCR) consists of cell surface IgM associated with the CD79 alpha/beta heterodimer. In this paper we describe a panel of monoclonal antibodies (mAbs) recognising the extracellular regions of human CD79 alpha and beta. FACS analysis demonstrated that the mAbs bind to a range of Burkitt's lymphoma lines, a mouse B-cell line (JO-72) transfected with human CD79 alpha and beta, and tumour biopsies from NHL patients. The specificity of the mAbs was confirmed by immunoprecipitation. The Ka for the binding of IgG from the anti-CD79 alpha mAbs to cell surface CD79 alpha on Ramos cells was 3 x 10(8) M-1, and their maximum level of binding, 1.7-2 x 10(5) molecules/cell, matched that obtained with anti-Fc mu and anti-Fd mu mAbs. All four anti-CD79 beta mAbs were of lower affinity. Interestingly, in growth arrest studies, we found that while all anti-Fc mu mAbs caused profound inhibition of proliferation of Ramos cells, a range of other anti-BCR mAbs, which included the anti-CD79, anti-Fab mu, anti-gamma and anti-idiotype reagents, all performed poorly giving a maximum of 25% inhibition. These differences in performance are believed to relate to the ability of anti-BCR mAbs to cross-link neighbouring surface BCR and suggest that, unlike anti-Fc mu which favours cross-linking, most of these mAbs are binding in a monogamous, non-cross-linking, union with the BCR.

Treatment of B-cell lymphomas with combination of bispecific antibodies and saporin.

We report the use of a bispecific F(ab')2 antibody to target the ribosome-inactivating protein saporin to the surface antigen CD22 in the treatment of low-grade, end-stage, B-cell lymphoma. Four patients were treated. Toxic effects were minimal (grade I), with mild fever, weakness, and myalgia for 1-2 days after treatment. One patient showed an antibody response to mouse Fab' and saporin. All patients showed rapid and beneficial responses to treatment with improvements in most disease sites and in peripheral blood cytopenia. The responses were short-lived (less than 28 days) but further study of this targeting system is warranted.

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.

Delivery of the ribosome-inactivating protein, gelonin, to lymphoma cells via CD22 and CD38 using bispecific antibodies.

It is well established that bispecific antibodies (BsAbs) can be used effectively in targeting the ribosome-inactivating protein (RIP), saporin, against neoplastic B cells. We have now extended this delivery system for use with gelonin. By measuring antigen-binding characteristics and epitope mapping a panel of anti-gelonin MAbs using the IAsys resonant mirror bisensor, we were able to rapidly select the most suitable for making BaAbs. The Fab' fragments from these MAbs were chemically conjugated with Fab' from either anti-CD22 or anti-CD38. Cytotoxicity assays showed that BsAbs were highly efficient at delivering gelonin to cultured Daudi cells and achieved levels of toxicity which correlated closely with the affinity of the BsAbs. Using pairs of anti-CD22 BsAbs we were able to generate bivalent BsAb-gelonin complexes which achieved IC50 values of 2 x 10(-11) M gelonin, a potency which is equivalent to that reached by saporin in this targeting system. However, because gelonin is 5-10 times less toxic than saporin, the therapeutic ratio for gelonin is superior, making it potentially a more useful agent for human treatment. Cytotoxicity assays and kinetic analysis showed that targeting gelonin via CD38 was 2-5 times less effective than delivery through CD22. However, with a pair of BsAbs designed to co-target gelonin via CD22 and CD38, the cytotoxicity achieved equalled that obtained with a pair of anti-CD22 BsAbs (IC50 = 1 x 10(-11) M). This important result suggests that the anti-CD38 helps bind the gelonin to the cell and is then 'dragged' or 'piggy-backed' into the cell by the anti-CD22 BsAb. The implication of these findings for cancer therapy is discussed.

Mechanisms in removal of tumor by antibody.

We report two preliminary trials of antibody treatment of B-cell lymphoma. Advanced lymphoma was treated with chimeric FabFc2, in which mouse Fab' gamma is linked to two human Fc gamma 1 fragments so as to recruit natural effectors to tumor targets. Terminal lymphoma was treated with bispecific antibody (BsAb) which recruits the ribosome-inactivating protein saporin. These different mechanisms led to interesting differences in patterns of tumor clearance. Eight patients were treated with chimeric antibody of two specificities, each at 12 mg/kg: anti-CD37, plus either anti-CD38 or anti-CD19 according to tumor phenotype. On completion of the 3-wk treatment, residual plasma antibody had a half-life exceeding 10 d. Tumor cells in blood disappeared rapidly. However significant reductions in solid masses occurred in only three patients, becoming apparent 3-4 wk after beginning treatment and then continuing slowly. Five patients were treated with preformed immune complexes of saporin and F(ab' gamma)2 BsAb. Although doses of saporin reached 10 mg weekly, contact with the tumor can only have been fleeting: plasma antibody was undetectable (< 0.5 micrograms/mL) 48 h after infusion, whereas the saporin disappeared even faster and was undetectable (< 4 ng/mL) at 24 h. Tumor cells disappeared from the blood more slowly than occurred with chimeric antibody. In contrast shrinkage of extravascular tumor was more rapid, and occurred in all patients, but proved less durable.

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.

Initial experience in treating human lymphoma with a combination of bispecific antibody and saporin.

Results are presented showing the use of bispecific F(ab')2 antibodies (bsAbs) in the delivery of saporin for the treatment of 2 human B-cell malignancies. BsAbs delivering saporin through CD22, but not through CD19, were effective at inhibiting the uptake of [3H]leucine by Daudi and Raji cells. Furthermore, a combination of 2 anti-CD22 bsAbs, selected to bind simultaneously to saporin, bound saporin 20 times more avidly and inhibited protein synthesis far more efficiently than any single bsAb. In the first patient, with end-stage chronic lymphocytic leukaemia (CLL), treatment with 10 mg of saporin complexed to 100 mg of anti-CD19 bsAb over 43 days showed no therapeutic effect. In contrast, the second patient, with end-stage non-Hodgkin's lymphoma (NHL), given 5 mg of saporin complexed with a pair (50 mg) of anti-CD22 bsAbs over 15 days showed a marked clinical response, including complete clearance of tumour from the blood, clearance of ascites and shrinkage of tumour masses. Neither patient experienced any toxic side-effects, either during or after treatment. However, the second patient developed a strong anti-mouse Fab (HAMA) response 28 days after the treatment started. No anti-saporin response could be detected.

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.

A study of GDP binding to purified thermogenin protein from brown adipose tissue.

1. The binding of GDP to purified thermogenin protein was studied by using fluorescence spectroscopy and equilibrium dialysis. 2. GDP binding to thermogenin diminished fluorescence emission in a concentration-dependent manner that exhibited saturation. 3. Kd values for binding of nucleoside di- and tri-phosphates were lower than those for nucleoside monophosphates. 4. The GDP-induced fluorescence quenching was decreased by increasing pH, but the apparent Kd was unaltered by pH changes. 5. Equilibrium dialysis showed a Kd change from 3 to 6 microM when the pH was increased from 6.6 to 8.5. 6. The apparent pK of the fluorescence changes induced by pH (8.3) was identical with the apparent pK of the GDP-binding response. 7. The data are consistent with the existence of protonated and non-protonated forms of thermogenin protein that both bind GDP.