Development, validation and application of ELISAs for pharmacokinetic and HACA assessment of a chimeric anti-CD40 monoclonal antibody in human serum.

As part of a Phase I chimeric anti-CD40 monoclonal antibody clinical trial, two enzyme-linked immunosorbent assays (ELISAs) were developed for secondary endpoints: 1) for the pharmacokinetic (PK) monitoring of serum antibody levels and 2) for immunogenic screening of human anti-chimeric antibody (HACA) responses. The ELISA is a well established immunoassay, with clear guidelines for validation when used as a quantitative assay. However, these parameters may not always be relevant for a semi-quantitative assay used to assess whether a sample is positive or negative for a novel marker such as an antibody developed against a therapeutic antibody. We report here the development of a quantitative PK ELISA and a semi-quantitative HACA ELISA, and the different approaches of validation to prove each assay are 'fit for purpose.' The parameters of linearity (R²>0.99), accuracy (±30%), lowest level of detection (4 µg/ml), intra-assay (coefficient of variation (CV) <20%) and inter-assay (CV<20%) variability were assessed for the quantitative PK assay. For the semi-quantitative HACA assay, parameters of linearity (R²>0.99), lowest level of detection, intra (CV<10%) and inter-assay (CV<30%) variability were assessed using a surrogate positive control. The validation outcome showed that each assay was robust, reliable and accurate to meet the requirements of the intended analytical application, that being to 1) quantitatively determine the concentration of antibody in the serum and 2) determine whether a sample is positive or negative for human anti-chimeric antibodies. Each assay has been successfully translated for use in a clinical trial with adequate quality controls and acceptance criteria set for monitoring consistency and performance.

Validation of an ELISA for the determination of rituximab pharmacokinetics in clinical trials subjects.

Rituximab is a chimeric anti-CD20 monoclonal antibody that has revolutionised the treatment of many B-cell malignancies, and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum rituximab levels are highly variable in patients receiving similar 'standard' approved doses. Little is known regarding the factors that affect serum rituximab concentration and that in turn may influence clinical outcome. In order to provide a tool that may ultimately enable patient specific dosing of rituximab therapy, we have validated a reliable, robust ELISA for the quantitation of serum rituximab levels to provide accurate pharmacokinetic (PK) data that will guide the optimisation of rituximab dosing regimes. Extensive validation of the assay was performed in order to utilise the assay for clinical applications. The within and between day plate coating reproducibility was tested and proved a robust starting platform for the assay. The within day precision for the assay was determined using spiked serum samples and was shown to have a coefficient of variation (CV) of <10% with an accuracy between 91 and 125%. The between day precision (CV) was <25% with an accuracy between 95 and 109%. Dilution linearity and parallelism were demonstrated. Spike recovery for all concentrations and donors was shown to be within +/-15% on average, with a CV below 10%. This assay is highly accurate and reproducible in determining the levels of rituximab in spiked serum samples. It meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating rituximab in the treatment of lymphoma. This assay represents a useful tool for clinical application of this widely used therapeutic.

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.

Therapeutic efficacy of FcgammaRI/CD64-directed bispecific antibodies in B-cell lymphoma.

CD64 (FcgammaRI) receptors represent highly potent trigger molecules for activated polymorphonuclear cells (PMN) and mediate lysis of a range of tumors in the presence of appropriate monoclonal antibodies. An huCD64 transgenic mouse model designed to analyze the therapeutic activity of a panel of bispecific F(ab')(2) (BsAb) in retargeting granulocyte-colony-stimulating factor (G-CSF)-activated PMN against syngeneic B-cell lymphomas is reported. This model allows careful analysis of the individual elements of the therapeutic process. BsAb were directed against immunoglobulin-idiotype (Id), major histocompatibility class II (MHC II), or CD19 on the tumors and huCD64 on the effectors. In vitro cytotoxicity assays and in vivo tumor tracking showed that, provided effectors were activated with G-CSF, all 3 derivatives destroyed and cleared lymphoma cells, with (huCD64 x MHC II) proving by far the most cytotoxic in vitro. However, though all derivatives delivered some survival advantage, only the [huCD64 x Id] BsAb provided long-term protection to tumor-bearing animals. These results demonstrate that CD64-recruited cytotoxic effectors operate in vivo but that the (huCD64 x Id) conferred an additional anti-tumor function essential for long-term protection. T-cell depletion studies demonstrated that this extra therapeutic activity with [huCD64 x Id] was totally dependent on CD4 and CD8 T cells and that mice, once "cured" with BsAb, were resistant to tumor rechallenge. These findings indicate that CD64 is an effective trigger molecule for delivering cytokine-activated PMN against tumor in vivo and that, provided tumor targets are selected appropriately, CD64-based BsAb can establish long-term T-cell immunity.

Triggering Fc alpha-receptor I (CD89) recruits neutrophils as effector cells for CD20-directed antibody therapy.

CD20 Abs induce clinical responses in lymphoma patients, but there are considerable differences between individual patients. In (51)Cr release assays with whole blood as effector source, RAJI cells were effectively killed by a mouse/human chimeric IgG1 construct of CD20 Ab 1F5, whereas ARH-77 proved resistant to killing by this Ab. When whole blood was fractionated into plasma, mononuclear cells, or granulocytic effector cells, RAJI cells were effectively killed in the presence of complement-containing plasma, whereas the mature B cell line ARH-77 proved complement resistant. However, with a bispecific Ab (BsAb) against the myeloid receptor for IgA (CD89; FcalphaRI) and CD20, a broad range of B cell lines were effectively killed. FcalphaRI is expressed on monocytes/macrophages, neutrophils, and eosinophils. As the numbers of these effector cells and their functional activity can be enhanced by application of G-CSF or GM-CSF, lysis via (FcalphaRI x CD20) BsAb was significantly enhanced in blood from patients during therapy with these myeloid growth factors. Interestingly, the major effector cell population for this BsAb were polymorphonuclear neutrophils, which proved ineffective in killing malignant B cells with murine, chimeric IgG1, or FcgammaRI- or FcgammaRIII-directed BsAbs against CD20. Experiments with blood from human FcalphaRI/FcgammaRI double-transgenic mice showed corresponding results, allowing the establishment of relevant syngenic animal models in these mice. In conclusion, the combination of myeloid growth factors and an (FcalphaRI x CD20) BsAb may represent a promising approach to improve effector cell recruitment for CD20-directed lymphoma therapy.

Rheumatoid arthritis T cells produce Th1 cytokines in response to stimulation with a novel trispecific antibody directed against CD2, CD3, and CD28.

Rheumatoid arthritis (RA) T cells respond poorly to conventional mitogens. We have examined the proliferative and cytokine responses of T cells to a synthetic trispecific antibody (Tsab) directed against CD2, CD3, and CD28. In 11 subjects RA T cells proliferated more, and secreted significantly more IL-2, in response to Tsab than did control peripheral blood (PB) cells. Very high levels of IL-2 were produced by 2 patients with aggressive disease. Measurement of intracellular IL-2, IFN-gamma, IL-4, and IL-5 by flow cytometry showed a Th1 pattern of cytokine production in 13 RA and 9 control subjects. We conclude that RA T cells are not irreversibly inactivated, and that spatial arrangement of stimulating molecules may be important in eliciting maximal responses.

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.

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.

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.

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.

Generation of HER-2/neu-specific cytotoxic neutrophils in vivo: efficient arming of neutrophils by combined administration of granulocyte colony-stimulating factor and Fcgamma receptor I bispecific antibodies.

Abs are able to induce inflammatory antitumor responses by recruiting IgG Fc receptor (FcgammaR)-bearing cytotoxic effector cells. We recently described the capacity of the high affinity FcgammaRI (CD64) to trigger cytotoxic activity of neutrophils (PMN) during granulocyte CSF (G-CSF) treatment. To take advantage of FcgammaRI as a cytotoxic trigger molecule on PMN, two Ab constructs were prepared. We show that a chimeric human IgG1 Ab (Ch520C9) and an anti-FcgammaRI bispecific Ab (BsAb; 22x520C9), both directed to the proto-oncogene product HER-2/neu, interact with FcgammaRI. In addition, both Ab constructs mediate enhanced lysis of HER-2/neu-expressing tumor cells by G-CSF-primed PMN. However, engagement of FcgammaRI by Ch520C9 was inhibited by human serum IgG, thereby abrogating the enhanced Ch520C9-mediated cytotoxicity. BsAb 22x520C9, which binds FcgammaRI outside the ligand binding domain, effectively recruits the cytotoxic potential of FcgammaRI on G-CSF-primed PMN regardless of the presence of human serum. These results indicate that under physiologic conditions, serum IgG impairs activation of FcgammaRI-mediated cytotoxicity by conventional antitumor Abs. The IgG blockade can be circumvented with anti-FcgammaRI BsAbs. Using human FcgammaRI transgenic mice we demonstrate that BsAb 22x520C9 is able to engage FcgammaRI in vivo. BsAb 22x520C9 injected i.v. was readily detected on circulating PMN of G-CSF-treated transgenic animals. In addition, we showed that PMN remain "armed" with BsAb 22x520C9 during migration to inflammatory sites, and that after isolation such PMN specifically lyse HER-2/neu-expressing tumor cells. These results point to the possibility of targeting anti-FcgammaRI BsAbs to G-CSF-primed PMN in vivo, endowing them with specific anti-tumor activity.

Bispecific Ab therapy of B-cell lymphoma: target cell specificity of antibody derivatives appears critical in determining therapeutic outcome.

Despite the success of mAb and bispecific (bs)Ab in the treatment of certain malignancies, there is still considerable uncertainty about the most appropriate format in which they should be used. In the current work we have investigated a panel of bsAb [IgG and F(ab)2] with dual specificity for T cells and neoplastic B cells. Throughout this work, anti-CD2 or anti-CD3 were used to bind the mouse T cells, and antibodies to surface IgM idiotype (Id), CD19, CD22, or MHC class II were used to target mouse B cell lymphomas BCL1 or A31. In vitro, killing was measured in a conventional cytotoxicity assay using 51Cr-labelled A31 and BCL1 cells as targets and activated mouse splenocytes as effectors. bsAb showed a wide range of cytotoxic activities, which could be ranked in the following order: [anti-CD3 x anti-class-II] > [anti-CD3 x anti-CD19] > [anti-CD3 x anti-Id] > [anti-CD3 x anti-CD22], with the [anti-CD2 x anti-Id] derivative showing relatively little cytotoxic activity. This hierarchy of activity indicates some correlation with the binding activity of the bsAb on target cells, but showed a much stronger parallel with the tendency of the anti-(target cells) mAb to undergo antigenic modulation (less modulation, more killing). In vivo, the situation was completely different and only the anti-Id derivatives, [anti-CD3 x anti-Id] and [anti-CD2 x anti-Id], were effective in prolonging the survival of tumour-bearing animals. Under optimal conditions Id-positive tumour was eradicated with a single treatment of bsAb. We conclude from this work that the target cell specificity of a bsAb is critical in determining therapeutic outcome and that in vitro cytotoxicity assays do not predict in vivo activity.

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.

Activation and preferential expansion of rat cytotoxic (CD8) T cells in vitro and in vivo with a bispecific (anti-TCR alpha/beta x anti-CD2) F(ab')2 antibody.

We show that a bispecific F(ab')2 Ab (BsAb), which cross-links the TCR to CD2 ([anti-CD2 x anti-TCR]) and is highly mitogenic in vitro, also induces marked T cell activation and proliferation when given as a small single dose (50 to 100 micrograms) to rats. Interestingly, the proliferation appeared selective for CD8 cells, increasing this compartment more than 20 times over 72 h. This resulted in a three- to fourfold increase in spleen weight, with histologic evidence of T-zone and red pulp expansion by CD8+ blast cells. Such changes were in striking contrast to those seen after a similar amount of the parent IgG anti-TCR mAb, R73, which, while inducing a transient increase in CD25+ cells, failed to alter the lymphocyte composition. The mitogenic activity of the BsAb was totally dependent on its ability to cross-link CD2 and the TCR, hence a mixture of anti-CD2 and anti-TCR F(ab')2 fragments was without effect in vitro or in vivo. Unlike normal rat T cells or those taken from R73 IgG-treated rats, blood and splenic T cells recovered from BsAb-treated rats were highly cytotoxic against R73 hybridoma targets that express surface anti-TCR mAb and consequently trigger the lytic activity of activated CTL or a rat lymphoma line, LAMA (Thy-1+), using a second BsAb, [anti-TCR x anti-Thy-1], to retarget the CTL. This latter assay provides the basis for a future two-stage targeting strategy for cancer immunotherapy in which a mitogenic BsAb would be given to patients to activate CTL nonspecifically, and then, at an appropriate time, a second BsAb [anti-TCR x antitumor] would be given to deliver activated cells to the tumor target cells.

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.

CD3 and CD2 antigen-mediated CD3 gamma-chain phosphorylation in permeabilized human T cells. Regulation by cytosolic phosphatases.

The role of cytosolic and membrane-associated phosphatases in regulating dephosphorylation of the CD3 antigen gamma-chain has been investigated using streptolysin-O-permeabilized T lymphoblasts and Jurkat T leukaemia cells. Permeabilization of T cells caused a rapid extrusion of cytosolic type 2A phosphatases, but a membrane-associated phosphorylase phosphatase activity remained inside the cells. This activity had the properties characteristic of type 2A phosphatases, being resistant to inhibition by type 1 phosphatase inhibitors, though it was inhibited in a time-dependent manner by ATP or by non-hydrolysable ATP analogues, but not by GTP, CTP, ITP or PPi. The membrane-associated type 2A phosphatase in permeabilized cells did not dephosphorylate the CD3 antigen gamma-chain, suggesting that cytosolic phosphatases dephosphorylate the gamma-chain in situ. Cross-linking the CD2 and CD3 antigens with a bivalent monoclonal antibody in the absence of cytosolic phosphatases induced marked phosphorylation of the CD3 gamma-chain, immunoprecipitated using a novel gamma-chain peptide analogue directed antiserum (TG1). Phosphorylation was inhibited by a protein kinase C (PKC) pseudosubstrate inhibitor, indicating that CD2/CD3-induced gamma-chain phosphorylation is a PKC-mediated event. Activation of T cells either with phorbol 12,13-dibutyrate or by CD2-CD3 cross-linking caused [32P]Pi incorporation into the same gamma-chain Ser residues. The site-mapping data suggested that PKC in situ may incorporate phosphate at the CD3 gamma-chain Ser-123 and Ser-126 residues, but that phosphate is rapidly lost from Ser-123 by cytosolic phosphatase action. Our findings underline the importance of the dual actions of kinases and phosphatases as potential regulators of T cell antigen-receptor complex function.

The cytotoxic action of lymphokine activated killer cells upon the human glioma cell line U251 is stimulated by bispecific monoclonal antibody (MoAb) constructs.

The ability of IL-2 stimulated mononuclear cells to kill the human glioblastoma cell line U251 has been investigated. Highest cytotoxic activity was generated in low cell density cultures incubated for 15 days with 250-1000 U/ml IL-2. Sub-optimal killing was noted, with cells only exposed to IL-2 for three days. Under the latter conditions, bispecific monoclonal antibodies (MoAbs) of either anti-CD3 or anti-CD16 and an anti-NCAM MoAb stimulated LAK cell activity markedly. Anti-CD16 conjugates were found more effective than anti-CD3 and (Fab')2 constructs more efficacious than those made with whole Ig molecules. Maximal stimulation of LAK cell activity was noted with bispecific MoAbs. Little effect was observed with either single or mixtures of monomeric MoAbs. Furthermore, no effect of bispecific MoAbs was observed when target cells lacked expression of NCAM. These results could be of clinical importance as it is not always feasible to screen LAK cells for optimal activity before administration to patients. Whilst bispecific MoAbs have no effect on optimally stimulated LAK cells, they are not inhibitory and can stimulate killing under sub-optimal IL-2 stimulation.

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.

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.

CD2 antigen mediated activation of the guanine nucleotide binding proteins p21ras in human T lymphocytes.

T cell stimulation via the TCR complex (TCR/CD3 complex) results in activation of the guanine nucleotide binding proteins encoded by the ras protooncogenes (p21ras). In the present study we show that the activation state of p21ras in T lymphocytes can also be controlled by triggering of the CD2 Ag. The activation state of p21ras is controlled by GTP levels on p21ras. In T cells stimulation of protein kinase C is able to induce an accumulation of "active" p21ras-GTP complexes due to an inhibitory effect of protein kinase C stimulation on the intrinsic GTPase activity of p21ras. The regulatory effect of protein kinase C on p21ras GTPase activity appears to be mediated via regulation of GAP, the GTPase activating protein of p21ras. In the present report, we demonstrate that the TCR/CD3 complex and the CD2 Ag control the accumulation of p21ras-GTP complexes via a regulatory effect on p21ras GTPase activity. The TCR/CD3 complex and CD2 Ag are also able to control the cellular activity of GAP. These data demonstrate that p21ras is part of the signal transduction responses controlled by the CD2 Ag, and reveal that the TCR/CD3 complex and CD2 Ag control the activation state of p21ras via a similar mechanism.