Engineered antibody for treating lymphoma.

One approach to improving the results of antibody treatment of posttransplant and other lymphomas is to alter the geometry of the antibody molecule so as to enhance its cytotoxicity. When antibody alights on a cell, cytotoxicity can be exerted by initiation of apoptotic signals at the cell surface, and by recruitment of effectors to specific sites on the antibody Fc region. Other routes to cytotoxicity have been described but their generality is debatable. The effectors consist of complement, and a series of potentially cytotoxic cells (macrophages, NK cells, neutrophils and others) bearing Fc-receptors (FcR). Recent evidence suggests that the FcR-bearing cells are the more important in dealing with antibody-coated autologous cells, including tumor cells. If the antibody happens to be of the host IgG class then its Fc region (Fcgamma) will contain a site for attachment of a non-cytotoxic FcR known as FcRB (the Brambell receptor): it is this receptor, present on endothelial cells, which recycles the molecule intact if it happens to be endocytosed and thus prolongs its metabolic life. Replacement of the mouse Fcgamma by human Fcgamma is expected to have three beneficial effects: better recruitment of human effector cells, better persistence of the antibody in extracellular fluid, and removal of a major source of immunogenic epitopes. Chemical manipulations of Fab'gamma and Fcgamma modules, linking them in different geometries via their hinge regions, offers constructs with further enhancements of cytotoxicity. These include Fab2Fc2, in which the presence of dual Fc regions enhances recruitment of both complement and cellular effectors; and bispecific antibody of the same modular formula in which one of the Fab arms is specific for tumor while the other is specific for, and recruits powerfully, a cellular effector.

Thioether-bonded constructs of Fab'gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds.

We describe a two-stage preparation of chemically engineered Ab constructs, employing as modules Fab'gamma from mAb or rAb, and Fc from human normal IgG1. A multivalent, optionally multispecific F(ab')(n) core is formed in stage one, and one or more Fc modules added in stage two. Examples include bispecific Fab(2)Fc(2) (for simplicity, primes and Greek letters are omitted from names of final constructs) and trivalent Fab(3)Fc(2), which are designed to kill neoplastic cells. An essential element in the construction is the availability of the Fab' in two reduced forms, Fab'(-sulfhydryl (SH))(5) and Fab'-SH. The first is obtained by full reduction of the interchain disulfide bonds (SS) in the F(ab')(2) fragment of IgG. Fab'-SH is obtained by disulfide-interchange reactions on Fab'(-SH)(5), whereby the gamma-light SS is reconstituted, an unusual intrachain SS forms in the gamma-chain hinge, and one hinge SH remains. F(ab')(2) and F(ab')(3) cores are built using partially reduced modules, being given intermodular thioether links that resist reduction. These cores are then fully reduced, making available SH groups for addition of the Fcgamma modules. In the final constructs, all intermodular links embody tandem thioether bonds arising at hinge-region cysteines. Cytotoxic activities of representative constructs, and some enhancements deriving from multiple modules, are assessed. In guinea pigs, catabolism of Fab(2)Fc(2) yielded a t(1/2) similar to that of human IgG1, although the serum Fab(2)Fc(2) revealed some proteolytic breakdown not shown by the IgG1. Immunotherapy of a guinea-pig leukemia confirmed the ability of these constructs to kill target cells in vivo.

Molecular markers of B-cell lymphoma.

Molecular mapping of the cell surface has probably proceeded further with the human lymphocyte than with any other mammalian cell, and the B lymphocyte yields a wide range of subtly varying neoplasms. These two bodies of knowledge are now readily correlated, given the widespread adoption of a modern lineage-based classification of lymphoma (Revised European-American). Studies of the markers of B-cell lymphoma have immediate practical importance in diagnosis, defining clonality, and detecting minimal residual disease. They also help to keep us abreast of lymphocyte physiology, and present new opportunities for treating these neoplasms.

Preparation of fcgamma for addition to sulfhydryl-expressing ligands with minimal disturbance of the hinge.

We described previously a scheme for linking functionally intact human normal Fcgamma1, via a thioether linkage emerging from its hinge, to any molecule expressing a free sulfhydryl group (SH). The scheme entails reducing the Fc to release four SH from the two inter-gamma disulfide bonds (SS) in the hinge, blocking one SH by a stochastic alkylation, restoring by SS-interchange the inter-gamma SS whose two SH are still available, and attaching a bismaleimide linker to the one remaining SH. One thereby obtains Fc with a single maleimide group (Fc-maleimide) for attachment to the SH-displaying partner. Restoration of the inter-gamma SS is necessary if the final chimeric construct is to be able to activate the classical complement pathway. However, during this preparation of Fc-maleimide, there is apparently some SS-formation between non-homologous SH, so that not all hinges emerge with a reconstituted inter-gamma SS. To reduce this error we have modified the preparative procedure after investigating an initial partial reduction of the hinge, and reviewing the conditions for stochastic alkylation. During partial reduction by dithiothreitol, the two hinge SS were cleaved apparently randomly: there was no evidence for one bond being more susceptible to reduction than the other, and little indication that the reduction of one bond enhanced the susceptibility of the other. By limiting reduction to an average of one SS per molecule, and alkylation to 0.8 SH per molecule, a final Fc-maleimide product is obtained in which most of the molecules have passed through the entire preparation with one of their hinge SS, and by inference much of the hinge conformation, remaining intact.

Conjugation of human Fc gamma in closed-hinge or open-hinge configuration to Fab'gamma and analogous ligands.

We describe a method for linking human normal Fc gamma1, via stable thioether bonds emerging from its hinge, to any molecule expressing a free sulfhydryl (SH) group. The Fc hinge may be closed by a disulfide (SS) bond or left open. Preparation begins with reduction of the Fc hinge to release four SH groups from its two parallel inter-gamma SS bonds. When the Fc is required in normal closed-hinge configuration, one SH group is alkylated with N-ethylmaleimide under limiting conditions, and one of the inter-gamma SS bonds is reconstituted by SS interchange. The residual SH group, to be used for linking, is left as a 4-dithiopyridyl group suitable for storage. When the Fc is required for conjugation the 4-dithiopyridyl is replaced by a metastable maleimidyl group, which reacts rapidly with SH on the partner molecule to form a tandem thioether link. If the partner is Ab Fab'gamma, linking to cysteines in the Fab'gamma hinge yields derivatives such as FabFc and FabFc2. Chimeric FabFc Abs (mouse Fab'gamma/human Fc gamma1) invoked cellular cytotoxicity in vitro, using human cell lines as targets and human lymphocytes as effectors, whether the Fc hinge was open or closed. The same Abs could kill the same targets by activating human complement, but only when the Fc hinge was closed. Both effector functions were enhanced by the presence of a second Fc in FabFc2. This method of Fc addition can be used to predict the performance of recombinant chimeric Abs and to provide novel molecular geometries.

Tumour cell killing using chemically engineered antibody constructs specific for tumour cells and the complement inhibitor CD59.

Immunotherapy using MoAbs is inefficient due to limited activation of human effectors by mouse antibodies and multiple protective mechanisms available to host cells against autologous complement. We have used chemically engineered antibody constructs and human complement in vitro to specifically target and kill neoplastic B lymphoid cells (Raji). Fab'gamma Fc gamma2 chimaeric antibody (specific for human CD37) was used to activate the classical pathway of human complement on Raji cells, whilst CD59 was neutralized using one of two different bispecific F(ab'gamma)2 antibody constructs which contained both cell-targeting (anti-CD19 or anti-CD38) and CD59-neutralizing moieties. When either bispecific construct was used to neutralize CD59, 15-25% of cells were lysed. If CD55 was also neutralized using specific antibody, Raji cells were efficiently killed (70% lysis). When added to a mixture of target (Raji) and bystander (K562) cells, one bispecific antibody (anti-CD38 x anti-CD59) could be specifically delivered to Raji, avoiding significant uptake on CD59-expressing bystander cells (K562). The second bispecific antibody (anti-CD19 x anti-CD59) bound equally well to either cell type. Cell-specific targeting was dependent upon combination of a low-affinity anti-CD59 Fab'gamma with a high-affinity anti-tumour cell Fab'gamma. When Raji and K562 cells were mixed and incubated with a combination of the engineered constructs and anti-CD55 antibodies, Raji cell lysis (30-40%) was observed in the absence of K562 killing. We propose that combinations of these constructs may be of use for treatments such as ex vivo purging of autologous bone marrow or in vivo targeting of tumour cells.

HLA class II as potential target antigen on malignant B cells for therapy with bispecific antibodies in combination with granulocyte colony-stimulating factor.

We have investigated the capacity of polymorphonuclear phagocytes (PMN) to lyse malignant B-cell lines using antibodies and antibody derivates to a range of different B-cell antigens. PMN were found to mediate lysis of all tested B-cell lines in the presence of HLA class II antibodies L227, L243, F3.3, and CR3/43. Target cell lysis was significantly enhanced when PMN isolated during granulocyte colony-stimulating factor (G-CSF) treatment were compared with PMN from healthy donors. Only G-CSF primed PMN, expressing Fc gamma RI (CD64), lysed B cells in the presence of monoclonal antibody (MoAb) 1D10 or Lym-1 to HLA class II related epitopes. Remarkably, PMN were consistently unable to kill malignant B cells with antibodies to the B-cell related antigens CD19, CD20, CD21, CD37, and CD38. These target antigen restriction was not observed with mononuclear effector cells, which mediated cytotoxicity with antibodies to HLA class II, but also with mouse/human chimeric constructs to CD19, CD37, and CD38. Blocking studies with Fc gamma RI antibodies and reverse antibody-dependent cellular cytotoxicity (ADCC) experiments against Fc gamma R antibody expressing hybridoma targets confirmed the pivotal role of Fc gamma RI in enhanced killing by G-CSF primed neutrophils. Bispecific antibodies (BsAb) with one specificity for Fc gamma RI, and another for a tumor associated antigen, offer an interesting approach to improve effector cell recruitment for immunotherapy. In our studies, very effective lysis was observed with G-CSF primed PMN and an [HLA class II x Fc gamma RI] BsAb. The therapeutic implications of these findings and the possible use of BsAb in combination with G-CSF are discussed.

Engineered anti-CD38 monoclonal antibodies for immunotherapy of multiple myeloma.

Multiple myeloma is a malignancy of plasma cells for which there is no effective treatment. To develop an immunotherapeutic agent, we have raised a high affinity mAb (AT13/5) against CD38, one of the few well-characterized surface Ags present on myeloma cells. Since murine monoclonals have many disadvantages as human therapeutics, we prepared two engineered forms of the Ab: a CDR-grafted humanized IgG1 and a chimeric FabFc2 (mouse Fab cross-linked to two human gamma 1 Fc). To retain affinity in the humanized Ab, a number of changes were required to the human framework regions of the heavy chain. In particular, through systematic mutagenesis and computer modeling, we identified a critical interaction between the side chains of residues 29 and 78, which may be important for the humanization of other Abs. The properties of the humanized IgG1 and FabFc2 constructs were compared in a series of in vitro tests. Both constructs efficiently directed Ab-dependent cellular cytotoxicity against CD38-positive cell lines, but C was activated only poorly. Neither construct caused down-modulation of CD38, nor did they affect the NADase activity of CD38. Despite their differing structures, both Abs showed similar activity in most assays, although the humanized IgG1 was more potent at inducing monocyte cytotoxicity. These data represent the first direct comparison of CDR-grafted and chimeric FabFc2 forms of the same Ab, and offer no support for the perceived advantages of the FabFc2. These Abs show promise for therapy of multiple myeloma and other diseases involving CD38-positive cells.

Linkage of silver to antibodies through 2-imino thiolane.

Earlier studies described the linkage of silver to antibodies using SH groups generated by the reduction of the SS groups using ascorbic acid (1) analogous to the Thakur and DeFulvio technique for linking technetium to antibodies. This work describes the linkage of silver to IgG after introducing SH groups by coupling the IgG to 2-imino thiolane. The protein was dissolved in sodium acetate buffer pH 4.5 containing 1 mM EDTA by dialysis/gel chromatography in a concentration of 20 mg/mL. 2-Imino thiolane dissolved in Tris-HCl acetate buffer, pH 8.2, 0.2M was added to give a final dilution of 0.2 mM 2-imino thiolane. The excess of 2-imino thiolane was removed by dialysis or G-25 Sephadex gel chromatography and then the protein was reacted with silver nitrate 0.1 mM. The unreacted SH groups were blocked by adding iodoacetamide to a concentration of 5 mM. The nonprotein reagents again were removed by dialysis or gel chromatography. The thiol groups were titrated using 1.5 mM 2 2-Py-SS-Py prior to and after addition of silver. It was observed that depending on the concentration of silver, 50-80% of the SH groups were coupled to silver. Higher concentrations of silver led to insoluble precipitates and should be avoided.

Design of trials for antibody treatment of tumour.

Our discussion is summarized in Fig. 1. Cytotoxic drugs are shown with the classical sigmoid relationship of response to log dose, with a parallel increase in toxicity which suits the Phase I approach. With antibody it is doubtful that clinical tumour ever responds to single doses with an asymptote of 100% ablation, so arbitrarily lower asymptotes have been used, higher for immunotoxins than for antibodies (n). The response curves have been made sigmoid simply by selecting, among the infinity of mathematical functions, two hypothetical functions of dose which yield sigmoid transformations of the data. After superimposing on the graphs the occurrence of toxicity, one discerns with antibodies (n) only a threshold dose, above which any toxicity will be highly dependent on the extent of encounter with readily accessible cellular or molecular antigen. Immunotoxins display an increasing toxicity with dose, but the positioning of the toxicity gradient in relation to dose is uncertain because of the ameliorating effect of uptake by tumour. The toxicity of an antibody and its derivatives can and should be clearly documented, but a case is presented here against trials of these reagents which systematically seek MTDs with only subsidiary attention to therapeutic effects. With concurrent evaluation of therapeutic effects. With concurrent evaluation of therapy and toxicity--in studies labelled Phase I/II in the current nomenclature--it may prove just as appropriate to relate toxicity to therapeutic efficacy as to dose.

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.

The role of apoptosis in antibody-dependent cellular cytotoxicity.

Apoptosis in three lymphoma cell lines has been studied following cytotoxicity induced in vitro by normal human blood lymphocytes utilizing either natural killer (NK) or antibody-dependent cellular cytotoxic (ADCC) mechanisms. Guinea-pig L2C leukaemic lymphocytes, but not the human cell lines Daudi and Jurkat, revealed a degree of time- and temperature-dependent apoptotic death upon simple culture in vitro. NK cytotoxicity at low effector:target ratios (E:T) induced both release of 51Cr and apoptosis. However NK cytotoxicity at higher E:T, and ADCC at all E:T, increased the level of 51Cr release while reducing the level of apoptosis. The findings were consistent with the apoptotic process being cut short by intervention of necrotic death. The same characteristics accompanied ADCC whether the effectors were recruited by Fc gamma regions of antibody coating the targets, or by bispecific antibodies attaching one arm to the targets and the other to Fc gamma receptors type III on effectors. This finding, and the high level of cytotoxicity elicited by the bispecific method, confirm the belief that NK cells, in addition to exerting NK cytotoxicity, represent the principal effectors for ADCC among blood mononuclear cells. Our results suggest that NK cells have both apoptotic and necrotic mechanisms available for killing their targets, but use only the latter for ADCC.

Subpopulations of guinea-pig T lymphocytes defined by isoforms of the leucocyte common antigen.

This report presents the characterization of three mouse monoclonal antibodies (mAb) reactive with the guinea-pig leucocyte common antigen (LCA); CD45 in the human nomenclature. One, IH-1, reacted with LCA on all leucocytes. The other two were more restricted: IH-2 recognized only the 220,000, 210,000 and 195,000 MW isoforms, and IH-4 the 220,000, 210,000 MW isoforms. Both IH-2 and IH-4 reacted with all B cells and all Kurloff cells [the putative guinea-pig natural killer (NK) cell]. IH-2, but not IH-4, reacted with monocytes and macrophages. Neither reacted with neutrophils. Most thymocytes expressed low levels of the IH-2 and IH-4 epitopes, with those expressing high levels located predominantly within the medulla. Most (90%) CD4+ T cells from newborn guinea-pigs expressed high levels of the IH-2 and IH-4 epitopes; this percentage decreased with age to 70% in 2-year-old animals. We have demonstrated that CD4+ T cells which express low levels of the IH-2 epitope also express low levels of the IH-4 epitope. CD8+ T cells can be divided into two subsets by IH-4 but not IH-2. The reactivities of IH-2 and IH-4 are remarkably similar to those of human anti-CD45RB and anti-CD45RA antibodies respectively. Analogies with man and other species suggest important functional differences for subpopulations of guinea-pig T lymphocytes defined by anti-CD45R antibodies.

Cytoplasmic calcium fluxes induced in cytotoxic effector cells by engagement of Fc gamma receptors I, II, and III.

Changes in the intracellular calcium ion concentration ([Ca2+]i) of monocytes, granulocytes, and NK cells have been studied following either (1) independent cross-linking of Fc gamma receptors (Fc gamma R) I, II, or III, with F(ab gamma')2 fragments of monoclonal antibodies; or (2) linking of a selected Fc gamma R to a tumour cell target with bispecific F(ab' gamma)2 antibodies. Upon cross-linking each Fc gamma R with antibody an increase in the [Ca2+]i was observed, although all receptors apart from Fc gamma RIII on NK cells required additional cross-linking with an anti-mouse Fab' gamma. These results indicate that each type of receptor can transduce signals to the cell independently. Bispecific antibodies (anti-Fc gamma R x anti-target) linking cytotoxic Fc gamma R-bearing effector cells to tumour target cells also mediated increases in [Ca2+]i for all Fc gamma R tested except for Fc gamma RIII on granulocytes. The failure to transduce a signal via this receptor may be related to the GPI link, which is in contrast to the transmembrane link of Fc gamma RIII on NK cells. Significant lysis of tumour cell targets occurred when bispecific antibodies recruited NK cells or monocytes, but not granulocytes, via Fc gamma R. Chelation of intracellular Ca2+ in the effector cells reduced the observed lysis, suggesting a role for Ca2+ in the pathways leading to cytotoxicity.

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.

Trispecific F(ab')3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells.

To investigate whether the retargeting of resting CTL can benefit from cooperative signaling between the TCR/CD3 complex and various accessory molecules, such as CD2, CD4, CD5, and CD8, we have constructed a series of trispecific F(ab')3 derivatives. Each derivative was designed to engage effector T lymphocytes with two Fab' arms, and tumor cells with a single Fab' arm. They were constructed by selective coupling of three mAb Fab' fragments, primarily via their hinge-region sulfhydryl groups, using the cross-linker o-phenylenedimaleimide. En route to the production of trispecific F(ab')3 antibodies a range of bispecific F(ab')2 derivatives was first prepared which could bind simultaneously to two different receptor molecules on T cells. Bispecific derivatives containing specificities for (CD2 (T11(1)) x CD3), (CD3 x CD4), (CD3 x CD8) or two epitopes on CD2, ((T11(1) x (T11(3)), all yielded two to three times the uptake of [3H]thymidine with fresh PBMC to that seen with intact IgG from anti-CD3 (OKT3). The exception to these findings was a bispecific F(ab')2 derivative with specificities for (CD3 x CD5) which caused slightly less proliferation than the control reagent, OKT3 IgG. When these bispecific antibodies were converted into trispecific antibodies (TsAb) by the addition of a Fab' from anti-CD37 they were then all able to retarget resting, unprimed, T cells from fresh PBMC for lysis of CD37+ tumor cells. However, the cytotoxic activity of these reagents fell into two distinct groups: group one, containing (anti-CD3 x anti-CD4 x anti-CD37), (anti-CD3 x anti-CD5 x anti-CD37), and (anti-CD3 x anti-CD8 x anti-CD37), gave minimal lysis and behaved in a similar way to the BsAb, (anti-CD3 x anti-CD37), i.e., no evidence of cooperative signaling for lysis; and group two, containing (anti-T11(1) x anti-CD3 x anti-CD37) and (anti-T11(1) x anti-T11(3) x anti-CD37), which were highly cytotoxic and gave up to 80% specific 51Cr-release. The failure of group one TsAb, in particular (anti-CD3 x anti-CD8 x anti-CD37) which should recruit CD8+ CTL, to give efficient lysis despite having anti-T cell arms that were mitogenic as a bispecific antibody, indicates that the cooperative signaling for proliferation is probably distinct from the signal(s) provided by group two TsAb that activate for both proliferation and lysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Bispecific F(ab'gamma)3 antibody derivatives for redirecting unprimed cytotoxic T cells.

Bispecific F(ab'gamma)3 derivatives have been constructed by selective coupling of monoclonal antibody Fab'gamma fragments, using the cross-linker o-phenylenedimaleimide attached to hinge-region sulfhydryl groups. All the derivatives were designed to redirect the cytotoxic activity of circulating human T lymphocytes, via their CD2, CD3 or CD5 molecules, against 51Cr-labeled chicken red blood cells (CRBC) or the human lymphoblastoid cell line, Namalwa. The composition of F(ab'gamma)3 molecules was controlled by the selection of Fab'gamma for initial attachment of cross-linker, allowing production of derivatives with either two anti-effector and one anti-target Fab'gamma arms or vice versa. Bispecific F(ab'gamma)3 derivatives, recruiting peripheral blood lymphocytes (PBL) via CD3 as effectors against CRBC, gave titers in redirected cellular cytotoxicity assays which were consistently 25-100 times higher than those given by an equivalent F(ab'gamma)2 reagent, with full activity being retained down to antibody concentrations of 1 ng/ml. The allocation of valencies--two anti-target/one anti-effector or the converse--was not important in determining the increase in titer. No significant lysis was seen when recruiting PBL via CD2 or CD5. It was notable that these CD3-specific derivatives yielded good tumor-cell lysis when using fresh, unprimed PBL as effectors. The increased activity over a bispecific F(ab'gamma)2 was apparent both in titer and the maximum level of lysis achieved. Blocking studies with excess Fab'gamma suggested that the potency of F(ab'gamma)3 derivatives lie in the strength of bridging between effector and target cells. This high avidity contact could favor metabolic activation in either the target or effector cell, or could increase the efficiency of lytic machinery already triggered.