Apparatus for ultrasound tissue characterization of excised specimens.

Apparatus is described for measurement of sound speed and ultrasound attenuation coefficients by the substitution technique in the frequency range 3 to 8 MHz. Phase-cancellation artifacts leading to overestimation of attenuation coefficients are avoided by use of an acoustoelectric transducer. Specimens confined by polystyrene windows can be interrogated by focused ultrasound beams at selected locations spaced on a grid of 3 x 3 mm voxels. Pulse time of flight is measured with an accuracy of 30 ns, yielding sound speeds accurate to +/- 6.7 m/s, for samples 10 mm thick. Uncertainties in measured insertion losses range from 0.1 dB in low-loss (10 dB) specimens to 0.5 dB in high-loss (25 dB) specimens.

Effect of linkage variation on pharmacokinetics of ricin A chain-antibody conjugates in normal rats.

We have investigated the pharmacokinetics of three ricin A chain-antibody conjugates having different bridging structures. Conjugate 1 has a disulphide linkage and was prepared with the N-succinimidyl-3-(2-pyridyldithio)propionate cross-linking reagent. Conjugate 2 has a protected disulphide linkage with a methyl group substituted on the carbon atom of the bridging structure adjacent to the disulphide linkage. Its preparation necessitated the synthesis of a new cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)-butyrate. Conjugate 3 has a sulphide linkage and was prepared with the cross-linking reagent N-succinimidyl 4-(iodoacetylamino)benzoate which was synthesized by a novel route. Conjugate 1 is reducible, conjugate 2 less easily reducible and conjugate 3 nonreducible. On administration to animals all three conjugates displayed biphasic kinetics. The reducibility of the bond had no significant effect on the early disappearance of the conjugate from the circulation. However, at the later time points ease of reduction of the bond was associated with a more rapid disappearance of conjugate.

Fate of an antibody-ricin A chain conjugate administered to normal rats.

The pharmacokinetics and catabolism of ricin A chain, a mouse monoclonal antibody (LICR-LOND-Fib 75) and a disulphide linked conjugate of the two have been studied following their intravenous administration to normal rats. Results indicate that the conjugate was removed from the circulation much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much more rapidly than the antibody but less quickly than the free ricin A chain. Disappearance of the conjugate from the circulation appeared to be biphasic with an early rapid initial phase followed by a much slower phase. The fate of a conjugate with a 125I iodide label in the antibody component was compared with that of a conjugate similarly labelled but in the ricin A chain component. The results indicate that breakdown of the conjugate involves both cleavage of the disulphide linkage and complete catabolism of the whole conjugate molecule with the release of 125I iodide. Rapid cleavage of the disulphide bond in the vasculature does not appear to be responsible for the initial rapid disappearance of the conjugate from the circulation.

Variants of lymphoid lines produced with ricin A-chain monoclonal antibody conjugates.

Conjugates of ricin A-chain with monoclonal anti-light chain antibodies specifically killed cells hearing kappa or lambda immunoglobulin (Ig) light chains. Exposure of cells from B-lymphoblastoid cell lines (B-LCL) to conjugate for less than 30 h had only a slight effect on cell growth, but on 48 h exposure a marked killing effect was achieved. After recovery of growth, cells were re-exposed to conjugate for 9-14 days. Treatment of cells from the EB4 line (sIgG lambda) in this way yielded 4 variants which showed a marked reduction in levels of surface Ig lambda and secreted Ig lambda with slight, or no, reduction in MHC class II expression and similar growth rates to the parent line. Variant lines retained their phenotype over long periods of culture.

An immunotoxin with therapeutic potential in T cell leukemia: WT1-ricin A.

A conjugate of the monoclonal antibody WT1 and ricin A-chain was studied for its suitability for purging marrow of leukemic T cells for autologous transplantation in T cell acute lymphocytic leukemia (T-ALL). The conjugate was powerfully cytotoxic to the human T-ALL cell line, GH1, which expresses the WT1 antigen at a high density. Treatment of the cells with the conjugate at 10(-11) M reduced their rate of protein synthesis by 50%, and the inclusion of 6 mM ammonium chloride in the cultures enhanced the potency of cytotoxic effect by 10-100-fold. Clonogenic assays indicated that less than 0.1% of GH1 cells survived 3-hr exposure to the conjugate in ammonium chloride. WT1 alone did not react with multipotent (CFU-GEMM) hematopoietic progenitors in normal human bone marrow, as measured by fluorescence-activated cell sorting. Under conditions giving maximal killing of GH1 cells, there was no toxicity to multipotential progenitors in normal human marrow.

Antibody-ricin conjugates: a method of linkage which blocks the galactose binding site of ricin.

Conjugates of the monoclonal antibody anti-Thy 1:1 (OX7) and ricin have been constructed, using a thioether bond, such that the ricin no longer can bind Sepharose or asialofetuin. These conjugates were found to be very toxic to Thy 1:1 expressing AKR-A cells whereas they showed little toxicity to Thy 1:2 expressing EL4 cells. By comparison, OX7-ricin conjugates which retained galactose-binding capability were still highly toxic to EL4 cells and this toxicity was antagonized by lactose. A second conjugate made from the W3/25 monoclonal antibody was constructed. The W3/25-ricin conjugate which had lost Sepharose-binding capacity was toxic to W3/25 antigen-expressing rat T-leukaemia cells. This is in sharp contrast to the disulphide linked W3/25-ricin A-chain conjugate which is totally inactive, suggesting that the role of the B-chain in membrane transport of the A-chain into the cytosol is independent of galactose recognition.

Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates.

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a beta-galactosyl matrix) and asialofetuin-Sepharose. Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy1.1-ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thy1.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2-5 pM. Comparable inhibition of EL4 cells was only achieved with 3000-7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thy1.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells. A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact ricin conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition. It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.

Delivery of ricin and abrin A-chains to human carcinoma cells in culture following covalent linkage to monoclonal antibody LICR-LOND-Fib 75.

With the object of generating specific cytotoxic agents, we have prepared covalent conjugates of the A-chains of ricin and of abrin with monoclonal antibody LICR-LOND-Fib 75 and investigated their toxicity toward a human tumor cell line in culture. Both conjugates proved to be potent cytotoxins toward cells carrying the appropriate antigen. The agent containing abrin A-chain was toxic at a significantly lower concentration and exerted its maximum effect more rapidly than the one containing ricin A-chain. Inclusion of chloroquine in the incubation medium significantly enhanced the toxic action of both conjugates without loss of immunospecificity. Because of the widespread occurrence on human tumor cell lines of the antigen recognized by Fib 75, these conjugates, particularly the one containing abrin A-chain, may find application in freeing human bone marrow ex vivo of infiltrated tumor cells prior to reinfusion as an autograft.

Ricin B chain converts a non-cytotoxic antibody-ricin A chain conjugate into a potent and specific cytotoxic agent.

We report the conversion of a non-cytotoxic antibody-ricin A chain conjugate to one displaying specific cytotoxic effects comparable with that of native ricin, by the addition of ricin B chain as a second stage reagent. The results suggest that this conversion is achieved by the association of the added B chain with the A chain of the conjugate, and not through a primary binding of B chain at the cell surface.

A comparison of the in vitro and in vivo activities of conjugates of anti-mouse lymphocyte globulin and abrin.

Anti-mouse lymphocyte globulin and normal immunoglobulin have been conjugated to abrin using two procedures, one involving linkage through an amide bond and a piperazine ring and the other the introduction of two amide bonds flanking a disulphide bridge. The four conjugates produced were equipotent as inhibitors of protein synthesis in rabbit reticulocyte lysates. Each antibody-containing conjugate was a more effective inhibitor of protein synthesis in cultured cells than the equivalent normal immunoglobulin-containing conjugate. In addition the conjugates with disulphide linkage groups were ten times more potent than their counterparts. The disulphide conjugates were also twice as toxic to mice in an acute toxicity test but when used to suppress their immune responses to sheep red blood cells it was the non-disulphide-linked conjugates that were superior. In all instances antibody-containing conjugates were more powerful immunosuppressants than those containing normal IgG. The results are taken to indicate a relative lack of stability of the disulphide conjugates in the tissues.

Cytotoxicity acquired by conjugation of an anti-Thy1.1 monoclonal antibody and the ribosome-inactivating protein, gelonin.

Gelonin, a plant protein which can powerfully reduce the protein-synthetic capacity of ribosome preparations, was covalently coupled to anti-Thy1.2 antibody. The conjugate was prepared using N-succinimidyl-3-(2-pyridyldithio)propionate which generates a disulphide linkage between the component molecules. Two conjugate fractions were obtained with Mr of 180 000 and greater than 200 000. After its linkage of the antibody, gelonin suppressed those Thy1.1-bearing T lymphocytes from AKR mice which will respond to phytohaemagglutinin and concanavalin A in tissue culture. The [3H]leucine incorporation with the T-cell mitogens was inhibited by 50% with the 180 000-Mr fraction at a concentration of 0.4 nM and with the greater than 200 000-Mr fraction of pM. Unconjugated gelonin induced comparable reductions in T-cell responsiveness but at concentrations of 30 nM. The conjugates exerted little or no effect upon B lymphocytes or T lymphocytes from CBA mice (Thy1.2 + ve). Thy1.1-expressing AKR lymphoma cell lines, AKR-A and BW5147, were found to be sensitive to the conjugates, albeit much less so than the normal T lymphocytes. The conjugates injected in vivo significantly prolonged the life of CBA mice bearing in an AKR-A lymphoma allograft. It is concluded that gelonin can, by its linkage to an antibody, be rendered cytotoxic with a potency to match or exceed those of the toxins abrin and ricin.

Abrogation of the non-specific toxicity of abrin conjugated to anti-lymphocyte globulin.

A covalent conjugate of abrin and anti-human lymphocyte globulin (AHLG) was prepared in an endeavour to create a cytotoxic agent with specificity for human lymphoid cells. The AHLG--abrin conjugate was found to be around 10-fold better able to inhibit 3H-leucine uptake by the human lymphoblastoid cell line, Daudi, in tissue culture than was the control conjugate comprising abrin and normal IgG (nIgG). Both materials were less potent than native abrin. Galactose, which is known competitively to antagonize the binding of abrin to cells, strongly inhibited the toxicities of abrin and the nIgG--abrin conjugate whereas that of ALG--abrin was unimpaired. Thus, at least for Daudi cells in tissue culture, abrin can be made selectively toxic, by linkage to AHLG, towards cells bearing antigens to which the antibody moiety of the conjugate can attach.

Increased toxicity of diphtheria toxin for human lymphoblastoid cells following covalent linkage to anti-(human lymphocyte) globulin or its F(ab')2 fragment.

Anti-(human lymphocyte) globulin was reacted with a mixed anhydride derivative of chlorambucil to give a product which was in turn reacted with diphtheria toxin. The resulting conjugate was partially purified and was found to possess an ability similar to that of the native antibody to bind to the human lymphoblastoid cell lines, CLA4 and Daudi. Daudi cells, as had been observed previously with CLA4 cells, lacked the high sensitivity to diphtheria toxin normally characteristic of cells of human origin. Thus treatment with free toxin at a concentration of 1 microgram/ml was without effect upon their ability to incorporate [3H]leucine. By contrast, Daudi cells were highly sensitive to toxin conjugated to anti-(human lymphocyte) globulin or to its F(ab')2 fragment. Exposure for 24 h to a solution of conjugate containing toxin at a concentration of 0.5 ng/ml caused a reduction of 50% in the leucine uptake by Daudi cells. The toxicity of the conjugate could be blocked by diphtheria antitoxin or by pretreatment of the cells with non-conjugated antibody. Toxin linked to normal horse IgG or to its F(ab')2 fragment was without cytotoxic effect upon Daudi cells. Furthermore both the conjugate with anti-(human lymphocyte) globulin and that with normal IgG were approximately 100-fold less able than non-conjugated toxin to inhibit protein synthesis by a human fibroblast cell line to which the antibody showed no appreciable binding. Thus the conjugates are relatively ineffective against cells which lack an antigen to which the antibody moiety can bind. In contrast with the greatly increased toxicity of diphtheria toxin for human lymphoblastoid cells following its linkage to anti-(human lymphocyte) globulin, a conjugate of toxin linked to anti-(mouse lymphocyte) globulin was ineffective against murine spleen cells in vitro.

Rh immunoglobulin utlization after spontaneous and induced abortion.

To monitor the utilization of Rh immunoglobulin (RhIG), we reviewed the charts of 389 spontaneous and 1350 induced abortion patients treated in 1975 at a metropolitan hospital. The rate of ascertainment of Rh type was significantly higher for induced (99.6%) than for spontaneous abortion patients (95.1%) (P less than 0.001). Utilization of Rh immunoglobulin (RhIG) also was significantly higher for induced (98.9%) than for spontaneous abortion patients (80.6%) (P less than 0.001). Women at risk who did not receive RhIG after spontaneous abortion were mostly young, of low gravidity, and at gestational ages (mean 14.4 weeks) associated with substantial risks of Rh sensitization. Eradication of Rh hemolytic disease requires improvement in the system of identifying and treating patients who need prophylaxis.

PIP: The charts of 389 spontaneous and 1350 induced abortion patients treated at a metropolitan hospital in 1975 were reviewed to assess the extent to which Rh immunoglobulin (RhIG) was utilized. The rate of determination of Rh type was 99.6% for cases of induced abortion and 95.1% for spontaneous abortion (p less than .001). RhIG was utilized in 98.9% of the induced abortion cases and in 80.6% of the cases of spontaneous abortion (p less than .001). Those women who did not receive RhIG after spontaneous abortion were primarily young, of low parity, and at those stages of pregnancy associated with a substantial risk of Rh sensitization (mean 14.4 weeks). The results point out the need for a better system for identifying and treating patients at risk of Rh hemolytic disease.