Philip M. Wallace

In vitro, when the B7 molecule on the surface of antigen-presenting cells binds to the T cell surface molecules CD28 and CTLA-4, a costimulatory signal for T cell activation is generated. CTLA4Ig is a soluble form of the extracellular domain of CTLA-4 and binds B7 with high avidity. CTLA4Ig treatment in vivo suppressed T cell-dependent antibody responses to sheep erythrocytes or keyhole limpet hemocyanin. Large doses of CTLA4Ig suppressed responses to a second immunization. Thus, costimulation by B7 is important for humoral immune responses in vivo, and interference with costimulation may be useful for treatment of antibody-mediated autoimmune disease.

Two new coupling agents were synthesized for making immunotoxins containing disulfide bonds with improved stability in vivo: sodium S-4-succinimidyloxycarbonyl-alpha-methyl benzyl thiosulfate (SMBT) and 4-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)tolue ne (SMPT). Both reagents generate the same hindered disulfide linkage in which a methyl group and a benzene ring are attached to the carbon atom adjacent to the disulfide bond and protect it from attack by thiolate anions. An immunotoxin consisting of monoclonal anti-Thy-1.1 antibody (OX7) linked by means of the SMPT reagent to chemically deglycosylated ricin A-chain had better stability in vivo than an immunotoxin prepared with 2-iminothiolane hydrochloride (2IT) which generates an unhindered disulfide linkage. About 48 h after i.v. injection into mice, one-half of the SMPT-linked immunotoxin present in the blood was in intact form and one-half as released free antibody, whereas equivalent breakdown of the 2IT-linked immunotoxin was seen at about 8 h after injection. Consequently, the blood levels of the SMPT-linked immunotoxin remained higher than those of the 2IT-linked immunotoxin despite loss of immunotoxin from the blood by other mechanisms. Forty-eight h after injection, 10% of the injected dose of the SMPT-linked immunotoxin remained in the bloodstream as compared with only 1.5% of the 2IT-linked immunotoxin. The ability of immunotoxins prepared with the new reagents to inhibit protein synthesis by Thy-1.1-expressing AKR-A/2 lymphoma cells in vitro was identical to that of immunotoxins prepared with 2IT or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Clonogenic assays showed that fewer than 0.01% of AKR-A/2 cells survived exposure to high concentrations of OX7-abrin A-chain immunotoxins prepared with SMBT, 2IT, or SPDP. Twelve clones of cells which had survived treatment with the SMBT-linked immunotoxin were isolated. None of the clones was selectively resistant to the SMBT-linked immunotoxin when retested in cytotoxicity assays. In conclusion, immunotoxins prepared with the new coupling agents should have improved antitumor activity in vivo because they are longer lived and do not break down so readily to release free antibody which could compete for the target antigens.

A monoclonal anti-Thy-1.1 antibody (OX7) was coupled to either native or chemically deglycosylated ricin A-chain (dgA) using one of two different cross-linking agents. One cross-linker, N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)tolu ene (SMPT), generates a sterically hindered disulfide bond which is relatively resistant to reduction, whereas the other, 2-iminothiolane hydrochloride, generates an unhindered disulfide bond with greater lability. A two-compartment pharmacokinetic model was used to analyze the blood levels of each immunotoxin and its breakdown product (free antibody) after i.v. injection into mice. Immunotoxins prepared with SMPT broke down in vivo 6.3-fold more slowly than those prepared with 2-iminothiolane hydrochloride, and immunotoxins containing native A-chain were cleared 2- to 3-fold more rapidly from the bloodstream than those containing dgA. As a result, 24 h after injection, 16% of the OX7-SMPT-dgA remained in the blood as compared with 0.4 to 2.5% of the other immunotoxins. Immunotoxins prepared with dgA were about 3-fold more toxic to mice than those prepared with native A-chain, whereas immunotoxins prepared with SMPT were only slightly more toxic than those prepared with 2-iminothiolane hydrochloride. When equivalent toxic doses of the immunotoxins were administered i.v. to mice which had been given injections of Thy-1.1+ AKR-A/2 lymphoma cells, the OX7-SMPT-dgA gave the best antitumor effect. A dose equivalent to one-seventh of the median lethal dose extended the survival time of the animals by the extent expected if 99.999% of the tumor cells had been eradicated. Furthermore, the tumors that did develop in the mice treated with OX7-SMPT-dgA were mutants which were resistant to all the immunotoxins. Some of the mutants were deficient in Thy-1.1 whereas others were not. In conclusion, both the use of the SMPT cross-linker and deglycosylation of the A-chain significantly improve the therapeutic index of the immunotoxins in AKR-A/2 tumor-bearing mice.

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigens, CD22 and CD19, were constructed using the monoclonal antibodies, HD6 and HD37, respectively. IT-As were prepared by coupling intact antibodies, F(ab')2, or Fab' fragments to native or chemically deglycosylated ricin A chain. The IT-As were then evaluated for cytotoxicity to normal and neoplastic human B-cells in vitro with the major objective of appraising their suitability for in vivo therapy of human B-cell tumors. The IT-As prepared with both the HD6 and HD37 antibodies were specifically toxic to normal B-cells and to most of the neoplastic B-cell lines tested. However, the IT-As prepared from HD6 were generally more potent than those prepared from HD37. On Daudi cells, to which the two antibodies bound in similar numbers and with similar affinities, IT-As prepared with intact HD6 antibody or its Fab' fragment were 10-fold and 1.5- to 4-fold more potent, respectively, than the corresponding HD37 IT-As. The IT-As constructed from intact HD6 antibody and native or deglycosylated A chain reduced protein synthesis in Daudi cells by 50% at a concentration of 1.2 X 10(-11) M indicating that they were only 5-fold less toxic to the cells than ricin itself. Intact HD37 IT-As produced equivalent inhibition of protein synthesis at 1.5 X 10(-10) M. With both antibodies, IT-As constructed from the Fab' fragments were 10- to 20-fold less potent than their intact antibody counterparts. Different neoplastic B-cell lines varied in sensitivity to the IT-As. In most cases, their sensitivity correlated with the levels of CD19 and CD22 antigens expressed. Neither HD6 nor HD37 IT-As affected the ability of normal human bone marrow cells to form granulocyte-macrophage colony-forming units in soft agar, suggesting that both antigens are absent from these progenitor cells. Examination of sections of frozen human tissues using immunoperoxidase staining procedures indicated that the antibodies did not bind to a panel of normal tissues lacking B-lymphocytes. These results suggest that HD6 and HD37 IT-As are candidates for in vivo therapy in humans with certain B-cell tumors. However, HD6 IT-As are more potent, reduce protein synthesis more completely, and hence appear to be the ITs of choice for treating tumors expressing the CD22 antigen.

Graft-versus-host disease (GVHD), a pathological condition associated with BMT, results from activation of donor T lymphocytes by host tissues. CD28 and CTLA-4 are structurally related T cell receptors for members of the B7 (CD80) gene family, which transmit important costimulatory signals for T cell activation in vitro and in vivo. Here we have investigated the effects of CTLA4Ig, a soluble form of CTLA-4, on lethal GVHD in a murine model. Lethal GVHD was induced by transfer of parent C57BL/6 bone marrow and spleen cells into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. Short courses of treatment with CTLA4Ig did not block engraftment, but prolonged survival of BMT recipients even when administration was delayed for 6 days after transplantation. CTLA4Ig-treated survivors of GVHD maintained body weight and did not exhibit visible signs of GVHD. However, treatment regimens that maximally prolonged survival did not detectably prevent T cell-mediated hematological abnormalities associated with GVHD, including pancytopenia and abnormal cellular composition of the spleen. Our data thus show that the lethality of acute GVHD in this model system is more dependent upon CD28/CTLA-4 costimulation than are other GVHD-associated abnormalities, and can be blocked for an extended period by brief treatment with CTLA4Ig.