Immunotoxin Research Articles

The Chronic Lymphocytic Leukemia Antigen (cCLLa) as Immunotherapy Target: Assessment of LD50 and MTD of Four Ricin-Based Anti-cCLLa Immunotoxins (ITs) in Balb/c Mice

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is a promising immunotherapy target given its disease-restricted expression, its highest prevalence among CLL surface antigens, and its lack of expression by normal T- and B-lymphocytes. The objectives of this study were to assess the 50% lethal dose (LD50) and the maximum tolerated dose (MTD) in Balb/c mice of four anti-cCLLa immunotoxins (ITs) derived from the intact monoclonal antibody (MoAb) or its Fab fraction, each conjugated to either ricin chain-A (RTA) or its deglycosylated derivative (dgA). The IgG fraction of anti-cCLLa monoclonal antibody CLL2m and its Fab fraction were conjugated to RTA or dgA to generate four ITs: IgG/RTA, IgG/dgA, Fab/RTA and Fab/dgA. Progressive concentrations of each IT (ranging between 2.60 mg/kg and 100.00 mg/kg) were injected intravenously into groups of 5 mice each. After injection, mice were monitored daily for 10 days for survival. Observed mortality data in each group were matched to those in Weil's tables for estimating LD50 (mg/kg) from the moving average interpolation method. Estimated LD50 (in mg/kg) were: IgG/RTA, 13.33; Fab/RTA, 25.53; IgG/dgA, 55.33; Fab/dgA, 55.33. Their respective MTD (mg/kg), defined as the highest dose level survived by all mice, were 8.78, 13.17, 29.63 and 29.63. Depending on the animal-to-human extrapolation method used, the calculated LD50 and MTD in humans ranged from 1.2 mg/kg and 0.8 mg/kg (IgG/RTA), to 55.6 mg/kg and 36.9 mg/kg (IgG/dgA and Fab/dgA), respectively. The following conclusions are drawn. 1. Antibody valence exerted little influence on either the LD50 or the MTD; 2. The LD50 to MTD ratios were approximately 2:1; 3. dgA-derived ITs were approximately one half as toxic as their RTA-derived counterparts; and 4. Extrapolation of LD50 and MTD mouse data to humans resulted in dose levels comparable to or exceeding those reported in most IT human trials. These data suggest the suitability of anti-cCLLa ITs for clinical immunotherapy trials.

The Chronic Lymphocytic Leukemia Antigen (cCLLa) as Immunotherapy Target: Pharmacokinetics and Biodistribution of Two Divalent, Ricin-Based Immunotoxins in Xenografted Athymic Mice

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is potentially suitable for targeted immunotherapy given its restriction to clonal CLL cells and lack of expression by normal lymphocytes. In order to assess the pharmacokinetics and biodistribution of two potent anti-cCLLa immunotoxins (ITs) were examined in the mouse model. The IgG fraction of anti-cCLLa monoclonal antibody CLL2m was conjugated with 125I-labeled intact (RTA) or deglycosylated (dgA) ricin chain A, injected intravenously into athymic mice engrafted with cCLLa-expressing human tumors, and monitored over 120 hours. Blood concentrations of CLL2m/125I-RTA and CLL2m/125I-dgA were best fit to biexponential equations but the latter exhibited a lower αT1/2 and βT1/2 (4.1 and 102 min v.s 5.9and 126 min), a smaller volume of distribution (5.1 g vs 9.7 g), and a lower blood clearance (2.2 g/hr vs 4.6 g/hr). Both ITs exhibited preferential tumor uptake that followed distinct kinetics: rising tumor uptake for 2 hrs post-injection (while tissue uptake decreased), reaching tumor/non-tumoral tissue uptake ratios up to 16.9; and slower dissociation rates of tumor- vs tissue-bound ITs (<45% vs >20% remaining tissue-bound 6 hrs post-injection, respectively). Non-specific liver uptake was not prominent for either IT. In vivo IT deconjugation reached 50% approximately 12 hours post-injection. The pharmacokinetics and biodistribution data in the mouse model suggest that ricin-based anti-cCLLa ITs are suitable for use in human trials.

The emerging role of immunotoxins in leukemia and lymphoma

Despite important advances in conventional cancer therapy, there is still a strong need for new approaches in order to reduce cancer death rates, which have not been drastically influenced in the past ten years. Since minimal residual disease is regarded as the major cause for relapses of malignant diseases, immunotherapeutic strategies utilizing monoclonal antibodies (MoAbs) or their conjugates to target 'dormant' tumor cells have increasingly attracted scientific interest. Immunotoxins (ITs) constructed by chemically linking plant or bacterial toxins to a MoAb can selectively kill their target cells when internalized after binding to specific cell surface receptors. Many different ITs against various blood-borne as well as solid malignancies have been successfully tested in vitro and in animal models. Chemically linked ITs and recombinant fusion toxins generated by using DNA technologies are currently being evaluated for their antitumor activity in several clinical phase I/II/III trials. While serious side effects are rare, there are still many problems to be solved, such as the immunogenicity of the toxin and/or antibody moiety or the poor capacity of ITs to penetrate large solid tumors. At the moment only heavily pretreated patients with massive tumor burden are admitted to early clinical studies, so that even minor responses after IT treatment are encouraging. However, minimal residual disease is expected to be most amenable to IT therapy.

Highly potent CD22-recombinant ricin A results in complete cure of disseminated malignant B-cell xenografts in SCID mice but fails to cure solid xenografts in nude mice.

The highly specific cytotoxic action of ribosome-inactivating protein (RIP) containing immunotoxins (ITs) makes IT therapy a promising approach to eliminating residual malignant cells. We investigated the cytotoxicity of the IT CD22-recombinant ricin A to the B-cell line Ramos in vitro and in vivo. Cytotoxicity of CD22-recombinant ricin A in vitro was very high as expressed by the very low 50% inhibition dose (ID50) of 3.5 x 10(-11) M. Cytotoxicity was increased 7 times in the presence of the cytotoxicity enhancer NH4Cl. The ultimate kill of Ramos cells by CD22-recombinant ricin A was high (2.7-log kill) and was increased strongly in the presence of NH4Cl (4.2-log kill). Anti-tumor activity in vivo was investigated by i.v. treatment of solid s.c. Ramos xenografts in nude BALB/c mice. A single dose did not inhibit tumor growth. Treatment on 5 consecutive days resulted in evident tumor reduction. In one mouse, tumor could no longer be detected on the 6th day after starting treatment. However, after 8 days tumor volumes increased again. Antitumor activity was more pronounced in a disseminated tumor model in SCID mice. IT treatment (i.v.) 7 days after i.v. inoculation with Ramos cells resulted in cure of all mice. Non-specific toxicity was low. Alanine aminotransferase (ALAT) levels in serum were elevated temporarily. Serum values of gamma-glutamyl transferase (gamma-GT), bilirubin and creatinin did not change. Body weight was also transiently reduced. The LD50 in SCID mice after i.v. administration was high (0.626 mg IT per mouse). The clearance rate in SCID mice, as determined by ELISA, was biphasic.

The use of cyclosporine, FK506, and SDZ NIM811 to prevent CD25- quiescent peripheral blood mononuclear cells from producing human immunodeficiency virus.

It has been shown that the combined use of two pharmacologic agents can inhibit human immunodeficiency virus (HIV) production by peripheral blood mononuclear cells in vitro. One, an anti-CD25 immunotoxin (IT), kills activated T cells that produce virus; the other, the immunosuppressive drug cyclosporine, prevents the quiescent cells, which harbor HIV, from becoming activated. The present study compares the antiviral activities of two agents, SDZ NIM811 and FK506, to that of cyclosporine. In combination with the anti-CD25 IT, these drugs significantly suppressed virus production. In the absence of prior addition of the IT, the ability of the drugs to inhibit virus production was much lower, suggesting that they work effectively in latently infected cells. In the case of SDZ NIM811, the inhibition of virus production was accompanied by a modest inhibition of cell proliferation. In contrast, FK506 exerted strong antiproliferative activity. Cyclosporine was both moderately antiproliferative and a potent antiviral agent.

Combination immunotoxin treatment and chemotherapy in SCID mice with advanced, disseminated Daudi lymphoma.

We describe the use of an immunotoxin (IT) cocktail (anti-CD22- and anti-CD19-ricin A chain) and any 1 of 3 chemotherapeutic drugs (doxorubicin, cytoxan or camptothecin) to treat advanced disseminated Daudi lymphoma in SCID mice (SCID/Daudi). In a previous report, we demonstrated that this regimen was curative when given the day following tumor cell inoculation. Here, we show that combination therapy in mice with advanced tumor significantly increased their survival, although it was not curative. Importantly, the outcome of therapy was dependent upon the temporal order in which IT and chemotherapy were administered. Thus, the best anti-tumor effect was achieved when an IT cocktail was given before or at the same time as chemotherapy. When the IT was given after chemotherapy, there was no additional therapeutic benefit. Our results confirm the rationale of using combination therapy in the treatment of advanced B-cell neoplasia and suggest that ITs should be administered prior to or during chemotherapy.

Anti-Graft-Versus-Host Disease Effect of DT390-Anti-CD3sFv, a Single-Chain Fv Fusion Immunotoxin Specifically Targeting the CD3∊ Moiety of the T-Cell Receptor

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.

A Phase I Study of Bolus Versus Continuous Infusion of the Anti-CD19 Immunotoxin, IgG-HD37-dgA, in Patients With B-Cell Lymphoma

IgG-HD37-SMPT-dgA is a deglycosylated ricin A chain (dgA)-containing immunotoxin (IT) prepared by conjugating the monoclonal murine (MoAb) anti-CD19 antibody, HD37, to dgA using the heterobifunctional hindered disulfide linker, N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio) toluene (SMPT). In this report, we have used two regimens for the administration of IgG-HD37-SMPT-dgA to patients with non-Hodgkin's lymphoma (NHL) in two concomitant phase I trials. One trial examined four intermittent bolus infusions administered at 48-hour intervals. The other studied a continuous infusion (CI) administered over the same 8-day period. In the intermittent bolus regimen, the maximum tolerated dose (MTD) was 16 mg/m2/8 d and the dose-limiting toxicity (DLT) consisted of vascular leak syndrome (VLS), aphasia, and evidence of rhabdomyolysis encountered at 24 mg/m2/8 d. Using the CI regimen, the MTD was defined by VLS at 19.2 mg/m2/8 d. At the MTD of both regimens, a novel toxicity, consisting of acrocyanosis with reversible superficial distal digital skin necrosis in the absence of overt evidence of systemic vasculitis, occurred in 3 patients. Of 23 evaluable patients on the bolus schedule, there was 1 persisting complete response (CR; > 40 months) and 1 partial response (PR). Of 9 evaluable patients on the continuous infusion regimen, there was 1 PR. Pharmacokinetic parameters for the bolus regimen at the MTD showed a mean maximum serum concentration (Cmax) of 1,209 +/- 430 ng/mL, with a median T1/2 beta for all courses of 18.2 hours (range, 10.0 to 80.0 hours), a volume of distribution (Vd) of 10.9 L (range, 3.1 to 34.5 L), and a clearance (CL) of 0.45 L/h (range, 0.13 to 2.3 L/h). For the CI regimen at MTD, the mean Cmax was 963 +/- 473 ng/mL, with a median T1/2 beta for all courses of 22.8 hours (range, 24.1 to 30.6 hours), a Vd of 9.4 L (range, 4.4 to 19.5 L), and a CL of 0.32 L/h (range, 0.12 to 0.55 L/h). Twenty-five percent of the patients on the bolus infusion regimen and 30% on the CI regimen made antibody against mouse Ig (HAMA) and/or ricin A chain antibody (HARA). We conclude that this IT can be administered safely and that both regimens achieve comparable peak serum concentrations at the MTD; these concentrations are similar to those achieved previously using other regimens with IgG-dgA ITs at their respective MTDs. Thus, toxicity is related to the serum level of the IT and does not differ with different targeting MoAbs.

Fibronectin inhibits the cytotoxic effect of ricin A chain on endothelial cells

We have previously reported that after in vitro treatment with deglycosylated ricin A chain (dgRTA), human umbilical vein endothelial cells (HUVECs) undergo changes in morphology including cell rounding and disruption of monolayers. The present studies were carried out to determine whether these changes were related to the disruptions in endothelial cell (EC) interactions with the extracellular matrix. To this end, we examined the effect of dgRTA on HUVECs in the presence of fibronectin (Fn), an extracellular matrix protein, which plays a role in the maintenance of vascular integrity. The addition of exogenous Fn greatly inhibited dgRTA-mediated morphological changes in HUVEC monolayers, dgRTA-mediated inhibition of [ 3H]thymidine incorporation in HUVECs and the binding of 125I-dgRTA to HUVECs. Should the same phenomenon occur with RTA-based immunotoxins (ITs) in vivo, this might shed light on the development of dgRTA-mediated vascular leak syndrome (VLS) during IT therapy and provide new insights into how to decrease this toxicity in patients.

Potent anti-tumor effects of an anti-CD24 ricin A-chain immunotoxin in vitro and in a disseminated human Burkitt's lymphoma model in SCID mice.

A new anti-CD24 immunotoxin (IT), SWA11.dgA, was constructed by coupling the MAb SWA11 via the bivalent linker SMPT to deglycosylated ricin A-chain (dgA). The effects of SWA11.dgA were evaluated in vitro against the B-precursor leukemia cell line REH, the non-B-non-T acute lymphoblastic leukemia cell line NALM-6 and the Burkitt's lymphoma cell lines BL-2 and BL-38. Binding of SWA11 to the CD24 antigen was assessed by flow cytometry demonstrating high affinity of the MAb for all cell lines tested. SWA11.dgA inhibited the protein synthesis of BL-38, NALM-6, REH and BL-2 cells by 50% at concentrations (IC50) of 4.0 x 10(-11) M, 6.0 x 10(-11) M, 8.0 x 10(-11) M and 3.0 x 10(-9) M, respectively. SWA11.dgA was subsequently used for the treatment of disseminated human BL-38 Burkitt's lymphoma in a newly developed SCID mouse model. The mean survival time (MST) of BL-38-bearing SCID mice was extended from 23 days in untreated controls to more than 230 days when 6 microg SWA11.dgA was applied intraperitoneally one day after tumor challenge. All of the animals achieved continuous complete remissions. SCID mice treated with SWA11.dgA 4 days after tumor cell challenge or a reduced dose of SWA11.dgA (67%) also had a significantly extended MST (45.0 and 51.4 days, respectively, as compared to 22.7 and 23.1 days in the controls). We conclude that SWA11.dgA might be of potential use for the treatment of lymphoma in man.

Immunotoxins: an update.

The use of immunotoxins (ITs) in the therapy of cancer, graft-vs-host disease (GvHD), autoimmune diseases, and AIDS has been ongoing for the past two decades. ITs contain a targeting moiety for delivery and a toxic moiety for cytotoxicity. Theoretically, one molecule of a toxin, routed to the appropriate cellular compartment, will be lethal to a cell. Newly developed MoAbs, toxins, and molecular biological technologies have enabled researchers to construct ITs that can effectively kill many different cell types. In fact, phase I/II clinical trials have given promising results. Although nonspecific toxicity and immunogenicity still limit the use of IT therapy, these agents hold enormous promise in an optimal setting to treat minimal disease.

P901 - Pretreatment of kidney allografts with anti-CD5 immunotoxin (IT) in cases of poor HLA histocompatibility

P901 - Pretreatment of kidney allografts with anti-CD5 immunotoxin (IT) in cases of poor HLA histocompatibility

Anti-CD30 (BER=H2) immunotoxins containing the type-1 ribosome-inactivating proteins momordin and PAP-S (pokeweed antiviral protein from seeds) display powerful antitumour activity against CD30+ tumour cells in vitro and in SCID mice.

The anti-CD30 immunotoxin (IT) Ber-H2/saporin is effective in patients with refractory Hodgkin's disease. However, responses are short and partial, one of the main reasons being the inability to repeat IT doses because of formation of human antibodies against the murine antibody and/or the toxin. To overcome this problem, we constructed two new anti-CD30 ITs by covalently linking the mouse monoclonal antibody Ber-H2 to the type 1 ribosome-inactivating proteins (RIPs) momordin (MOM) and pokeweed antiviral protein from seeds (PAP-S), which do not cross-react with each other or with saporin. Both ITs inhibited protein synthesis by Hodgkin's disease and anaplastic large-cell lymphoma (ALCL)-derived CD30+ target cell lines with a very high efficiency (IC50 ranging from < 5 x 10(-13) M to 2.75 x 10(-11) M, as RIP). In a SCID mouse model of xenografted CD30+ human ALCL, a 3d treatment with non-toxin doses of Ber-H2/MOM (50%LD50), started 24 h after transplantation, prevented tumour development in about 40% of the animals and significantly delayed tumour growth rate in the others. Main toxicity signs in mice and rabbits were dose-related increase of serum transaminases (AST and ALT) and creatine phosphokinase (CPK). LD50 (as RIP) in Swiss mice was 7 mg/kg for Ber-H2/MOM and 0.45 mg/kg for Ber-H2/PAP-S. Sequential administration of two anti-CD30 ITs (Ber-H2/MOM and Ber-H2/saporin) was well tolerated and did not result in formation of antibodies cross-reacting and with the two plant toxins. The results presented in this paper suggest that in the future, sequential administration of anti-CD30 humanized antibodies linked to antigenically distinct type 1 RIPs (saporin, MOM, PAP-S) should be feasible.

Combined in vitro and in vivo T lymphocyte depletion for the control of graft-versus-host disease following haploidentical marrow transplant.

Most patients requiring allogeneic bone marrow transplantation (BMT) lack a human leukocyte antigen genotypically identical sibling and require an alternative donor. This carries an increased risk of graft failure and acute graft-versus-host disease (GVHD). We sought to overcome these problems with transplants by using grafts obtained from the most readily available source: the haploidentical, partially mismatched, related donor. This study of 40 patients used a novel approach combining in vitro and in vivo T cell depletion with T lymphocyte targeted monoclonal antibodies (mAb) and intensified conditioning therapy, including fractionated total body irradiation before etoposide, cytoside arabinoside, cyclophosphamide, and methylprednisolone. Grafts were treated with T10B9.1A-31 mAb, directed against the alpha-beta heterodimer of the T cell receptor, and rabbit complement. In vivo depletion was attempted with an anti-CD5 mAb-Ricin A-chain (H65-RTA) immunotoxin (IT). Study patients were compared with a historical control group of 17 patients not given H65-RTA. Rates of engraftment were not significantly different (93% vs. 100%, P=0.12), although patients receiving IT engrafted more rapidly. The incidence of > grade I GVHD was significantly lower in the study group (36% vs. 100%, P=0.0001), as well as for severe grade III-IV GVHD (19% vs. 92%, P=0.0001). Five-year survival tended to be improved in the study group (40% vs. 18%, P=0.21). Transplant from haploidentical family members is indicated for patients without a matched sibling in whom allogeneic BMT offers the best opportunity to achieve cure.

Therapy for Ongoing Graft-Versus-Host Disease Induced Across the Major or Minor Histocompatibility Barrier in Mice With Anti-CD3F(ab′)2-Ricin Toxin A Chain Immunotoxin

A new pharmacologic agent, anti-CD3F(ab')2-ricin toxin A chain (RTA), was synthesized for the purpose of targeting T cells and as a means of treating established graft-versus-host disease (GVHD). The Fc region of anti-CD3 monoclonal antibody (MoAb) was removed to prevent its ability to activate T cells. The resulting F(ab')2 fragments were conjugated to deglycosylated RTA (dgRTA), a catalytic and potent phytotoxin. The resulting immunotoxin (IT) was potent (greater than 95% inhibition) and selective in inhibiting T-cell mitogenesis in vitro. In vivo, the IT depleted 80% of T cells in mice receiving bone marrow (BM) transplants. Transplantation in an aggressive acute GVHD model using C57BL/6 donor cells and H-2 disparate B10.BR recipients resulted in an infiltration of CD3-expressing cells and a median survival time (MST) of 20 to 30 days. A 5-day course of anti-CD3F(ab')2-RTA (30 micrograms/d intraperitoneally) beginning 7 days after GVHD induction was beneficial in treating established GVHD in these mice, as evidenced by significantly prolonged survival (MST, greater than 80 days), superior mean weight values, and improved clinical appearance. Neither intact anti-CD3, unconjugated anti-CD3 F(ab')2 fragments, nor a mixture of anti-CD4 and anti-CD8 MoAbs (which are highly effective in prophylactic models) were as effective. F(ab')2 fragments made from anti-Lyt-1 (the murine homologue of human anti-CD5) linked to RTA were also not effective, despite the fact that both anti-CD3F(ab')2-RTA and anti-Lyt-1F(ab')2-RTA had similar half-lives of about 9 hours. The IT also increased MST in two aggressive models of GVHD across non-H-2 minor histocompatibility barriers, indicating that the usefulness of anti-CD3F(ab')2-dgRTA is not limited to a single-strain combination. This agent should be further investigated as an alternative to current strategies for treating steroid refractory GVHD.

The in vivo anti-tumor activity of immunotoxins containing two versus one deglycosylated ricin A chains

We have compared the in vitro and in vivo activities of three deglycosylated ricin A chain (dgA)-containing immunotoxins (ITs) with one versus two molecules of dgA per molecule of IgG. Two of the ITs (anti-CD19-dgA 2 and anti-CD25-dgA 2 ) had 3-0-fold improvements over their dgA 1 counterparts in vitro. However, the in vivo anti-tumor activity of both constructs of each IT was the same. In contrast, anti-CD22-dgA 2 was 7-fold more potent in vitro and significantly more potent in vivo than its dgA 1 counterpart. These studies suggest that monoclonal antibodies must be individually tested to determine whether the addition of two versus one dgA will improve cytotoxic activity in vivo.

The in vivo anti-tumor activity of immunotoxins containing two versus one deglycosylated ricin A chains

We have compared the in vitro and in vivo activities of three deglycosylated ricin A chain (dgA)-containing immunotoxins (ITs) with one versus two molecules of dgA per molecule of IgG. Two of the ITs (anti-CD19-dgA 2 and anti-CD25-dgA 2) had 3-0-fold improvements over their dgA 1 counterparts in vitro. However, the in vivo anti-tumor activity of both constructs of each IT was the same. In contrast, anti-CD22-dgA 2 was 7-fold more potent in vitro and significantly more potent in vivo than its dgA 1 counterpart. These studies suggest that monoclonal antibodies must be individually tested to determine whether the addition of two versus one dgA will improve cytotoxic activity in vivo.

Cocktails of ricin a‐chain immunotoxins against different antigens on hodgkin and sternberg‐reed cells have superior anti‐tumor effects against H‐RS cells in vitro and solid Hodgkin tumors in mice

Three ricin A-chain immunotoxins (ITs) recognizing different antigens on Hodgkin-Reed/Sternberg (H-RS) cells were evaluated for their anti-tumor effects when used in combination as "cocktails". These ITs, BB10.dgA (CD25), HRS3.dgA (CD30), and IRac.dgA (70 kDa), strongly inhibited the growth of L540Cy H-RS cells in vitro. The protein synthesis of this cell line was reduced more efficiently by the combination of 2 of these ITs than by BB10.dgA, HRS3.dgA or IRac.dgA alone. A cocktail of all 3 ITs was most effective in vitro. This was at least in part due to the non-homogeneous distribution of CD25, CD30 or IRac on the L540Cy H-RS target cells and to the fact that subpopulations deficient in one antigen nevertheless expressed appreciable levels of the other target antigens. IT cocktails were also superior as anti-tumor agents in nude mice with solid L540Cy tumors. Ninety percent of mice treated with cocktails containing 2 or 3 ITs had continuous complete remissions (CCR), as compared with only 40% of mice treated with the same dose of a single IT. Analysis of 7 L540Cy sub-lines re-established ex vivo from mice that relapsed after having achieved complete remission (CR) after therapy with a single IT showed that the surviving tumor cells were antigen-deficient variants which were resistant to the original IT, but which could be killed by ITs directed against other target antigens. Thus, IT cocktails give superior results against human H-RS cells, both in vitro and in vivo.

Development of new ricin A-chain immunotoxins with potent anti-tumor effects against human Hodgkin cells in vitro and disseminated Hodgkin tumors in SCID mice using high-affinity monoclonal antibodies directed against the CD30 antigen.

The lymphocyte activation marker CD30 has been shown to be an excellent target for the immunotherapy of human Hodgkin's lymphoma. In order to develop new potent immunotoxins (ITs) against CD30, we chemically linked 6 recently described monoclonal antibodies (MAbs) via SMPT to deglycosylated ricin A-chain (dgA). Cross-blocking experiments demonstrated that these MAbs, termed Ki-2 to Ki-7, recognize 3 different clusters on the CD30 antigen: Ki-2, Ki-4, Ki-5 and Ki-7 recognize cluster A; Ki-6 recognizes cluster B; Ki-3 binds to cluster C. Staining of 29 sections of normal human organs revealed no major cross-reactivity of any MAbs tested. Binding to the CD30 antigen on L540Cy Hodgkin cells was assessed by flow cytometry, and demonstrated high affinities for Ki-2, Ki-3 and Ki-4. The concentration giving 50% of the mean fluorescence intensity (MFI50) was 0.58 micrograms/ml to 0.78 micrograms/l. MAbs Ki-5, Ki-6, and Ki-7 bound much more weakly. The staining intensity of the MAbs correlated with the cytotoxicity of the corresponding ITs. Ki-2.dgA, ki-3.dgA and Ki-4.dgA inhibited the protein synthesis of L540Cy cells by 50% at concentrations (IC50) of 3.5 x 10(-10)M to 4.0 x 10(-11)M. The most effective IT, Ki-4dgA, is 5-fold more potent than previously reported CD30 ricin A-chain ITs. Ki-4.dgA was subsequently used for the treatment of disseminated human Hodgkin's lymphoma in a SCID mouse model. The mean survival time (MST) of lymphoma-bearing SCID mice was extended from 42 days in untreated controls to more than 132 days when Ki-4.dgA was applied one day after tumor challenge. Ki-4.dgA is a new potent IT suitable for further evaluation against Hodgkin's lymphoma in man.

Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection.

The present studies were designed to further determine whether the CD25 marker could distinguish between cells productively and latently infected with HIV. This was accomplished by combining immunotoxin (IT)-mediated killing of CD25+ cells, highly sensitive indirect immunofluorescence to detect remaining CD25+ cells, and PCR-mediated amplification of proviral DNA in immunotoxin-treated vs untreated HIV-infected cells. Our results demonstrate that: 1) By direct immunofluorescence 3 to 8% of PBMCs are CD25+, whereas by indirect immunofluorescence 30% are CD25+. The increased number of CD25+ cells is due to their detection by the highly sensitive indirect immunofluorescence assay. Up to 60% of the CD25+ cells are CD4+ and 12% are CD8+. 2) Treatment of HIV-infected PBMCs with an anti-CD25 IT for 6 days eliminated both CD25high and CD25low cells and decreased the production of p24 by 99%. 3) Differences in the HIV proviral genome were detected in the unfractionated PBMCs vs PBMCs from which CD25+ cells had been eliminated by IT treatment. Hence, PBMCs containing both CD25+ and CD25- cells express all intermediate proviral species and full-length double-stranded proviral DNA. In contrast, CD25- quiescent cells contain predominantly intermediate species. These results confirm and extend our previous observations that expression of CD25 can distinguish latently infected cells from cells producing virus.