Antitumor activity of Fab' and IgG-anti-CD22 immunotoxins in disseminated human B lymphoma grown in mice with severe combined immunodeficiency disease: effect on tumor cells in extranodal sites

The antitumor effects of two anti-CD22 ricin A chain-containing immunotoxin (IT) constructs were compared in mice with severe combined immunodeficiency disease with human Daudi cell tumors (SCID-Daudi mice). SCID-Daudi mice develop disseminated lymphoma that clinically resembles African Burkitt's lymphoma, i.e., extranodal disease including infiltration of the vertebral column and spinal canal. In the absence of treatment, the mean survival time of SCID-Daudi mice was 45.9 +/- 4.3 days. The mice was given injections of a dose of IT equal to 40% of the 50% lethal dose. The ITs consisted of either IgG or Fab' fragments of mouse anti-CD22 antibody coupled to deglycosylated ricin A chain (dgA). Both ITs were potent and specific and inhibited protein synthesis in Daudi cells in vitro by 50% at concentrations of 1.2 x 10(-12) (IgG-dgA) and 1.3 x 10(-11) M (Fab'-dgA). When administered to mice beginning 1 day after inoculation with tumor cells, both ITs extended the mean survival time, to 87.2 +/- 18.9 days (IgG-dgA) or 57.9 +/- 3.8 days (Fab'-dgA). The latter represented the killing of 2 logs of Daudi cells, and the former 4 logs. IgG antibody alone killed 1 log of tumor cells. The IgG-dgA had an antitumor effect even when administered 20-23 days after tumor inoculation. Gross and histological examinations of IT-treated tumor-bearing mice showed a marked decrease in the number and size of neoplastic foci in both lymphoid organs and extranodal sites.     

Antitumor activity of L6-ricin immunotoxin against the H2981-T3 lung adenocarcinoma cell line in vitro and in vivo.

The monoclonal antibody L6 recognizes a determinant that is expressed on lung, breast, colon, and ovarian carcinomas and is present only at trace levels in normal tissues. L6 was covalently linked to intact ricin by a thioether bond to produce an immunotoxin (IT). Gel analysis revealed that this IT was heterogeneous, but mostly one monoclonal antibody molecule linked to one ricin molecule. The L6-ricin IT selectively bound and was selectively toxic to L6-positive H2981-T3 adenocarcinoma cells in protein synthesis inhibition assays in which lactose was added to block the native ricin binding site. Clonogenic studies showed that 1 microgram/ml L6-ricin could inhibit about 99.99% of H2981-T3 growth in a limiting dilution assay, even in the presence of a 20-fold excess of human bone marrow cells. Treatment of bone marrow cells with the same dose of L6-ricin resulted in the growth of ample numbers of bone marrow progenitor cells (colony-forming units-mixed, colony-forming units granulocyte/macrophage, and blast-forming units erythroid) after 14 days. We also evaluated the antitumor effect of L6-ricin administered intratumorally with lactose against established H2981-T3 tumors in a nude mouse model. Thirty % of the tumor-bearing animals responded completely to single-dose treatment, while 60% gave partial responses. The in vivo effects were not absolutely specific, since irrelevant anti-CD5 IT also induced tumor regression in this model (10% responded completely, while 30% gave partial responses). However, irrelevant IT gave higher systemic toxicity (50% mortality) than L6-ricin (23% mortality). The nonspecific activity of IT was possibly due to Fc binding, which was demonstrated in vitro, or due to ricin B-chain binding. Ricin alone was too toxic for sustained tumor protection. Unconjugated L6 had no antitumor effect. The data suggest that L6-ricin may be useful for in vitro purging of autologous bone marrow from patients with solid tumors and marrow involvement and for in vivo regional therapy of L6-positive carcinomas.     

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. © 1996 Wiley-Liss, Inc.     

Synergistic potentiation of in vivo antitumor activity of anti-human T-leukemia immunotoxins by recombinant alpha-interferon and daunorubicin

In the present study, immunotoxins (ITs) containing ricin A chain (RA) and anti-human T leukemia monoclonal antibodies SN1 and SN2 were used with or without alpha-interferon (IFN) and/or daunorubicin (DNR) for in vivo tumor suppression. SN1 and SN2 are directed toward two unique human T-leukemia-associated cell surface antigens, TALLA and GP37, respectively. As the tumor model, we used nude mice bearing ascitic tumors of Ichikawa, a human T acute lymphoblastic leukemia cell line. In initial studies, we investigated the effect of the IT injection schedule on the efficacy of ITs in the in vivo suppression of the ascitic tumors. Four doses of 20 micrograms each of SN1-RA and SN2-RA completely suppress the tumor growth in 100% of the treated mice when the IT treatment is initiated either 1 or 2 days after tumor inoculation of 1.6 x 10(7) Ichikawa cells into the mice. Subsequently, we investigated the potentiating effects of IFN and DNR on the in vivo antitumor activity of ITs. To this end, we chose to initiate the treatment 4 days after the tumor inoculation when IT treatment alone is only partially effective. ITs (10 micrograms each of SN1-RA and SN2-RA) plus IFN (2 x 10(5) IU) or ITs plus IFN plus DNR (5 micrograms) completely suppress tumor growth in 100% of the treated mice while similar treatment with any one of the three agents is only partially effective. Similar treatment with ITs plus DNR or IFN plus DNR results in complete suppression of tumor growth in 80% of the treated mice. These results were reproducible in a repeated experiment. To gain information about the mechanisms involving the IFN potentiation of IT activity, we carried out several experiments. The cell surface expression of TALLA and GP37 was slightly augmented by the in vitro incubation of Ichikawa cells with IFN as measured by fluorescence-activated cell sorter analysis. The degree of the increase in either TALLA or GP37 was significantly smaller than that of HLA class I antigens in the same experiment. In in vitro experiments, IFN did not show any significant cytotoxic activity against Ichikawa cells or augment the cytotoxic activity of ITs against Ichikawa cells. On the other hand, injections of IFN into nude mice augmented activity of macrophages and NK cells; however, Ichikawa leukemia cells were rather resistant to the NK cell lysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sensitivity of fresh leukemic cells to T101 ricin A-chain immunotoxin: a comparative study between Fab fragment and whole Ig conjugates

We compared the cell killing potency of a whole Ig ricin A-chain immunotoxin (T101 IgG-RTA) against its Fab fragment counterpart (T101 Fab-RTA) on both CEM cells and fresh malignant lymphoid cells. A dye exclusion assay (DEA), was used to evaluate the kinetics of leukaemia cell viability mediated in vitro by each immunotoxin (IT). This study found that in the absence of ammonium chloride (NH4Cl), used as an enhancer agent, T101 Fab-RTA was significantly more toxic to both CEM and fresh leukaemia cells than T101 IgG-RTA. In the presence of NH4Cl (10(-2) M), while no differences could be found between the two IT on CEM cells, T101 Fab-RTA was clearly superior to T101 IgG-RTA on fresh leukaemia cells. These results suggest that T101 Fab-RTA may offer an excellent alternative to T101 IgG-RTA for IT treatment of CD5 positive leukaemia patients.     

Combined immunochemotherapy of human solid tumors in nude mice

In vivo immunochemotherapy of human solid tumors was studied in a nude mouse model. Large tumors (3 to 6 cm3) were induced by s.c. injection of the acute lymphoblastic leukemia T-cell line CEM. Transient tumor inhibition could be achieved by intratumoral injection of an intact-ricin immunotoxin that specifically recognizes a determinant CD5 (T,p67) expressed on the cell surface. Injection of the in vitro active cyclophosphamide congener mafosfamid had little effect on the progression of tumor growth. A combination regimen of immunotoxin and mafosfamid induced the most dramatic antitumor effect; a 72 to 100% reduction in tumor volume was observed within 3 to 4 days posttreatment. However, tumors relapsed within 5 to 13 days. Persistent, tumor regression was observed only when protocols using successive injections of combined immunotoxin/mafosfamid were used. All seven mice undergoing this treatment had a precipitous decrease in tumor size, and 86% survived greater than 30 days posttreatment. No residual tumor was detectable on Day 30 in five of seven mice. Regression was partly attributed to the selective activity of immunotoxin, since successive injections of an irrelevant control immunotoxin coupled to ricin in combination with mafosfamid did not reduce tumor size. Thus, we have demonstrated that a combination of anticancer chemotherapy and immunotoxin therapy yielded a greater antitumor effect than either therapy alone.     

Influence of cytotoxicity enhancers in combination with human serum on the activity of CD22-recombinant ricin A against B cell lines, chronic and acute lymphocytic leukemia cells.

Despite the strong in vitro activity of some immunotoxins (ITs), clinical application did not result in complete cure. The outcome of therapy may be improved by combining ITs with IT-cytotoxicity enhancing agents. We studied the effect of various agents that influence the intracellular routing of ITs on the activity of the anti-B cell IT CD22-recombinant (rec) ricin A. In protein synthesis inhibition assays the carboxylic ionophores monensin and nigericin enhanced the activity of the IT 117- and 382-fold, respectively, against the cell line Daudi, and 81- and 318-fold, respectively, against the cell line Ramos. IT activity to Daudi and Ramos was enhanced to a lesser extent by the lysosomotropic amines chloroquine (14- and 11-fold, respectively) and NH4Cl (nine- and 10-fold, respectively). However, the combination of NH4Cl and chloroquine induced more than an additive effect (145- and 107-fold, respectively). Cytotoxicity was not influenced by brefeldin A, all-trans retinoic acid (ATRA), verapamil and perhexiline maleate. Bacitracin enhanced the IT cytotoxicity in contrast to the other protease inhibitors aprotinin, leupeptin and soybean trypsin inhibitor, albeit enhancement was weak (two-fold). The enhancers exerted only a negligible effect on bone marrow progenitor cells. We recently developed a flow cytometric cytotoxicity assay in which cell elimination can be assessed. In order to detect enhancement in this assay, we used 5 x 10(-11) M IT (approximately the 50% protein synthesis inhibiting dose (ID50)). This concentration killed 41% of the Daudi cells and 42% of the Ramos cells. In the presence of 10 nM monensin the IT killed 74% and 99% and in the presence of 10 nM nigericin 96% and 99% of the Daudi and Ramos cells, respectively. At 10(-8) M, CD22-rec ricin A eliminated malignant cells originating from three patients with B-CLL (0.42 log) and two with B-ALL (0.19 log) patients. Cytotoxicity to malignant cells was enhanced by NH4Cl, chloroquine, monensin and nigericin. The combination of NH4Cl and chloroquine enhanced the activity most effectively (up to 2.06 log). To determine the applicability of the IT in combination with enhancers in vivo we investigated the effect of human serum. Human serum inhibited IT activity which could not be restored by monensin and nigericin because of complete inhibition of these enhancers by serum. In contrast, chloroquine partially restored the activity of CD22-rec ricin A in the presence of human serum. We conclude that monensin, nigericin and the combination of NH4Cl and chloroquine can be used instead of NH4Cl to potentiate CD22-rec ricin A activity in purging autologous bone marrow transplants contaminated with malignant B cells. Chloroquine might be a promising enhancer of CD22-rec ricin A for treating patients in vivo.     

Efficacy of an anti-CD7-ricin A chain immunoconjugate in a novel murine model of human T-cell leukemia.

In vivo efficacy testing of monoclonal antibody-based drugs specific for human leukemias is hampered by the paucity of suitable animal models, due in part to the inability of many anti-human monoclonal antibodies to cross-react with antigens expressed in animal tissues or cells. Moreover, human leukemic cells have proven difficult to establish in immunosuppressed mice except as solid tumors. We report here the establishment of a murine model for human leukemia displaying features of human disease, such as growth of malignant cells and localization of such cells to lymphoid compartments, and the effective depletion of leukemic cells from these mice by an immunoconjugate. Human T-leukemia cells (CEM) injected into cyclophosphamide-pretreated NIH-III mice engrafted in all mice (n = 41), with CEM cells detected in the bone marrow, spleen, and blood 4 weeks after injection. There was no evidence of solid tumors. Treatment of CEM-engrafted mice with 4A2-RTA30, an immunoconjugate of an anti-CD7 monoclonal antibody and ricin A chain (RTA30), resulted in a 100- to 200-fold overall depletion of CEM cells from the spleen and the bone marrow (P less than 0.02). This depletion was specific and toxin-dependent, as a control immunoconjugate had no demonstrable effect (P greater than 0.5). Depletion of CEM cells was also observed after treatment with unconjugated anti-CD7 mAb, but this effect was not significantly different from controls (P greater than 0.1). Therefore, significant depletion of CEM cells required the presence of the ricin A chain moiety. Further investigations revealed that CEM cells recovered from NIH-III mice expressed less CD7 antigen, but remained sensitive to subsequent in vitro exposure to 4A2-RTA30. In conclusion, we have established a model for studying the efficacy of immunoconjugates and have successfully depleted human T-leukemic cells from lymphoid tissues in immunodeficient mice by treatment with an anti-CD7-RTA30 immunoconjugate.     

Tumor suppressive efficacy through augmentation of tumor-infiltrating immune cells by intratumoral injection of chemokine-expressing adenoviral vector

Our goal in the present study was to evaluate antitumor effects and frequency of tumor-infiltrating immune cells upon intratumoral injection of RGD fiber-mutant adenoviral vector (AdRGD) encoding the chemokines CCL17, CCL19, CCL20, CCL21, CCL22, CCL27, XCL1, and CX3CL1. Among eight kinds of chemokine-expressing AdRGDs, AdRGD-CCL19 injection most efficiently induced infiltration of T cells into established B16BL6 tumor parenchyma, whereas most of these T cells were perforin-negative in immunohistochemical analysis. Additionally, the growth of AdRGD-CCL19-injected tumors decreased only slightly as well as that of other tumors treated with each chemokine-expressing AdRGD, which indicated that accumulation of naive T cells in tumor tissue does not effectively damage the tumor cells. Tumor-bearing mice, in which B16BL6-specific T cells were elicited by dendritic cell-based immunization, demonstrated that intratumoral injection of AdRGD-CCL17, -CCL22, or -CCL27 could considerably suppress tumor growth and attract activated T cells. On the other hand, AdRGD-CCL19-injection in the immunized mice showed slight increase of tumor-infiltrating T cells compared to treatment using control vector. Collectively, although AdRGD-mediated chemokine gene transduction into established tumors would be very useful for augmentation of tumor-infiltrating immune cells, a combinational treatment that can systemically induce tumor-specific effector T cells is necessary for satisfactory antitumor efficacy.     

Evidence for absence of toxicity of T101 immunotoxin on human hematopoietic progenitor cells prior to bone marrow transplantation

T101-ricin A-chain immunotoxin is a hybrid molecule made up of the T101 monoclonal antibody bound to the A-chain of ricin. It specifically destroys cells expressing the cell surface T65 antigen. We have designed a preclinical study to evaluate its possible use for the in vitro treatment of T-cell hematological cancers prior to autologous bone marrow transplantation. The data presented here show that conditions previously defined to produce high tumor cell killing, i.e., a 20-hr incubation at 37 degrees in the presence of T101-ricin A-chain immunotoxin up to 10(-7) M in a 10 mM ammonium chloride solution, do not affect the in vitro proliferative capacity of human hematopoietic stem cells studied by means of semisolid medium cultures (granulocyte-macrophage progenitors, burst-forming units-erythrocyte) and continuous liquid cultures (pre-granulocyte-macrophage progenitors). Therefore, autologous bone marrow transplantation with T101-ricin A-chain immunotoxin-treated graft should be feasible.     

Parameters affecting tumor-specific delivery of anti-CD5 antibody-ricin A chain immunotoxins in vivo.

The tumor localization of various anti-CD5 immunotoxins (ITs) was studied in vivo in BALB/c nude mice bearing human ascitic Ichikawa tumor cells. Plasma and ascitic clearance of IT as well as the number of intact IT molecules taken up per tumor cell was measured with a highly sensitive immunoenzymometric assay. This assay allowed the quantification of as few as 400 molecules per cell, either bound to cell surface antigens or internalized. Tumor cell localization of an i.v.-administered IT composed of a whole IgG linked by a disulfide bond to native ricin A chain (RTA) was extremely small. The highest binding level of this IT to ascitic cells did not exceed 1,600 molecules per cell, whereas up to 25,000 molecules per cell were quantified after i.p. injection. To improve IT localization after i.v. injection, both the size of the monoclonal antibody moiety and glycosylation of the ricin A chain were examined. Two major additive improvements were obtained by (a) Fc removal and (b) partial deglycosylation of RTA. The localization of F(ab')2-deglycosylated RTA IT was 5.5 times greater than that of IgG-RTA IT, resulting from reduced plasma clearance and better extravasation of the conjugate. However, the IT targeting observed in vivo after i.v. administration was unexpectedly lower than the theoretical number of bound IT molecules, calculated from the concentration of soluble IT molecules in the ascitic fluid. This discrepancy was found to be related to a dramatic decrease in the antigen binding capacity of the antibody moiety in ascitic fluid.     

白细胞介素2质粒复合物治疗后小鼠肝癌的组织学变化

目的 探讨瘤体内直接注射白细胞介素2质粒／阳离子脂质体复合物治疗小鼠肝癌的效果和机制。方法 将小鼠白介素2表达质粒（VR1110）与阳离子脂质体（Transfectam)按适当比例混合而形成复合物（VR1110／Transfectam)。通过瘤体内接注射此复合物治疗小鼠肝癌模型,观察治疗后肿瘤积变化、组织学变化及表达情况。结果 瘤体内注射VR1110／Transfectam后,可在肿瘤组织中检测到小鼠IL－2mRNA的表达。VR1110／与Tansfectam治疗组肿瘤生长较其它对照组明显减慢,第1次治疗后第12天肿瘤体积明显小于其它组（P＜0．05）。治疗组小鼠均可见肿瘤组织大量坏死,炎性细胞浸润,及CD4^ 、CD8 淋巴细胞。结论 瘤体内直接注射VR1110／Transfectam复合物对小鼠肝癌的治疗作用明显,增强了小鼠机体对肝癌细胞的免疫学反应。     

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 (CD2S), 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 sub-populations 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.     

In vivo therapy of the BCL1 tumor: effect of immunotoxin valency and deglycosylation of the ricin A chain

The in vivo therapeutic effects of Fab' and IgG anti-delta (anti-IgD)-ricin A chain immunotoxins were compared in mice bearing the surface IgD-positive BCL1 leukemia. The immunotoxins were prepared with either native or deglycosylated ricin A chain. Immunotoxin therapy was assessed both by the number of cells bearing the BCL1 immunoglobulin idiotype which remained in the spleen 24-48 h after injection with immunotoxin and by adoptive transfer of these spleen cells into normal mice. Immunotoxins prepared with either Fab'-anti-delta or IgG-anti-delta and native A chain induced a dose-dependent reduction in the number of idiotype-positive BCL1 cells present in the spleens of the tumor-bearing mice. The maximal therapeutic response was achieved with 250 micrograms of immunotoxin (containing 80-100 micrograms A chain) per mouse for both immunotoxins, resulting in tumor reduction of approximately 90%. This represents an elimination of 3-4 X 10(8) tumor cells. Reduction in the number of tumor cells was not observed with control reagents including antibody alone, antibody mixed with A chain, or an immunotoxin of irrelevant specificity. A Fab' immunotoxin prepared with deglycosylated ricin A chain was approximately 5-fold more effective as an antitumor reagent than the same immunotoxin prepared with native A chain; thus, optimal therapy was achieved after injection of 50 micrograms of immunotoxin (containing 15-20 micrograms A chain). Since the immunotoxins prepared with deglycosylated A chain were only 2-3-fold more toxic to the mice than those prepared with native A chain, the former resulted in a 2-3-fold increase in the therapeutic index.     

Therapy of human T-cell acute lymphoblastic leukaemia with a combination of anti-CD7 and anti-CD38-SAPORIN immunotoxins is significantly better than therapy with each individual immunotoxin

Severe combined immunodeficient (SCID) mice injected i.v. with the human T-ALL cell line CCRF CEM (SCID-CEM mice) develop within 50 days life-threatening multi-organ growth of leukaemia cells. The development of leukaemia in SCID-CEM mice treated with three 10 microg i.v. doses of the anti-CD7 immunotoxin (IT) HB2-SAPORIN or the anti-CD38 IT OKT10-SAPORIN was significantly delayed compared with PBS sham-treated animals but 90% of animals treated with either IT eventually developed disseminated leukaemia cell growth. In contrast treatment of SCID-CEM mice with a combination of both ITs led not only to a significantly greater delay in time to leukaemia development but also in the numbers of animals remaining leukaemia free (60%). The native HB2 and OKT10 antibodies (both murine IgG1antibodies) exerted significant, though relatively weak therapeutic effects, probably mediated through an antibody-dependent cellular cytotoxicity (ADCC) mechanism. Moreover, there was no in vivo additivity of therapeutic effect when both antibodies were used in combination. Apparent, however, was that the combination of HB2-SAPORIN IT with OKT10 antibody led to an intermediate therapeutic effect that was significantly greater than that obtained when either was used alone but significantly less than that obtained when the two IT combination was utilized. This was similarly the case for the combination of OKT10-SAPORIN IT with HB2 antibody though the effect was less pronounced in this instance. This result suggests that the therapeutic effect of IT + antibody treatment results from an additivity between antibody-mediated delivery of saporin combined with a SCID mouse NK cell-mediated ADCC attack on the target cell directed through target cell bound antibody Fc engagement with FcgammaRIII on the NK cell surface. The combination of both ITs however gave the best therapeutic outcome in SCID-CEM mice probably as the result of (i) delivery of greater amounts of saporin to target CEM cells positive for both CD7 and CD38, (ii) delivery of an effective dose of saporin to CEM cells downregulated or negative for one of the target antigens and (iii) through ADCC mechanisms that interact additively with IT action. We have previously proposed that combination IT therapy would be one means of overcoming the problem of heterogeneity of antigen expression within a global tumour cell population and these additional findings support this and provide a further strengthening of the rationale for employing cocktails of ITs for the treatment of human malignancies.     

Evaluation of four CD22 antibodies as ricin A chain-containing immunotoxins for the in vivo therapy of human B-cell leukemias and lymphomas

Ricin A chain-containing immunotoxins (IT-As) specific for the human B-cell antigen, CD22, were prepared from 4 monoclonal antibodies (MAbs) or their Fab' fragments: RFB4, HD6, UV22-I and UV22-2. The ITs were tested for their ability to kill cells from the Burkitt lymphoma line, Daudi, the pre-B-cell leukemia line, NALM-6, and the myeloma cell line, ARH-77. Daudi expresses high levels of CD22, whereas NALM-6 and ARH-77 express low levels of CD22. The IgG-RFB4-A was highly toxic to all 3 cell lines; it killed 50% of the Daudi cells at a concentration of 1.2 x 10(-12) M and 50% of NALM-6 and ARH-77 cells at concentrations of 1.5 to 2.1 x 10(-11) M. IgG-RFB4-A was 10-30 times more toxic to Daudi cells than were the IgG-As constructed from the other 3 CD22 MAbs and 10 times more toxic than ricin itself. IT-As constructed from the Fab' fragments of the 4 CD22 antibodies were 2 to 5 times less toxic to Daudi cells than their IgG-A counterparts. Fab'-RFB4-A was twice as toxic to Daudi cells as ricin, whereas the other Fab'-As were about 7 times less toxic than ricin. Scatchard analyses of the binding of the radio-iodinated antibodies to Daudi cells showed that the intact RFB4 antibody bound 3-10 times more strongly than the other antibodies, whereas the Fab'-RFB4 bound 1.2 to 3.5 times more strongly than the Fab' fragments prepared from the other antibodies. Thus, the potent cytotoxic activity of the RFB4-As appears to derive, in part, from their superior binding affinity. Prior studies have shown that UV22-I and HD6 cross-react with certain normal human tissues lacking cells of B-cell lineage, whereas UV22-2 and RFB4 are B-cell-specific. This fact, together with its superior potency as an IT-A, suggests that RFB4 is the antibody of choice for preparing Fab'-As or IgG-As for in vivo therapy of human B-cell leukemias and lymphomas.     

Antitumor effect of intratumoral administration of bone marrow-derived dendritic cells transduced with wild-type p53 gene.

PURPOSE: Dendritic cells (DCs) are attractive effectors for cancer immunotherapy because of their potential to function as professional antigen-presenting cells for initiating cellular immune responses. The tumor suppressor gene p53 is pivotal in the regulation of apoptosis, and approximately 50% of human malignancies exhibit mutation and aberrant expression of p53. We investigated the antitumor effect of intratumoral administration of bone marrow-derived dendritic cells transduced with wild-type p53 gene. EXPERIMENTAL DESIGN: We examined whether intratumoral administration of DCs infected with recombinant adenovirus expressing murine wild-type p53 (Ad-mp53) could induce systemic antitumor responses against mutant p53-expressing tumors, highly immunogenic MethA, or weakly immunogenic MCA-207 implanted in syngeneic mice. RESULTS: Accumulation of wild-type p53 protein in bone marrow-derived murine DCs could be successfully achieved by Ad-mp53 infection. Treatment with intratumoral injection of Ad-mp53-transduced DCs caused a marked reduction in the in vivo growth of established MethA and MCA-207 tumors with massive cellular infiltrates. Administration of p53-expressing DCs suppressed the growth of both injected MCA-207 tumors and untreated distant MCA-207 tumors, but not unrelated Lewis lung carcinoma tumors, suggesting the augmentation of systemic immunogenicity against MCA-207 tumor cells. Moreover, intratumoral injection of p53-expressing DCs had a greater antitumor effect than did s.c. immunization. CONCLUSIONS: Our results indicate that intratumoral administration of DCs expressing murine wild-type p53 leads to significant systemic immune responses and potent antitumor effects in mutant p53-expressing murine cancer models. These findings raise the possibility of using this strategy of intratumoral injection of p53-expressing DCs for human cancer treatment.     

Comparative biochemical, cytotoxic and pharmacokinetic properties of immunotoxins made with native ricin A chain, ricin A1 chain and recombinant ricin A chain

Immunotoxins were constructed by attaching native ricin A chain, ricin A1 chain and recombinant ricin A chain to the mouse monoclonal IgG2a antibody Fib75 by means of a disulphide linkage using the hetero-bifunctional cross-linker SPDP. The Fib75 immunotoxins were of similar composition and exerted identical cytotoxic effects against the EJ human bladder carcinoma cell line in tissue culture. All 3 immunotoxins broke down to the same extent upon incubation with glutathione in vitro. The clearance of the immunotoxins from the circulation of normal Wistar rats was determined following i.v. administration. The concentration of intact immunotoxin in serum samples taken at various intervals up to 48hr after injection was measured by a ricin A chain-specific ELISA. The Fib75 immunotoxin made with native ricin A chain was removed from the circulation most rapidly. Fib75-recombinant ricin A chain persisted in the circulation at a higher level than Fib75-ricin A1 chain. A higher proportion of the ricin A1 chain immunotoxin was lost from the bloodstream during the alpha-phase. The beta-phase half-lives of Fib75-recombinant ricin A chain and Fib75-ricin A1 chain were similar, consistent with the identical susceptibility of the immunotoxins to cleavage by glutathione. The presence of the complex-type oligosaccharide side-chain on the A1 chain may have accelerated the clearance of the A1 chain immunotoxin in relation to that of the immunotoxin made with the aglycosyl recombinant A chain.     

The antitumor effects of human lymphoblastoid interferon on human renal cell carcinoma in athymic nude mice

Summary The antitumor effects of human lymphoblastoid interferon (HLBI) on human renal cell carcinomas transplanted in nude mice, i.e., KU-2 and RCC-1, were investigated and compared with those on other human tumors, viz. HeLa (cervical carcinoma), KB (nasopharyngeal carcinoma), H.Ep#2 (laryngeal carcinoma), and MX-1 (breast cancer). A pharmacokinetic study on HLBI was also carried out in non-tumor-bearing nude mice.HLBI therapy was performed with a dose of 10⁵ IU/mouse by daily SC or IT (intratumoral) injection for 2–4 weeks. Two renal cell carcinomas, KU-2 and RCC-1, proved to be highly sensitive to HLBI. The growth of these tumors was inhibited not only by IT but also by SC injection of HLBI. In contrast, HLBI exerted only a slight effect or none at all on the other human tumors, namely, MX-1, KB, H.Ep#2, and HeLa, even when given by IT injection. The data show that the antitumor effects of HLBI depend on the types of human tumors and may be relevant to the clinical observation that renal tumors are sensitive to HLBI.The serum HLBI reached a peak level of 4,390 U/ml 1 h after a single SC injection at a dose of 10⁵ IU/mouse and declined with a half-life of 4h to 128 U/ml 24 h later. This time-course was not affected by 10 consecutive daily injections of HLBI. In nude mice, the consecutive administration of HLBI at this dose level appears to result in neither accumulation nor rapid clearance due to antibody formation. From this range of serum HLBI levels and its in vitro anticellular activity, the in vivo antitumor effects of HLBI in nude mice seemed to depend on its direct anticellular action.     

Treatment of SCID/human B cell precursor ALL with anti-CD19 and anti-CD22 immunotoxins.

The anti-CD19 (HD37-dgRTA) and anti-CD22 (RFB4-dgRTA) immunotoxins (ITs) are murine IgG(1) monoclonal antibodies (Mabs) conjugated to a deglycosylated ricin A chain (dgRTA). They are effective in killing B-lineage non-Hodgkin's lymphoma (NHL) cells in vitro, in vivo and in adult patients with B-lineage NHL. The potential of these agents for the treatment of childhood B-precursor acute lymphoblastic leukemia (ALL) is unknown. The anti-CD19 and anti-CD22 ITs should have anti-tumor activity against childhood B-lineage ALL since both target antigens are expressed on the surface of these cells. We have previously shown that, in vitro these two ITs selectively kill leukemia cells obtained from children with leukemia. To evaluate the efficacy of our ITs in an in vivo model we injected the human pre-B ALL cell line, NALM-6-UM1, into severe combined immunodeficient (SCID) mice. We tested the ability of two ITs to prolong survival or cure mice of both early and advanced tumors. In early disease, treatment with HD37-dgRTA, RFB4-dgRTA, or Combotox (an equimolar concentration of the two ITs) significantly improved their survival. In advanced disease, treatment with RFB4-dgRTA or Combotox significantly improved survival. Overall there were 10 long-term survivors who were cured, as determined by survival beyond 150 days with no evidence of disease as determined by polymerase chain reaction (PCR) analysis.