Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ～ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ～ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.     

Efficacy and toxicity of plasma-cell-reactive monoclonal antibodies B-B2 and B-B4 and their immunotoxins

 Immunotherapy based on the delivery of toxic agents to the tumor site using monoclonal antibodies (mAb) may be a promising modality in the treatment of hematological malignancies. In the selection of mAb, both for ex vivo but even more for in vivo therapy, not only their reactivity to the neoplastic cells should be considered, but also reactivity to other body constituents. Here we describe the screening of two human plasma-cell-reactive mAb B-B2 and B-B4, which may be used for immunotherapy of multiple myeloma. Cross-reactivity of B-B2 and B-B4 was determined by immunohistochemistry on a series of tissues. This revealed for both B-B2 and B-B4 a strong staining of epithelial cells in various organs, e.g. lung, liver, skin, kidney and gut, while only a weak and diffuse staining was seen with endothelial cells. In bone marrow reactivity was only found with plasma cells and not with hemopoietic precursors (CD34⁺ cells). Immunotoxins from B-B2 and B-B4 were constructed by coupling them to the plant-derived ribosome-inactivating protein saporin. Both B-B2 and B-B4 immunotoxins appeared to be efficient in specific inhibition of protein synthesis in plasma cell lines (IC₅₀ respectively 1 nM and 0.1 nm). The immunotoxins were also tested on epithelial cell line A431, on liver cell line HepG2 and on human umbilical vein endothelial cells. The epithelial cell line A431 was reactive with both B-B2 and B-B4, but was only inhibited by B-B4 immunotoxin. Cell line HepG2 was reactive with both mAb, but was not inhibited by either immunotoxin. The endothelial cells showed no reactivity with B-B2 and B-B4 and were not inhibited by either immunotoxin. Bone marrow treated with B-B2 and B-B4 immunotoxin did not show a decrease in colonies of hemopoietic precursor cells. Incubation of multiple-myeloma-derived bone marrow with these immunotoxin resulted in a clear decrease of the number of plasma cells. Received: 13 March 1996 / Accepted: 21 May 1996     

Blocked and non-blocked ricin immunotoxins against the CD4 antigen exhibit higher cytotoxic potency than a ricin A chain immunotoxin potentiated with ricin B chain or with a ricin B chain immunotoxin

Summary An immunotoxin consisting of ricin A chain linked to the monoclonal antibody M-T151, recognising the CD4 antigen, was weakly toxic to the human T-lymphoblastoid cell line CEM in tissue culture. The incorporation of [³H]leucine by CEM cells was inhibited by 50% at an M-T151-ricin-A-chain concentration (IC₅₀) of 4.6 nM compared with an IC₅₀ of 1.0 pM for ricin. In contrast, immunotoxins made by linking intact ricin to M-T151 in such a way that the galactose-binding sites of the B chain subunit were either blocked sterically by the antibody component or were left unblocked, were both powerfully cytotoxic with IC₅₀ values of 20–30 pM. The addition of ricin B chain to CEM cells treated with M-T151—ricin-A-chain enhanced cytotoxicity by only eight-fold indicating that isolated B chain potentiated the action of the A chain less effectively than it did as an integral component of an intact ricin immunotoxin. Ricin B chain linked to goat anti-(mouse immunoglobulin) also potentiated weakly.Lactose completely inhibited the ability of isolated ricin B chain to potentiate the cytotoxicity of M-T151—ricin-A-chain and partially (3- to 4-fold) inhibited the cytotoxicity of the blocked and non-blocked ricin immunotoxins. Thus, in this system, the galactose-binding sites of the B chain contributed to cell killing regardless of whether isolated B chain was associated with the A chain immunotoxin or was present in blocked or non-blocked form as part of an intact ricin immunotoxin. The findings suggest that the blocked ricin immunotoxin may become unblocked after binding to the target antigen to re-expose the cryptic galactose-binding sites. However, the unblocking cannot be complete because the maximal inhibition of [³H]leucine incorporation by the blocked immunotoxin was only 80% compared with greater than 99% inhibition by the non-blocked immunotoxin.     

Comparison of multiple anti-CEA immunotoxins active against human adenocarcinoma cells

Summary Anti-carcinoembryonic antigen (CEA) immunotoxins constructed with multiple anti-CEA antibodies (goat and baboon polyclonal, and three murine monoclonal antibodies) by covalently linking them to the A chain of ricin via a disulfide bond all function as potent and specific toxins for CEA-bearing cells, suggesting that the CEA molecule is capable of directing productive internalization of ricin A chain. The high potency of anti-CEA immunotoxins apparently makes addition of ricin B chain unnecessary for high toxic efficiency, as in some other systems, because presence of the B chain reduces target cell specificity. Several characteristics of the immunotoxins which might account for their cytotoxic potency were studied. Equilibrium association constants of the goat, baboon, and murine monoclonal C-19 antibodies with fluid-phase CEA were determined by using Langmuir plots and were found to be 8.79, 6.61, and 8.13×10⁹ M ^–1, respectively, indicating the high and similar affinities of the three antibodies toward CEA. Radioimmunoassay binding studies of the three immunotoxins with ¹²⁵I-CEA showed that the antibody portions of the molecules retained the ability to form complexes with CEA after conjugation to ricin A chain. The maximum number of anti-CEA antibody molecules bound per cell, as demonstrated by ¹¹¹In-labeled C-19 binding assays with CEA-bearing cell lines, varied from 2.65×10⁵ per cell for HT29 to 2.01×10⁶ for LoVo, with an intermediate value of 1.17×10⁶ per cell for WiDr. Cytotoxicity of the immunotoxins was assessed by inhibition of protein synthesis and expressed as a median inhibitory dose (ID₅₀). Comparison of the ID₅₀'s of each immunotoxin on the three cell lines has shown that the immunotoxin made of the monoclonal C-19 antibody is in general 6 to 7 times more cytotoxic than the goat and baboon antibody immunotoxins. The affinity of CEA-antibody binding is probably an important, but not a sole factor in determining the immunotoxin potency. The fact that the antibodies with very similar affinity toward fluid phase CEA make immunotoxins of different potency might indicate that interactions with membrane-bound CEA are more complex and/or the efficiency of internalization of various immunotoxins is different. An important factor in immunotoxin action appears to be the CEA content in target adenocarcinoma cells.Supported in part by the NIH BRSG grant SO7RRO5712, the American Cancer Society, Mass. Div. Research Grant 1543-C-1, and by the Aid for Cancer Research (Boston) award to L. V. L., and by RO1 CA 29160 and RO1 CA 39748 grants to T. W. G.     

Antibody-induced activation of p185HER2 in the human lung adenocarcinoma cell line Calu-3 requires bivalency

In the present study we utilized two previously described monoclonal antibodies (mAb), and their respective Fab portions, directed against the extracellular domain of p185^HER2, a transmembrane glycoprotein with intrinsic tyrosine kinase activity coded by theHER2/neu oncogene, to study the mechanism of mAb-induced receptor internalization and phosphorylation. Fluorescence scan analysis and direct binding of radiolabelled mAb and their Fab fragments showed that entire MGR2 and MGR3 mAb were reactive with similar binding affinity on two cell lines (Calu-3 and Sk-Br-3) overexpressing the p185^HER2 receptor, and unreactive on unrelated cells. The corresponding Fab fragments were positive on the related cells, but bound with diminished intensity and affinity. Entire MGR2 and MGR3 induced internalization in both Calu-3 and Sk-Br-3 cells, whereas their Fab portions were not internalized. When the bivalency of the MGR2 Fab fragment was artificially reconstituted by incubation with rabbit anti-(mouse IgG), internalization was obtained. Monovalent binding of the entire labelled antibodies, obtained in the presence of a saturating amount of unlabelled antibody, decreased both the rate and the final amount of internalized antibody. Metabolic labelling and immunoblotting experiments showed that incubation with entire MGR3 amplified the basal phosphorylation of the p185^HER2 receptor in Calu-3 and Sk-Br-3 cells, whereas MGR3 Fab decreased the signal. Taken together, our data indicate that antibody-mediated activation of p185^HER2 in Calu-3 and Sk-Br-3 cells occurs through the dimerization of receptor molecules and that bivalency of the activating antibody is mandatory for induction of internalization and phosphorylation of the receptor. Our data support an allosteric model of activation for the p185^HER2 receptor.     

Surface distribution and internalization of erbB-2 proteins.

We report the localization over the cell surface and the early steps of antibody-induced internalization of the product of the erbB-2 proto-oncogene, structurally related to the epidermal growth factor receptor (EGFR). We show that erbB-2/p 185 is mostly excluded from endocytic pits on the cell surface. Incubation at 37 degrees C with an anti-erbB-2/p185 monoclonal antibody induces the rapid entry of the protein into the cell. Similar internalization is shown by a chimeric molecule EGFR/erbB-2 in response to EGF. Both the timing and the pathway of internalization followed by the erbB-2/p185 appear totally similar to those described for the EGFR. At variance with the normal erbB-2/p185, two mutant activated erbB-2 proteins are frequently localized within endocytic pits of the cell surface, indicating that mutations in the transmembrane regions may determine constitutive internalization of the protein.     

Immunotoxin sensitivity of Chinese hamster ovary cells expressing human transferrin receptors with differing internalization rates

 Previous studies have shown that immunotoxin action is dependent upon selective binding to the target cell, internalization and then passage into the cytosol. It is important to define precisely how these critical steps are controlled so that the underlying relationship of each to high cytotoxic effectiveness is understood. In order to evaluate the contribution of internalization rate and receptor number on immunotoxin potency, the effects of an anti-(transferrin receptor, TfR)/ricin A chain immunotoxin, 7D3-A, were assessed on a parent Chinese hamster ovary cell line developed in our laboratory with no TfR (TfR^neg) and two lines transfected with either wild-type TfR (Tfr^wt) or an internalization-deficient (TfR^{δ7 – 58del}) mutated human TfR. Potent, receptor-mediated cytotoxicity resulted from the action of 7D3-A on TfR^wt cells (ID₅₀<1 nM) while both TfR^neg cells and TfR^{δ7 – 58del} were only minimally affected (ID₅₀>100 nM). Butyrate up-regulation substantially increased receptor expression on the TfR^wt and TfR^{δ7 – 58del} cells, but no corresponding rise in sensitivity to 7D3-A was observed. In contrast, immunotoxin potency was increased by co-treatment of TfR^wt cells with the carboxylic ionophore monensin and the effect was even more pronounced for TfR^{δ7 – 58del} cells. We conclude that internalization rate or intracellular destination is a much more important determinant of immunotoxin efficacy than receptor number. Received: 15 March 1996 / Accepted: 28 May 1996     

In vitro synergism between hybrid immunotoxins and chemotherapeutic drugs: relevance to immunotherapy of prostate carcinoma

Summary Cultured prostate carcinoma cells incubated in the presence of a novel hybrid immunotoxin and ricin A chain exhibited synergy with the chemotherapeutic drugs vinblastine, methotrexate, and bleomycin. No cooperative effect was noted with adriamycin. Under conditions where individual components of immunotoxin or chemotherapeutic drug mixtures were nontoxic or minimally toxic the immunotoxin-drug mixture exhibited marked impact on ¹⁴C amino acid incorporation into prostate carcinoma cells. Analysis of drug-treated cells by flow cytometry indicated that cells exposed to vinblastine and bleomycin bound hybrid immunotoxin antibody to a greater extent than cells not exposed to these drugs. Adriamycin did not exhibit synergistic cytotoxicity with hybrid immunotoxin. Also, adriamycin did not enhance antibody binding as evaluated by flow cytometry. The fact that hybrid monoclonal antibody-ricin A chain (HIT-RAC) conjugates inhibited uptake of ¹⁴C amino acids 3 to 10-fold within 48 h of incubation with target cells and that this inhibition was further increased 2 to 3-fold in conjunction with three out of four chemotherapeutic drugs tested may be attributed to the unique cytotoxicity imposed by the hybrid immunotoxins. The RAC moiety is not chemically coupled to antibody but instead occupies one of the antigen-combining sites of the molecule. In this manner, RAC is closely juxtaposed to the cell membrane of the target cell and is anchored in this position via binding of the remaining antigen-combining site to p40 prostate restricted antigen.     

Endocytosis of an antibody ricin A-chain conjugate (immuno-A-toxin) adsorbed on colloidal gold. Effects of ammonium chloride and monensin

An immunotoxin (IT) formed by a specific antibody coupled to the ricin A chain was adsorbed on colloidal gold particles (IT-Au). Binding and internalization of IT-Au in human lymphoblastic CEM cells were studied using electron microscopy. IT-Au showed specific cytotoxic activity toward the target cells. After 1 h at 4 degrees C, IT-Au were linked diffusely to the plasma membrane with 45% of the particles regrouped in clusters. Upon transfer to 37 degrees C, the particles carrying the ligand were regrouped more frequently and internalized into the cell by endocytosis through smooth microinvaginations or coated pits of the plasma membrane. After 15 min, IT-Au was observed in endocytic vacuoles, or receptosomes, in tubular structure near the Golgi apparatus and in lysosomes. Entry of IT-Au into lysosomes was rapid (around 50% of intracellular IT-Au particles after 30 min). NH4Cl or monensin, well-known potentiators of immunotoxin activity, when present in incubation medium, altered neither the processes nor the rate of IT-Au endocytosis. In the presence of either of these substances, IT-Au accumulated in the normal or often enlarged endocytic vacuoles, and entry into the lysosomes was slowed down (50% of particles after 2 h 15 min). We conclude that this intense slowing-down in the speed of IT-Au transportation into lysosomes and the functional modifications of these organelles help to explain the increased efficacy of immunotoxins in the presence of potentiators.     

In vitro efficacy of anti-HIV immunotoxins targeted by various antibodies to the envelope protein.

Six different anti-HIV envelope antibodies and one irrelevant control antibody were coupled to ricin A chain and tested for their efficacy in inhibiting HIV tissue culture infections. The anti-HIV antibodies consisted of five monoclonals, three of murine and two of human origin, and one polyclonal preparation prepared by affinity purifying pooled serum antibodies from HIV-infected humans on rgp160. The binding specificity of the antibodies was defined by ELISA by using recombinant envelope proteins and synthetic peptides, and by flow cytometry on HIV-infected cells. The in vitro efficacy of the antibodies was tested by the abilities of the immunotoxins to inhibit protein synthesis in persistently infected cell lines and by their abilities to inhibit HIV production during both acute and persistent infection as measured with an HIV-specific focal immunoassay. The immunotoxins were tested against a panel of distinctly different HIV isolates. The results indicate the following: 1) A mAb to the immunodominant neutralizing loop was highly effective against homologous strains of HIV, but had no activity against heterologous HIV. 2) The efficacy of anti-gp41 mAb varied depending upon the epitope recognized and possibly the affinity of binding to gp41. 3) The polyclonal human anti-gp160 antibodies produced the immunotoxin with the broadest specificity for different HIV strains and the greatest specific activity. This is related to the polyclonal nature of the preparation rather than an increase in relative avidity of the antibody. 4) Activity of an immunotoxin is not a direct function of the binding of the antibody to the surface of infected cells. 5) The ability of an immunotoxin to halt the spread of infection through a tissue culture cell population is dependent upon the ability of the antibody to neutralize the virus as well as the activity of the toxin. Our data suggest that efficacious immunotoxins for the treatment of AIDS may be made with polyclonal anti-envelope antibodies derived from the serum of patients who have been infected with HIV or with appropriately chosen anti-gp41 antibodies.     

Different cytotoxic activity and intracellular fate of an anti-CD5-momordin immunotoxin in normal compared to tumour cells

We investigated the different sensitivity of peripheral blood mononuclear cells (PBMC) and human T cell leukaemias (Jurkat and CEM) to an anti-CD5-momordin immunotoxin. In a short-term assay, the immunotoxin displayed different cytotoxic activity on normal and tumour cells: for leukaemic cell lines an incubation time of 72 h was necessary for the immunotoxin to reach the IC₅₀ of 41–53 pM, compared to the 1 h sufficient for 6 pM immunotoxin to inhibit 50% of PBMC protein synthesis. In a long-term clonogenic assay (15 days), the immunotoxin demonstrated a compareble efficacy of clonogenic cell killing for both cell types. We investigated the immunotoxin internalization pathway by a flow-cytometric method and our data seem to indicate that the molecules meet a different intracellular fate in the two cell populations. It may be assumed that the low cytotoxic activity of immunotoxins on tumour cells, detected in the shortterm assay, is due to infefficient delivery to their cytoplasmatic target, while a longer exposure of the cells to the immunotoxin promotes adequate intracellular distribution.     

Comparison of the cytotoxic potency of T101 Fab, F(ab')2 and whole IgG immunotoxins

The in vitro killing of the human CEM cell line was studied by using ricin A-chain immunotoxins constructed with either the whole IgG or the Fab and F(ab')2 fragments of the same T101 (anti-CD5) antibody. In the presence of ammonium chloride as an activator, the "whole" immunotoxin as well as the "fragment" immunotoxins did not show any significant difference in the cell killing efficacy. In contrast, without the activator, the efficacy of the T101 immunotoxin was greatly improved when fragments were used. Indeed, at a saturating dose, a cytoreduction of three orders of magnitude was obtained with the fragment immunotoxins vs less than one order of magnitude for the whole immunotoxin, as assessed in a clonogenic assay. This enhancing effect was related to better cell killing kinetics, because with a similar amount of A-chain molecules bound per cell, T101 fragment immunotoxins achieved a twofold faster protein synthesis inactivation rate than the corresponding whole IgG immunotoxin. No significant difference in activity was shown between monovalent (Fab) and divalent (F(ab')2) forms of fragment immunotoxins. The observation that T101 fragment immunotoxins were more potent than intact immunotoxins was extended to another fragment immunotoxin constructed with an antibody (F111.98) directed against a different epitope of the CD5 Ag. In another model (anti-CD22 1G11 antibody on Raji cells), the fragment immunotoxin did not show any superiority over the IgG immunotoxin which was by itself very potent, strongly suggesting an Ag-dependent phenomenon.     

Cellular resistance to the antimelanoma immunotoxin ZME-gelonin and strategies to target resistant cells

 The development of cellular resistance to immunotoxins has been demonstrated in a variety of models and can involve a number of mechanisms. For the present study, an immunotoxin was utilized composed of an antimelanoma antibody ZME-018 recognizing a 240-kDa surface glycoprotein (gp 240) and the plant toxin gelonin. Human melanoma cells (A375-M) were grown in the presence of increasing amounts of ZME-gelonin and a clonal variant (A-375-ZR) was developed that was 100-fold resistant to ZME-gelonin compared to parental cells. Scatchard analysis showed that the A375-M parental cells had 260×10³ ZME-gelonin-binding sites/cell with relatively low affinity (5 nM). In contrast, resistant A375-ZR cells demonstrated a reduced number of low-affinity sites (160×10³/cell), but showed a small number (47×10³) of higher-affinity sites (0.8 nM). Internalization rates and degradation rates of ¹²⁵I-labeled ZME-gelonin were identical in both the parental and resistant cells. A375-ZR cells were found to be more resistant to vincristine and doxorubicin than were parental cells. Both cell lines were almost equally sensitive to native gelonin, 5-fluorouracil (5-FU), cisplatin, melphalan, carmustine, interferon γ (IFNγ) and IFNα. In addition, both cell lines were equally sensitive to another gelonin-antibody conjugate that binds to cell-surface, GD₂ (antibody 14G₂A). However, resistant cells were twice as sensitive to the cytotoxic effects of etoposide than were parental cells. Finally, a variety of agents were tested in combination with ZME-gelonin against A375-ZR cells in an attempt to identify agents to augment immunotoxin cytotoxic effects against resistant cells. The agents 5-FU, cisplatin, IFNγ, IFNα, and etoposide were the most effective in augmenting the cytotoxicity of ZME-gelonin against resistant cells. These studies suggest that development of resistance to one immunotoxin does not cause development of cross-resistance to other gelonin immunotoxins. Further, specific biological response modifiers and chemotherapeutic agents may be effective in augmenting the effectiveness of immunotoxins and specifically targeting or reducing the emergence of immunotoxin-resistant cells. Received: 15 March 1995 / Accepted: 28 November 1995     

Degradation of radioiodinated B cell monoclonal antibodies: inhibition via a FCγ -receptor-II-mediated mechanism and by drugs

 Our aim is to treat patients with B cell malignancies with radioimmunotherapy using monoclonal antibodies (mAb) such as CD19, CD20 and CD22. In this study we investigated the rate of internalization and catabolism of these mAb. After 24 h at 37°C, 20% – 25% of initially cell-bound ¹²⁵I-CD19 mAb and ¹²⁵I-CD22 mAb was degraded in B cells, whereas almost no degradation occurred after binding of ¹²⁵I-CD20 mAb. For B cells expressing Fcγ receptor II (FcγRII), isotype-dependent degradation was noted as the CD19 IgG1 mAb showed an enhanced degradation rate compared to the switch variant IgG2a. The effect of various pharmaceutical agents that delay the internalization or subsequent degradation of mAb was evaluated. The degradation was inhibited most effectively by a combination of etoposide and vinblastine, resulting in accumulation of radioactivity in the target cell. Also the simultaneous application of CD20 or CD22 with ¹²⁵I-CD19 mAb or of CD20 with ¹²⁵I-CD22 mAb proved to be a potent inhibitor of the rapid degradation of these mAb, by inhibiting internalization via an FcγRII-mediated mechanism. Both methods of reducing the degradation of radioiodinated mAb are expected to prolong irradiation of malignant B cells and consequently result in an enhanced therapeutic effect in vivo. Received: 22 September 1995 / Accepted: 13 November 1995     

In vivo cytotoxic efficacy of immunotoxins prepared from anti-CD5 antibody linked to ricin A-chain

Summary The antitumoral efficacy of various anti-CD5 immunotoxins, prepared with whole monoclonal antibody (mAb), F(ab)₂ or Fab fragment linked to native ricin A-chain (RTA) or partially deglycosylated ricin A-chain (dRTA), was examined in vivo in ascitic nude mice bearing a large burden of Ichikawa human tumour cells. We first demonstrated that after systemic administration of IgG-RTA or F(ab)₂-dRTA, the cytotoxic activity of immunotoxin molecules specifically bound to tumour cells was preserved. Secondly we showed, by using different immunotoxins with various targeting capacities, that their cytotoxic effect in vivo was related to the number of immunotoxin molecules bound per cell. However, even when antigen saturation was achieved after i.p. injection, the cytotoxic effect did not exceed 53% of the tumour burden. By contrast, when the immunotoxin was administered i.p. or i.v. with the enhancer monensin conjugated to human serum albumin and injected i.p., 90% of the tumour cells were killed. This potentiating effect was demonstrated even when the tumour localisation was as low as 5% of the saturation level. Such an effect could be completely prevented by addition of unconjugated monoclonal antibody, demonstrating the specificity of the immunotoxin-induced cytotoxicity in the presence of the enhancer. However this enhancement was demonstrated whatever the route of immunotoxin administration, i.p. or i.v., but was only observed when the enhancer was injected i.p. and not i.v.. These results emphasize the importance of optimizing the therapeutic course to improve the antitumoral efficacy of immunotoxins.     

Anti-CD30 immunotoxins with native and recombinant dianthin 30

Immunotoxins were prepared with a Ber-H2 (anti-CD30) monoclonal antibody and native or recombinant dianthin 30, a ribosome-inactivating protein fromDianthus caryophyllus (carnation). Both immunotoxins selectively inhibited protein synthesis by CD30⁺ cell lines D430B (lymphoblastoid, infected with Epstein-Barr virus). L428 and L540 (both from Hodgkin's lymphoma). IC₅₀ values (concentrations, as dianthin, causing 50% inhibition) ranged from 324 pM to 479 pM (immunotoxin with native dianthin 30) or from 45 pM to 182 pM (immunotoxin with recombinant dianthin 30). The effect of either immunotoxin on protein synthesis by the CD30^– cell line K562 (from a chronic myeloid leukaemia) was not different from that of free dianthin (IC₅₀ higher than nM).     

Internalization of the cytotoxic molecules of T101 F(ab')2-(ricin-A-chain) immunotoxin into human T-leukemic cells

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab')2-T101(ricin-A-chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells. We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy-dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate-limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low. Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.     

抗癌胚抗原免疫毒素生物学功能的研究

为了研究免疫毒素发挥生物学作用的全过程,构建了两种不同形式的基于假单胞菌外毒素(Pseudomonas exotoxin A,PE)的抗癌胚抗原（carcinoembryonic antigen, CEA）免疫毒素CEA(Fv)/PE38/KDEL和PE35/CEA(Fv)/KDEL.用流式细胞术经间接免疫荧光法测定免疫毒素的抗原结合活性.免疫毒素的内化(internalization)通过间接免疫荧光标记的方法在激光共聚焦显微镜下进行检测,采用流式细胞术进行免疫毒素内化的定量分析.细胞凋亡采用FITC-annexin V/PI双荧光染色方法进行分析.用噻唑蓝(MTT)法测定免疫毒素的靶细胞杀伤功能.对两种形式的免疫毒素在各个功能阶段的性质进行研究和比较,全面展现了免疫毒素发挥生物学功能的过程及各个生物作用过程之间的相互影响,为详尽的机制研究和免疫毒素的进一步优化改造提供了重要的依据.     

Releasable PEGylation of mesothelin targeted immunotoxin SS1P achieves single dosage complete regression of a human carcinoma in mice

Recombinant immunotoxins exhibit targeting and cytotoxic functions needed for cell-specific destruction. However, antitumor efficacy, safety, and pharmacokinetics of these therapeutics might be improved by further macromolecular engineering. SS1P is a recombinant anti-mesothelin immunotoxin in clinical trials in patients with mesothelin-expressing tumors. We have modified this immunotoxin using several PEGylation strategies employing releasable linkages between the protein and the PEG polymers, and observed superior performance of these bioconjugates when compared to similar PEG derivatives bearing permanent linkages to the polymers. PEGylated derivatives displayed markedly diminished cytotoxicity on cultured mesothelin-overexpressing A431-K5 cells; however, the releasable PEGylated immunotoxins exhibited increased antitumor activity in A431-K5 xenografts in mice, with a diminished animal toxicity. Most significantly, complete tumor regressions were achievable with single dose administration of the bioconjugates but not the native immunotoxin. Pharmacokinetic analysis of the releasable PEGylated derivatives in mice demonstrated an over 80-fold expansion of the area under the curve exposure of bioactive protein when compared to native immunotoxin. A correlation in degree of derivatization, release kinetics, and polymer size with potency was observed in vivo, whereas in vitro cytotoxicity was not predictive of efficacy in animal models. The potent antitumor efficacy of the releasable PEGylated mesothelin-targeted immunotoxins was not exhibited by similar untargeted PEG immunotoxins in this model. Since the bioconjugates can also exhibit the attributes of passive targeting via enhanced permeability and retention, this is the first demonstration of a pivotal role of active targeting for immunotoxin bioconjugate efficacy.     

Affinity-matured recombinant immunotoxin targeting gangliosides 3′-isoLM1 and 3′,6'-isoLD1 on malignant gliomas

About 60 percent of glioblastomas highly express the gangliosides 3′-isoLM1 and 3′,6′-isoLD1 on the cell surface, providing ideal targets for brain tumor immunotherapy. A novel recombinant immunotoxin, DmAb14m-(scFv)-PE38KDEL (DmAb14m-IT), specific for the gangliosides 3′-isoLM1 and 3′,6′-isoLD1, was constructed with improved affinity and increased cytotoxicity for immunotherapeutic targeting of glioblastoma. We isolated an scFv parental clone from a previously established murine hybridoma, DmAb14, that is specific to both 3′-isoLM1 and 3′,6′-isoLD1. We then performed in vitro affinity maturation by CDR hotspot random mutagenesis. The binding affinity and specificity of affinity-matured DmAb14m-IT were measured by surface-plasmon resonance, flow cytometry, and immunohistochemical analysis. In vitro cytotoxicity of DmAb14m-IT was measured by protein synthesis inhibition and cell death assays in human cell lines expressing gangliosides 3′-isoLM1 and 3′,6′-isoLD1 (D54MG and D336MG) and xenograft-derived cells (D2224MG). As a result, the K_D of DmAb14m-IT for gangliosides 3′-isoLM1 and 3′,6′-isoLD1 was 2.6 × 10⁻⁹M. Also, DmAb14m-IT showed a significantly higher internalization rate in cells expressing 3′-isoLM1 and 3′,6′-isoLD1. The DmAb14m-IT IC₅₀ was 80 ng/mL (1194 pM) on the D54MG cell line, 5 ng/ml (75 pM) on the D336MG cell line, and 0.5 ng/ml (7.5 pM) on the D2224MG xenograft-derived cells. There was no cytotoxicity on ganglioside-negative HEK293 cells. Immunohistochemical analysis confirmed the specific apparent affinity of DmAb14m-IT with 3′-isoLM1 and 3′,6′-isoLD1. In conclusion, DmAb14m-IT showed specific binding affinity, a significantly high internalization rate, and selective cytotoxicity on glioma cell lines and xenograft-derived cells expressing 3′-isoLM1 and 3′,6′-isoLD1, thereby displaying robust therapeutic potential for testing the antitumor efficacy of DmAb14m-IT at the preclinical level and eventually in the clinical setting.