Ricin A-chain conjugated with monoclonal anti-L1210 antibody

Summary In studies of antitumor antibody-cytotoxic agent conjugates as potential antitumor agents with improved tumor specificity, the toxic subunit A-chain of ricin was conjugated with a monoclonal antibody to a tumor-associated antigen expressed weakly on murine leukemia L1210 cells and strongly on L1210/GZL cells, a guanazole-resistant subline of L1210, employing N-succinimidyl 3-(2-pyridyldithio)propionate as cross-linking agent. The conjugate (anti-L1210 conjugate) exhibited a potent concentration-dependent cytotoxicity against cultured L1210/GZL cells, and inhibited cell growth at concentrations over 0.8 g/ml. The conjugate killed all L1210/GZL cells at a concentration of 100 g/ml. Neither nonimmune conjugate similarly prepared from mouse nonimmune IgG nor unconjugated anti-L1210 IgG alone showed cytotoxicity against L1210/GZL cells. When (BALB/c×DBA/2)F₁ mice inoculated with 1 × 10⁵ L1210/GZL cells were treated with IP injections of 27 g anti-L1210 conjugate 1 h and 5 days after tumor cell inoculation, a life-prolonging effect was observed. [Lifespan in treated animals as percentage of that in controls (T/C)=146%]. However, when the dose per injection was increased to 50 g per mouse, survival was the same as in the control group. Postmortem examination of mice that had been treated with 50 g anti-L1210 conjugate revealed lesions with necrosis and hemorrhage in the liver parenchyma and the intestinal epithelium, respectively. A similar toxic effect on the host mice was also observed with nonimmune conjugate.     

Ricin A-chain activity on stem-loop and unstructured DNA substrates

Ricin toxin A-chain (RTA) depurinates a single adenylate on a GAGA stem-loop region of eukaryotic 28S RNA, making it a potent toxin. Steady state rate analysis is used to establish the kinetic parameters for depurination of short RNA, DNA, and RNA-DNA hybrids of GAGA linear segments and stem-loop regions as substrates for RTA. Both stem and tetraloop structures are essential for action on RNA. For DNA stem-loop substrates, stem stability plays a small role in enhancing catalytic turnover but can enhance binding by up to 3 orders of magnitude. DNA sequences of d[GAGA] without stem-loop structures are found to be slow substrates for RTA. In contrast, equivalent RNA sequences exhibit no activity with RTA. Introduction of a deoxyadenosine at the depurination site of short RNA oligonucleotides restores catalytic function. NMR analysis indicates that the short, nonsubstrate GAGA is converted to substrate in GdAGA by the presence of a more flexible ribosyl group at the deoxyadenosine site. Conversion between C2'-endo and C2'-exo conformations at the deoxyadenosine site moves the 3'- and 5'-phosphorus atoms by 1.1 A, and the former is proposed to place them in a catalytically favorable configuration. The ability to use short RNA-DNA hybrids as substrates for RTA permits exploration of related structures to function as substrates and inhibitors.     

Cytotoxicity of a conjugate of ricin A-chain and monoclonal anti-allotypic antibody

A conjugate was developed between ricin A-chain and monoclonal antiallotypic antibody specific for L-chains of the rat Ig. Incubation of spleen cells with the conjugate (10(-7) M) resulted in a strong decrease of 1a-positive B-lymphocyte growth. At the same time spleen cells that lacked this antigen were not affected by immunotoxin (IT). Both the isolated antibodies and ricin A-chain did not inhibit cell growth in the same concentrations. IT injection to 10-day-old heterozygous rats (1a/1b) resulted in a partial suppression of Ig production with 1a-allotype. The conjugate was 4.5 times more effective than the isolated antibodies used to form it. The in vivo and in vitro differences in IT cytotoxicity were probably connected with insufficient efficacy of conjugate targeting in vivo.     

Target-selective cytotoxicity of methotrexate conjugated with monoclonal anti-MM46 antibody

Summary In studies on antitumor antibody-cytotoxic drug conjugates as potential tumor-selective cytotoxic agents, methotrexate (MTX) was conjugated via its active ester derivative with a murine monoclonal antibody (aMM46) to a mouse mammary tumor antigen (MM antigen) on syngeneic, ascitic C3H/He mouse mammary tumor MM46 cells. The conjugate retained full antibody activity, as assayed by complement-dependent cytolysis. The target-selective cytotoxicity of aMM46-MTX was verified by the observations that this conjugate showed greater cytotoxicity than the corresponding normal mouse immunoglobulin (nIg) conjugate to MM46 cells, neither aMM46 nor nIg being cytotoxic, and that it showed less cytotoxicity to MM antigen negative mouse mammary tumor MM48 cells than to MM46 cells, its cytotoxicity to MM48 cells being similar to that of the nIg conjugate. From the results of assays of cell binding and uptake of ¹³¹I-labeled aMM46 and aMM46-³H-MTX, aMM46 and aMM46-MTX were internalized after their binding to MM46 cell surface antigen. Leupeptin, an inhibitor of the lysosomal endopeptidase cathepsin, decreased the cytotoxicity of aMM46-MTX, supporting the involvement of lysosomal degradation of the conjugate in its action.     

Ricin A-chain inhibitors resembling the oxacarbenium ion transition state

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.     

Prevention of myosin-induced autoimmune myocarditis in mice by anti-L3T4 monoclonal antibody

This study was aimed at studying the effect of the induction of immune tolerance to swine cardiac myosin from anti-L3T4 monoclonal antibody injection and whether the immune tolerance could protect mice with myosin-induced myocarditis from myocardial injury. Twenty-four Balb/c mice were divided into two groups at random. All of the mice were immunized with swine cardiac myosin on the 1st day, 14th, 28th, 42nd, and 52nd day. Immune tolerance was induced by triplicate injections of 400 microg anti-L3T4 McAb on the 0 day (intravenous), 1st day, and 2nd day (intraperitoneal) in McAb-treated group. In the saline-treated group, saline of the same volume as anti-L3T4 monoclonal antibody was used as a control. The sera and hearts biopsies of all mice were collected on the 58th day. The anti-cardiac myosin antibody was examined with ELISA, and pathological changes of heart were observed by light microscope. It was shown that mice immunized with swine cardiac myosin could produce anti-myosin antibody and the anti-cardiac myosin antibody was positive in most of the saline-treated group but negative in the McAb-treated group. Morphologically, myocardial degeneration, necrosis, and infiltration of inflammatory cells were found in the saline-treated group but not in the McAb-treated group. In conclusion, this study indicated that the immune tolerance to cardiac myosin was induced by the anti-L3T4 monoclonal antibody, and accordingly myocardial injury could be prevented by induction of immune tolerance.     

Treatment with anti-L3T4 (CD4) monoclonal antibody reduces the inflammatory response in toxoplasmic encephalitis

Toxoplasma gondii is an important cause of disease of the central nervous system in patients with AIDS. Among the variety of immunologic disorders encountered by AIDS patients is a depletion of CD4+ subpopulation of lymphocytes. In order to determine the role of this population of T lymphocytes in the generation of toxoplasmic encephalitis, mice chronically infected with T. gondii were treated with mAb GK1.5 directed against the cell surface glycoprotein L3T4 (CD4) of T lymphocytes. Histopathologic sections of brains of control and treated animals were examined at regular intervals during and after completion of treatment. The results demonstrated significantly less inflammation in brains of mice during treatment with GK1.5 mAb. In addition, recrudescence of the inflammatory process occurred after discontinuation of treatment. Similar results were observed in experiments in which different strains of mice and T. gondii were used.     

Ricin A-chain substrate specificity in RNA, DNA, and hybrid stem-loop structures

Ricin toxin A-chain (RTA) is the catalytic subunit of ricin, a heterodimeric toxin from castor beans. Its ribosomal inactivating activity arises from depurination of a single adenine from position A(4324) in a GAGA tetraloop from 28S ribosomal RNA. Minimal substrate requirements are the GAGA tetraloop and stem of two or more base pairs. Depurination activity also occurs on stem-loop DNA with the same sequence, but with the k(cat) reduced 200-fold. Systematic variation of RNA 5'-G(1)C(2)G(3)C(4)[G(5)A(6)G(7)A(8)]G(9)C(10)G(11)C(12)-3' 12mers via replacement of each nucleotide in the tetraloop with a deoxynucleotide showed a 16-fold increase in k(cat) for A(6) --> dA(6) but reduced k(cat) up to 300-fold for the other sites. Methylation of individual 2'-hydroxyls in a similar experiment reduced k(cat) by as much as 3 x 10(-3)-fold. In stem-loop DNA, replacement of d[G(5)A(6)G(7)A(8)] with individual ribonucleotides resulted in small kinetic changes, except for the dA(6) --> A(6) replacement for which k(cat) decreased 6-fold. Insertion of d[G(5)A(6)G(7)A(8)] into an RNA stem-loop or G(5)A(6)G(7)A(8) into a DNA stem-loop reduced k(cat) by 30- and 5-fold, respectively. Multiple substitutions of deoxyribonucleotides into RNA stem-loops in one case (dG(5),dG(7)) decreased k(cat)/K(m) by 10(5)-fold, while a second change (dG(5),dA(8)) decreased k(cat) by 100-fold. Mapping these interactions on the structure of GAGA stem-loop RNA suggests that all the loop 2'-hydroxyl groups play a significant role in the action of ricin A-chain. Improved binding of RNA-DNA stem-loop hybrids provides a scaffold for inhibitor design. Replacing the adenosine of the RTA depurination site with deoxyadenosine in a small RNA stem-loop increased k(cat) 20-fold to 1660 min(-1), a value similar to RTA's k(cat) on intact ribosomes.     

Ricin A-chain requires c-Jun N-terminal kinase to induce apoptosis in nontransformed epithelial cells

Ricin is a toxin isolated from castor beans that has potential as a weapon of bioterrorism. This glycoprotein consists of an A-chain (RTA) that damages the ribosome and inhibits protein synthesis and a B-chain that plays a role in cellular uptake. Ricin activates the c-Jun N-terminal kinase (JNK) and p38 signaling pathways; however, a role for these pathways in ricin-induced cell death has not been investigated. Our goals were to determine if RTA alone could activate apoptosis and if the JNK and p38 pathways were required for this response. Comparable caspase activation was observed with both ricin and RTA treatment in the immortalized, nontransformed epithelial cell line, MAC-T. Ribosome depurination and inhibition of protein synthesis were induced in 2–4 h with 1 μg/ml RTA and within 4–6 h with 0.1 μg/ml RTA. Apoptosis was not observed until 4 h of treatment with either RTA concentration. RTA activated JNK and p38 in a time- and concentration-dependent manner that preceded increases in apoptosis. Inhibition of the JNK pathway reduced RTA-induced caspase activation and poly(ADP-ribose) polymerase cleavage. In contrast, inhibition of the p38 pathway had little effect on RTA-induced caspase 3/7 activation. These studies are the first to demonstrate a role for the JNK signaling pathway in ricin-induced cell death. In addition, the MAC-T cell line is shown to be a sensitive in vitro model system for future studies using RTA mutants to determine relationships between RTA-induced depurination, ribotoxic stress, and apoptosis in normal epithelial cells.     

Generation,characterization and preclinical studies of a human anti-L1CAM monoclonal antibody that cross-reacts with rodent L1CAM

L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (K_D) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.     

Ricin A-chain structural determinant for binding substrate analogues: A molecular dynamics simulation analysis

Ricin A-chain is a cytotoxic protein that attacks ribosomes by hydrolyzing a specific adenine base from a highly conserved, single-stranded rRNA hairpin containing the tetraloop sequence GAGA. Molecular-dynamics simulation methods are used to analyze the structural determinant for three substrate analogues bound to the ricin A-chain molecule. Simulations were applied to the binding of the dinucleotide adenyl-3′,5′-guanosine employing the x-ray crystal structure of the ricin complex and a modeled CGAGAG hexanucleotide loop taken from the NMR solution structure of a 29-mer oligonucleotide hairpin. A third simulation model is also presented describing a conformational search of the docked 29-mer structure by using a simulated-annealing method. Analysis of the structural interaction energies for each model shows the overall binding dominated by nonspecific interactions, which are mediated by specific arginine contacts from the highly basic region on the protein surface. The tetraloop conformation of the 29-mer was found to make specific interactions with conserved protein residues, in a manner that favored the GAGA sequence. A comparison of the two docked loop conformations with the NMR structure revealed significant positional deviations, suggesting that ricin may use an induced fit mechanism to recognize and bind the rRNA substrate. The conserved Tyr-80 may play an important confirmational entropic role in the binding and release of the target adenine in the active site. Proteins 27:80–95 © 1997 Wiley-Liss, Inc.     

Optical imaging of disseminated leukemia models in mice with near-infrared probe conjugated to a monoclonal antibody

Background

The assessment of anticancer agents to treat leukemia needs to have animal models closer to the human pathology such as implantation in immunodeficient mice of leukemic cells from patient samples. A sensitive and early detection of tumor cells in these orthotopic models is a prerequisite for monitoring engraftment of leukemic cells and their dissemination in mice. Therefore, we developed a fluorescent antibody based strategy to detect leukemic foci in mice bearing patient-derived leukemic cells using fluorescence reflectance imaging (FRI) to determine when to start treatments with novel antitumor agents.

Methods

Two mAbs against the CD44 human myeloid marker or the CD45 human leukocyte marker were labeled with Alexa Fluor 750 and administered to leukemia-bearing mice after having verified the immunoreactivity in vitro. Bioluminescent leukemic cells (HL60-Luc) were used to compare the colocalization of the fluorescent mAb with these cells. The impact of the labeled antibodies on disease progression was further determined. Finally, the fluorescent hCD45 mAb was tested in mice engrafted with human leukemic cells.

Results

The probe labeling did not modify the immunoreactivity of the mAbs. There was a satisfactory correlation between bioluminescence imaging (BLI) and FRI and low doses of mAb were sufficient to detect leukemic foci. However, anti-hCD44 mAb had a strong impact on the tumor proliferation contrary to anti-hCD45 mAb. The use of anti-hCD45 mAb allowed the detection of leukemic patient cells engrafted onto NOD/SCID mice.

Conclusions

A mAb labeled with a near infrared fluorochrome is useful to detect leukemic foci in disseminated models provided that its potential impact on tumor proliferation has been thoroughly documented.     

Therapeutic effect of anti-L3T4 monoclonal antibody GK1.5 on cutaneous leishmaniasis in genetically-susceptible BALB/c mice

The effect of in vivo treatment with anti-L3T4 monoclonal antibody (mAb) on the course of experimentally-induced cutaneous leishmaniasis was studied in genetically susceptible BALB/c mice. Administration of anti-L3T4 mAb resulted in a pronounced therapeutic effect as reflected by both resolution of lesions and a dramatic reduction in the parasite load in treated mice. This effect was specific for anti-L3T4 mAb and correlated with a selective depletion of L3T4+ T cells in the lymphoid tissues of treated mice. Moreover, both the therapeutic effect of anti-L3T4 mAb on cutaneous leishmaniasis and the concurrent depletion of L3T4+ T cells in the lymphoid tissues of treated mice were dependent upon the isotype of the anti-L3T4 mAb employed.     

Specific killing of a human breast carcinoma cell line by a monoclonal antibody coupled to the A-chain of ricin

Summary We coupled monoclonal IgM_k antibodies directed against human breast carcinoma cells to the A-chain of the plant toxin ricin. These molecular hybrids maintain both their antibody-binding activity and the toxic activity of the A-chain. Thus, they specifically bind to and kill the breast carcinoma cells in vitro.     

Regional assignment of human liver-type 6-phosphofructokinase to chromosome 21q22.3 by using somatic cell hybrids and a monoclonal anti-L antibody

Summary The three structural loci encoding human phosphofructokinase, a key regulatory enzyme of glycolysis, are located on separate chromosomes. The gene coding for the liver-type subunit PFKL has previously been assigned to chromosome 21. We have used a subunit- and human-specific monoclonal antibody to liver PFK to detect the expression of human PFKL in hamster x human hybrid cell lines. A cell line carrying an 8;21 translocation which contains all of chromosome 21 except the band 21q22.3 was negative for the expression of PFKL whereas cell lines carrying the reciprocal 8;21 translocation were positive. In addition, a cell line with a ring chromosome 21 containing a breakpoint which excluded the distal part of the q22.3 band was negative for expression of PFKL. These results indicate that human PFKL is located on chromosome 21q22.3.     

In vivo effects of monoclonal anti-L3T4 antibody on immune responsiveness of mice infected with Schistosoma mansoni. Reduction of irradiated cercariae-induced resistance

Mice can be partially protected against challenge infections of Schistosoma mansoni cercariae by either single or multiple exposure to irradiated cercariae (x-cerc). The participation of L3T4+ lymphocytes on this resistance phenomenon was evaluated by selectively depleting this cell population through in vivo administration of mAb anti-L3T4 (hybridoma GK 1.5) at three different times in relationship to the challenge infections. Treatment with anti-L3T4 before challenge such that depletion was effective during the time of cercarial skin penetration and dermal/s.c. residence significantly reduced the level of resistance induced by x-cerc sensitization. When treatment was delayed until after challenge, depletion of L3T4+ cells coincided with either the lung or post-lung/liver phases of schistosomular migration, and normal levels of x-cerc-induced resistance were induced. In contrast to once-immunized mice, mice hyperimmunized by five exposures to x-cerc and then depleted of L3T4+ cells at the time of challenge (skin phase) still expressed resistance to the challenge. These data suggest that when mice are sensitized only once with x-cerc the challenge infection provides a necessary immunologic boost which requires L3T4+ cells for effective expression of resistance. The requirement for this anamnestic effect by the challenge infection can be circumvented by hyperimmunization. Evaluation of the immune response of one-time sensitized or hyperimmunized mice demonstrated that cellular Ag-specific proliferative responses and mitogen-induced lymphokine production were abrogated after any of the various in vivo regimens of anti-L3T4 antibody. In contrast, immunoblot analysis of humoral responsiveness revealed a correlation between the expression of resistance and the ability of sera from immunized and anti-L3T4 treated mice to recognize a 75-kDa parasite antigenic component.     

In vivo effects of GK1.5 (anti-L3T4a) monoclonal antibody on induction and expression of delayed-type hypersensitivity

The in vivo effects of monoclonal GK1.5 antibody, directed against the L3T4a determinant expressed on Class II-restricted T cells, on the induction and expression of murine delayed-type hypersensitivity (DTH) responses were examined. Development and expression of both hapten (2,4-dinitrofluorobenzene and 2,4,6-trinitrochlorobenzene)- and protein antigen poly(Glu60Ala30Tyr10)-specific DTH are significantly inhibited by injection of monoclonal anti-L3T4a antibody. The inhibitory effects of anti-L3T4a were most pronounced when administered during the afferent (induction) phase of the DTH response, leading to the functional inhibition of the generation of both polyclonal lymph node T-proliferative cells (Tprlf) and DTH effector cells (TDH). The in vivo inhibitory effect is apparently unrelated to preferential induction of suppressor T cells as GK1.5 inhibited DTH induction in cyclophosphamide-treated as well as normal recipients. L3T4a expression on the various T-cell subsets involved in DTH induction and elicitation was also examined. The data show that three functionally distinct, antigen-specific T-cell subsets, Tprlf, TDH, and Th cells involved in DTH induction, bear the Lyt 1+2-, L3T4+ phenotype. Possible mechanisms where in vivo injection of anti-L3T4a inhibits Class II-restricted T-cell subsets involved in DTH induction and expression, including immune depletion and inhibition of T-cell-receptor/ligand interactions, are discussed.     

A monoclonal antibody for the detection of conjugated forms of abscisic acid in plant tissues

A mouse monoclonal antibody against abscisic acid (ABA) was produced and characterized. It was raised using ABA conjugated to the carrier protein through the carboxyl (Cl) group as immunogen. It did not discriminate between free ABA or its ester derivatives. This antibody, which is the first monoclonal against Cl-conjugated ABA, shows interesting characteristics. It has high affinity (K_a=1.5 × 10⁹ L/mol) and specificity. Compounds structurally similar to ABA, such as phaseic acid, dihydrophaseic acid, and both the 2,trans-isomer and the (R)-enantiomer of ABA, are not reactive. The narrow linear range of the standard curve (0.018–1.8 pmol) ensures great precision of the assay. This monoclonal antibody has been used for the quantification of ABA conjugates in crude aqueous extracts of bean leaves by radioimmuno-assay (RIA). The fractionation of the extracts by high-performance liquid chromatography (HPLC) confirmed the absence of cross-reacting compounds. Because of its affinity and specificity, in combination with antibodies against free ABA, this antibody should be a sound tool for studying the metabolism and immunolocalization of ABA in plant tissues.