Antigenic modulation by anti-CD5 immunotoxins

We evaluated the modulation of T101 immunotoxins (IT) and free T101 antibody from the surface of normal and leukemic cells to determine whether the presence of toxin on antibody affected antigenic modulation. Reagents were made by conjugating T101, which binds to the T cell antigen CD5, to either intact ricin or purified ricin A chain. We found that T101-A chain modulated CD5 more efficiently than T101-ricin, which modulated CD5 more efficiently than T101 alone. Kinetic studies showed that maximal modulation of IT was reached within 3 hr. When toxicity of the reagents was tested in protein synthesis inhibition assays, T101-ricin in the presence of lactose inhibited 99% of the protein synthesis of CEM cells. T101-A chain was less toxic, inhibiting protein synthesis only 23 to 43%. The addition of the potentiating agent monensin nearly doubled the toxicity of T101-A chain, but did not affect T101-A chain modulation. To determine the fate of bound IT, T101 and T101-ricin were labeled with 125I. Cells were incubated under modulating conditions in the presence of radiolabeled reagents. T101 and T101-ricin were internalized into CEM cells. In contrast, T101, but not T101-ricin, appeared to be shed from peripheral blood mononuclear cells. Our findings show clearly that: 1) the presence of toxin on antibody does not inhibit--and may actually enhance--modulation; 2) T101-IT are internalized, not shed from the cell surface; 3) the lack of toxicity of T101-A chain is not attributed to inability to modulate; 4) there is no correlation between enhancement of T101-A chain toxicity by monensin and antigenic modulation by A chain reagents; and 5) modulation, which is undesirable in monoclonal antibody therapy, may be advantageous in the therapeutic use of IT.     

Cellular processing of a ricin-antibody conjugate. A kinetic analysis of the rate-limiting step

Upon exposure to holoricin-antibody conjugates, target cells display a period of no measurable intoxication followed by a concentration-dependent exponential decline in protein synthesis. The rate of this decline is increased by conjugate structural variables such as addition of a second ricin to the antibody or introduction of a peptide spacer between the toxin and antibody. Additionally, the rate is enhanced by the addition of ammonia or monensin. The relationship between immunotoxin concentration and the rate of protein synthesis inhibition is shown to obey typical Michaelis-Menten kinetics. By displacing bound immunotoxin with native antibody, it was demonstrated that the rate-limiting saturable process is not due to antibody-surface antigen interaction. Although addition of a second ricin as well as addition of ammonia elevated the measured rate of protein synthesis inhibition, curve-fitting techniques indicated that they did not elevate the maximal rate; however, introduction of a peptide spacer into the conjugate or addition of monensin increased the maximal rate of this saturable process. Additionally, the kinetic examination of the potentiation effects of ammonia and monensin indicates that they operate on different cellular processes involved with the intracellular routing toward intoxication. An equational model is developed which helps interpret the known features of the conjugate intoxication process.     

Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a beta-galactosyl matrix) and asialofetuin-Sepharose. Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy1.1-ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thy1.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2-5 pM. Comparable inhibition of EL4 cells was only achieved with 3000-7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thy1.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells. A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact ricin conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition. It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.     

Somatic cell mutants resistant to ricin, diphtheria toxin, and to immunotoxins

The human B-cell line Namalwa expresses the common acute lymphoblastic leukemia antigen (CALLA). Frame-shift mutants in Namalwa cell cultures were generated with ICR-191, and mutants were then selected for resistance to ricin or resistance to a conjugate of ricin with the anti-CALLA antibody J5 in the presence of lactose. Three mutants were found that were resistant to ricin and were in addition shown to be resistant to diphtheria toxin, to a J5-ricin conjugate, and to a conjugate between ricin B-chain and gelonin. The mutants, however, were sensitive to a J5-gelonin conjugate. These mutants expressed high levels of CALLA and/or receptors for ricin, and their cell-free translation systems appeared to be as sensitive to the inhibitory action of ricin A-chain and of gelonin as the translation system of wild-type Namalwa cells. The behavior of these mutants was consistent with the hypothesis that these cells possess an alteration of their surface that impedes the passage of ricin and diphtheria toxin across the plasma membrane. A fourth mutant was found to bind reduced quantities of ricin and was resistant to ricin but was sensitive to J5-ricin. The properties of this cell line provide evidence that the binding of antibody-ricin conjugates to cells via the ricin moiety may be prevented without impeding the cytotoxicity of the conjugates.     

Influence of endogenous Thy1.1 cells upon the efficacy of an anti-Thy1.1 antibody-diphtheria toxin conjugate.

The chemical conjugation of antibodies to protein toxins results in cell-specific cytotoxic agents that can be defined in terms of in vitro potency and efficacy; however, it is the in vivo utilities that are largely being pursued in clinical trials. The nature of in vivo target cell depletion by toxin conjugates is largely unknown. The anti-murine Thy1.1 antibody-diphtheria toxin conjugate possesses high in vitro efficacy, and because mice are remarkably resistant to the native toxin, the conjugate possesses in vivo efficacy. When administered intravenously, the conjugate is shown to deplete peripheral blood Thy1.1+ target cells in a concentration-dependent fashion. When the log kill of Thy1.1+ tumor cells was analyzed by the life span extension method, it was determined, however, that the log kill is inversely proportional to the number of target cells. That is, the presence of an endogenous cell population, which is expressing the same surface antigen targeted by the antibody conjugate as on the pathological cell, may drastically lower the clinical efficacy of the immunotoxin. Thus, the greatest potential for antibody-toxin conjugates will be for low target cell burdens and for pathogenic cell populations expressing unique surface antigens. These are important considerations in the design of bioconjugates to insure high in vivo efficacy in elimination of intended target cells.     

Improved binding of a bivalent single-chain immunotoxin results in increased efficacy for in vivo T-cell depletion.

Anti-CD3 immunotoxins exhibit considerable promise for the induction of transplantation tolerance in pre-clinical large animal models. Recently an anti-human anti-CD3epsilon single-chain immunotoxin based on truncated diphtheria toxin has been described that can be expressed in CHO cells that have been mutated to diphtheria toxin resistance. After the two toxin glycosylation sites were removed, the bioactivity of the expressed immunotoxin was nearly equal to that of the chemically conjugated immunotoxin. This immunotoxin, A-dmDT390-sFv, contains diphtheria toxin to residue 390 at the N-terminus followed by VL and VH domains of antibody UCHT1 linked by a (G(4)S)(3) spacer (sFv). Surprisingly, we now report that this immunotoxin is severely compromised in its binding affinity toward CD3(+) cells as compared with the intact parental UCHT1 antibody, the UCHT1 Fab fragment or the engineered UCHT1 sFv domain alone. Binding was increased 7-fold by adding an additional identical sFv domain to the immunotoxin generating a divalent construct, A-dmDT390-bisFv (G(4)S). In vitro potency increased 10-fold over the chemically conjugated immunotoxin, UCHT1-CRM9 and the monovalent A-dmDT390-sFv. The in vivo potency of the genetically engineered immunotoxins was assayed in the transgenic heterozygote mouse, tgepsilon 600, in which the T-cells express human CD3epsilon as well as murine CD3epsilon. T-cell depletion in the spleen and lymph node observed with the divalent construct was increased 9- and 34-fold, respectively, compared with the monovalent construct. The additional sFv domain appears partially to compensate for steric hindrance of immunotoxin binding due to the large N-terminal toxin domain.     

Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants

We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).     

Insulin-cholera toxin binding unit conjugate: a hybrid molecule with insulin biological activity and cholera toxin binding specificity

The polypeptide hormone insulin and the binding unit of cholera toxin (CTB) were coupled via a disulfide bond. This hybrid molecule had 1/30 the ability of native insulin to bind to the insulin receptor and 1/30 the biological activity of native insulin in H35 rat hepatoma cells and rat adipocytes. Thus, in these two cell types that are very sensitive to insulin, the biological activity of the hybrid molecule was as predicted on the basis of the ability of the molecule to interact with the insulin receptor. In contrast, in HTC rat hepatoma cells and rat thymocytes, two poorly responsive cell types, the insulin-CTB conjugate had 1/3 the biological activity of native insulin, a value 10 times greater than its insulin receptor binding potency. This increased activity of the conjugate did not appear to be due to cholera toxin in the preparation, since a control of uncoupled CTB had no biological activity. Furthermore, native cholera toxin increased intracellular levels of cAMP by 20-fold, whereas the conjugate had no effect on cAMP levels. The CTB moiety did, however, contribute to the biological activity of the conjugate, since the activity of the hybrid molecule, like cholera toxin, was inhibited by gangliosides, whereas the activity of native insulin was not. Finally, the binding to thymocytes of insulin-CTB conjugate, but not insulin, was inhibited by gangliosides. Thus, a hybrid hormone molecule has been constructed which has insulin-like biological activity with the receptor specificity of cholera toxin in poorly responsive cells.     

Cytotoxic ribonuclease chimeras. Targeted tumoricidal activity in vitro and in vivo.

Monoclonal antibodies to the transferrin receptor or to the T cell antigen, CD5, were chemically linked to mammalian RNase A and found to specifically inhibit protein synthesis in antigen-positive cells. Antibody-mediated specificity of these cytotoxic ribonuclease chimeras (CRCs) was demonstrated in three ways. 1) Toxicity was due to the chemical linkage of RNase to antibody, as the individual components added separately or in combination did not inhibit protein synthesis; 2) the anti-transferrin receptor CRCs inhibited protein synthesis in those cells expressing the human transferrin receptor (K562, U251, Jurkat cells) but had no detectable toxicity to cells lacking the human transferrin receptor (Vero or NIH 3T3 cells); 3) free antibody to either the human transferrin receptor (454A12 or 5E-9) or to the T cell antigen, CD5 (T101), blocked the cytotoxicity of the respective CRC. Two CRC species, designated P1 and P2, that differed in size and stoichiometry of RNase A to antibody, were purified by size-exclusion high performance liquid chromatography. The higher molecular weight P1 conjugate had an IC50 of 20-30 nM, whereas the P2 conjugate had a higher IC50 of 300-500 nM. Bioactivity could be reversibly increased more than 10-fold by freezing. The cytotoxicity of the CRCs was examined in vivo in a solid tumor animal model. Intratumoral injections of an anti-transferrin receptor CRC into established U251 human glioblastoma tumors grown in the flanks of nude mice prevented tumor growth, whereas RNase A mixed with antibody was ineffective. CRCs, therefore, express cytotoxicity in vitro and in vivo. Mammalian nucleases coupled to antibodies may be utilized as cell type-selective cytotoxins and have potential as pharmacologic reagents. The systemic toxicity and immunogenicity observed with mammalian derived cytotoxins may be significantly less than that of the currently employed plant- and bacterial-derived immunotoxins.     

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.     

Tandem copies of a human rotavirus VP8 epitope can induce specific neutralizing antibodies in BALB/c mice

The VP8 subunit protein of human rotavirus (HRV) plays an important role in viral infectivity and neutralization. Recombinant peptide antigens displaying the amino acid sequence M(1)ASLIYRQLL(10), a linear neutralization epitope on the VP8 protein, were constructed and examined for their ability to generate anti-peptide antibodies and HRV-neutralizing antibodies in BALB/c mice. Peptide antigen constructs were expressed in E. coli as fusion proteins with thioredoxin and a universal tetanus toxin T-cell epitope (P2), in order to enhance the anti-peptide immune response. The peptide antigen containing three tandem copies of the VP8 epitope induced significantly higher levels of anti-peptide antibody than only a single copy of the epitope, or the peptide co-administered with the carrier protein and T-cell epitope. Furthermore, the peptide antigen containing three copies of the peptide produced significantly higher virus-neutralization titres, higher than VP8, indicating that a peptide antigen displaying repeating copies of the amino acid region 1-10 of VP8 is a more potent inducer of HRV-neutralizing antibodies than VP8 alone, and may be useful for the production of specific neutralizing antibodies for passive immunotherapy of HRV infection.     

Immunogenicity of a promiscuous T cell epitope peptide based conjugate vaccine against benzo[a]pyrene: redirecting antibodies to the hapten

The prototype polycyclic aromatic hydrocarbon benzo[a]pyrene (B[a]P) is an environmental pollutant and food contaminant of epidemiological importance. To protect against adverse effects of this ubiquitous carcinogen, we developed an immunoprophylactic strategy based on a B[a]P-protein conjugate vaccine to induce B[a]P specific antibodies (Grova et al., Vaccine. 2009;27:4142-51). Here, we investigated in mice the efficacy of B[a]P-peptide conjugates based on promiscuous T cell epitopes (TCE) into further improve this approach. We showed that B[a]P-peptide conjugates induced very different levels of hapten-specific antibodies with variable functional efficacy, depending on the carrier. In some cases peptide carriers induced a more efficient antibody response against B[a]P than tetanus toxoid as a protein carrier, with the capacity to sequester more B[a]P in the blood. Reducing the carrier size to a single TCE can dramatically shift the antibody bias from the carrier to the B[a]P. Conjugates based on the TCE FIGITEL induced the best anti-hapten response and no antibodies against the carrier peptide. Some peptide conjugates increased the selectivity of the antibodies for the activated metabolite 7,8-diol-B[a]P and B[a]P by one or two orders of magnitude. The antibody efficacy was also demonstrated in their ability to sequester B[a]P in the blood and modulate its faecal excretion (15-56%). We further showed that pre-existing immunity to the carrier from which the TCE was derived did not reduce the immunogenicity of the peptide conjugate. In conclusion, we showed that a vaccination against B[a]P using promiscuous TCEs of tetanus toxin as carriers is feasible even in case of a pre-existing immunity to the toxoid and that some TCE epitopes dramatically redirect the antibody response to the hapten. Further studies to demonstrate a long-term protection of an immunoprophylactic immunisation against B[a]P are warranted.     

The extracellular domain of receptor activity-modifying protein 1 is sufficient for calcitonin receptor-like receptor function

A functional calcitonin gene-related peptide (CGRP) receptor requires dimerization of calcitonin receptor-like receptor (CRLR) with receptor activity-modifying protein 1 (RAMP 1). To determine the function of the three domains (extracellular, ECD; transmembrane, TM; and tail domains) of human RAMP 1, three mutants were constructed: RAMP 1 without the cytoplasmic tail, a chimera consisting of the ECD of RAMP 1 and the TM and tail of the platelet-derived growth factor receptor, and the ECD of RAMP 1 alone. These RAMP 1 mutants were examined for their ability to associate with CRLR to effect CGRP-stimulated cAMP accumulation, CGRP binding, CRLR trafficking, and cell surface expression. All RAMP 1 mutants were able to associate with CRLR with full efficacy for CGRP-stimulated cAMP accumulation. However, the RAMP 1/platelet-derived growth factor receptor chimera demonstrated a 10-fold decrease in potency for CGRP signaling and binding, and the RAMP 1-ECD mutant had a 4000-fold decrease in potency. In conclusion, the ECD of RAMP 1 is sufficient for normal CRLR association and efficacy. The presence of a TM domain and the specific sequence of the RAMP 1 TM domain contribute to CGRP affinity and potency. The C-terminal tail of RAMP 1 is unnecessary for CRLR function.     

Monoclonal antibody mediated intracellular targeting of tallysomycin S(10b)

The potency of tallysomycin S(10b) (TLM S(10b)) an analogue bleomycin was enhanced by up to 875-fold when it was conjugated to the internalizing antibody BR96. Attachment to the antibody is achieved via a Cathepsin B cleavable linker. The enhancement in potency is believed to be a result of cellular uptake of the conjugate upon antigen binding followed by rapid release of the drug inside the lysosome. This method provides a novel approach for increasing the potency and therapeutic index of nominally moderately-active cytotoxic agents.     

Synthesis and molecular characterization of a biotinylated analog of [Lys]bradykinin.

We report the synthesis and molecular characterization of a biotinylated analog of kallidin, [Lys]bradykinin. Bradykinin was prepared by solid phase peptide synthesis. Before cleavage from the resin, a biotin moiety was coupled to the epsilon amino group of a lysine in the zeroth position of the bradykinin peptide. An omega-amino caproic acid spacer was incorporated between the biotin group and the N-terminal lysine. The biotinylated peptide was deprotected, cleaved from the resin and purified by RP-HPLC. The identity of this analog was confirmed by amino acid analysis and FAB-mass spectrometry. Biotinyl [Lys]bradykinin (BLBK, mol, wt. = 1528) inhibited [3H]-bradykinin binding to guinea pig ileum homogenates dose dependently, with an IC50 of 28.9 +/- 6 nM. The IC50 for [Lys]bradykinin was approximately 10-fold lower, 3.2 +/- 0.6 nM. BLBK induced contractility in an isolated guinea pig smooth muscle preparation with an EC50 of 129 +/- 14 nM; the corresponding value for [Lys]bradykinin was 29 +/- 8 nM. These data are consistent with the difference in binding potency observed for BLBK compared to [Lys]bradykinin. In an ELISA assay using BLBK and affinity-purified rabbit anti-bradykinin antibody, BLBK bound to anti-bradykinin antibody with an EC50 = 1.21 +/- 0.54 nM. Rank order potencies for several bradykinin peptide analogs suggest that the epitope on bradykinin recognized by the antibody is likely to be at the carboxy terminus of the peptide.     

Kinetic comparison of ricin immunotoxins: biricin conjugate has potentiated cytotoxicity

The plant toxin ricin was chemically coupled to an anti-Thy-1.1 antibody, and the resultant conjugates were fractionated by gel filtration. The cytotoxicity of the conjugate possessing two ricin molecules per immunoglobulin, yielding a first-order inactivation rate of protein synthesis of -0.4 log/h at 200 ng/mL, was well above that expected just from the increase in ricin per unit mass of conjugate, when compared to a conjugate possessing only one ricin per immunoglobulin. On a conjugate molar scale the biricin immunotoxin was determined to be 8 times more potent than the monoricin conjugate; thus, relative to the number of ricin molecules, the coupling of a second ricin to the immunoglobulin quadrupled the observed potency. The concentration of immunotoxin and the resultant inactivation rates of protein synthesis were found to be related through a power function. Additionally, the inactivation kinetics of these conjugates were found to be similar to those of native ricin.     

Determinants of potency on alpha-conotoxin MII, a peptide antagonist of neuronal nicotinic receptors

Alpha-conotoxin MII, a peptide toxin isolated from Conus magus, antagonizes a subset of neuronal nicotinic receptors. Rat alpha3beta2 receptors, expressed in Xenopus oocytes, are blocked with an IC(50) of 3.7 +/- 0.3 nM. To identify structural features that determine toxin potency, a series of alanine-substituted toxins were synthesized and tested for the ability to block the function of alpha3beta2 receptors. Circular dichroism and protein modeling were used to assess the structural integrity of the mutant toxins. Three residues were identified as major determinants of toxin potency. Replacement of asparagine 5, proline 6, or histidine 12 with alanine resulted in >2700-fold, 700-fold, and approximately 2700-fold losses in toxin potency, respectively. A decrease in pH improved toxin potency, while an increase in pH eliminated toxin blockade, suggesting that, in the active form of the toxin, histidine 12 is charged. The imidazole ring of histidine 12 protrudes from one side, while asparagine 5 and proline 6 are located at the opposite end of the toxin structure. The side chains of these three residues are exposed on the surface of the toxin, suggesting that they directly interact with the alpha3beta2 receptor.     

Single-chain immunotoxins directed at the human transferrin receptor containing Pseudomonas exotoxin A or diphtheria toxin: anti-TFR(Fv)-PE40 and DT388-anti-TFR(Fv).

Two single-chain immunotoxins directed at the human transferrin receptor have been constructed by using polymerase chain reaction-based methods. Anti-TFR(Fv)-PE40 is encoded by a gene fusion between the DNA sequence encoding the antigen-binding portion (Fv) of a monoclonal antibody directed at the human transferrin receptor and that encoding a 40,000-molecular-weight fragment of Pseudomonas exotoxin (PE40). The other fusion protein, DT388-anti-TFR(Fv), is encoded by a gene fusion between the DNA encoding a truncated form of diphtheria toxin and that encoding the antigen-binding portion of antibody to human transferrin receptor. These gene fusions were expressed in Escherichia coli, and fusion proteins were purified by conventional chromatography techniques to near homogeneity. In anti-TFR(Fv)-PE40, the antigen-binding portion is placed at the amino terminus of the toxin, while in DT388-anti-TFR(Fv), it is at the carboxyl end of the toxin. Both these single-chain immunotoxins kill cells bearing the human transferrin receptors. However, anti-TFR(Fv)-PE40 was usually more active than DT388-anti-TFR(Fv), and in some cases it was several-hundred-fold more active. Anti-TFR(Fv)-PE40 was also more active on cell lines than a conjugate made by chemically coupling the native antibody to PE40, and in some cases it was more than 100-fold more active.     

Protein kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino acid substitutions for phenylalanine 10. Inhibition of cAMP-dependent protein kinase

The minimal structure in the heat-stable inhibitor protein of cAMP-dependent protein kinase required for a low nanomolar potency of inhibition is the peptide Thr6-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-Ala-+ ++Ile22-NH2 (PKI-(6-22)-amide). While primary structural determinants for interaction with the protein kinase are distributed throughout the 17 residues of this peptide, we have previously shown that phenylalanine 10 in the NH2-terminal portion is a particularly important determinant for high affinity binding (Glass, D. B., Cheng, H.-C., Mende-Mueller, L., Reed, J., and Walsh, D. A. (1989) J. Biol. Chem. 264, 8802-8810). To investigate this requirement further, peptide analogs of PKI-(6-22)-amide in which various natural and nonstandard amino acids are substituted for phenylalanine 10 have been synthesized and tested for inhibitory potency against the catalytic subunit of the protein kinase. Consistent with the importance of the hydrophobicity of phenylalanine, an alanine 10 substitution analog exhibited a 270-fold decrease in inhibitory potency, whereas the leucine 10 analog lost only 33-fold in activity as compared to the parent peptide PKI-(6-22)-amide. Peptides containing the spatial conformation analogs D-phenylalanine, homophenylalanine, or phenylglycine were 60-120-fold less potent than the parent peptide. Peptides containing various para-substituted phenylalanines at position 10 were only 5-11-fold less potent. One exception to this was (4'-azidophenylalanine 10)PKI-(6-22)-amide, which was nearly equipotent with the parent inhibitor. The most potent analogs were those peptides containing highly aromatic residues at position 10. The 2'-thienylalanine 10, tryptophan (formyl) 10, tryptophan 10, and the 1'-naphthylalanine 10 analogs were 3-fold less potent, equipotent, slightly more potent, and 4-fold more potent than the parent peptide inhibitor, respectively. We conclude that phenylalanine 10 in PKI-(6-22)-amide, and presumably in the native protein inhibitor, interacts through specific hydrophobic and/or aromatic binding to a hydrophobic pocket or cleft near the active site of the protein kinase.     

The role of the diphtheria toxin receptor in cytosol translocation

The role of the receptor in the transport of diphtheria toxin (DT) to the cytosol was examined. A point-mutant form of DT, CRM 107 (CRM represents cross-reacting material), that has an 8,000-fold lower affinity for the DT receptor than native toxin was conjugated to transferrin and monoclonal antibodies specific for the cell-surface receptors T3 and Thy1. Conjugating the binding site-inactivated CRM 107 to new binding moieties reconstituted full toxicity, indistinguishable from native DT linked to the same ligand, indicating that the entry activity of the DT B chain can be fully separated from the receptor binding function. Like DT, the toxin conjugates exhibited a dose-dependent lag period before first-order inactivation of protein synthesis. Inactivation of the binding site of the toxin portion of the conjugate was found to have no effect on the kinetics of protein synthesis inactivation. The receptor used by the toxin determined the length of the lag period relative to the killing rate. Comparing the potency of CRM 107 conjugates with native DT, standardized for receptor occupancy, shows that new receptors can be as or more efficient than the DT receptor in transporting DT to the cytosol. The transferrin-CRM 107 conjugate, unlike native DT, was highly toxic to murine cells. All the data presented are consistent with a model that the DT receptor, other than initiating rapid internalization of the toxin to low pH compartments, is unnecessary for transport of the toxin to the cytosol and that membrane translocation activity is expressed by the DT B subunit independent of the receptor-binding site.