Background: The epidermal growth factor receptor (EGFR) plays a key role in the growth and survival of many human tumors of epithelial origin. The monoclonal antibody, 40H3, targeting overexpressed EGFR and the truncated form EGFR variant III (EGFRvIII) on tumor cells, was conjugated to small molecules with potent cytotoxic activity, generating five antibody-drug conjugates (ADCs). Their lethality for tumor cells was evaluated in vitro and in vivo. Materials and methods: ADC construction: Purified 40H3 antibody was conjugated to five different payloads: two tubulin inhibitors, monomethyl auristatin E (MMAE) and DM1, two topoisomerase inhibitors, SN38 and deruxtecan (DXd1) and a PBD dimer (SG-3199). Binding assay: The binding affinity constants of the various 40H3-based ADCs were determined against the His-tagged EGFR peptide loop (aa 287– 302) immobilized on Ni-NTA biosensors with the Octet Red96 analyzer. Bystander assay: Cocultured F98npEGFRvIII and F98 cells were treated with media, free payload (SG-3199), 40H3 antibody, 40H3-tesirine or IgG-tesirine at the indicated concentrations. After 48 hours, the cells were labeled with cetuximab-PE and SYTOXTM Red viability dye and then analyzed for bystander killing by flow cytometry. In vivo studies: MDA-MB-468 or BT-20 tumor xenografts were treated with unmodified 40H3 antibody or 40H3-tesirine. Tumor volumes and mouse weights were measured at least three times weekly. Results: The five ADCs retained antigen binding activity of the unmodified 40H3 antibody. They showed a potent cytotoxicity on a panel of EGFR-expressing cells including three triple negative breast cancer lines. Cell killing was correlated to the number of binding sites for the 40H3 antibody. The 40H3 conjugate with the PBD dimer (40H3-tesirine) was the most active killing agent and it also exhibited bystander killing of cells not expressing human EGFR. Moreover, in vivo, on two different models of tumor xenografts, treatment with 40H3-tesirine achieved complete remissions. Conclusions: The 40H3 antibody is a valid delivery agent for toxic payloads. Among the five ADCs, 40H3-tesirine showed the highest cytotoxic effect toward EGFR-expressing tumor cells in vitro and in vivo. No conflict of interest.

The routes by which antibody-based therapeutics reach malignant cells are poorly defined. Tofacitinib, an FDA-approved JAK inhibitor, reduced tumor-associated inflammatory cells and allowed increased delivery of antibody-based agents to malignant cells. Alone, tofacitinib exhibited no antitumor activity, but combinations with immunotoxins or an antibody-drug conjugate resulted in increased antitumor responses. Quantification using flow cytometry revealed that antibody-based agents accumulated in malignant cells at higher percentages following tofacitinib treatment. Profiling of tofacitinib-treated tumor-bearing mice indicated that cytokine transcripts and various proteins involved in chemotaxis were reduced compared with vehicle-treated mice. Histological analysis revealed significant changes to the composition of the tumor microenvironment, with reductions in monocytes, macrophages, and neutrophils. Tumor-associated inflammatory cells contributed to non-target uptake of antibody-based therapeutics, with mice treated with tofacitinib showing decreased accumulation of therapeutics in intratumoral inflammatory cells and increased delivery to malignant cells. The present findings serve as a rationale for conducting trials where short-term treatments with tofacitinib could be administered in combination with antibody-based therapies.

An improved understanding of immunologic events associated with immunization, the identification of promising new antigens, and an increased capacity to generate these antigens through chemical and biotechnology methods have led to many new vaccine opportunities. Inappropriate antigen exposure, however, can result in unwanted outcomes, such as incomplete protection, allergic reactions, autoimmunity, infection, or even tolerization. Thus, proper antigen delivery is critical for achieving the desired outcome. A number of vaccination approaches have now been described with varied degrees of success. The relative success of these approaches can be correlated with antigen delivery to specific presentation cells and stimulation of the immune system at sites where protective immunity is most appropriate. In addition, a greater understanding of mechanisms involving cells and effector molecules in the events of immunity may allow for improved possibilities for initiating, augmenting, and maintaining the response to a delivered antigen. This review provides insights into the various strategies currently being explored to optimize antigen delivery and the immune response to that antigen.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage. TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Pseudomonas exotoxin has been genetically modified so that it targets cancer cells. This was accomplished by deleting its cell binding domain and replacing it with Fv fragments of antibodies that react with breast, colon, and other cancers. Several recombinant immunotoxins are now in clinical trials.

The ability of a chimeric toxin containing transforming growth factor α (TGFα) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated. TGFα-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC50 value of 0.5–3 μg/ml. Similarly, other forms of the fusion protein, TGFα-PE38 and TGFα-PE40Asp553, which have active TGFα binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC50 values of 0.1–2 and 0.05–0.5 μg/ml respectively. TGFα-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED50 value of 1–30 ng/ml. TGFα-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED50 values of 3–50 ng/ml whereas TGFα-PE40Asp553, which lacks ADP ribosylation activity, had an ED50 > 100 ng/ml. TGFα-PE40 inhibited colony formation of NSCLC cells with an LD50 value of 0.008–0.1 ng/ml. Similarly, TGFα-PE38 inhibited NSCLC colony formation with LD50 values of 0.002–0.1 ng/ml whereas TGFα-PE40Asp553 had an LD50 > 10 ng/ml. Also, TGFα-PE40 and TGFα-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGFα-PE40Asp553 was inactive. These data suggest that TGFα-PE40 and TGFα-PE38 may be useful agents to inactive NSCLC cells.

Pseudomonas exotoxin (PE) is a bacterial toxin that kills mammalian cells by gaining entry to the cytosol and inactivating protein synthesis. The toxin binds and enters cells via the alpha 2-macroglobulin receptors. Within cells, the toxin is processed in several steps to produce an enzymatically active 37-kDa C-terminal fragment which translocates to the cytosol and ADP-ribosylates elongation factor 2. Because PE is a very potent toxin, derivatives of it have been produced which, when joined to various binding ligands, are capable of killing specific target cells. It is hoped that this strategy will lead to the development of effective therapeutic agents for the treatment of human diseases such as cancer, AIDS, and various immunologic disorders.

The synthesis, standardization, and immunogenicity in young outbred mice and clinical evaluation in adult volunteers of investigational vaccines designed to induce serum antibodies to the type 5 and type 8 capsular polysaccharides (CPs) of Staphylococcus aureus are described. Conjugates composed of the type 5 CP and a sonicated preparation of a high-molecular-weight type 8 CP bound to a nontoxic recombinant protein derived from Pseudomonas aeruginosa exotoxin A (rEPA) were synthesized. The conjugates were nontoxic and elicited serum CP antibodies after two subcutaneous injections into young outbred mice; a third injection elicited a booster response. The lower-molecular-weight type 8 CP was not immunogenic in the mice, and the high-molecular-weight type 8 CP elicited low levels of antibodies without a booster effect. In the volunteers, neither the conjugates nor the type 8 CP alone caused significant local reactions or fever. The conjugates elicited type-specific antibodies of both the immunoglobulin M (IgM) and IgG classes after the first injection; a second injection 6 weeks later did not stimulate a booster effect. The high-molecular-weight type 8 CP alone, injected once only, elicited levels of IgG and IgM type-specific antibodies similar to those of the conjugate. The vaccine-induced CP antibodies were mostly of the IgG1 and IgG2 subclasses and had opsonophagocytic activity. The conjugates elicited IgG antibodies to the native exotoxin A with neutralizing activity. In summary, the type 5 and type 8 conjugates were safe and elicited biologically active antibodies to both the CP and rEPA components.

The 37-kDa C-terminal fragment of Pseudomonas exotoxin A (PE; termed PE37 and composed of aa 280-613 of PE) translocates to the cell cytosol to cause cell death. PE37 requires a C-terminal endoplasmic reticulum retention sequence to be cytotoxic, indicating that the toxin may translocate to the cytosol from the endoplasmic reticulum. We show here that the N-terminal region of nascent PE37 can be inserted into the membrane of canine pancreatic microsomes by the preprocecropin signal sequence but then is exported or released from microsomes. The 34 N-terminal amino acids of the toxin fragment are sufficient to arrest translocation and prevent the microsomal accumulation of nascent chains that otherwise are sequestered into microsomes. These data support a role for the N-terminal region of PE37 in the translocation of the toxin from the endoplasmic reticulum to the cytosol in mammalian cells.

The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.

Chimeric toxins are generated by removing or crippling the receptor-binding domains of bacterial or plant toxins and substituting in their place proteins or peptides that bind mammalian cells. The cell-binding protein will then dictate which cell type the chimeric toxin will bind and kill. Gene fusions are then introduced into an appropriate expression system and recombinant chimeric toxins produced. Recombinant chimeric toxins have been made with diphtheria toxin and Pseudomonas exotoxin in combination with a variety of hormones, antibodies, and other cell-binding proteins. To be cytotoxic, chimeric toxins must have binding, translocating, and enzymatic activities. Of the chimeric toxins examined, most bind 3to 10-fold less well than the corresponding native ligands (34,38,45). The chimeric toxin IL-2-PE40 is toxic for cells and cell lines that display IL-2 receptors on their surface. IX A major clinical focus for the use of chimeric toxins is the treatment of cancer and immunological disorders.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.

Pseudomonas exotoxin (PE) is a 66-kDa bacterial toxin that is proteolytically cleaved by cells to produce an N-terminal fragment of 28 kDa and a C-terminal 37-kDa fragment which translocates to the cytosol and inhibits protein synthesis (M. Ogata, V.K. Chaudhary, I. Pastan, and D.J. FitzGerald, J. Biol. Chem. 265:20678-20685, 1990). When cells were broken by homogenization, the appropriate proteolytic activity was found associated with cellular membranes and not in a soluble fraction. Proteolysis of PE by crude membranes was stimulated by divalent cations, was ATP independent, and had a pH optimum of 5.5. When cells were disrupted by nitrogen cavitation and fractionated on Percoll gradients, proteolytic activity was present in fractions corresponding to the density of plasma membranes or endosomes but not in fractions containing lysosomes. Proteolytic activity was recovered in detergent extracts after crude membranes were treated with Nonidet P-40 or octylglucoside. Proteolysis of PE by either crude membranes or detergent extracts generated fragments of 28 and 37 kDa. The sizes of these fragments resembled those produced by intact cells. However, when the nontoxic mutant, PEgly276, which cannot be cleaved appropriately by intact cells, was incubated with membranes or extracts there was no production of the 28- and 37-kDa fragments.

Staphylococcus aureus type 8 capsular polysaccharide (CP) was conjugated either to diphtheria toxoid or to Pseudomonas aeruginosa recombinant exoprotein A by using adipic acid dihydrazide (ADH) or N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) as the joining reagent. The polysaccharide/protein ratios of these two pairs of conjugates were similar. The two synthetic schemes bound the linker to the carboxyls of the type 8 CP by carbodiimide-mediated condensation. ADH was bound to the carboxyls of the protein, whereas SPDP reacted with the amino groups of the protein. Intermolecular linking of the carrier protein, caused by the carbodiimide during the conjugation reaction with the type 8 CP derivative, probably accounts for the larger size of the conjugates formed with ADH compared with those formed with SPDP. Both conjugates synthesized with ADH elicited higher levels of CP antibodies, especially after the first immunization, than did those prepared with SPDP. Similar levels of exoprotein A antibodies were elicited by both conjugates. Higher levels of diphtheria toxoid antibodies were elicited by the conjugate prepared with SPDP than by the one prepared with ADH. The basis for the differences in the immunogenicities of these two pairs of S. aureus type 8 CP conjugates is discussed.

Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.

BACKGROUND:Restenosis after percutaneous transluminal coronary angioplasty is associated with activation of medial smooth muscle cells (SMCs); they proliferate, migrate to the subintima, and narrow the vessel lumen. Cancer cells often express more cell surface receptors than do normal cells. This has allowed tumor cells to be specifically targeted using cytotoxic agents. We have examined whether a similar concept can be applied to rapidly proliferating but nontransformed SMCs. Pseudomonas exotoxin (PE; MW, 66 kDa) is a potent toxin that kills cells by inhibiting protein synthesis; its toxicity is diminished when its cell recognition domain is deleted to produce a 40-kDa protein (PE40).METHODS AND RESULTS:A complementary DNA encoding transforming growth factor alpha (TGF alpha) was ligated to that encoding PE40 and the chimeric toxin TGF alpha-PE40, which is cytotoxic to cancer cells displaying epidermal growth factor (EGF) receptors, was expressed in Escherichia coli. The ability of this toxin to kill proliferating SMCs was tested. When cells were seeded at low density (2,500 cells/cm2) and grown in medium supplemented with 10% fetal bovine serum, they were found to be rapidly proliferating; these cells were very sensitive to the cytotoxic effects of TGF alpha-PE40 (ID50, 4.0 +/- 0.17 ng/ml). In contrast, cytotoxicity was 30-fold less (ID50, 125 +/- 23 ng/ml; p less than 0.0004) when cells were in a quiescent state (grown in medium supplemented with 0.5% fetal bovine serum).CONCLUSIONS:Competition studies using excess EGF indicated that the cytotoxic effects of TGF alpha-PE40 are specifically mediated by the EGF receptor. EGF receptor binding analysis demonstrated that rapidly proliferating SMCs display 10-fold more EGF receptors than do quiescent SMCs in vitro. Thus, a chimeric toxin targeted toward the EGF receptor can selectively kill rapidly proliferating SMCs. Whether this toxin or other chimeric toxins directed against other cell surface receptors will effectively inhibit SMCs proliferating in vivo or be useful in preventing restenosis remains to be determined.

We examined the action of a chimeric protein, IL-2-PE40, on the development of a T cell-mediated disease of the central nervous system with numerous similarities to multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). EAE is caused by IL-2 receptor-bearing T cells specific for myelin basic protein (BP). We report here that the treatment of Lewis rats with IL-2-PE40 delayed and shortened the course of EAE induced by BP in adjuvant and dramatically prevented EAE mediated by anti-myelin basic protein T line cells. The absence of paralytic signs, the absence of cell infiltration in the central nervous system, and the abatement of cellular immunity to myelin basic protein in the treated rats are direct consequences of the specific mechanism of action of IL-2-PE40. Our data support the notion that IL-2-PE40 may be efficient as an immunosuppressive agent for those disorders in which activated T cells play a crucial role.

TGF-alpha-PE40 is a chimeric toxin made by replacing domain Ia of Pseudomonas exotoxin (PE) with transforming growth factor-alpha (TGF-alpha). We have now replaced a portion of domain Ib of PE with different polypeptides or an extra domain III of PE in transforming growth factor alpha-PE40 and maintained cell killing. Thus, TGF-alpha-PE40 can be used to transport foreign protein sequences into the cytosol of cells.