#immunotoxins

Immunological Applications

Which toxin is commonly used in immunotoxins?

Selecting optimal toxin component for immunoconjugate involves potency, intracellular stability, lack of mammalian cell surface receptors to avoid nonspecific uptake, and ability to produce recombinant fusion maintaining disulfide integrity. Diphtheria toxin secreted by toxigenic Corynebacterium diphtheriae, 58 kDa single polypeptide proteolytically cleaved into 21 kDa catalytic A fragment and 37 kDa binding-translocation B fragment linked by single disulfide Cys186-Cys201. Catalytic activity transfers ADP-ribose to elongation factor 2 with turnover number approximately 1000 per minute. Native receptor heparin binding EGF-like precursor widely expressed, so receptor-binding domain residues 1-389 deleted to generate DT388 retaining translocation domain helices capable of endosomal membrane insertion at acidic pH after furin cleavage. Similarly Pseudomonas exotoxin A 66 kDa secreted by Pseudomonas aeruginosa, ADP-ribosylates EF2 after binding CD91 receptor, truncated to PE38 removing domain Ia binding region retaining domain II translocation and domain III catalytic with C-terminal REDLK endoplasmic reticulum retrieval motif. Plant toxins like ricin A chain or gelonin inactivate ribosomes via depurination but require chemical conjugation via heterobifunctional crosslinkers SPDP forming disulfide bridge.

Ref: Weldon & Pastan FEBS J 2011 PE38 DT388 catalytic domain; Collier EF2 ADP-ribosylation translation arrest mechanism.

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Immunological Applications

Immunotoxins kill target cells mainly by:

Mechanism by which protein toxins arrest protein synthesis culminates in rapid programmed cell death via mitochondrial pathway capable of killing quiescent cells. Diphtheria toxin fragment A catalyzes ADP-ribosylation of unusual post-translationally modified histidine diphthamide at position 699 of eukaryotic elongation factor 2 present exclusively on domain IV, using oxidized NAD+ as ADP-ribose donor releasing nicotinamide. Modified elongation factor cannot mediate translocation step of peptidyl-tRNA from A site to P site on 60S ribosomal subunit, halting elongation after single round. Ricin A chain functions as RNA N-glycosidase hydrolyzing N-glycosidic bond of adenine 4324 in 28S ribosomal RNA alpha sarcin-ricin loop critical for EF2 GTPase activation, depurinating single base rendering 60S subunit unable to bind elongation factors. Both events cause accumulation of stalled ribosomes triggering ribotoxic stress response via MAP3K ZAK activating JNK and p38, plus depletion of short-lived anti-apoptotic proteins Mcl-1 half-life thirty minutes, XIAP, c-FLIP required to restrain caspase cascade, tipping balance toward Bax Bak oligomerization forming mitochondrial outer membrane pores releasing cytochrome c, Smac DIABLO, formation of Apaf-1 apoptosome activating initiator caspase 9 then executioners caspase 3 and 7 cleaving PARP leading to chromatin condensation within hours even in nondividing cells resistant to mitotic inhibitors.

Ref: Collier Annu Rev Biochem 1975 diphtheria ADP-ribosyl EF2; Olsnes Pharmac Ther ribosome N-glycosidase ricin apoptosis.

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Immunological Applications

Immunotoxins are composed of:

Immunotoxins constructed to overcome lack of selectivity of conventional chemotherapy achieve tumor specific delivery of ultrapotent protein toxins that cannot enter mammalian cells unaided. Design consists of targeting moiety typically single-chain variable fragment scFv or disulfide stabilized Fab derived from murine or humanized antibody recognizing tumor antigen CD22 on hairy cell leukemia, CD25 on adult T cell leukemia, mesothelin on mesothelioma, linked via flexible glycine-serine peptide or reducible disulfide bond formed between engineered cysteines to effector toxin devoid of native receptor binding domain to prevent off target internalization. Toxin moiety from Pseudomonas aeruginosa exotoxin A truncated to 38 kDa PE38 containing ADP-ribosylation domain plus translocation domain or diphtheria toxin DT388 lacking receptor domain 1-389, or plant toxin ricin A chain 32 kDa. This mechanistic insight supports diagnostic and therapeutic applications while reinforcing core immunological and cell biology principles taught in advanced curricula. This mechanistic detail underpins practical applications in diagnostics, vaccine design, and biopharmaceutical manufacturing.

Ref: Pastan et al Nature Rev Cancer 2006 6:559 immunotoxins PE38; Kreitman Clin Cancer Res 2009 fusion toxin de-immunized.

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Immunological Applications

Which toxin is commonly used in immunotoxins?

Diphtheria toxin produced Corynebacterium diphtheriae lysogenized temperate corynephage beta carrying tox gene regulated iron-dependent repressor DtxR provides ideal warhead immunotoxin because catalytic mechanism well characterized potency extreme one molecule sufficient kill cell enzymatic turnover inactivating millions ribosomes extensive clinical experience vaccine toxoid. AB architecture A domain catalytic 21 kDa active site Glu148 critical nucleophile performing NAD-dependent ADP-ribosylation EF2 B domain receptor-binding translocation B composed receptor-binding subdomain C-terminal 482-535 binding HBEGF ubiquitously expressed mediating endocytosis plus translocation domain hydrophobic helices forming pore acidic endosomal membrane pH 5.5 facilitating cytosolic entry. Wild-type B domain causes nonspecific toxicity liver peripheral nerves limiting therapeutic window. Immunotoxin construction receptor-binding subdomain deleted generating DT388 variant 1-388 retaining translocation helices TH8 TH9 catalytic preserving ability translocate abrogating native tropism reducing off-target 1000-fold. Fusion anti-CD22 scFv flexible linker produces BL22 CAT-3888 moxetumomab pasudotox improved affinity 14-fold mutagenesis heavy chain CDR3. Upon endocytosis furin cleaves arginine-rich loop disulfide reduction releasing A chain cytosol. Clinical immunotoxins denileukin diftitox Ontak IL-2 DT389 CD25 cutaneous T-cell lymphoma Tagraxofusp IL3 DT blastic plasmacytoid dendritic neoplasm illustrate successful retargeting achieving FDA approvals objective response 70 percent.

Ref: Pastan Diphtheria Toxin Immunotoxin Clinical Development 2009; FDA Ontak Tagraxofusp DT Fusion Mechanism Label; Collier Diphtheria Toxin Structure Function Catalysis Enzymology.

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Immunological Applications

Immunotoxins kill target cells mainly by:

Protein toxins employed immunotoxin design kill primarily through catalytic inactivation protein synthesis machinery requiring only few molecules cytosol triggering irreversible apoptosis. Diphtheria toxin fragment A 21 kDa ADP-ribosyltransferase modifies diphthamide residue unique post-translationally modified histidine 699 eukaryotic elongation factor 2 synthesized seven enzymes DPH1-7 adding 3-amino-3-carboxypropyl trimethylation diphthine amidation transferring ADP-ribose oxidized NAD imidazole inhibiting translocation peptidyl tRNA A site P site elongation cycle 80S ribosome mediated GTP hydrolysis. Pseudomonas exotoxin A domain III similarly ADP-ribosylates same residue furin cleavage toxin endosome separating catalytic domain. Ricin A chain N-glycosidase hydrolyzes N-glycosidic bond depurinating adenine 4324 sarcin-ricin loop GAGA tetraloop 28S rRNA preventing binding elongation factors eEF1 aminoacyl tRNA eEF2 interfering factor-dependent GTPase. Arrest translation rapidly depletes short-lived antiapoptotic proteins MCL-1 half-life 2h XIAP continuous turnover releasing Bak Bax oligomerization forming pores outer mitochondrial membrane cytochrome c release activating apoptosome caspase-9 caspase-3 cascade executing death concentrations 10^-11 M explaining extraordinary potency exceeding conventional small-molecule chemotherapeutics lacking enzymatic amplification catalytic turnover.

Ref: Collier 1967 Diphtheria Toxin ADP-ribosylation EF2 Discovery; Pastan PE38 Protein Synthesis Inhibition Mechanism; Lodish Protein Synthesis eEF2 Function Chapter 7.

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Immunological Applications

Immunotoxins are composed of:

Immunotoxins chimeric proteins integrating targeting domain derived monoclonal antibody and cytotoxic domain protein toxin to achieve picomolar potency against malignant cells expressing defined surface antigen. Antibody moiety typically Fab prime 50 kDa scFv 25 kDa composed heavy variable light variable domains connected glycine serine linker disulfide-stabilized Fv dsFv recognizing tumor antigens CD22 135 kDa hairy cell leukemia CD25 IL-2 receptor alpha cutaneous T-cell lymphoma mesothelin 40 kDa GPI-anchored mesothelioma pancreatic adenocarcinoma HER2 with affinity 1-10 nM. Toxin component truncated Pseudomonas exotoxin PE38 38 kDa lacking domain Ia binding LRP1 ubiquitous retaining domain II translocation domain III ADP-ribosylating diphtheria toxin DT388 receptor-binding domain deleted but translocation catalytic retained ricin A chain N-glycosidase depurinating 28S RNA ribosome-inactivating protein saporin Saponaria. Components genetically fused peptide linker GGS chemically conjugated thioether preserving binding enzymatic functions. Upon antigen-mediated endocytosis clathrin-coated pits binding trafficking early endosomes pH 6.0 furin cleavage trans-Golgi network generating heterodimer retrograde via KDEL receptor ER translocation Sec61 cytosol where one molecule sufficient killing cell protein synthesis inhibition triggering apoptosis.

Ref: Pastan Annu Rev Med 2007 Immunotoxin Construction Principles Review; FDA Lumoxiti Moxetumomab Design Label; Janeway Immunotoxin Antibody Toxin Fusion Mechanism.

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