Saporin Uses in Immunology

One of the more common methods of using Streptavidin-ZAP is to couple the complex with biotinylated antibodies. However, there are also many instances of biotinylated ligands, peptides, and even biotinylated major histocompatibility complex (MHC) tetramers in immunology fields.

B-Cell Targeting. An example of utilizing Streptavidin-ZAP to study ligands that target CD22 were shown by Collins et al. [1]. CD22 is a potential target for immunotherapy of B-cell lymphomas. The authors examined the equilibrium between CD22 and the cis and trans forms of its ligands using high affinity sialoside probes. They also demonstrated that a biotinylated probe specific for CD22, when combined with Streptavidin-ZAP, can eliminate several different lymphoma cell lines.

Figure 5. Selective cytotoxicity by enzymatic activation of CCP1-SA-ZAP conjugates. Schematic representation of streptavidin-ZAP bound to the CXP1 peptides (A) and the expected toxicity of the different SA-ZAP conjugates to ACPA expressing B cells (B). Percentage of living ACPAexpressing B cells (C) and TT-specific B cells (D) after 4 days of treatment with antigen-toxin conjugates [2].

Rheumatoid arthritis is a chronic disease that is accompanied with anti-citrullinated protein antibodies (ACPA) produced by autoreactive B cells. A study in 2018 used a synthesized cyclic citrullinated peptide (CCP) antigen suitable for B-cell receptor binding and demonstrated that binding by ACPA was impaired upon manipulation of the residue [2]. The data were generated using biotinylated CCP mixed with Streptavidin-ZAP in cell viability assays. The results marked an important step towards antigen-selective B-cell targeting in general, and more specifically in rheumatoid arthritis.

T-Cell Targeting. An example of using Streptavidin-ZAP to deplete specific T cells was published in 2010; Akiyosi et al. used the dendritic cell-associated heparan sulfate proteoglycan- dependent integrin ligand (DC-HIL) as the targeting agent. DC-HIL is exclusively associated with syndecan-4 (SD-4) which is expressed on some activated T cells [3]. A similar study was done with Sézary syndrome cells that overexpress syndecan-4 [4].

Hess et al. investigated whether pathogenic T cells could be depleted via Streptavidin-ZAP coupled to MHC class I tetramers to kill antigen-specific CD8+ T cells [5]. Their work showed the therapeutic potential for using cytotoxic tetramers to eliminate specific T cells. This same strategy was employed in vivo to delay diabetes in non-obese diabetic mice [6]. The Hess group also used biotinylated peptide-MHC class I tetramers with Streptavidin-ZAP to selectively deplete a population of alloreactive T cells in mice to determine that toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo cytotoxic T lymphocyte assay and reduced the frequency of corresponding T cells [7]. More research towards T-cell depletion utilizing Streptavidin-ZAP and biotinylated MHC tetramers came from Sims et al. where they found that following a significant transient depletion of cells, the population rebounded and reached a higher percentage of total CD8+ T cells than before the depletion. This research provides helps further understanding of the ‘flexibility and turnover’ of these cells [8].

Figure 1. Frequency and function of MCMV-specific CD8+ T cells and depletion of M38- specific CD8+ T cells. C57BL/6 mice were infected i.v. with 1 x 106 pfu MCMV. D) Representative flow cytometry plots of M38-specific CD8+ T cells and M45-specific CD8+ T cells 50 days after MCMV infection, 1 and 6 days after either M38-tetramer-saporin (red box) or M38-tetramer-PE injection (orange box). Data are shown as mean ± SEM and are from one representative plot out of 6/group) [8].

Figure 7. Sequential treatment with mAb 440c and saporin depletes IPCs in vitro. (A) Expression of 440c on bone marrow cells cultured in FLT3-L for 10 days. CD11c  cells were excluded from the gate. 440c is highly expressed on B220  cells, which represent fully developed IPCs. (B-C) Depletion of CD11c /B220  cells from FLT3-L–derived bone marrow IPCs by treatment with 440c-saporin. Cells were stained on ice with biotinylated-440c (B) or a biotinylated control antibody (C), followed by avidin-saporin, cultured for an additional 36 hours, and analyzed for residual presence of CD11c /B220  cells. The percentage of gated cells is indicated. Results are representative of 3 separate experiments [10].

Other Applications. Outside of T cells and B cells, Streptavidin-ZAP has also been used to deplete dendritic cells (DC). Alonso et al. depleted inflammatory DCs with biotinylated anti- CD209 via intravenous injection in a mouse animal model of induced inflammatory DC formation [9]. The authors suggest that the depletion of inflammatory DCs could be useful in understanding inflammatory diseases such as psoriasis. Depletion of natural interferon-producing cells (IPCs) was demonstrated with an IPC-specific biotinylated antibody in vitro [10].

Figure 3. CCL28 promotes tumour growth through attracting CCR101 Treg cells. h) CCR10 depletion eliminates most CD41CD251FOXP31 cells but not CD81 T cells, as determined by flow cytometric analysis. CCR3 depletion eliminates both populations (numbers inside plots refer to the boxed areas: left column, % Treg cells; right column, % CD31CD41 cells (left) and % CD31CD81 cells (right)).or M38-tetramer-PE injection (orange box). Data are shown as mean ± SEM and are from one representative plot out of 6/group) [8].

Facciabene et al. made immunotoxins with Streptavidin-ZAP to biotinylated antibodies for CCR10 and CCR3 (Anti-CC10-Saporin and Anti-CCR3-Saporin). They investigated whether a direct link between tumor hypoxia and tolerance occurs through the recruitment of regulatory cells [11]. Their findings showed that peripheral immune tolerance and angiogenesis programs are closely connected and cooperate to sustain tumors.


  1. Collins BE, Blixt O, Han S, Duong B, Li H, Nathan JK, Bovin N, Paulson JC (2006) High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells. J Immunol 177(5):2994-3003. doi: 10.4049/jimmunol.177.5.2994 PMID: 16920935
  2. Lelieveldt LPWM, Kristyanto H, Pruijn GJM, Scherer HU, Toes REM, Bonger KM (2018) Sequential prodrug strategy to target and eliminate ACPA-selective autoreactive B cells. Mol Pharm 15(12):5565-5573. doi: 10.1021/acs.molpharmaceut.8b00741 PMID: 30289723
  3. Akiyoshi H, Chung JS, Tomihari M, Cruz PD, Jr., Ariizumi K (2010) Depleting syndecan-4+ T lymphocytes using toxin-bearing dendritic cell-associated heparan sulfate proteoglycan-dependent integrin ligand: A new opportunity for treating activated T cell-driven disease. J Immunol 184:3554-3561. doi: 10.4049/jimmunol.0903250 PMID: 20176742
    Read the Targeting Trends Article.
  4. Chung JS, Shiue LH, Duvic M, Pandya A, Cruz PDJ, Ariizumi K (2011) Sezary syndrome cells overexpress syndecan-4 bearing distinct heparan sulfate moieties that suppress T-cell activation by binding DC-HIL and trapping TGF-beta on the cell surface. Blood 117(12):3382-3390. doi: 10.1182/blood-2010-08-302034 PMID: 21252093
  5. Hess PR, Barnes C, Woolard MD, Johnson MD, Cullen JM, Collins EJ, Frelinger JA (2007) Selective deletion of antigen-specific CD8+ T cells by MHC class I tetramers coupled to the type I ribosome-inactivating protein saporin. Blood 109:3300-3307. doi: 10.1182/blood-2006-06-028001 PMID: 17179221
    Read the Targeting Trends Article.
  6. Vincent BG, Young EF, Buntzman AS, Stevens R, Kepler TB, Tisch RM, Frelinger JA, Hess PR (2010) Toxin-coupled MHC class I tetramers can specifically ablate autoreactive CD8+ T cells and delay diabetes in nonobese diabetic mice. J Immunol 184(8):4196-4204. doi: 10.4049/jimmunol.0903931 PMID: 20220085
  7. Hess SM, Young EF, Miller KR, Vincent BG, Buntzman AS, Collins EJ, Frelinger JA, Hess PR (2013) Deletion of naive T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance. Transpl Immunol 29(1-4):138-145. doi: 10.1016/j.trim.2013.10.005 PMID: 24161680
  8. Sims S, Bolinger B, Klenerman P (2015) Increasing inflationary T-cell responses following transient depletion of MCMV-specific memory T cells. Eur J Immunol 45:113-118. doi: 10.1002/eji.201445016 PMID: 25331015
  9. Alonso M, Gregorio J, Davidson M, Gonzalez J, Engleman E (2014) Depletion of inflammatory dendritic cells with anti-CD209 conjugated to saporin toxin. Immunol Res 58:374-377. doi: 10.1007/s12026-014-8511-6 PMID: 24781193
  10. Blasius A, Vermi W, Krug A, Facchetti F, Cella M, Colonna M (2004) A cell-surface molecule selectively expressed on murine natural interferon-producing cells that blocks secretion of interferon-alpha. Blood 103(11):4201-4206. doi: 10.1182/blood-2003-09-3108 PMID: 14695235
  11. Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang L-P, Gimotty PA, Gilks CB, Lal P, Zhang L, Coukos G (2011) Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and Treg cells. Nature 475:226-230. doi: 10.1038/nature10169 PMID: 21753853

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