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. 2004 Nov 16;101(46):16298-303.
doi: 10.1073/pnas.0405271101. Epub 2004 Nov 1.

The Regulated Long-Term Delivery of Therapeutic Proteins by Using Antigen-Specific B Lymphocytes

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Free PMC article

The Regulated Long-Term Delivery of Therapeutic Proteins by Using Antigen-Specific B Lymphocytes

Katalin Takács et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Memory lymphocytes are important mediators of the immune response. These cells are long-lived and undergo clonal expansion upon reexposure to specific antigen, differentiating into effector cells that secrete Ig or cytokines while maintaining a residual pool of memory T and B lymphocytes. Here, the ability of antigen-specific lymphocytes to undergo repeated cycles of antigen-driven clonal expansion and contraction is exploited in a therapeutic protocol aimed at regulating protein delivery. The principle of this strategy is to introduce genes encoding proteins of therapeutic interest into a small number of antigen-specific B lymphocytes. Output of therapeutic protein can then be regulated in vivo by manipulating the size of the responder population by antigen challenge. To evaluate whether such an approach is feasible, we developed a mouse model system in which Emu- and Iglambda-based vectors were used to express human erythropoietin (hEPO) gene in B lymphocytes. These mice were then immunized with the model antigen phycoerythrin (PE), and immune splenocytes (or purified PE-specific B lymphocytes) were adoptively transferred to normal or mutant (EPO-deficient) hosts. High levels of hEPO were detected in the serum of adoptively transferred normal mice after PE administration, and this responsiveness was maintained for several months. Similarly, in EPO-deficient anemic recipients, antigen-driven hEPO expression was shown to restore hematocrit levels to normal. These results show that antigen-mediated regulation of memory lymphocytes can be used as a strategy for delivering therapeutic proteins in vivo.

Figures

Fig. 1.
Fig. 1.
Expression of therapeutic proteins by mouse lymphocytes. (a) The construction of Eμ- and λ1-based vectors used to generate transgenic mice, where Eμ indicates the Ig heavy chain enhancer, and Igλ1 Pro labels the Ig λ1 promoter. β-globin 3′+UMS denotes a fragment encompassing the 3′ end of human β-globin gene and transcription terminator UMS, followed by λ1-3′HS1-4, the 3′ Igλ1 enhancer. As a model therapeutic gene, hEPO was inserted into the Eμ- and λ1-based cassettes as shown. (b) Northern blots of total RNA extracted from different transgenic tissues; spleen (Sp), thymus (Th), liver (Li), kidney (Kd), brain (Br), as well as LPS-stimulated splenocytes. The arrows indicate the major 1.8-kb hEPO transgene-derived transcripts. 18S ribosomal RNA bands stained with ethidium bromide are shown to illustrate the equivalence of RNA loading between samples. (Transgene expression was confirmed in two independent Eμ-hEPO and λ1-hEPO lines, respectively.) (c) hEPO expression by polyclonally stimulated transgenic splenocytes. hEPO concentrations detected in the supernatants of 48 h in vitro cultures of 3 × 106 LPS-stimulated splenocytes isolated from independent hEPO transgenic lines with different numbers of integrated transgene copies are shown. Mice used in these experiments were age-matched, and the mean values obtained from two independent cell cultures are shown. (d) Antigen-induced hEPO production in hEPO transgenic mice in vivo. The shaded histogram profile illustrates hEPO levels detected in a transgenic mouse (line 339) immunized with PE on day 1. Serum hEPO levels of an untreated, hEPO transgenic littermate are shown for comparison (open histograms). A representative profile of three independent experiments is shown.
Fig. 2.
Fig. 2.
Regulated production of hEPO in normal mice after the adoptive transfer of antigen-specific hEPO transgenic lymphocytes. (a) The different responses of mice transferred with 3 × 107 unfractionated, PE-primed λ1-hEPO splenocytes, challenged with either soluble PE (filled circles) or PBS (open circles) at different time points after cell transfer (indicated by arrows). Serum hEPO levels were monitored for 145 days. (b) 3 × 106 CD43-depleted lymphocytes purified from a PE-primed λ1-hEPO transgenic mouse were transferred into two normal recipients that were injected with soluble PE at different times after transfer (arrows), and the level of hEPO in their sera was monitored for 56 days. (c) Transfer of 4 × 104 PE-binding (filled circles) and PE-nonreactive (open circles) λ1-hEPO transgenic B cells into two normal recipients. Both mice were boosted with soluble PE at the indicated times (arrows), and serum hEPO levels were monitored for 98 days.
Fig. 3.
Fig. 3.
Correcting anemia in mutant mice by transferring bone marrow cells or PE-specific B lymphocytes from λ1-hEPO transgenic donors. (a) 5 × 106 CD43-depleted B cells purified from PE-immunized hEPO transgenic (filled circles) or wild-type (open circles) donors were injected into two groups of four heterozygous Epo-TAg anemic mice. Both sets of mice were injected with PE on the day of cell transfer and additionally on days 16, 40, 51, 79, and 94 (arrows). Their hematocrit values were monitored between days 45 and 120. The data shown are the mean ± SD of hematocrits. (b) The differing responses of heterozygous Epo-TAg mice that received 107 purified B220-positive (filled circles) or B220-negative (open circles) PE-primed hEPO transgenic splenocytes. The hematocrit values and PE immunization schedule (arrows) are shown. (c) Five severely anemic homozygous Epo-TAg mice were adoptively transferred with 5 × 106 B220-positive λ1-hEPO transgenic B lymphocytes separated from PE-immunized donors. The recipients were injected with PE on the day of cell transfer, 2 and 7 weeks later (arrows). Mean hematocrit values of five mice and standard deviations are shown (filled circles). A control Epo-TAg mouse (open circles) was transferred with 5 × 106 B cells derived from PE-immunized wild-type donor and injected with PBS (arrows).
Fig. 4.
Fig. 4.
Schematic representation of the immunoregulated gene therapy approach.

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