Outline

  • Abstract
  • Introduction
  • Methods
  • Results
  • Conclusion
  • Keywords
  • 1. Introduction
  • 2. Materials and Methods
  • 2.1. Materials
  • 2.2. Antibody Reduction
  • 2.3. Synthesis of the Tricarbonyl Precursor [m(oh2)3(co)3]+ (m=99mtc/188re)
  • 2.4. Radiolabeling of the Antibody with 99mtc/188re(co)3
  • 2.5. in Vitro Stability of 99mtc/188re(co)3-Rtxred
  • 2.6. Cell Culture
  • 2.7. Immunoreactivity of 99mtc/188re(co)3-Rtxred
  • 2.8. Binding Affinity of 99mtc/188re(co)3-Rtxred
  • 2.9. in Vivo Studies of 99mtc/188re(co)3-Rtxred
  • 2.10. Ex Vivo Autoradiography
  • 2.11. in Vitro Autoradiography
  • 3. Results
  • 3.1. Evaluation of Non-Reduced 99mtc(co)3-Rtxwt
  • 3.2. Labeling Study of Reduced 99mtc/188re(co)3-Rtxred
  • 3.3. Cysteine and Histidine Challenge of 99mtc/188re(co)3-Rtxred
  • 3.4. Plasma Stability of 99mtc/188re(co)3-Rtxred
  • 3.5. Immunoreactivity and Binding Affinity of 99mtc/188re(co)3-Rtxred
  • 3.6. Biodistribution of 99mtc/188re(co)3-Rtxred
  • 3.7. Ex Vivo and in Vitro Autoradiography
  • 4. Discussion
  • 5. Conclusion
  • Acknowledgment
  • References

رئوس مطالب

  • چکیده
  • 1. مقدمه
  • 2. مواد و روش ها
  • 2.1 مواد
  • 2.2 کاهش آنتی بادی
  • 2.3 سنتز پیش ماده سه کربنیله [M(OH2)3(CO)3]+ (M = 99mTc/188Re)
  • 2.4. نشان دار کردن رادیویی آنتی بادی با 99mTc/188Re(CO)3
  • 2.5. پایداری 99mTc/188Re (CO)3.RTXred در شرایط آزمایشگاهی
  • 2.6 کشت سلولی
  • 2.7. واکنش سیستم ایمنی 99mTc/188Re (CO)3.RTXred
  • 2.8 تمایل به اتصال 99mTc/188Re (CO)3.RTXred
  • 2.9 مطالعات 99mTc / 188Re (CO)3.RTXred در vivo
  • 2.10 رادیوگرافی خودکار خارج vivo
  • 2.11 رادیوگرافی خودکار در vitro
  • 3. نتایج
  • 3.1 ارزیابی 99mTc (CO)3.RTXwt بدون کاهش
  • 3.2 بررسی نشان دار کردن 99mTc/188Re (CO)3.RTXred کاهش یافته
  • 3.3. چالش سيستئين و هيستيدين 99mTc/188Re(CO)3.RTXred
  • 3.4. پایداری 99mTc/188Re(CO)3.RTXred در پلاسما
  • 3.5 واکنش دستگاه ایمنی و تمایل به اتصال 99mTc/188Re(CO)3.RTXred
  • 3.6. توزیع بیولوژیکی 99mTc/188Re(CO)3.RTXred
  • 3.7 رادیوگرافی خودکار درون بدن و در آزمایشگاه
  • 4. بحث
  • 5. نتیجه گیری

Abstract

Introduction

The most successful clinical studies of immunotherapy in patients with non-Hodgkin’s lymphoma (NHL) use the antibody rituximab (RTX) targeting CD20+ B-cell tumors. Rituximab radiolabeled with β− emitters could potentiate the therapeutic efficacy of the antibody by virtue of the particle radiation. Here, we report on a direct radiolabeling approach of rituximab with the 99mTc- and 188Re-tricarbonyl core (IsoLink technology).

Methods

The native format of the antibody (RTXwt) as well as a reduced form (RTXred) was labeled with 99mTc/188Re(CO)3. The partial reduction of the disulfide bonds to produce free sulfhydryl groups (–SH) was achieved with 2-mercaptoethanol. Radiolabeling efficiency, in vitro human plasma stability as well as transchelation toward cysteine and histidine was investigated. The immunoreactivity and binding affinity were determined on Ramos and/or Raji cells expressing CD20. Biodistribution was performed in mice bearing subcutaneous Ramos lymphoma xenografts.

Results

The radiolabeling efficiency and kinetics of RTXred were superior to that of RTXwt (99mTc: 98% after 3 h for RTXred vs. 70% after 24 h for RTXwt). 99mTc(CO)3-RTXred was used without purification for in vitro and in vivo studies whereas 188Re(CO)3-RTXred was purified to eliminate free 188Re-precursor. Both radioimmunoconjugates were stable in human plasma for 24 h at 37°C. In contrast, displacement experiments with excess cysteine/histidine showed significant transchelation in the case of 99mTc(CO)3-RTXred but not with pre-purified 188Re(CO)3-RTXred. Both conjugates revealed high binding affinity to the CD20 antigen (Kd=5–6 nM). Tumor uptake of 188Re(CO)3-RTXred was 2.5 %ID/g and 0.8 %ID/g for 99mTc(CO)3-RTXred 48 h after injection. The values for other organs and tissues were similar for both compounds, for example the tumor-to-blood and tumor-to-liver ratios were 0.4 and 0.3 for 99mTc(CO)3-RTXred and for 188Re(CO)3-RTXred 0.5 and 0.5 (24 h pi).

Conclusion

Rituximab could be directly and stably labeled with the matched pair 99mTc/188Re using the IsoLink technology under retention of the biological activity. Labeling kinetics and yields need further improvement for potential routine application in radioimmuno diagnosis and therapy.

Conclusions

An alternative route for the direct radiolabeling of the anti-CD20 mAb rituximab with both 99mTc and 188Re using the tricarbonyl technique was developed and assessed. Acceptable radiolabeling yields could only be achieved after reduction of the antibody. Unfortunately, the exact site and mode of coordination of the M(CO)3 core are not clear. It can only be speculated but not proven at this time whether the metal core is primarily coordinated via the newly accessible histidines of the Fab portion (after a moderate unfolding of the tertiary protein structure as a consequence of reduction of the disulfide bonds) or in fact via the newly available cysteine side chains (or a combination of both). One also has to keep in mind that RTXred per se represents a heterogeneous product, since the number and site of reduced disulfide bonds vary. Thus, also M(CO)3-RTXred is presumably a heterogeneous mixture of different species. In vitro binding studies revealed full retention of biological activity for both 99mTc and 188Re immunoconjugates. Both 99mTc(CO)3-RTXred and 188Re(CO)3-RTXred revealed an almost similar pharmacokinetic profile in vivo. However, the radiolabeling procedure of the reduced antibody with 188Re needs further improvement to consider these radioimmunoconjugates as a suitable alternative to the 111In/90Y-Zevalin system. Notably, the necessity of reduction of the antibody and the long incubation time to achieve reasonable radiolabeling yields is prohibitive for a convenient application of this approach in a clinical environment.

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