Thomas Gribbin

A V3 loop haptenic peptide sequence, when tandemly repeated, enhances immunogenicity by facilitating helper T-cell responses to a covalently linked carrier protein

Subunit immunogens containing tandemly repeated copies of T- and B-cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. It has been unclear, however, whether the increased immunogenicity of a tandemly repeated B-cell epitope necessarily results from increased helper T-cell responses to intrinsic T-cell epitopes in the tandemly repeated sequences, or to neodeterminant T-cell epitopes created at the junction of adjacent repeat sequences. We examined this question by comparing the immunogenicity in mice of two immunogens containing one or eight tandemly repeated copies of an HIV-1 V3 loop haptenic sequence. Our results show that the tandemly repeated haptenic sequence potentiates the immunogenicity of the protein construct, likely through the facilitation of enhanced B-cell interaction with the tandem repeat construct and the consequent elicitation of increased carrier protein-specific helper T-cell responses.

A CD20 tandem-epitope immunogen elicits antibody in mice that binds murine cell surface CD20 and depletes splenic B cells in vivo.

CD20 is an important target for monoclonal antibody therapy of B-cell malignancies and for some autoimmune disorders. Though there is interest in evaluating the induction of active immunity to CD20 in the mouse model, the CD20 extracellular domain (ECD) has significant secondary and tertiary structure which make it difficult to target using peptide immunogens. We constructed, expressed, and purified a recombinant immunogen, CD20ECD-6, which displays six tandemly repeated copies of the C-terminus of the murine CD20 ECD covalently linked to maltose-binding protein. Analysis of the purified protein suggested a complex conformation as the protein migrated in significantly retarded fashion by SDS-PAGE analysis. Immunization of mice and rabbits with the CD20ECD-6 led to the induction of antibodies reactive with the C-terminal ECD peptide sequence by ELISA and more importantly, with native cell surface CD20 on the murine B-cell lymphomas, 38C13 and A20. Immunoprecipitation using the rabbit antisera and non-denaturing detergent confirmed the identity of the bound cell surface protein as murine CD20 and suggested that the cell-binding antibodies were specific for the native, folded conformation. Finally, immunization of mice with the CD20ECD-6 using Freunds or QS-21 adjuvants was shown to exert significant biological effects in vivo with the pronounced depletion of splenic B cells. The tandem-epitope immunogen represents a promising tool for eliciting and studying active autoimmunity to CD20, as a basis for potential development of new immunotherapeutics for cancer and autoimmune diseases.

Abstract B98: Final results of a randomized phase Ib study of fractionated 90Y-clivatuzumab tetraxetan in patients with metastatic pancreatic cancer having at least two prior therapies

Background: Prior clinical studies in the first and second line setting showed radioimmunotherapy (RAIT) is a promising therapy for pancreatic cancer that avoids the side effects of further chemotherapy. This multicenter study evaluated the contribution of low radiosensitizing doses of gemcitabine (GEM) to fractionated doses of 90 Y-clivatuzumab tetraxetan in patients with metastatic pancreatic ductal cancer after having received at least 2 prior systemic therapies. Methods: Fifty-eight patients (33 males, 25 females; median age 63.5 years), 1.6 median years from diagnosis and with a median of 3 (2-7) prior treatments, were randomized to Arm A (N=29, 4-week cycles: 200 mg/m 2 GEM, weekly, combined with 6.5 mCi/m 2 90 Y-clivatuzumab tetraxetan, weekly the last 3 weeks) or Arm B (N=29, 3-week cycles: 6.5 mCi/m 2 90 Y-clivatuzumab tetraxetan alone, once-weekly), repeating cycles after 4-week delays. Safety and efficacy were evaluated. Results: None of the patients had infusion reactions, and as expected, cytopenias (predominantly thrombocytopenia) were the only significant toxicities, but mostly transient and manageable with infrequent hematologic support and little evidence of increased infection or bleeding. Patients terminated treatment cycles due to disease progression or clinical deterioration, not treatment toxicity. Fifty-three patients (27 Arm A, 26 Arm B, 91% overall) completed ≥1 full treatment cycle and thus were evaluable for efficacy, with 23 (12 Arm A, 11 Arm B; 40%) receiving multiple cycles, including 7 (6 Arm A, 1 Arm B; 12%) given 3-7 cycles. Two patients in Arm A had PRs by RECIST criteria. Karnofsky performance status (90-100 v 70-80), number of prior therapies, and tumor burden estimates (summed length of index lesions, serum CA 19-9 levels) correlated with overall survival (OS), but appear balanced between arms. Kaplan-Meier median OS was 3.9 months (1.0-16.7) in Arm A v 2.8 months (0.9-9.4) in Arm B (hazard ratio 0.54, 95% CI: 0.27-0.87; P=0.020, log-rank). The median OS for Arm A v Arm B increased to 7.9 v 3.4 months with multiple cycles (P= 0.004) and 3 patients in Arm A still being observed (11 – 17 months). Conclusions: This randomized trial demonstrated the feasibility of performing clinical studies in metastatic pancreatic cancer patients after having at least 2 prior therapies (3rd line and beyond). With significant survival advantage and favorable safety profile, fractionated RAIT with 90 Y-clivatuzumab tetraxetan and low-dose GEM appears promising in this difficult population, supporting Phase 3 studies of this combination now being initiated. Citation Format: Vincent J. Picozzi, Ramesh K. Ramanathan, Maeve A. Lowery, Allyson J. Ocean, Edith P. Mitchell, Bert H. O9Neil, Michael J. Guarino, Paul R. Conkling, Steven J. Cohen, Nathan Bahary, Richard C. Frank, Tomislav Dragovich, Benjamin B. Bridges, Marie Lee, Ronald L. Korn, Neeta Pandit-Taskar, Stanley J. Goldsmith, Charles M. Intenzo, Arif Sheikh, Timothy C. Manzone, Michael L. Miller, Michael Yu, Judith M. Joyce, Edward B. Strauss, Susan Passalaqua, Ronald V. Dorn, III, Michael J. Anderson, Michael Holt, Fadi S. Braiteh, Fa-Chyi Lee, Thomas E. Gribbin, Donald A. Richards, Alexander N. Starodub, Wegener A. William, Eileen M. O9Reilly, Daniel D. Von Hoff, David M. Goldenberg. Final results of a randomized phase Ib study of fractionated 90Y-clivatuzumab tetraxetan in patients with metastatic pancreatic cancer having at least two prior therapies. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B98.