CD20-directed therapy with rituximab is effective in many patients with malignant lymphoma or follicular lymphoma. However, relapse frequently occurs within 1 year, and patients become increasingly refractory to retreatment. Our purpose was to produce a compact, single-chain CD20-targeting immunotherapeutic that could offer therapeutic advantages in the treatment of B-cell lymphoma.Rituximab is a chimeric antibody containing two heavy chains and two light chains. Here, we describe the properties of TRU-015, a small modular immunopharmaceutical specific for CD20, encoded by a single-chain construct containing a single-chain Fv specific for CD20 linked to human IgG1 hinge, CH2, and CH3 domains but devoid of CH1 and CL domains.TRU-015 mediates potent direct signaling and antibody-dependent cellular cytotoxicity but has reduced size and complement-mediated cytotoxicity activity compared with rituximab. TRU-015 is a compact dimer of 104 kDa that comigrates with albumin in size exclusion chromatography and retains a long half-life in vivo. TRU-015 induced growth arrest in multiple B lymphoma cell lines in vitro and showed effective antitumor activity against large, established subcutaneous Ramos or Daudi xenograft tumors in nude mice. TRU-015 also showed rapid, dose-dependent, and durable depletion of peripheral blood B cells following single-dose administration to nonhuman primates.These results indicate that TRU-015 may improve CD20-directed therapy by effectively depleting embedded malignant B cells and nonmalignant pathogenic B cells and do so with reduced complement activation.
AbstractWe report the functional characterization of a single-chain Fv (scFv) constructed from an anti-CD44 mAb (S5) that abrogates marrow rejection in a mismatched canine donor transplant model. The variable light chain (VL) and variable heavy chain (VH) domains of the parent anti-CD44 antibody were cloned and exact match PCR primers designed that spliced the mature variable domains together through a 15 amino acid [Gly4Ser]3 linker-encoding sequence. This gene was put under the control of a T7 promoter and expressed in Escherichia coli in insoluble inclusion bodies. The scFv was refolded in a cystine/cysteine redox buffer and purified to homogeneity using anion exchange chromatography. The concentrationdependent binding isotherm of the S5 scFv was determined using both direct binding and competitive inhibition flow cytometry assays. S5 scFv effectively blocked FITC-conjugated MAb S5 binding to canine peripheral blood mononuclear cells (PBMC), possessing a mean EC50 (15 nM) equivalent to Fab1 fragments of parental S5 (14.7 nM) and approximately twofold higher than Mab S5 (6 nM). It also binds directly to canine PBMC and possesses a mean EC50 similar to that of the Fab1 fragments (1.01 nM vs 1.03 nM). The recombinant S5 scFv also retains the potent biological activity of the parent Mab, stimulating the activation of natural killer (NK) cell activity and the release of tumor necrosis factor alpha (TNFα) in canine PBMC. Like the parent antibody, scFv crossreacted with human CD44 as examined by direct binding to human PBMC in the flow cytometry assay as well as direct binding to human CD44 immunoglobulin fusion protein in an enzyme-linked immunosorbent assay (ELISA). It was also able to induce TNFα release in human PBMC. These results support previous work suggesting that monovalent binding is sufficient to generate the in vitro biological activity of S5 (1). The scFv S5 antibody will thus serve as a useful model for elucidating the mechanism of antibody abrogated marrow rejection and may serve as a human therapeutic agent.
Abstract Humanized Abs are created by combining, at the genetic level, the complementarity-determining regions of a murine mAb with the framework sequences of a human Ab variable domain. This leads to a functional Ab with reduced immunogenic side effects in human therapy. In this study, we report a new approach to humanizing murine mAbs that may reduce immunogenicity even further. This method is applied to humanize the murine anti-human CD28 Ab, 9.3. The canonical structures of the hypervariable loops of murine 9.3 were matched to human genomic V gene sequences whose hypervariable loops had identical or similar canonical structures. Framework sequences for those human V genes were then used, unmodified, with the 9.3 complementarity-determining regions to construct a humanized version of 9.3. The humanized 9.3 and a chimeric 9.3 control were expressed in Escherichia coli as Fab. The humanized Fab showed a moderate loss in avidity in a direct binding ELISA with immobilized CD28-Ig fusion protein (CD28-Ig). Humanized 9.3 blocked ligation of CD28-Ig to cells expressing the CD28 receptor CD80. Lastly, the humanized 9.3 showed biological activity as an immunosuppressant by inhibiting a MLR.
Abstract Background: Effective treatment of metastatic, triple-negative breast cancer (TNBC) remains a highly unmet medical need. We have developed ES425, a bispecific ADAPTIR™ (modular protein technology) molecule that redirects T-cell cytotoxicity to tumor cells expressing ROR1 (receptor tyrosine kinase-like orphan receptor 1), an oncofetal antigen expressed on TNBC and other malignancies. Results are presented for studies run to examine in vitro and in vivo activity of ES425 in preclinical models of TNBC. Materials and Methods: Target-dependent cytotoxic activity was examined in vitro by treating ROR1(+) cell lines and ROR1(−) cell lines with ES425 in the presence of purified human T cells or human peripheral blood mononuclear cells (PBMCs). Cytotoxic activity was determined using chromium release assays. T cells were assessed for activation and proliferation using multi-color flow cytometry. Pharmacokinetics of ES425 in NOD/SCID gamma (NSG) mice was determined using single intravenous dose of approximately 10 mg/kg. Serum concentrations at time points ranging from 15 minutes to 504 hours were used to calculate the terminal elimination half-life of ES425. To assess activity in vivo, NOD/SCID mice were implanted subcutaneously with the ROR1(+) TNBC tumor cell line MDA-MB-231 and purified human T cells and treated with ES425. This model was run twice with T cells from different human donors. Tumor growth was assessed by measuring tumor volume. Results: ES425 efficiently redirected T cell cytotoxicity against ROR1(+) cell lines at low picomolar concentrations in vitro. Cytotoxic activity was dependent on expression of ROR1 by the target cells. T cells were activated and proliferated in response to ES425 in the presence of ROR1(+) target cells; proliferation was not observed in response to ROR1(−) cells. In vivo, pharmacokinetic analysis showed a serum half-life of approximately 7 days in NSG mice, and ES425 inhibited growth of MDA-MB-231 tumors in mouse xenografts. Repeat experiments showed similar inhibition of tumor growth and an improvement in overall survival. Conclusions: These studies show that ES425 may be an efficient agent for redirecting T cell cytotoxicity in preclinical TNBC models and merits investigation as a potential therapeutic in TNBC and other malignancies. Citation Format: John W. Blankenship, Lynda Misher, Danielle Mitchell, Nicole Zhang, Philip Tan, Gabriela H. Hoyos, Padma Ravikumar, Robert Bader, Catherine J. McMahan, Robert E. Miller, Jeannette Bannink, Hang Fang, Lara Parr, Maria Dasovich, David Bienvenue, Megan Aguilar, Carina Xu, Mollie Daugherty, Brian Woodruff, Jane A. Gross. anti-ROR1 x anti-CD3 ADAPTIR™ molecule, ES425, redirects T-cell cytotoxicity and inhibits tumor growth in preclinical models of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4995.
Bispecific and multivalent therapeutics are compelling candidates for next-generation oncology treatments. B cell depletion with agents such as rituximab has proven successful in the treatment of hematological malignancies in the clinic, but patients still relapse and become refractory. Strategies to improve efficacy of B-cell directed therapies include boosting the cytotoxicity of agents and expanding to targets beyond CD20. Following a screen to rank candidate bispecific B-cell therapies, an agent that targets CD79B and DR (MHC class II) was developed. This bispecific molecule demonstrated highly potent activities against B lymphoma cells in vitro. A matrix approach was taken to identify the optimal targets for direct killing of B cell lymphoma by a bispecific molecule. When selected sets of antibodies were tested on tumor cell lines, several combinations showed synergistic activities. Of the targets tested, CD79B and DR were among the leading pairs showing enhanced cytotoxicity. A SCORPION molecule is a single-chain bispecific immunopharmaceutical comprised of independent binding domains (BDs) on each end of the molecule, flanking an IgG CH2 and CH3 domain. For the Scorpion molecule directed against CD79B and DR, BDs were derived from hybridomas generated against these receptors. Following transient expression in HEK-293 cells, CD79BxDR dimerized in solution and was purified as a single species that specifically bound both receptors on target cells. The activities of the CD79BxDR SCORPION protein were gauged against those of each monospecific molecule (Small Modular Immunopharmaceutical or SMIPTM protein) having the same IgG domain as the SCORPION protein. Targeting either CD79B or DR alone led to distinct cytotoxic activities. The DR SMIP protein promoted Fc-dependent cell mediated cytotoxicity (FcDCC), whereas the CD79B SMIP protein exhibited strong direct killing and only modest FcDCC activity. Advantages of a bispecific approach were evident in the activities of CD79BxDR, which had strong FcDCC as well as highly potent direct killing. In assays against the NHL-derived DoHH2 cell line, CD79BxDR not only outperformed each component SMIP molecule, but consistently gave IC50 values that were two orders of magnitude more potent than rituximab. Upon 48 hour incubation with primary human peripheral blood mononuclear cells, CD79BxDR showed selective depletion of B (CD19+) but not T (CD3+) cells. The selectivity and high potency of the bispecific CD79BxDR SCORPION molecule shows promise as a therapeutic for B cell depletion, particularly in disease refractory to CD20-targeted therapies. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5465.
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