A breakthrough novel method to resolve the drug and target interference problem in immunogenicity assays.

Biological matrix interference in detection and quantitation immunoassays remains a major challenge in the field of bioanalysis. For example, circulating drug may interfere with the detection of anti-drug antibodies (ADA) and drug target, or ADA may interfere with quantitation of drug levels in PK/TK analysis. Monoclonal antibody drug interference, especially for human IgG4 drugs, presents an additional challenge for ADA analysis due to its longer half-life and higher dose. Assay tolerance to such interference may depend on assay platform and reagents. Various approaches have been used to improve drug tolerance in ADA analysis but limited success was observed. We have developed a breakthrough novel method that uses Precipitation and Acid dissociation (PandA) to overcome drug interference in the ADA assay. The method principle is based on four components for detection of total ADA (free ADA and drug bound ADA) in the presence of drug in patient samples: (1) use excess drug to saturate free ADA to form drug bound ADA as drug:ADA complexes, (2) precipitate the complex using an agent such as PEG, (3) acid dissociate ADA from drug and immobilize (capture) free ADA (and free drug) under acidic conditions (without neutralization) onto a large capacity surface, and (4) detect free ADA (not the captured drug) using specific anti-human Ig detection reagent. In this manuscript, we are describing case studies for three humanized monoclonal antibodies (an IgG1 and two IgG4 drugs). The three drug specific PandA ADA assays resulted in complete recovery of ADA in samples containing drug levels in excess of those expected in patients, in contrast to the commonly used acid dissociation approach in ECL bridging assays. This breakthrough novel method shows significant improvement over the current approaches. In fact, the drug interference or under detecting of ADA in all three cases was eliminated. This assay principle could be used not only for ADA assays but also PK and biomarker (drug target) analysis in the presence of interference factors.

Antibody targeting of stem cells to infarcted myocardium.

Hematopoietic stem cell (HSC) therapy for myocardial repair is limited by the number of stem cells that migrate to, engraft in, and proliferate at sites of injured myocardium. To alleviate this limitation, we studied whether a strategy using a bispecific antibody (BiAb) could target human stem cells specifically to injured myocardium and preserve myocardial function. Using a xenogeneic rat model whereby ischemic injury was induced by transient ligation of the left anterior descending artery (LAD), we determined the ability of a bispecific antibody to target human CD34+ cells to specific antigens expressed in ischemic injured myocardium. A bispecific antibody comprising an anti-CD45 antibody recognizing the common leukocyte antigen found on HSCs and an antibody recognizing myosin light chain, an organ-specific injury antigen expressed by infarcted myocardium, was prepared by chemical conjugation. CD34+ cells armed and unarmed with this BiAb were injected intravenously in rats 2 days postmyocardial injury. Immunohistochemistry studies showed that the armed CD34+ cells specifically localized to the infarcted region of the heart, colocalized with troponin T-stained cells, and colocalization with vascular structures. Compared to unarmed CD34+ cells, the bispecific antibody improved delivery of the stem cells to injured myocardium, and such targeted delivery was correlated with improved myocardial function 5 weeks after infarction (p < .01). Bispecific antibody targeting offers a unique means to improve the delivery of stem cells to facilitate organ repair and a tool to study stem cell biology.

Human T cells armed with Her2/neu bispecific antibodies divide, are cytotoxic, and secrete cytokines with repeated stimulation.

PURPOSE: Cancer immunotherapy has been limited by anergy of patient T cells, inadequate numbers of precursor tumor-specific CTL, and difficulty in producing therapeutic doses of CTL. To overcome these limitations, bispecific antibodies have been used to create artificial antibody receptors that direct polyclonal activated T cells (ATC) to target tumor antigens. Studies reported herein were designed to characterize bispecific antibody-armed ATC functions during multiple rounds of targeted cell stimulation. EXPERIMENTAL DESIGN: ATCs were generated from human peripheral blood mononuclear cells (PBMC) by culture with anti-CD3 and interleukin 2 for 14 days and armed with anti-CD3 x anti-Her2 bispecific antibody (Her2Bi). In vitro, Her2Bi-armed ATC were examined for a range of functions after repeated stimulation with the Her2/neu-expressing breast cancer cell line SK-BR-3. PBMC isolated from cancer patients treated with Her2Bi-armed ATC were tested ex vivo for cytotoxicity against SK-BR-3. RESULTS: In vitro, armed ATC divided, maintained surface Her2Bi, and expressed a range of activities for extended periods of time. Perforin-mediated cytotoxic activity by armed ATC continued for at least 336 hours, and cytokines and chemokines (i.e., IFN-gamma and regulated on activation, normal T-cell expressed and secreted protein [RANTES]) were secreted during successive rounds of stimulation. Furthermore, PBMC isolated from patients over their courses of immunotherapy exhibited significant cytolytic activity against SK-BR-3 as a function of Her2Bi-armed ATC infusions. CONCLUSIONS: These studies show that armed ATC are specific, durable, and highly functional T-cell populations in vitro. These previously unappreciated broad and long-term functions of armed ATC are encouraging for their therapeutic use in treating cancer.

Preclinical studies comparing different bispecific antibodies for redirecting T cell cytotoxicity to extracellular antigens on prostate carcinomas.

INTRODUCTION: Bispecific antibodies (BiAbs) are used to enhance targeting of T cells and other cytotoxic agents to tumors while minimizing non-specific tissue toxicities. This study compares the targeting efficacy of 3 BiAbs derived from chemically heteroconjugating a T cell-directed monoclonal antibody (mAb) to 9184, 9187 or 9189, which are mAbs directed at extracellular antigens expressed on human prostate carcinoma cell lines. MATERIALS AND METHODS: 9184 (anti-Her2/neu), 9187 (anti-gp55) and 9189 (anti-gp42) were each heteroconjugated to anti-CD3 to produce BiAbs capable of binding to ("arming") anti-CD3 activated T cells (ATC) and redirecting their cytotoxicity to prostate cancer cells expressing the respective antigen. ATC from cancer patients and/or normal subjects were armed with each BiAb and tested in co-cultures with PC-3, DU 145, and LNCaP cells for binding, cytotoxicity, and cytokine secretion. RESULTS: All 3 tumor-directed mAbs bound to each of the prostate cancer cell lines. ATC armed with 9184Bi statistically augmented cytotoxicity directed at PC-3 and increased IFN-gamma, TNF-alpha, and GM-CSF secretion as well as induced IFN-gamma EliSpots above that seen for 9187Bi, 9189Bi, ATC alone or ATC armed with an irrelevant BiAb. 9184Bi-armed ATC mediated significant cytotoxicity against LNCaP and DU 145 cells as well. When we armed ATC from 6 cancer patients with 9184Bi, 9184Bi markedly enhanced cytotoxicity of ATC from 5 of the 6 patients. CONCLUSION: Arming ATC with BiAbs augments cytotoxicity directed at prostate cancer lines expressing the target antigens. Arming with 9184Bi was the most effective at redirecting cytotoxicity at PC-3 cells and inducing cytokine secretion. As an alternative to mAb therapy with anti-HER2, the HER2 antigen may provide a suitable target for redirecting anti-cancer immune cells, immunobiologicals, or other agents to HRPC.

T cells armed with anti-CD3 x anti-CD20 bispecific antibody enhance killing of CD20+ malignant B cells and bypass complement-mediated rituximab resistance in vitro.

OBJECTIVE: Resistance to rituximab, a chimeric monoclonal antibody that binds to CD20, is a major limitation for the successful treatment of patients with non-Hodgkin lymphoma and other CD20+ B-cell malignancies. To circumvent rituximab resistance in these patient populations, we have constructed a bispecific antibody (BiAb), anti-CD3 x anti-CD20 (CD20Bi), that combines rituximab targeting with non-major histocompatibility complex (non-MHC)-restricted cytotoxicity mediated by activated T cells (ATC). MATERIALS AND METHODS: Activated T cells were obtained from anti-CD3 activated peripheral blood mononuclear cells (PBMC) of normal donors or the leukapheresis products of patients by culturing in the presence of interleukin-2 for 6-14 days. After ATC expansion, the cells were armed with CD20Bi. Killing activity was evaluated by 51Cr-release assay. RESULTS: Arming ATC with as little as 5 ng CD20Bi/10(6) cells significantly increased cytotoxicity above unarmed ATC. CD20Bi-armed ATC (50 ng/10(6) cells) efficiently lysed CD20+ cell lines at E:T of 6.25-50, but not the nonhematologic, CD20- SK-BR-3 cell line. High levels of cytotoxicity mediated by CD20Bi-armed ATC (p < 0.05) could not be blocked by an 8000-fold excess of soluble rituximab. CD20Bi-armed ATC in the presence of complement killed ARH-77 cells, a rituximab-complement pathway-resistant multiple myeloma, significantly (p < 0.05) better than rituximab or unarmed ATC, suggesting that CD20Bi-armed ATC may be clinically effective for treatment of rituximab-resistant CD20+ hematologic malignancies. CONCLUSIONS: Our findings demonstrate that CD20Bi-armed ATC enhance cytotoxicity against CD20+ B-cell lines and circumvent complement-mediated rituximab resistance, providing a strong rationale for this immune-based strategy for the treatment of rituximab-refractory CD20+ B-cell malignancies.