Single-chain Antibody Construct Research Articles

A single-chain antibody construct with specificity of a natural IgM antibody reduces hepatic ischemia reperfusion injury in mice.

Natural immunoglobulin M (IgM) antibodies have been shown to recognize post-ischemic neoepitopes following reperfusion of tissues and to activate complement. Specifically, IgM antibodies and complement have been shown to drive hepatic ischemia reperfusion injury (IRI). Herein, we investigate the therapeutic effect of C2 scFv (single-chain antibody construct with specificity of a natural IgM antibody) on hepatic IRI in C57BL/6 mice. Compared with PBS-treated mice, C2 scFv-treated mice displayed almost no necrotic areas, significant reduction in serum ALT, AST and LDH levels, and significantly reduced in the number of TUNEL positive cells. Moreover, C2 scFv-treated mice exhibited a notable reduction in inflammatory cells after hepatic IRI than PBS-treated mice. The serum IL-6, IL-1β, TNF-α and MPC-1 levels were also severely suppressed by C2 scFv. Interestingly, C2 scFv reconstituted hepatic inflammation and IRI in Rag1-/- mice. We found that C2 scFv promoted hepatic cell death and increased inflammatory cytokines and infiltration of inflammatory cells after hepatic IRI in Rag1-/- mice. In addition, IgM and complement 3d (C3d) were deposited in WT mice and in Rag1-/- mice reconstituted with C2 scFv, indicating that C2 scFv can affect IgM binding and complement activation and reconstitute hepatic IRI. C3d expression was significantly lower in C57BL/6 mice treated with C2 scFv compared to PBS, indicating that excessive exogenous C2 scFv inhibited complement activation. These data suggest that C2 scFv alleviates hepatic IRI by blocking complement activation, and treatment with C2 scFv may be a promising therapy for hepatic IRI.

Trial in Progress: A Phase 2 Single Arm, Multicenter Trial to Evaluate the Efficacy of the BiTE ® Antibody Blinatumomab (Blincyto) and Vincristine Sulfate Liposomal Injection (Marqibo) in Adult Subjects with Relapsed/Refractory Philadelphia Negative CD19+ Acute Lymphoblastic Leukemia

Trial in Progress: A Phase 2 Single Arm, Multicenter Trial to Evaluate the Efficacy of the BiTE ® Antibody Blinatumomab (Blincyto) and Vincristine Sulfate Liposomal Injection (Marqibo) in Adult Subjects with Relapsed/Refractory Philadelphia Negative CD19+ Acute Lymphoblastic Leukemia

Increasing the efficacy and safety of a human complement inhibitor for treating post-transplant cardiac ischemia reperfusion injury by targeting to a graft-specific neoepitope

Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. The complement system is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor, CR1, to a post-transplant ischemia-induced neoepitope. A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety. CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts. At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact. We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety.

Construction and characterization of porcine single-chain fragment variable antibodies that neutralize transmissible gastroenteritis virus in vitro.

Transmissible gastroenteritis virus (TGEV) infection causes severe diarrhea in piglets and imposes a significant economic burden on pig farms. Single-chain fragment variable (scFv) antibodies effectively inhibit virus infection and could be a potential therapeutic reagent for preventing disease. In this study, a recombinant scFv antibody phage display library was constructed from peripheral blood lymphocytes of piglets infected with TGEV. The library was screened with four rounds of biopanning using purified TGEV antigen, and scFv antibodies that bound to TGEV were obtained. The scFv gene was subcloned into the pET-28a(+), and the constituted plasmid was introduced into Escherichia coli BL21 (DE3) for protein expression. All three scFv clones identified had neutralizing activity against TGEV. An immunofluorescence assay and western blot analysis demonstrated that two scFv antibodies reacted with the spike protein of TGEV. These results indicate that scFv antibodies provide protection against viral infection in vitro and may be a therapeutic candidate for both prevention and treatment of TGEV infection in swine.

Natural immunoglobulin M initiates an inflammatory response important for both hepatic ischemia reperfusion injury and regeneration in mice.

Complement plays a role in both hepatic ischemia reperfusion (IR) injury (IRI) and liver regeneration, but it is not clear how complement is activated in either process. We investigated the role of self-reactive immunoglobulin M (IgM) antibodies in activating complement after hepatic IR and liver resection. Natural IgM antibodies that recognize danger-associated molecular patterns (neoepitopes) activate complement following both hepatic IR and liver resection. Antibody-deficient Rag1-/- mice were protected from hepatic IRI, but had increased hepatic injury and an impaired regenerative response after 70% partial hepatectomy (PHx). We identified two IgM monoclonal antibodies (mAbs) that specifically reversed the effect of Rag1 deficiency in both models; B4 (recognizes Annexin IV) and C2 (recognizes subset of phospholipids). Focusing on the B4-specific response, we demonstrated sinusoidal colocalization of IgM and C3d in Rag1-/- mice that were reconstituted with B4 mAb, and furthermore that the Annexin IV neoepitope is specifically and similarly expressed after both hepatic IR and PHx in wild-type (WT) mice. A single-chain antibody construct (scFv) derived from B4 mAb blocked IgM binding and reduced injury post-IR in WT mice, although, interestingly, B4scFv did not alter regeneration post-PHx, indicating that anti-Annexin IV antibodies are sufficient, but not necessary, for the regenerative response in the context of an entire natural antibody repertoire. We also demonstrated expression of the B4 neoepitope in postischemic human liver samples obtained posttransplantation and a corollary depletion in IgM recognizing the B4 and C2 neoepitopes in patient sera following liver transplantation. Conclusion: These data indicate an important role for IgM in hepatic IRI and regeneration, with a similar cross-species injury-specific recognition system that has implications for the design of neoepitope targeted therapeutics. (Hepatology 2018;67:721-735).

Abstract 585: Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy

Abstract BiTE® therapeutics are single chain antibody constructs harboring two binding moieties: one directed at a tumor associated antigen and one directed at the CD3e protein, which trigger T cell dependent cellular cytotoxicity (TDCC) against targeted cancer cells. Here, we evaluated the suitability of ENPP3 as a potential BiTE® target. The ENPP3 mRNA is highly differentially expressed in clear cell Renal Cell Carcinoma (ccRCC) and the ENPP3 protein is detected with high uniformity and intensity in ccRCC tumor samples by immunohistochemistry. We demonstrated the surface expression of this protein in Renal Cancer cell lines and confirmed that the ENPP3 protein was highly restricted to the luminal side of normal epithelial layers in which it was detected (proximal renal tubules, bronchial epithelium, salivary glands). We developed highly potent anti-ENPP3 BiTE® molecules and demonstrated the in vitro TDCC activity of these molecules. Two anti-ENPP3 BiTE® molecules further demonstrated tumor formation inhibition activity in a human T cell / human target cell admixture mouse xenograft model. Citation Format: Olivier Nolan-Stevaux, Flordeliza Fajardo, Lily Liu, Suzanne Coberly, Patricia McElroy, Aaron Nazarian, Franziska Bott, Elisabeth Nahrwold, Tobias Raum, Roman Kischel, Ralf Lutterbuese, Patrick Hoffmann, Peter Kufer. Assessing ENPP3 as a renal cancer target for bispecific T-cell engager (BiTE) therapy. [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 585.

Impact of Diverse Immune Evasion Mechanisms of Cancer Cells on T Cells Engaged by EpCAM/CD3-Bispecific Antibody Construct AMG 110.

BackgroundBispecific T cell engager (BiTE®) are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They can elicit a polyclonal cytotoxic T cell response that is not restricted by T cell receptor (TCR) specificity, and surface expression of MHC class I/peptide antigen complexes. Using human EpCAM/CD3-bispecific BiTE® antibody construct AMG 110, we here assessed to what extent surface expression of PD-L1, cytoplasmic expression of indoleamine-2,3-deoxygenase type 1, Bcl-2 and serpin PI-9, and the presence of transforming growth factor beta (TGF-β), interleukin-10 (IL-10) and adenosine in culture medium can impact redirected lysis by AMG 110-engaged T cells.MethodsThe seven factors, which are all involved in inhibiting T cell functions by cancer cells, were tested with human EpCAM-expressing Chinese hamster ovary (CHO) target cells at levels that in most cases exceeded those observed in a number of human cancer cell lines. Co-culture experiments were used to determine the impact of the evasion mechanisms on EC50 values and amplitude of redirected lysis by AMG 110, and on BiTE®-induced proliferation of previously resting human peripheral T cells.FindingsAn inhibitory effect on redirected lysis by AMG 110-engaged T cells was seen upon overexpression of serpin PI-9, Bcl-2, TGF-βand PD-L1. An inhibitory effect on induction of T cell proliferation was only seen with CHO cells overexpressing IDO. In no case, a single evasion mechanism rendered target cells completely resistant to BiTE®-induced lysis, and even various combinations could not.ConclusionsOur data suggest that diverse mechanisms employed by cancer cells to fend off T cells cannot inactivate AMG 110-engaged T cells, and that inhibitory effects observed in vitro may be overcome by increased concentrations of the BiTE® antibody construct.

Construction of human single-chain variable fragment antibodies of medullary thyroid carcinoma and single photon emission computed tomography/computed tomography imaging in tumor-bearing nude mice.

Medullary thyroid carcinoma (MTC) is a rare tumor of the endocrine system with poor prognosis as it exhibits high resistance against conventional therapy. Recent studies have shown that monoclonal antibodies labeled with radionuclide have become important agents for diagnosing tumors. To elucidate whether single-chain fragment of variable (scFv) antibody labeled with 131I isotope is a potential imaging agent for diagnosing MTC. A human scFv antibody library of MTC using phage display technique was constructed with a capacity of 3x10(5). The library was panned with thyroid epithelial cell lines and MTC cell lines (TT). Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to identify the biological characteristics of the panned scFv. Methyl thiazolyl tetrazolium (MTT) assay was also used to explore the optimal concentration of the TT cell proliferation inhibition rate. They were categorized into TT, SW480 and control groups using phosphate-buffered saline. Western blotting showed that molecular weight of scFv was 28 kDa, cell ELISA showed that the absorbance of TT cell group was significantly increased (P=0.000??) vs. the other three groups, and MTT assay showed that the inhibition rate between the two cell lines was statistically significantly different (P<0.05) when the concentration of scFv was 0.1, 1 and 10 µmol/l. The tumor uptake of 131I-scFv was visible at 12 h and clear image was obtained at 48 h using the single photon emission computed tomography. scFv rapidly and specifically target MTC cells, suggesting the potential of this antibody as an imaging agent for diagnosing MTC.

Solitomab, an EpCAM/CD3 bispecific antibody construct (BiTE®), is highly active against primary uterine and ovarian carcinosarcoma cell lines in vitro.

BackgroundUterine and ovarian carcinosarcomas (CS) are rare but highly aggressive gynecologic tumors which carry an extremely poor prognosis. We evaluated the expression levels of EpCAM and the in vitro activity of solitomab, a bispecific single-chain antibody construct which targets epithelial-cell-adhesion-molecule (EpCAM) on tumor cells and also contains a CD3 binding region, against primary uterine and ovarian CS cell lines.MethodsEpCAM expression was evaluated by flow cytometry in a total of 5 primary CS cell lines. Sensitivity to solitomab-dependent-cellular-cytotoxicity (ADCC) was tested against the panel of primary CS cell lines expressing different levels of EpCAM in standard 4 h 51Cr release-assays. The proliferative activity, activation, cytokine secretion (i.e., Type I vs Type II) and cytotoxicity of solitomab in autologous tumor-associated-T cells (TAL) in the pleural fluid of a CS patient were also evaluated by CFSE and flow-cytometry assays.ResultsSurface expression of EpCAM was found in 80.0 % (4 out of 5) of the CS cell lines tested by flow cytometry. EpCAM positive cell lines were found resistant to NK or T-cell-mediated killing after exposure to peripheral blood lymphocytes (PBL) in 4-h chromium-release assays (mean killing ± SEM = 1.1 ± 1.6 %, range 0–5.3 % after incubation of EpCAM positive cell lines with control BiTE®). In contrast, after incubation with solitomab, EpCAM positive CS cells became highly sensitive to T-cell-cytotoxicity (mean killing ± SEM of 19.7 ± 6.3 %; range 10.0-32.0 %; P < 0.0001). Ex vivo incubation of autologous TAL with EpCAM expressing malignant cells in pleural effusion with solitomab, resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (i.e., CD25 and HLA-DR), and a reduction in number of viable CS cells in the exudate (P < 0.001).ConclusionsSolitomab may represent an effective treatment for patients with recurrent/metastatic and/or chemo-resistant CS overexpressing EpCAM.

Solitomab, an EpCAM/CD3 bispecific antibody construct (BiTE), is highly active against primary uterine serous papillary carcinoma cell lines in vitro

Uterine serous carcinoma is an aggressive form of endometrial cancer that carries an extremely poor prognosis. Solitomab is a novel bispecific single-chain antibody construct that targets epithelial cell adhesion molecule on tumor cells and also contains a CD3 binding region. We evaluated the expression levels of epithelial cell adhesion molecule and the invitro activity of solitomab against primary uterine serous carcinoma cell lines invitro and ex-vivo in the ascites of patients with uterine serous carcinoma. The purpose of this study was to determine the frequency of expression of epithelial cell adhesion molecule on uterine serous carcinoma cell lines and the ability of solitomab to modulate immune responses (T-cell proliferation, activation, cytokine production, and tumor killing) to tumor cells when it is combined with lymphocytes and epithelial cell adhesion molecule-positive cell lines or epithelial cell adhesion molecule-positive ascitic fluid invitro. Epithelial cell adhesion molecule expression was evaluated by flow cytometry in a total of 14 primary uterine serous carcinoma cell lines. Sensitivity to solitomab-dependent cellular-cytotoxicity was tested against a panel of primary uterine serous carcinoma cell lines that express different levels of epithelial cell adhesion molecule in standard 4-hour chromium release assays. The proliferative activity, activation, cytokine secretion (ie, type I vs type II), and cytotoxicity of solitomab in autologous tumor-associated T cells in the ascitic fluid of patients with uterine serous carcinoma was also evaluated by carboxyfluorescein succinimidyl ester and flow-cytometry assays. Differences in epithelial cell adhesion molecule expression, solitomab-dependent cellular-cytotoxicity levels were analyzed with the use of an unpaired ttest. T-cell activation marker increase and cytokine release were analyzed by a paired t test. Surface expression of epithelial cell adhesion molecule was found in 85.7% (12 of 14) of the uterine serous carcinoma cell lines that were tested by flow cytometry. Epithelial cell adhesion molecule-positive cell lines were found resistant to natural killer cells or T-cell-mediated killing after exposure to peripheral blood lymphocytes in 4-hour chromium-release assays (mean killing ± standard of the mean, 2.7% ± 3.1% after incubation of epithelial cell adhesion molecule-positive cell lines with control bispecific antibody construct). In contrast, after incubation with solitomab, epithelial cell adhesion molecule-positive uterine serous carcinoma cells became highly sensitive to T-cell cytotoxicity (mean killing, 25.7% ± 4.5%; P < .0001) by peripheral blood lymphocytes. Exvivo incubation of autologous tumor-associated lymphocytes with epithelial cell adhesion molecule that expressed malignant cells in ascites with solitomab resulted in a significant increase in T-cell proliferation in both CD4+ and CD8+ T cells, increase in T-cell activation markers (ie, CD25 and HLA-DR), and a reduction in number of viable uterine serous carcinoma cells in ascites (P < .001). Solitomab induces robust immunologic responses invitro that result in increased T-cell activation, proliferation, production of cytokines, and direct killing of tumor cells. These findings suggest that solitomab may represent a novel, potentially effective agent for the treatment of recurrent/metastatic and/or chemo-resistant uterine serous carcinoma-overexpressing epithelial cell adhesion molecule.

Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab.

We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 μg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.

Abstract 4734: Impact of various immune escape mechanisms on redirected lysis by T cells engaged via EpCAM/CD3 bispecific BiTE® antibody AMG 110 .

Abstract BiTE antibodies are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. They are capable of temporarily mounting a polyclonal T cell response that is not restricted by T cell receptor specificity, presence of MHC class I, generation and presentation of peptide antigen, or the addition of T cell co-stimuli. Examples are the CD19/CD3-bispecific BiTE antibody blinatumomab, which has been reported to show high response rates in leukemia and lymphoma patients, and EpCAM/CD3-bispecific BiTE antibody solitomab (AMG 110), which is in a dose-escalating phase 1 study in patients with solid tumors. Because specific T cell responses against cancer cells are frequently hampered by a variety of immune escape mechanisms, we have here assessed to what extent surface expression of PD-L1 (B7-H1) and CD73, cytoplasmic expression of IDO and serpin PI-9, and secretion of TGF-ß and IL-10 can protect target cells from redirected lysis by AMG 110-engaged T cells. The six human proteins, which are all known to be associated with immune escape of cancer cells by inhibition of T cell function, were stably expressed in human EpCAM-expressing Chinese hamster ovary (CHO) cells. In all cases, expression levels of the six proteins in stable CHO cell lines exceeded those found in human cancer cell lines. Characterized CHO cell lines and the parental EpCAM+ CHO line were used in co-culture assays as target cells for analysis of EC50 values for redirected lysis, and induction of proliferation of resting human peripheral T cells in the presence of various AMG 110 concentrations. High level cytosolic expression of the tryptophan metabolizing enzyme IDO had no effect on redirected lysis but reduced AMG 110-induced T cell proliferation, while expression of the granzyme inhibitor PI-9 increased the EC50 value for redirected lysis but had no effect on T cell proliferation. High level expression of the membrane-bound ligand for PD-1, PD-L1, and of secreted IL-10 and TGF-β all had no impact on AMG 110-induced T cell proliferation. The EC50 value for redirected lysis was however impacted by expression of TGF-ß and PD-L1 as determined by a FACS-based cytotoxicity assays. In no case, transfected CHO cell lines became completely resistant to BiTE-induced lysis despite expressing the immune escape proteins at higher levels than observed in tumor cell lines. Our data suggest that BiTE-engaged T cells are relatively insensitive to proteins expressed by cancer cells to fend off T cells, and that inhibitory effects can be compensated by higher concentrations of the BiTE antibody. Citation Format: Wibke Deisting, Tobias Raum, Peter Kufer, Patrick A. Baeuerle, Markus Muenz. Impact of various immune escape mechanisms on redirected lysis by T cells engaged via EpCAM/CD3 bispecific BiTE® antibody AMG 110 . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4734. doi:10.1158/1538-7445.AM2013-4734

Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production.

Construction of single-chain variable fragment antibodies against MCF-7 breast cancer cells

A phage display library of single chain variable fragment (scFv) against MCF-7 breast cancer cells was constructed from C3A8 hybridoma cells. RNA from the C3A8 was isolated, cDNA was constructed, and variable heavy and light immunoglobulin chain gene region were amplified using PCR. The variable heavy and light chain gene regions were combined with flexible linker, linked to a pCANTAB 5E phagemid vector and electrophoresed into supE strain of Escherichia coli TG1 cells. Forty-eight clones demonstrated positive binding activity to MCF-7 breast cancer cell membrane fragments and the strongest of 48 clones was selected for analysis. The anti-MCF-7 library evaluated by SfiI and NotI digests demonstrated that anti-MCF-7 scFv antibodies possess individual patterns that should be able to recognize distinct human breast cancer cells. The C3A8 scFv, with an apparent molecular weight of 32 kDa, showed high homology (99%) with single chain antibody against rice stripe virus protein P20. In summary, the anti MCF-7 scFv antibody can be used for pretargeting breast cancer for clinical diagnosis of patients; it also has potential for therapeutic applications.

Abstract 4622: A novel CD33/CD3-bispecific BiTE antibody can effectively recruit autologous T cells from AML-patients for in vitro cell lysis of CD33+ blasts

Abstract Bispecific T cell-engaging (BiTE®) antibodies are single-chain antibody constructs which combine dual specifity against CD3 on T cells and against a tumor-associated antigen. Phase 1 and 2 clinical trials with the CD19/CD3-bispecific BiTE antibody blinatumomab have shown very high response rates in patients suffering from non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33 in redirecting autologous T cells for in vitro lysis of blasts from acute myeloid leukemia (AML) patients. Initial antibody titration experiments showed that CD33 BiTE concentrations at low picomolar ranges mediate effective lysis of leukemic cell lines. We next analysed primary samples from AML patients, which were co-cultured with mononuclear cells (MNC) from healthy donors in presence or absence of specific BiTE antibodies at an E:T ratio of 1:1. After 48h of incubation in the presence of 1ng/ml CD33 BiTE we observed a decrease of both CD33+ AML blasts as well as CD33+ monocytes when compared to samples with control BiTE or without BiTE. 1ng/ml CD33 BiTE specifically induced strong upregulation of activation markers CD25 and CD69 on the vast majority of all CD4+ and CD8+ T cells. We furthermore investigated whether T cells from AML patients were able to mediate lysis of CD33+ leukemia cells by CD33 BiTE. T cells purified from peripheral blood or bone marrow of newly diagnosed AML patients were tested for BiTE mediated lysis of U-937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls. In consequence of this we studied blast lysis and T cell behaviour in a completely autologous setting using cryo-conserved samples from AML patients. Despite strong activation of T cells only moderate lysis of myeloid cells was observed after 48h, possibly due to low E:T ratios in these samples. Therefore, to allow autologous T cell expansion, cells were cultured for up to 144h. After this prolonged incubation, CD33 BiTE-mediated redirected lysis of AML blasts and myeloid cells was observed in most but those patient samples that had a very low initial effector-to-target (E:T) cell ratio. To adress this problem, we stimulated autologous T cells in a first step by incubating AML samples in presence of 1ng/ml CD33 BiTE for 7 days. After purification of the expanded T cells we performed autologous cultures with high initial E:T ratio (1:1). This led to fast, effective and almost complete lysis of CD33+ target cells. Taken together, autologous T cells from AML patients are effectively engaged and activated by the novel CD33 BiTE and redirected for the elimination of myeloblasts at low picomolar concentrations in vitro and may thereby constitute a promising novel therapeutic option for the treatment of patients with CD33+ acute myeloid leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4622. doi:1538-7445.AM2012-4622

Abstract 4841: Lysis of cancer cells by highly purified T regulatory cells engaged via an EpCAM/CD3-bispecific BiTE antibody

Abstract BiTE antibodies are single-chain bispecific antibody constructs with dual specificity for CD3 on T cells and a surface antigen on target cells. By bridging cytotoxic T cells and cancer cells, BiTE antibodies are capable to temporarily mount a polyclonal T cell response that is not restricted by T cell receptor specificity, presence of MHC class I, generation and presentation of peptide antigen, or the need for T cell co-stimulation. Examples are the CD19/CD3-bispecific BiTE antibody blinatumomab, which has shown high response rates and durable remissions in leukemia and lymphoma patients, and EpCAM/CD3-bispecific BiTE antibody solitomab (MT110), which is in a dose-escalating phase 1 study in patients with solid tumors. CD8+ and CD4+ effector memory T cells were found to be the most potent effector cells for BiTE antibodies. Here, we have assessed whether highly purified T regulatory cells (T regs) can also serve as effector cells for BiTE antibody MT110, despite multiple reports suggesting that T regs rather suppress the anti-tumor activity of cytotoxic T cells. T regs were purified from human peripheral blood mononuclear cells (PBMC) using anti-CD25 magnetic beads and fluorescence-activated cell sorting (FACS) for expression of CD4 and CD45RA markers, followed by expansion in the presence of IL-2 (Hoffmann P. et al., Methods Mol. Biol. 677:15-30, 2011). Purified naïve CD4+/CD25high/CD45RA+/FOXP3+ T regs had a purity of 96-99.6%, and contained no detectable CD8+ T cells. In co-culture experiments, a small fraction of purified T regs up-regulated the expression of granzyme B and perforin when stimulated with MT110 in the presence of EpCAM+ target cells. As shown by chromium release assay and phase contrast microscopy, T regs caused a MT110 dose-dependent, redirected lysis of target cells. No target cell lysis was observed with a BiTE antibody solely binding to CD3 on T cells, and by T regs alone. We then tested the activity of T regs against spheroids formed in vitro from colorectal cancer stem cells. Only in the presence of MT110, T regs could significantly reduce the size and integrity of spheroids. In that respect, T regs had a similar activity as PBMC. Our study suggests that T regs, which often are associated with immune escape of tumors, can be effectively engaged for redirected cancer cell lysis by a BiTE antibody. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4841. doi:1538-7445.AM2012-4841

Blinatumomab Monotherapy Shows Efficacy in Patients with Relapsed Diffuse Large B Cell Lymphoma

Abstract 1637Blinatumomab (MT103) is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®).A phase I trial with indolent and mantle cell lymphoma patients established a maximal tolerable dose (MTD) at 60 μg/m2/d. The trial was subsequently amended to evaluate blinatumomab in patients with diffuse large B cell lymphoma (DLBCL). Patients were treated by 4–8-week continuous i.v. administration with the following dosing regimen: first week at 5 μg/m2/d, second week at 15 μg/m2/d and for the remaining treatment period at 60 μg/m2/d. Two cohorts each with 6 DLBCL patients were enrolled. The two cohorts solely differed by the dose and schedule of corticosteroid medication administered at the beginning of blinatumomab infusion for mitigation of adverse events. In the first cohort 100 mg prednisolone was applied 1 hour prior to start; and in the second cohort patients received dexamethasone on days 1, 2, and 3. Three sequential patients received dexamethasone also 6–12 hours prior to start of infusion. Out of the twelve patients, 5 were male and 7 female. The median age was 57 years (range from 26 to 78 years). Patients had received a median of 4 prior regimens (range from 2–6). All patients had been exposed to rituximab. Eight of the 12 patients had undergone autologous stem cell transplantation (ASCT). International prognostic index (IPI) at screening ranged from 1 to 3 with a median of 2.The most common clinical adverse events (AEs) regardless of causality (>30%) were pyrexia (81.8%), fatigue (54.5%), constipation (36.4%), headache (36.4%), tremor (36.4%) and weight increase (36.4%). The most frequent laboratory AEs regardless of causality (>30%) were hyperglycemia (63.6%), lymphopenia (54.5%), C-reactive protein increase (45.5%), gamma-glutamyltransferase increase (45.5%) and thrombocytopenia (36.4%). Most AEs occurred early and were reversible. Four of 12 patients discontinued infusion due to fully reversible CNS events, 2 of which qualified as dose limiting toxicities (DLTs). Although just one DLT (reversible CNS event grade 3) occurred in the prednisolone cohort, a further cohort applying prophylactic dexamethasone was opened to optimize management of CNS events. A further refinement of the dexamethasone schedule, starting longer time prior to start of blinatumomab, was introduced after one early patient in the cohort receiving dexamethasone had experienced a reversible CNS event leading to discontinuation. All three patients treated in this manner completed the first blinatumomab cycle without discontinuations. Only one showed a grade 1 tremor, and no other CNS AEs were reported in these three patients.Two of 12 patients were not exposed to 60 μg/m2/d due to early discontinuations and 1 patient is too early in treatment for response evaluation. Five out of the remaining 9 evaluable patients (56%) showed objective clinical responses (4 CR/CRu; 1 PR). Three out of the 5 patients with CR/CRu or PR had prior ASCT. Two patients achieved objective responses (1 CR, 1 PR) despite of discontinuation at 60 μg/m2/d. The median response duration is +182 days (longest current duration +428 days), with 4 out of 5 responses still ongoing.Further evaluation of the last cohort will refine the recommended phase II dose, and the intensity and timing of dexamethasone comedication. The observation of lasting CRs after blinatumomab monotherapy in DLBCL patients is promising and warrants further exploration in a phase II study. Disclosures:Krause:Micromet: Research Funding. Mackensen:Micromet Inc.: Research Funding. Topp:Micromet: Consultancy, Honoraria. Scheele:Micromet Inc.: Employment, Equity Ownership, Patents & Royalties. Nagorsen:Micromet Inc.: Employment, Equity Ownership, Patents & Royalties. Zugmaier:Micromet: Employment. Degenhard:Micromet Inc: Employment. Schmidt:Micromet AG: Employment. Kufer:Micromet Inc: Employment, Equity Ownership. Libicher:Micromet Inc.: Consultancy, Honoraria. Bargou:Micromet: Consultancy, Honoraria.

Construction and molecular characterization of mouse single-chain variable fragment antibodies against Burkholderia mallei and Burkholderia pseudomallei

We have selected two lipopolysaccharide (LPS) specific Burkholderia mallei mouse monoclonal antibodies (mAbs) and four anti-capsular B. pseudomallei-specific mAbs to generate mouse single-chain variable fragment (scFv) antibodies. This selection was made through extensive in vitro and in vivo assay from our library of mAbs against B. mallei and B. pseudomallei. We initially generated the mouse immunoglobulin variable heavy chain (VH) and light chain (VL) regions from each of these six selected mAbs using a phage display scFv technology. We determined the coding sequences of the VH and VL regions and successfully constructed two B. mallei-specific scFv phage antibodies consisting of two different VH (VH1 and VH2) and one Vλ1 families. Four scFvs constructed against B. pseudomallei had two VH (VH1 and VH6) and two VL (Vκ4/5 and Vκ21) families. All of six scFv antibodies constructed demonstrated good binding activity without any rounds of biopanning against B. mallei (M5D and M18F were 0.425 and 0.480 at OD405nm) and B. pseudomallei (P1E7, P2I67, P7C6, and P7F4 were 0.523, 0.859, 0.775, and 0.449 at OD405nm) by ELISA, respectively. A comparison of the immunoglobulin gene segments revealed that the gene sequences in complementarity-determining regions (CDRs) of three out of four B. pseudomallei-specific scFvs are highly conserved. We determined that the two B. mallei-specific scFvs have different CDRs in the VH, but the amino acid sequences of CDRs in the VL are conserved. This high sequence homology found in CDRs of VH or VL of these mAbs contributes to our better understanding and determination of binding to the specific antigenic epitope(s). The scFv phage display technology may be a valuable tool to develop and engineer mAbs with improved antigen-binding affinity.

Treatment of Patients with Non-Hodgkin Lymphoma (NHL) with CD19/CD3 Bispecific Antibody Blinatumomab (MT103): Double-Step Dose Increase to Continuous Infusion of 60 μg/m2/d Is Tolerable and Highly Effective

AbstractAbstract 2880Blinatumomab (MT103) is a single-chain bispecific antibody construct with specificity for CD19 and CD3 belonging to the class of bispecific T cell engager (BiTE®). We have previously reported that blinatumomab delivered as single agent to patients with relapsed NHL and B-precursor acute lymphoblastic leukemia by continuous intravenous (CIV) infusion over 4–8 weeks depleted peripheral B cells, expanded T effector cells, and resulted in clinical responses.In this phase I study 52 patients (41 males, 11 females) have been treated, 21 with FL, 21 with MCL and 10 with other subtypes of lymphoma (MZL, SLL, LPL, CLL). Patients have received a median of 3 prior regimens (range 1 to 12). Ninety percent of the patients had prior exposure to rituximab and 45% to fludarabine. Patients were treated at a dose range from 0.5 to 90 μg/m2/d.The most common adverse events (AEs) occurred early, were transient, reversible and did not require discontinuation of treatment. The most common clinical AEs regardless of causality were pyrexia (75%), headache (45%) and fatigue (37%). The most common laboratory abnormality AEs regardless of causality were lymphopenia (75%), leukopenia (57%), thrombocytopenia (39%), C-reactive protein increase (53%) and fibrin D dimer increase (37%).The medically most important AEs that resulted in permanent discontinuation were CNS events. Signs and symptoms observed included kinetic tremor, speech impairment, disorientation, apraxia and seizure. All CNS events were fully reversible without sequelae and no pathological findings by MRI imaging were reported. Out of the 52 patients treated, 9 had to discontinue treatment permanently in the first cycle due to these CNS events. At a dose of 90 μg/m2/d two DLTs were observed which were CNS events during the DLT period of the first 2 weeks of treatment. Therefore 60μg/m2/d is the currently recommended dose.A low B to T cell ratio (<1:8) determined by FACS analysis in peripheral blood was found to be a risk factor for CNS events. No CNS events requiring treatment discontinuation occurred in 32 out of 34 patients with a high B:T cell ratio (≥1:8) up to 60μg/m2/d. In the 18 patients with low B:T cell ratio, 7 had CNS events requiring treatment discontinuation. Patients with low B:T cell ratio were subsequently treated with an alternative dosing schedule in which reduced doses of either 5 and 15 μg/m2/d were sequentially administered for 1 week respectively followed by an increase to 60 μg/m2/d. First single-step dosing was applied. However, CNS events leading to treatment discontinuation were still observed. Therefore a double-step regimen was implemented; in the initial cohort of 3 patients with low B:T cell ratio there were no treatment discontinuations due to CNS events.Overall 18 patients with FL or MCL were treated with constant or step dosing regimens at or reaching 60 μg/m2/d dose level. Eight out of the 9 patients with constant dosing showed an objective response by Cheson criteria. All responders had a high B:T cell ratio. One patient with a low B:T cell ratio was discontinued due to a CNS AE. Six out of 9 patients with low B:T cell ratio enrolled for step dosing showed an objective response. One patient (single step dosing) discontinued treatment because of a CNS AE and 2 patients discontinued because of tumor progression. As of June 15th 2010, response duration ranged from 1 to 30+ months. Median for response duration was 26 months with 5 out of 14 responses ongoing.Table 1:Response evaluation in patients enrolled in 60 μg/m2/d cohorts.Dose Level CohortPatientsComplete ResponsePartial ResponseOverall ResponseFLMCLTotalFLMCLTotalFLMCLTotalFLMCLTotal60 μg/m2/d Constant6392134156285 or 15 then 60 μg/m2/d Single step3362021123145/15/60 μg/m2/d Double Step213000202202Total1171841572911314These data confirm high single-agent activity of blinatumomab with long lasting remissions. Initial data with double step dosing demonstrate that this approach maintains clinical activity without discontinuations due to CNS AEs. Evaluation of safety and clinical efficacy of this uniform double step schedule including other subtypes of NHL is ongoing. Disclosure:Scheele:Micromet Inc.: Employment. Zugmaier:Micromet Inc.: Employment. Nagorsen:Micromet Inc.: Employment. Klinger:Micromet Inc.: Employment. Schmidt:Micromet Inc.: Employment. Klappers:Micromet. Inc: Employment. Kufer:Micromet Inc.: Employment. Bargou:Micromet Inc.: Consultancy.

Abstract 5621: MCSP/CD3-bispecific single-chain antibody construct engages CD4+ and CD8+ T cells for lysis of MCSP-expressing human uveal melanoma cells

Abstract Uveal melanoma is the most common cancer of the eye and metastasizes in approximately 50% of patients. None of the current therapies prevents the development of metastases or extends the survival time of these patients. Bi-specific T cell engager (BiTE) are based on recombinant single-chain antibody constructs. These molecules can transiently connect T cells to tumor cells, leading to concomitant T cell activation and serial lysis of tumor cells. Recently, a BiTE antibody bi-specific for CD19 and CD3 (blinatumomab) has shown very high response rates in patients with refractory non-Hodgkin's B cell lymphoma (100%) and B-precursor acute lymphocytic leukemia (80%). These findings suggest that the mode of BiTE antibody action may also be useful for treatment of solid tumors. Human melanoma-associated chondroitin sulfate proteoglycan (MCSP, also known as HMW-MAA, gp240, or CSPG4), is expressed on the surface of &amp;gt;90% of human cutaneous melanoma lesions and cell lines, with restricted distribution in normal tissues. MCSP expression has also been detected in uveal melanoma tumor-tissue sections and could serve as a target antigen for immunotherapeutic approaches. Here we report that primary and metastatic uveal melanoma cell lines express MCSP and that CD4+ and CD8+ T cells are activated to lyse MCSP-expressing uveal melanoma cells by a novel BiTE antibody, which is bispecific for MCSP and CD3 (MCSP-BiTE). The surface of uveal melanoma cells was stained by anti-MCSP monoclonal antibodies when analyzed by flow cytometry. Seven out of 9 primary and 1 out of 3 metastatic uveal melanoma cell lines expressed MCSP at various expression levels. For functional analysis, CD4+ and CD8+ T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors using magnetic bead sorting. Both PBMC and unstimulated CD8+ T cells specifically lysed MCSP+ uveal melanoma cells in the presence of MCSP-BiTE as determined by 51Cr-release assays. As detected by ELISA, unstimulated CD4+ T cells produced IFNγ in response to MCSP-expressing uveal melanoma cells in the presence of MCSP-BiTE, and redirected lysis was evident by microscopy. In contrast, co-culture of T cells with MCSP-expressing uveal melanoma cells alone, or in the presence of a control BiTE, did not result in cytolytic activity or IFNγ release. Furthermore, naïve CD4+ and CD8+ T cells upregulated expression of T cell activation markers CD25 and CD137 solely in response to MCSP-expressing uveal melanoma cells decorated with MCSP-BiTE. BiTE-induced T cell activation required the expression of MCSP, as co-culture of T cells with MCSP-negative uveal melanoma cells in combination with MCSP-BiTE did not result in T cell activation. In conclusion, MCSP-BiTE can induce CD4+ and CD8+ T cells against MCSP-expressing uveal melanoma cells and should therefore be tested as a novel immunotherapy for uveal melanoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5621.