Fc Part Of IgG1 Research Articles

Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

Affinity Maturation of B7-H6 Translates into Enhanced NK Cell-Mediated Tumor Cell Lysis and Improved Proinflammatory Cytokine Release of Bispecific Immunoligands via NKp30 Engagement.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6-derived NK cell engagers revealed an up to 45-fold-enhanced affinity for NKp30 and significantly improved NK cell-mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC50 killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6-based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell-activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy.

Effective Targeting of PRAME-Positive Tumors with Bispecific T Cell-Engaging Receptor (TCER®) Molecules

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

The Epitope-Specific Anti-human CD4 Antibody MAX.16H5 and Its Role in Immune Tolerance.

T cell modulation in the clinical background of autoimmune diseases or allogeneic cell and organ transplantations with concurrent preservation of their natural immunological functions (e.g., pathogen defense) is the major obstacle in immunology. An anti-human CD4 antibody (MAX.16H5) was applied intravenously in clinical trials for the treatment of autoimmune diseases (e.g., rheumatoid arthritis) and acute late-onset rejection after transplantation of a renal allograft. The response rates were remarkable and no critical allergic problems or side effects were obtained. During the treatment of autoimmune diseases with the murine MAX.16H5 IgG1 antibody its effector mechanisms with effects on lymphocytes, cytokines, laboratory and clinical parameters, adverse effects as well as pharmacodynamics and kinetics were studied in detail. However, as the possibility of developing immune reactions against the murine IgG1 Fc-part remains, the murine antibody was chimerized, inheriting CD4-directed variable domains of the MAX.16H5 IgG1 connected to a human IgG4 backbone. Both antibodies were studied in vitro and in specific humanized mouse transplantation models in vivo with a new scope. By ex vivo incubation of an allogeneic immune cell transplant with MAX.16H5 a new therapy strategy has emerged for the first time enabling both the preservation of the graft-vs.-leukemia (GVL) effect and the permanent suppression of the acute graft-vs.-host disease (aGVHD) without conventional immunosuppression. In this review, we especially focus on experimental data and clinical trials obtained from the treatment of autoimmune diseases with the murine MAX.16H5 IgG1 antibody. Insights gained from these trials have paved the way to better understand the effects with the chimerized MAX.16H5 IgG4 as novel therapeutic approach in the context of GVHD prevention.

Physicochemical Characterization, Glycosylation Pattern and Biosimilarity Assessment of the Fusion Protein Etanercept.

Etanercept is a soluble fusion protein of the tumor necrosis factor receptor (TNFR) extracellular domain, linked to an Fc part of IgG1. It possesses three N- and 13 O-glycosylation sites. Due to its complex structure, an analytical challenge is facing the development and approval of biosimilars. In the current study, physicochemical characterization using state-of-the-art analytics was performed to analyze intact and subunit masses, post-translational modifications (PTMs), higher order structure and potency of Etanercept originator Enbrel® and its biosimilar Altebrel™ (AryoGen Pharmed) in accordance to critical quality attributes of biopharmaceuticals. Intact mass and subunit analysis revealed a size of about 126kDa for both biologicals. Similar glycoprotein species for the complete monomer and the Fc domain of originator and follow-on product were observed, however, small differences in lysine variants and oxidation were found. N-Glycopeptide analysis with UHPLC-QTOF-MSE confirmed the N-glycosylation sites (N149, N171 and N317) as well as Fc-specific glycosylation on N317, and TNFR-specific highly sialylated glycans on N149 and N171 on both investigated products. Small quantitative variations in the N-glycan profile were detected, although the N-glycans were qualitatively similar. Four different O-glycopeptides bearing core 1-type glycans were detected. For both, N- and O-glycopeptide analysis, determination was achieved without prior cleavage of the sialic acid residues for the first time. In addition, ion mobility spectrometry data confirmed close similarity of higher-order structure of both biologics. Furthermore, a neutralization assay, investigating the impact of altered PTMs on potency, indicated that the differences within all batches are still in the acceptable range for biosimilarity.

Development and Preclinical Characterization of an Fc-Optimized CD133 Antibody for Induction of NK Cell Reactivity Against Myeloid Leukemia

NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity and largely contribute to the efficacy of allogenic stem cell transplantation (SCT) in leukemia. Another clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of monoclonal antibodies (mAb) like Rituximab, a feature considered critical for the therapeutic success of antibody treament. Modifications of the human IgG1 Fc-part in anti-tumor antibodies lead to markedly improved capability to recruit Fc-receptor bearing effector cells as highlighted by the improved clinical efficacy of the Fc-engineered CD20 antibody Ofatumumab as compared to its unmodified counterpart Rituximab in CLL. So far, no immunotherapeutic antibodies are available for the treatment of myeloid leukemias. Here we report on the development and preclinical characterization of an Fc-optimized mAb directed towards CD133, which is expressed on a wide variety of malignant cell types. As a first step we evaluated binding of three different mouse anti-human CD133 mAbs (clones AC133, W6B3 and 293C3) to 20 primary AML and 6 primary CML samples in order to identify a clone with optimal binding characteristics. AC133 and W6B3 comparably bound to the leukemic cells in 11/20 AML and 5/6 CML samples. In contrast, binding of 293C3 was observed in 18/20 AML cases and 5/6 CML cases. Thus, 293C3 recognizes a different epitope than the other two antibody clones, which is expressed in a high proportion of myeloid leukemia cases. Accordingly, 293C3 was selected for generating chimeric mAbs with either a wildtype Fc part (293C3-WT) or a variant containing distinct modifications (S239D/I332E) to enhance its affinity to the activating Fc receptor CD16 (293C3-SDIE). The binding specificity of 293C3-WT and 293C3-SDIE was validated by FACS in analyses with CD133 transfectants and mock controls. When comparing 293C3-WT and 293C3-SDIE with regard to their immunostimulatory properties, we found that already 293C3-WT induced NK cell ADCC against primary leukemia cells as revealed by analyses of degranulation and target cell lysis. These effects were by far exceeded by treatment with 293C3-SDIE, confirming the functional relevance of the SDIE modification in its Fc part. Notably, treatment with 293C3-SDIE also enhanced the reactivity of NK cells against CD133-positive AML cells in an autologous setting. Considering the expression of CD133, among others, on healthy hematopoietic progenitor cells, we further performed colony forming unit assays with healthy bone marrow cells, which did not reveal any toxicity of 293C3-SDIE at the level of committed hematopoietic progenitor cells. Thus, 293C3-SDIE constitutes an attractive immunotherapeutic compound which we envisage in particular for the elimination of minimal residual disease in CD133 bearing leukemia, especially in the context of allogenic SCT. DisclosuresNo relevant conflicts of interest to declare.

Abstract 649: Development and preclinical characterization of an Fc-optimized CD133 antibody for improved induction of NK cell reactivity against myeloid leukemia

Abstract NK cells are cytotoxic lymphocytes that play a major role in anti-tumor immunity and largely contribute to the efficacy of allogenic stem cell transplantation (SCT) in leukemia. Another clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies (mAb) such as Rituximab. Modifications of the human IgG1 Fc-part of anti-tumor antibodies may lead to markedly improved capability to recruit Fc-receptor bearing effector cells. Here we report on the development and preclinical characterization of an Fc-optimized mAb directed towards CD133, a molecule expressed on a wide variety of malignant cell types. Initially we evaluated binding of three different mouse anti-human CD133 mAbs (clones AC133, W6B3 and 293C3) to 20 AML and 6 CML samples to identify a clone with optimal binding characteristics. Binding of AC133 and W6B3 to the leukemic cells in 11/20 AML and 5/6 CML samples was comparable. In contrast, binding of 293C3 was observed in 18/20 AML cases and 5/6 CML cases. This indicates that 293C3 recognizes a particular epitope, which is expressed in a high proportion of AML cases. Therefore mAb 293C3 was selected for generation of chimeric mAbs with either a wildtype Fc part (293C3-WT) or a variant containing distinct modifications (S239D/I332E) to enhance its affinity to the Fc receptor CD16 (293C3-SDIE). The binding specificity of 293C3-WT and 293C3-SDIE was validated in analyses with CD133 transfectants and the respective mock controls. In cytotoxicity assays and analyses of NK cell degranulation, 293C3-WT clearly induced NK cell ADCC against primary leukemia cells. However, ADCC activity of the Fc-optimized mAb 293C3-SDIE markedly exceeded that of its counterpart containing the wild type Fc part. Considering the expression of CD133 on healthy hematopoietic progenitor cells, we presently evaluate potential toxicity of the antibodies towards these cells and other healthy tissue. We envisage that 293C3-SDIE might be an attractive immunotherapeutic reagent for the elimination of minimal residual disease in CD133 expressing leukemia, particularly in the context of allogenic SCT. Citation Format: Samuel Koerner, Julia Leibold, Ludger Grosse-Hovest, Hans-Jörg Bühring, Helmut R. Salih, Gundram Jung. Development and preclinical characterization of an Fc-optimized CD133 antibody for improved induction of NK cell reactivity against myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 649. doi:10.1158/1538-7445.AM2014-649

Induction of NK Cell Reactivity Against AML Cells by Fc-Engineered RANK-Ig Fusion Proteins.

AbstractAbstract 2625NK cells are cytotoxic lymphocytes that play an important role in anti-tumor immunity. A clinically important feature of NK cells is their ability to mediate antibody-dependent cellular cytotoxicity (ADCC) upon application of anti-tumor antibodies. In acute myeloid leukemia (AML) NK cells largely contribute to the therapeutic efficacy allogenic stem cell transplantation (SCT). Recently we demonstrated that AML cells functionally express the TNF family member RANK ligand (RANKL) which impairs NK cell anti-leukemia reactivity (Schmiedel et al., ASH annual meeting 2011). Here we developed a strategy to combine blocking of the NK inhibitory effects of RANKL with targeting of AML cells for NK cell ADCC. To this end we generated fusion proteins consisting of the extracellular domain of RANK and a human IgG1 Fc part that was modified by amino acid exchange. Compared to wild type RANK-Fc fusion protein (RANK-Fc-WT), our mutant RANK-Fc-ADCC (S239D/I332E) displayed highly enhanced affinity to FcγRIIIa (CD16) on NK cells. Primary AML cells expressed substantial levels of RANKL in 53 of 78 (68%) investigated patient cases, and our RANK-Ig fusion proteins bound to AML cells in a target antigen-specific manner. Treatment with both RANK-Fc-WT and RANK-Fc-ADCC clearly reduced the release of RANKL-induced immunomodulatory factors like TNF, IL-6, IL-8 and IL-10 by AML cells. When the effects of the fusion proteins on NK cell ADCC were studied we found that treatment with RANK-Fc-WT only slightly enhanced NK cell reactivity against RANKL-positive patient AML cells. However, RANK-Fc-ADCC potently induced NK cell ADCC and cytokine production in response to AML targets in a target antigen-dependent manner due to the functional properties of its engineered Fc moiety. Taken together, our Fc-engineered RANK-Fc-ADCC fusion protein may serve to modulate the cytokine milieu involved in AML pathophysiology and target RANKL-expressing leukemia cells for NK anti-tumor reactivity. Thus, RANK-Fc-ADCC constitutes an attractive immunotherapeutic means for the treatment of AML, e.g. for elimination of minimal residual disease after conventional therapy including SCT. Disclosures:No relevant conflicts of interest to declare.

The Stalk Domain and the Glycosylation Status of the Activating Natural Killer Cell Receptor NKp30 Are Important for Ligand Binding

The natural cytotoxicity receptors are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The human natural cytotoxicity receptor family comprises the three type I membrane proteins NKp30, NKp44, and NKp46. Especially NKp30 is critical for the cytotoxicity of NK cells against different targets including tumor, virus-infected, and immature dendritic cells. Although the crystal structure of NKp30 was recently solved (Li, Y., Wang, Q., and Mariuzza, R. A. (2011) J. Exp. Med. 208, 703-714; Joyce, M. G., Tran, P., Zhuravleva, M. A., Jaw, J., Colonna, M., and Sun, P. D. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 6223-6228), a key question, how NKp30 recognizes several non-related ligands, remains unclear. Therefore, we investigated the parameters that impact ligand recognition of NKp30. Based on various NKp30-hIgG1-Fc fusion proteins, which were optimized for minimal background binding to cellular Fcγ receptors, we identified the flexible stalk region of NKp30 as an important but so far neglected module for ligand recognition and related signaling of the corresponding full-length receptor proteins. Moreover, we found that the ectodomain of NKp30 is N-linked glycosylated at three different sites. Mutational analyses revealed differential binding affinities and signaling capacities of mono-, di-, or triglycosylated NKp30, suggesting that the degree of glycosylation could provide a switch to modulate the ligand binding properties of NKp30 and NK cell cytotoxicity.

Detection of weak receptor–ligand interactions using IgM and J‐chain‐based fusion proteins

Detection of weak receptor–ligand interactions using <scp>I</scp>g<scp>M</scp> and <scp>J</scp>‐chain‐based fusion proteins

Abatacept in the treatment of polyarticular JIA: development, clinical utility, and place in therapy

Juvenile idiopathic arthritis (JIA) is a group of chronic arthritides affecting children. The polyarthritis category, affecting five or more joints in the first six months, tends to be more aggressive, leading to a destructive joint disease with significant morbidity, disability, and costs to society. The current treatment regimen, which primarily combines methotrexate and tumor necrosis factor alpha (TNF-α) blockade, still leaves a significant group of patients with an inadequate response. Therefore, the development of new medications that act via other mechanisms of pathogenesis is necessary. T cell lymphocytes are key components in the immune reaction in JIA. Cytotoxic lymphocyte-associated antigen-4 (CTLA-4) is a potent inhibitor of the costimulation pathway necessary to activate T cells. Abatacept is a recombinant fusion protein comprising the extracellular part of human CTLA-4 connected to a modified Fc part of IgG-1. In a randomized, multinational, blinded withdrawal study in children with polyarticular JIA, abatacept was found to be effective in about 70% of the patients, including 39% of TNF-α blockade failures, with significantly fewer flares occurring during the withdrawal phase than in patients receiving placebo. Abatacept continued to show good efficacy in a three-year open-label extension study, with a beneficial effect on health-related quality of life. The safety profile of abatacept is generally good. In 2008, the US Food and Drug Administration approved abatacept for use in children over six years of age with JIA and a polyarticular course. In 2010, the European Medicines Agency gave approval for abatacept to be used in combination with methotrexate for those who fail at least one disease-modifying medication and TNF-α blockade.

Generation, Functional Characterization and Clinical Application of a Novel Fc-Optimized Anti-FLT3 Antibody for the Treatment of Acute Myeloid Leukemia

AbstractAbstract 2187For more than a decade, chimeric/humanized second generation monoclonal antibodies (mAb) are used in cancer therapy. While their success e.g. in lymphoma therapy is undisputed, mAb are not established as treatment for acute myeloid leukemia (AML). Moreover, the therapeutic activity of available mAb leaves ample room for improvement. In the recent years, modifications of the human IgG1 Fc-part have enabled the development of third generation mAb with markedly improved capability to recruit Fc-receptor bearing immune effector cells. We here report on the development and evaluation of an Fc-optimized mAb directed to FLT3, an antigen expressed on leukemic cells of the majority of AML patients. In vitro, this antibody termed 4G8-SDIEM effectively induced antibody-dependent cellular cytotoxicity (ADCC) against FLT3-expressing AML cells at concentrations as low as 10ng/ml. Compared to the parental humanized antibody, the ADCC activity of 4G8-SDIEM was increased by a factor of 100. FLT3 expression on primary AML cells (range, 500–5000 molecules) was considerably higher as compared to healthy hematopoietic cells (several hundred molecules). 4G8-SDIEM did not induce relevant ADCC against healthy cells and did not decrease the CFU-forming capacity of bone marrow (BM) cells in vitro. The mAb was then produced in pharmaceutical quality and quantity at a university-owned production unit and used for compassionate need treatment of a 30 year old AML patient (FAB M0, complex kariotype with 45, XY, inv(3)(q21q26), -7, 9q-) with relapse after haploidentical and unrelated donor stem cell transplantation (SCT). Preclinical testing revealed that 4G8-SDIEM effectively induced ADCC of the patients peripheral blood (PB) mononuclear cells (PBMC) against NALM16 leukemia cells and autologous leukemic blasts. Directly before initiation of treatment, the percentage of leukemic blasts among the patients PBMC was 11% and 38% in PB and BM, respectively, and his AML cells displayed homogeneous FLT3 expression. The percentage of NK cells (CD56+CD3-) was 7% and 5% in PB and BM, respectively, with less than 2% displaying an activated phenotype (CD69+). 4G8-SDIEM was applied in escalating doses (d1: 10μg; d2: 100μg; d3: 1mg; d4: 2mg; d5, 7, 10: 10mg). Several hours after the first 10 mg-dose 5×108 CD3/CD19-depleted donor lymphocytes were infused. Besides elevated temperature (max. 38.2°C), no relevant side effects of treatment were observed. After the first first 10mg dose, BM blasts were nearly saturated with mAb as judged by crosscompetition assays. Serum concentration of 4G8-SDIEM reached 1.0μg/ml 1h after the first 10mg dose declining to 0.4 μg/ml 24h later. Upon treatment, serum levels of the index cytokines TNF and IL-6 (peak d4, 60 and 27pg/ml, respectively) and the percentage of activated NK cells (peak d6, 42%) in PB increased rapidly. Already at day 4, leukemic blasts were nearly completely cleared from the PB (<2%), while effects in BM were less pronounced (down to 15%) indicating that lower effector to target cell ratios in the BM limit the therapeutic activity of the mAb. In any case, the anti-leukemic effects of 4G8-SDIEM remained transient, as 10 days after initiation of therapy blast counts in PB and BM reached and later exceeded pre-treatment levels. Thus we conclude that although 4G8-SDIEM clearly exerts anti-leukemic effects in vivo, it may not be capable of inducing long-lasting responses of AML in proliferative phase. Rather, we envisage application of the mAb in settings with suitably high effector to target cell ratios such as minimal residual disease in morphological complete remission, possibly in combination with adoptive NK cell transfer. As of now, development of 4G8-SDIEM cost |CE2 million and required 2.5 years from bench to bedside. Thus, development and early clinical evaluation of novel mAb can be achieved at academic institutions at reasonable costs and time before initiation of larger clinical Phase II/III studies by the pharmaceutical industry. Such an approach may not only accelerate the developmental process for anti-tumor mAb, but rather for innovative biological drugs in general. Disclosures:Hofmann: University of Tuebingen, Germany: Patents & Royalties. Grosse-Hovest: University of Tuebingen, Germany: Patents & Royalties. Aulwurm: University of Tuebingen, Germany: Patents & Royalties. Buehring: University of Tuebingen, Germany: Patents & Royalties. Jung: University of Tuebingen, Germany: Patents & Royalties.

The structure of the unliganded extracellular domain of the interleukin-6 signal transducer gp130 in solution

Interleukin-6 (IL-6) plays an important role in immune responses and signals via two different pathways. When IL-6 binds to its non-signalling membrane-bound receptor (IL-6R), a non-covalent dimer of the ubiquitous interleukin-6 signal transducer gp130 is recruited to initiate intracellular signalling cascades. This so-called classical signalling pathway is restricted to cells expressing the membrane-bound IL-6R, such as hepatocytes and certain leukocytes. In addition, an alternative trans-signalling pathway uses soluble forms of IL-6R (sIL-6R) in complex with IL-6 to activate cells expressing gp130, but not membrane-bound IL-6R. In both cases, a tetrameric or hexameric signalling complex consisting of two gp130 molecules and one or two molecules each of IL-6 and (s)IL-6R is formed. The structure of the hexameric complex of the ligand-binding domains of gp130 (D1–D3) with IL-6 and sIL-6R has been solved by X-ray crystallography as well as the full-length extracellular part of gp130 (D1–D6) as a monomer. Since gp130 exists as a preformed dimer on the cell surface, we used a sgp130Fc fusion protein – consisting of two extracellular gp130 regions (D1–D6) dimerised by an IgG1-Fc part – to study the structure of unliganded gp130 extracellular domains in solution by small-angle X-ray scattering (SAXS). The SAXS data indicated that sgp130Fc forms a rigid molecule in solution. The low resolution structural model reveals an elongated assembly with an Fc base and two gp130 arms, whereby the orientation of the ligand-binding domains D1–D3 with respect to the membrane-proximal domains D4–D6 differs from that in the crystallographic monomer. Functional implications of these findings are discussed.

A Transmembrane Tight Junction Protein Selectively Expressed on Endothelial Cells and Platelets

Searching for cell surface proteins expressed at interendothelial cell contacts, we have raised monoclonal antibodies against intact mouse endothelial cells. We obtained two monoclonal antibodies, 1G8 and 4C10, that stain endothelial cell contacts and recognize a protein of 55 kDa. Purification and identification by mass spectrometry of this protein revealed that it contains two extracellular Ig domains, reminiscent of the JAM family, but a much longer 120-amino acid cytoplasmic domain. The antigen is exclusively expressed on endothelial cells of various organs as was analyzed by immunohistochemistry. Immunogold labeling of ultrathin sections of brain as well as skeletal muscle revealed that the antigen strictly colocalizes in capillaries with the tight junction markers occludin, claudin-5, and ZO-1. Upon transfection into MDCK cells, the antigen was restricted to the most apical tip of the lateral cell surface, where it colocalized with ZO-1 but not with beta-catenin. In contrast to JAM-1, however, the 1G8 antigen does not associate with the PDZ domain proteins ZO-1, AF-6, or ASIP/PAR-3, despite the presence of a PDZ-binding motif. The 1G8 antigen was not detected on peripheral blood mouse leukocytes, whereas similar to JAM-1 it was strongly expressed on platelets and megakaryocytes. The 1G8 antigen supports homophilic interactions on transfected Chinese hamster ovary cells. Based on the similarity to the JAM molecules, it is plausible that the 1G8 antigen might be involved in interendothelial cell adhesion.

An interleukin-2-IgG-Fas ligand fusion protein suppresses delayed-type hypersensitivity in mice by triggering apoptosis in activated T cells as a novel strategy for immunosuppression.

Cell-mediated immune responses can be down-regulated by induction of apoptosis of immunoreactive lymphocytes. In the present study, we have tested the feasibility of a strategy for immunosuppression by the selective induction of apoptosis in activated, interleukin (IL)-2 receptor-positive lymphocytes, using a triple IL-2-IgG-FasL fusion protein. The IL-2-IgG-FasL fusion protein combines IL-2 for the selection of activated T cells, with the extracellular domain of the FasL molecule for inducing T-cell apoptosis. These components were separated by the Fc part of IgG1 serving as a spacer as well as for half-life prolongation. The gene for the chimeric protein was created by fusing DNA sequences encoding for the three functional components: human IL-2, the Fc part of human IgG1, and the extracellular domain of murine FasL. When the fusion gene was expressed in murine J558L cells, we obtained soluble dimeric immunoglobulin-like proteins in the supernatant. After analyzing the function of the IL-2 and FasL portions individually in vitro, a delayed-type hypersensitivity (DTH) reaction to sheep red blood cells as model for cell-mediated immune responses was investigated to evaluate the IL-2-IgG-FasL-mediated immunosuppression in vivo. In vitro, the IL-2-IgG-FasL fusion protein supported IL-2-dependent proliferation of Fas-resistant CTLL-2 cells, whereas concanavalin A-T blasts were induced to undergo apoptosis by the FasL portion. In vivo, this fusion protein potently inhibited a murine DTH. This was associated with an increased rate of apoptosis in activated lymphocytes in the spleen, even at very low doses of the fusion protein. Furthermore, a second antigen challenge 10 days after IL-2-IgG-FasL treatment still failed to elicit a DTH response. The abrogation of a standard T cell-dependent immune response in vivo demonstrates that IL-2-IgG-FasL can be successfully exploited to trigger the death of deleterious T cells, presenting a potentially useful strategy in the management of autoimmune diseases and allotransplant rejections.

A MOUSE MODEL FOR THE PRECLINICAL EVALUATION OF IMMUNOSUPPRESSIVE EFFECTOR FUNCTIONS OF HUMAN ISOTYPES

Antibodies against T cells are widely used as immunosuppressive agents in clinical therapy. As effector functions of chimeric or humanized anti-T cell antibodies cannot be predicted in vitro, we compared T cell-depleting effects of human isotypes in vivo with their immunosuppressive consequences in a mouse BMT model. This system is based on chimeric antibodies with a mouse pan T cell specificity and human constant regions. To secure optimal immunosuppression, the specificity for Thy-1.2--one of the best-characterized T cell antigens--was selected, as Thy-1.2-specific antibodies prevent graft-versus-host disease in fully mismatched mice. Chimeric mouse anti-Thy-1.2 antibody with the human IgG1 Fc part was found to be equally effective in preventing graft-versus-host disease mortality as the highly protective anti-Thy-1.2 mouse IgG2a isotype, while human IgG3 was far less effective. This was not predictable by measuring the degree of T cell depletion in peripheral blood. T cell depletion in lymph nodes, however, exactly reflected the results obtained in the BMT system. In addition, this system offers the advantage of assessing the influence of reduced antigen density by using heterozygous Thy-1.2 mice.