Dina Segal

Recombinant human single-chain MHC-peptide complexes made from E. coli By in vitro refolding: functional single-chain MHC-peptide complexes and tetramers with tumor associated antigens.

Soluble recombinant MHC‐peptide complexes are valuable tools for molecular characterization of immune responses as well as for other functional and structural studies. In this study, soluble recombinant single‐chain human MHC (scMHC)‐peptide complexes were generated by in vitro refolding of inclusion bodies from bacterially expressed engineered HLA‐A2 in the presence of tumor‐associated or viral peptides. The scMHC molecule was composed of β2‐microglobulin connected to the first three domains of the HLA‐A2 heavy chain through a 15‐amino acid flexible linker. Highly purified scMHC‐peptide complexes were obtained in high yield using several peptides derived from the melanoma antigens gp100 and MART‐1 or a viral peptide derived from HTLV‐1. The scMHC complexes were characterized in detail and were found to be correctly folded and able to specifically bind HLA‐A2‐restricted peptides. We also generated scMHC‐peptide tetramers, which were biologically functional; they induced a peptide‐specific CTL clone to be activated and secrete IFN‐γ, and were able to stain specifically CTL lines. Such recombinant soluble scMHC‐peptide complexes and tetramers should prove of great value for characterization of immune responses involving CTL, for visualization of antigen‐specific immune responses, for in vitro primary CTL induction, and for peptide binding assays and structural studies.

Antibody-mediated targeting of human single-chain class I MHC with covalently linked peptides induces efficient killing of tumor cells by tumor or viral-specific cytotoxic T lymphocytes

Soluble forms of human MHC class I HLA-A2 were produced in which the peptide binding groove was uniformly occupied by a single tumor or viral-derived peptides attached via a covalent flexible peptide linker to the N terminus of a single-chain β-2-microglobulin-HLA-A2 heavy chain fusion protein. A tetravalent version of this molecule with various peptides was found to be functional. It could stimulate T cells specifically as well as bind them with high avidity. The covalently linked single chain peptide-HLA-A2 construct was next fused at its C-terminal end to a scFv antibody fragment derived from the variable domains of an anti-IL-2R α subunit-specific humanized antibody, anti-Tac. The scFv–MHC fusion was thus encoded by a single gene and produced in E. coli as a single polypeptide chain. Binding studies revealed its ability to decorate Ag-positive human tumor cells with covalent peptide single-chain HLA-A2 (scHLA-A2) molecules in a manner that was entirely dependent upon the specificity of the targeting Antibody fragment. Most importantly, the covalent scHLA-A2 molecule, when bound to the target tumor cells, could induce efficient and specific HLA-A2-restricted, peptide-specific CTL-mediated lysis. These results demonstrate the ability to generate soluble, stable, and functional single-chain HLA-A2 molecules with covalently linked peptides, which when fused to targeting antibodies, potentiate CTL killing. This new approach may open the way for the development of new immunotherapeutic strategies based on antibody targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.

Antibody Engineering for Targeted Therapy of Cancer Recombinant Fv-Immunotoxins

Recombinant Fv-immunotoxins are a new class of biologic anticancer agents composed of a recombinant antibody fragment linked to a very potent bacterial toxin. These potent molecules are designed to specifically bind and kill cancer cells that express a specific target antigen on their cell surface. Recombinant Fv-immunotoxins are an excellent example for the concept of rational drug design. They combine the progress in understanding cancer biology, -the recent knowledge on the mechanisms of malignant transformation and the special properties of cancer cells, -with the enormous developments in recombinant DNA technology and antibody engineering. Recombinant Fv immunotoxins were developed for solid tumors and hematological malignancies and have been characterized intensively for their biological activity in vitro and in vivo in animal models. The excellent in vitro and in vivo activities of recombinant Fv-immunotoxins have lead to their pre-clinical development and to the initiation of clinical trial protocols. Recent trials have demonstrated potent clinical efficacy in patients with malignant diseases that are refractory to traditional modalities of cancer treatment. It is thus suggested that this strategy can be developed into a separate modality of cancer treatment with the basic rationale of specifically targeting cancer cells on the basis of their unique surface markers combined with potent effective biological toxic agents that directly kill the cancer cell. Efforts are now being made to improve the current molecules and to develop new agents with better clinical efficacy. In this review, we will describe the rationale in designing Fv-immunotoxins and will review current progress made in using these agents for cancer treatment.

Recruitment of CTL Activity by Tumor-Specific Antibody-Mediated Targeting of Single-Chain Class I MHC-Peptide Complexes

The MHC class I-restricted CD8 CTL effector arm of the adaptive immune response is uniquely equipped to recognize tumor cells as foreign and consequently initiates the cascade of events resulting in their destruction. However, tumors have developed sophisticated strategies to escape immune effector mechanisms; their most well-known strategy is down-regulation of MHC class I molecules. To overcome this and develop new approaches for immunotherapy, we have constructed a recombinant molecule in which a single-chain MHC is specifically targeted to tumor cells through its fusion to cancer-specific recombinant Ab fragments. As a model we used a single-chain HLA-A2 molecule genetically fused to the variable domains of an anti-IL-2Rα subunit-specific humanized Ab, anti-Tac. The construct, termed B2M-aTac(dsFv), was expressed in Escherichia coli, and functional molecules were produced by in vitro refolding in the presence of HLA-A2-restricted antigenic peptides. Flow cytometry studies revealed the ability to decorate Ag-positive, HLA-A2-negative human tumor cells with HLA-A2-peptide complexes in a manner that was entirely dependent upon the specificity of the targeting Ab fragment. Most importantly, the B2M-aTac(dsFv)-mediated coating of the target tumor cells made them susceptible for efficient and specific HLA-A2-restricted, melanoma gp100 peptide-specific CTL-mediated lysis. These results demonstrate the concept that Ab-guided, Ag-specific targeting of MHC-peptide complexes on tumor cells can render them susceptible and more receptive and thus potentiate CTL killing. This type of approach may open the way for the development of new immunotherapeutic strategies based on Ab targeting of natural cognate MHC ligands and CTL-based cytotoxic mechanisms.

New substrates of acetylcholinesterase

Volume 134, number 1 FEBS LETTERS November 1981 NEW SUBSTRATES OF ACETYLCHOLINESTERASE Meira NAVEH, Zmira BERNSTEIN, Dina SEGAL and Yechiel SHALITIN* Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000, Israel Received 2 September 1981 1. Introduction Acetylcholinesterase (ACHE) has been the subject of intensive studies in the last 4 decades [1,2]. Occu- pying a central role in transmission of nervous signals and being an efficient hydrolase model enzyme, AChE attracted much interest, and a vast number of compounds were studied as substrates and inhibitors of the enzyme, for both theoretical and applied rea- sons. Our aim was to examine the ability of new classes of compounds to serve as AChE substrates. A variety of compounds were tested as possible substrates of the Electrophorus enzyme and several highly active substrates were found: (1) Phenothiazine carbonyl chloride was found to be a potent covalent inhibitor of acetylcholinesterase and could be used to titrate the enzyme active site ; * To whom correspondence should be addressed (2) Enol acetates (vinyl acetate and its derivatives) are good substrates of the enzyme, yielding ace- tate and aldehyde upon hydrolysis; (3) Alkylidenediacetates are hydrolyzed by acetyl- cholinesterase, yielding also acetate and aldehyde; (4) Tropolone acetate is an excellent substrate of the enzyme; (5) Several acetanilide derivatives are susceptible to acetylcholinesterase hydrolysis, although at a considerably slower rate than analogous esters. 2. Materials and methods Acetylcholinesterase from electric eel and acetyl- choline chloride were purchased from Sigma. Pheno- thlazine-10-carbonyl chloride (PTCC, (a) in fig.l) was the product of Aldrich. Commercially available ace- tate esters were of high purity grade. Other acetates were prepared according to reported procedures. For example, 1-butenylacetate was prepared from

A Novel Postpriming Regulatory Check Point of Effector/Memory T Cells Dictated through Antigen Density Threshold-Dependent Anergy

CTLs act as the effector arm of the cell-mediated immune system to kill undesirable cells. Two processes regulate these effector cells to prevent self reactivity: a thymic selection process that eliminates autoreactive clones and a multistage activation or priming process that endows them with a license to kill cognate target cells. Hitherto no subsequent regulatory restrictions have been ascribed for properly primed and activated CTLs that are licensed to kill. In this study we show that CTLs possess a novel postpriming regulatory mechanism(s) that influences the outcome of their encounter with cognate target cells. This mechanism gauges the degree of Ag density, whereupon reaching a certain threshold significant changes occur that induce anergy in the effector T cells. The biological consequences of this Ag-induced postpriming control includes alterations in the expression of cell surface molecules that control immunological synapse activity and cytokine profiles and induce retarded cell proliferation. Most profound is genome-wide microarray analysis that demonstrates changes in the expression of genes related to membrane potential, TCR signal transduction, energy metabolism, and cell cycle control. Thus, a discernible and unique gene expression signature for anergy as a response to high Ag density has been observed. Consequently, activated T cells possess properties of a self-referential sensory organ. These studies identify a new postpriming control mechanism of CTL with anergenic-like properties. This mechanism extends our understanding of the control of immune function and regulation such as peripheral tolerance, viral infections, antitumor immune responses, hypersensitivity, and autoimmunity.

Selective antibody-mediated targeting of class I MHC to EGFR-expressing tumor cells induces potent antitumor CTL activity in vitro and in vivo

Epidermal growth factor receptor (EGFR) is highly overexpressed in many tumor types. We present a new fusion molecule that can target solid tumors that express EGFR. The fusion molecule combines the advantage(s) of the well‐established tumor targeting capabilities of high affinity recombinant fragments of antibodies with the known efficient, specific and potent killing ability of CD8 T lymphocytes directed against highly antigenic MHC/peptide complexes. A recombinant chimeric molecule was created by the genetic fusion of the scFv antibody fragment derived from the anti‐EGFR monoclonal antibody C225, to monomeric single‐chain HLA‐A2 complexes containing immunodominant tumor or viral‐specific peptides. The fusion protein can induce very efficiently CTL‐dependent lysis of EGFR‐expressing tumor cells regardless of the expression of self peptide‐MHC complexes. Moreover, the molecule exhibited very potent antitumor activity in vivo in nude mice bearing preestablished human tumor xenografts. These in vitro and in vivo results indicate that recombinant scFv‐MHC‐peptide fusion molecules might represent a novel and powerful approach to immunotherapy of solid tumors, bridging antibody and T lymphocyte attack on cancer cells.

Simultaneous monitoring of binding to and activation of tumor-specific T lymphocytes by peptide-MHC

The recent advent of peptide-MHC tetramers has provided a new and effective tool for studying antigen-specific T cell populations through monitoring tetramer binding to T cells by flow cytometry. Yet information regarding T cell activation induced by the bound tetramers cannot be deduced from binding studies alone; complementary methods are needed to bridge this gap. To this end, we have developed a new approach that now enables monitoring both binding to and activation of T cells by peptide-MHC tetramers at the single-cell level. For this purpose, we have employed the CellScan, a non-flow cytometer designed for repetitive measurements of optical parameters (e.g., fluorescence intensity and polarization) of individual living cells. A melanoma-specific MART1 CTL line and a gp100-specific CTL clone were incubated with specific and control single-chain peptide-MHC tetramers for 45 min. Subsequently, the fluorescence intensity and polarization were measured by the CellScan. Specific binding of fluorescently labeled peptide-MHC tetramers to CTLs, recorded by the CellScan, was comparable to that measured by flow cytometry. CellScan monitoring of the degree of fluorescence polarization of fluorescein diacetate-labeled CTLs that were reacted with tetramers revealed specific activation of the CTLs, which was confirmed by cytokine (INF gamma) production. These results provide a new means of monitoring both the binding to and activation of T lymphocytes by cognate peptide-MHC complexes at the single-cell level, which can now be applied to distinguish between cognate responding and anergic T cells.

The interaction of alkynyl carboxylates with serine enzymes a potent new class of serine enzyme inhibitors

The recently reported alkynyl esters, propynyl benzoate and propynyl p‐methoxybenzoate, were found to interact with a variety of serine enzymes. α‐Chymotrypsin was inhibited very rapidly by an equivalent amount of the esters. Trypsin, elastase and pronase were also inhibited by the esters. On the other hand, liver esterase started to hydrolyse the alkynyl esters rapidly, but the enzyme became inhibited during the course of reaction. The inhibited enzymes exhibited slow reactivation which could be considerably enhanced by hydroxylamine.

The interaction of amiloride with acetylcholinesterase and butyrylcholinesterase.

The diuretic drug amiloride was found to be a powerful inhibitor of the reaction of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with their specific choline ester substrates. The inhibition constant is in the micromolar range. On the other hand, when added to a mixture of cholinesterase (AChE and BChE) and neutral substrates, amiloride, in some cases, enhanced the reaction rate. The rate of the reaction of butyrylcholinesterase with p‐nitrophenyl butyrate was increased up to 12 fold by amiloride.