Monoclonal antibodies against IREM-1: potential for targeted therapy of AML.

IREM-1 is an inhibitory cell surface receptor with an unknown function and is expressed on myeloid cell lineages, including cell lines derived from acute myeloid leukemia (AML) patients. We have generated a series of monoclonal antibodies (mAbs) against the extracellular domain of IREM-1 and further assessed its expression in normal and AML cells. IREM-1 was restricted to cells from myeloid origin and extensive expression analysis in primary cells obtained from AML patients showed IREM-1 expression in leukemic blasts of 72% (39/54) of samples. We therefore searched for specific IREM-1 mAbs with activity in functional complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). Lead mAbs against IREM-1 showed specific cytotoxic activity against a variety of AML-derived cell lines and freshly isolated blasts from AML patients. Internalization of mAbs upon IREM-1 binding was also shown. In vivo anticancer activity of lead mAbs was observed in an established HL-60 xenograft model with a tumor growth delay of up to 40% and in a model using primary human AML cells, where treatment with anti-IREM-1 mAb resulted in a significant reduction of engrafted human cells. These results demonstrate IREM-1 as a potential novel target for immunotherapy of AML.

A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.

Controlling chemical reactivity with antibodies.

The remarkable specificity of an antibody molecule has been used to accomplish highly selective functional group transformations not attainable by current chemical methods. An antibody raised against an amine-oxide hapten catalyzes the reduction of a diketone to a hydroxyketone with greater than 75:1 regioselectivity for one of two nearly equivalent ketone moieties. The antibody-catalyzed reaction is highly stereoselective, affording the hydroxyketone in high enantiomeric excess. Similarly, the reduction of ketones containing branched and aryl substituents, including the highly symmetrical 1-nitrophenyl-3-phenyl-2-propanone, was enantioselective. The simple strategy presented herein may find general applicability to the regio- and stereoselective reduction of a broad range of compounds.

An efficient antibody-catalyzed aminoacylation reaction.

An antibody generated against a neutral phosphonate diester transition-state analog was found to catalyze the aminoacylation of the 3'-hydroxyl group of thymidine with an alanyl ester. A comparison of the apparent second-order rate constant of the antibody-catalyzed reaction [5.4 x 10(4) molar-1 minute-1 (M-1 min-1)] with that of the uncatalyzed reaction (2.6 x 10(-4) M-1 min-1) revealed this to be a remarkably efficient catalyst. Moreover, although the concentration of water (55 M) greatly exceeds that of the secondary alcohol, the antibody selectively catalyzes acyl transfer to thymidine. The antibody exhibits sequential binding, with Michaelis constants of 770 microM and 260 microM for acyl acceptor and donor, respectively, and a dissociation constant of 240 pM for hapten. This antibody-catalyzed reaction provides increased insight into the requirements for efficient aminoacylation catalysts and may represent a first step toward the generation of "aminoacyl transfer RNA synthetases" with novel specificities.

Reactivity of monoclonal antibody 17.13. with human squamous cell carcinoma and its application to tumor diagnosis.

Monoclonal antibody 17.13., derived from a fusion of splenocytes of a BALB/c mouse immunized with a surgically resected poorly differentiated human laryngeal recurrent squamous cell carcinoma (SCC) with mouse Sp2/0 cells, is an IgM-K which recognizes a cytoplasmic component of basal cells. Tissue sections of malignant and normal squamous epithelium, tumors of nonsquamous origin, and normal and malignant cytological specimens were tested with an immunoperoxidase assay. Seventy-nine of 81 (98%) SCC of the head and neck, 26 of 26 (100%) SCC of the cervical and female gynecological tract, 29 of 30 (97%) SCC of the lung, 19 of 19 (100%) SCC of the oral cavity, and 17 of 17 (100%) SCC-involved neck lymph nodes reacted strongly. Various carcinomas from breast, colon, ovary, and others were unreactive. In normal squamous epithelial tissues, monoclonal antibody 17.13. reacts only with basal cells but not the cells above the basal layers. Normal tissues from heart, liver, spleen, kidney, bladder, colon, ovary, stomach, pancreas, breast, lung, prostate, thyroid, and lymph nodes were unreactive with the exception of myoepithelial cells. Monoclonal antibody 17.13. may be useful in the diagnosis and management of SCC.

A monoclonal antibody to squamous cell carcinoma.

Monoclonal antibody 17.13.Cl.10 is a murine IgM kappa monoclonal antibody (Mab) that stains frozen section squamous cell carcinoma (SCC) homogeneously and intensely with a sensitivity greater than 98%, including 106/107 SCC specimens from the head and neck. It was produced using a human laryngeal SCC as immunogen and screened using frozen section human tissue. Monoclonal antibody 17.13.Cl.10 faintly stains the basal layer of normal squamous epithelium, does not stain normal organ tissue other than myoepithelial cells, and reacts with few non-SCC tumors. It, therefore, may be a useful adjunct to standard histopathologic criteria for the diagnosis of SCC. It may prove helpful in the investigation of tumor-associated antigens. Despite major technical and immunologic problems, monoclonal antibodies to functional tissue-specific tumor-associated antigens have the potential to play a major role in imaging and in treatment in the future.