Hisaaki Saeki

A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunotherapy for bile duct carcinoma.

Abstract. In cancer immunotherapy research, many bispecific antibodies (BsAbs) have been developed for directing T cells toward tumor cells. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems. Therefore, recombinant BsAbs, termed diabodies, have attracted particular attention. We have previously produced an anti-MUC1 × anti-CD3 diabody (M×3 diabody) in an Escherichia coli (E. coli) expression system. In order to reinforce the antitumor effects of the M×3 diabody, mutated superantigen staphylococcal enterotoxin A (SEA) D227A was genetically fused to the M×3 diabody. The SEA D227A fusion M×3 diabody (SEA D227A-M×3 diabody) thus constructed showed remarkable MUC1-specific antitumor effects when used with effector cells (lymphokine-activated killer cells with T-cell phenotype [T-LAK] and peripheral blood mononuclear cells [PBMCs]). In the bile duct carcinoma (BDC)-xenografted severe combined immunodeficient (SCID) mouse model, it also demonstrated strong antitumor activity when administered i.v. together with T-LAK cells and interleukin-2 (IL-2). In this experiment, the complete disappearance of tumors was observed in 3 out of 6 mice, and the other 3 showed marked retardation of tumor growth. Therefore, the SEA D227A-M×3 diabody is considered to be a promising reagent in specific targeted immunotherapy for BDC and other MUC1-positive carcinomas. This is the first report on a diabody that is effective in treating human solid cancers in the xenografted SCID mouse experimental model.

Specific and effective targeting cancer immunotherapy with a combination of three bispecific antibodies.

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we previously constructed two kinds of bispecific antibodies (bsAbs), anti-MUC1 x anti-CD3 (M x 3) and anti-MUC1 x anti-CD28 (M x 28), which activate T cells and form bridges between them and MUC1-expressing tumor cells. In our previous studies [Cancer Res. 56 (1996) 4205] specific targeting therapy (STT) consisting of i.v. administration of lymphokine activated killer cells with a T cell phenotype (T-LAK) sensitized with two kinds of bsAbs to human BDC-grafted severe combined immunodeficient (SCID) mice demonstrated remarkable inhibition of tumor growth. However, complete cures could not be obtained. In order to improve antitumor efficacy, we have paid attention to anti-CD2 monoclonal antibodies (mAbs), thought to play an important roles in signal transduction in T cell activation or control of T cell receptor (TCR)-driven activation. Therefore, we developed another bsAb, anti-MUC1 x anti-CD2 (M x 2), in order to examine if this would show synergism with the two previously described bsAbs. The combination of the three bsAbs (M x 3, M x 28 and M x 2 bsAbs) showed highest cytotoxicity against MUC1-expressing BDC cells when given simultaneously with peripheral blood mononuclear cells (PBMCs) or T-LAK cells in vitro. When 2 x 10(7) T-LAK cells sensitized with different combinations of bsAbs were administered four times i.v. to BDC-grafted SCID mice, the best therapeutic result was obtained with a combination of all three bsAbs. These results indicate usefulness of combination of three bsAbs for targeting cancer immunotherapy.

Transformation fo glucosamine to glycogen and lactate by ascites tumor cells

Abstract 1. 1. Ascites tumors, including Yoshida sarcoma, when incubated in vitro , phosphorylate exogenous glucosamine readily and transform a considerable part of the glucosamine 6-phosphate formed into lactate and glycogen. 2. 2. The metabolic fate of glucosamine differs strikingly according to the concentration of glucosamine employed. When Yoshida sarcoma cells are incubated with 0.5 mM glucosamine, the formation of glucosamine 6-phosphate is slow, and the major end-product is glycogen. There is a lag period of about 5 min before the onset of glycogen and lactate formation. The cellular level of adenine nucleotides is little affected. 3. 3. When the initial concentration of glucosamine is 2 mM, glucosamine is phosphorylated rapidly, and a marked fall in the level of ATP (unaccompanied by a rise in ADP) as well as a large accumulation of glucosamine 6-phosphate results. The major end-product is lactate, the formation of which starts without appreciable lag period. These metabolic differences due to difference in glucosamine concentration arise primarily from a low affinity of hexokinase towards glucosamine. 4. 4. Ascites tumors contain phosphoglucosamine isomerase in much greater amounts than does liver. The enzyme appears to determine the rate of glucosamine conversion to glycogen, lactate and CO 2 in tumor cells.

A simple and improved method to generate human hybridomas.

By improving our previous method for production of human hybridomas, we developed a simple and remarkably efficient method for production of human hybridomas. We reformed our previous method in the following three points: (1) we added irradiated (30 Gy) myeloma cells as feeder cells to culture of fusion cells; (2) ouabain concentration in the selective medium was reduced from 2 x 10(-6) M to 1 x 10(-6) M; (3) selective medium (GIT-HAT-OL) was added to the culture after overnight cultivation of fusion cells. Consequently, we obtained higher fusion frequency (1/700 vs. 1/5500) compared with our previous method. By our present method, only so small number of human B cells (EBV-LCL) is required, so that the time necessary to establish human hybridomas is reduced.

A novel human monoclonal antibody directed to a tumor-associated antigen.

Twelve human monoclonal antibodies (HuMAb) were established by the fusion of (mouse × human) heteromyeloma cells with B‐lymphoblastoid cells derived from the regional lymph nodes of three patients with squamous cell carcinoma of the lung. They were tested for reactivity to two kinds of proteins (purified protein derivatives and bovine serum albumin) by ELISA, Sq‐19 (squamous cell carcinoma) culture cells by indirect membrane immunofluorescence tests, and Sq‐19 tumor xenograft by immunohistological study. Among them, one HuMAb 904F (IgM, λ) was selected. In indirect membrane immunofluorescence tests, this 904F antibody reacted with various kinds of cell lines, e.g. lung cancer, esophageal cancer, endometrial cancer, and stomach cancer. It did not react with malignant hematopoietic and diploid fibroblast cell lines. Immunohistologically, it stained the tumor nests of squamous cell carcinoma, adenocarcinoma, and large cell carcinoma of the lung. It also stained those of esophagus and colon, but not those of small cell carcinoma of lung, or stomach. On frozen‐section specimens of normal tissues from various organs, it showed only limited areas of positive staining. Limited positive findings were observed at a reticular zone of the adrenal gland, at the esophagus as weak staining, and at islets of the pancreas as very weak staining. Western blotting analysis demonstrated that it recognized a 54 kDa trypsin‐sensitive molecule which is expressed on the surface of tumor cells. These results suggest the 904F monoclonal antibody detects a novel tumor‐associated antigen which is recognized by the human immune system.

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma.

Abstract. For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1×anti-CD3) and BsAb (anti-MUC1×anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.

Presence of 3-amino-β-carbolines (3-amino-9H-pyrido-[3,4-b]indoles), effectors in the induction of sister-chromatid exchanges, in protein pyrolysates

3-Amino-9H-pyrido[3,4-b]indole (3-aminonorharman) and 3-amino-1-methyl-9H-pyrido[3,4-b]indole (3-aminoharman), effectors for induction of sister-chromatid exchanges in human lymphoblastoid cells, were identified in pyrolysates of casein by high-performance liquid chromatography and UV spectrophotometry, together with some other mutagenic/carcinogenic carbolines. In pyrolysates from a preparation of zein, which contained little tryptophan, only norharman and harman were found in small amounts.

A new and efficient method to generate human IgG monoclonal antibodies reactive to cancer cells using SCID-hu mice.

Human monoclonal antibodies (mAbs) are very useful for treatment of cancer, but they are difficult to obtain since immunization of humans is not a practical proposition at present. As an approach to circumvent this problem, we have simultaneously inoculated cancer tissues and regional lymph node cells obtained from lung cancer patients into SCID mice to allow in vivo stimulation of human lymphocytes against autologous cancer tissues. Human immunoglobulins, especially IgG, were observed in the SCID-hu sera, and some showed high reactivity to lung cancer cell lines. Testing of human B-lymphoblastoid cell lines obtained from SCID-hu spleen and thymus for antibody activity revealed 16-45% of them to be reactive to lung cancer cells. These percentages are high as compared with previous reports. Furthermore, we could establish 4 human IgG mAbs reactive to lung cancer cell lines. These results indicate successful stimulation of specific human lymphocytes in vivo, which thereby enables efficient generation of human monoclonal antibodies using SCID-hu mice.

Construction of a human B cell line, TKHMY, suitable for production of stable human hybridomas

In order to produce stable hybridomas for generation of human monoclonal antibodies (HMAbs), an attempt was made to construct a suitable human parental B cell line with double selection markers, the strategy being to produce HMAbs by fusing a parental B cell line with EBV-transformed B cells producing specific antibodies. The resultant TKHMY cell line, constructed by transfecting the Blasticidin S resistant (bsr) gene into the HAT sensitive human B cell line LTR 228 could be used as a fusion partner for very efficient production of human hybridomas. Secretion of IgG monoclonal antibodies to antigens on lung cancer cells by one such hybridoma was very stable, indicating that this TKHMY cell line has practical advantage in the area of antibody production.

Nucleotide sequence analysis of a human monoclonal antibody 22-13 reactive with lung tumor-associated antigen

A human monoclonal antibody (HuMAb) 22-13 (IgG1, kappa) recognizes a cytoplasmic antigen associated primarily with human lung tumors. This study reports the primary nucleotide and deduced amino acid sequences of the rearranged heavy and light chains of the HuMAb 22-13. This HuMAb uses a VH gene member of the V(H)Ia gene family, 51P1 and is productively rearranged with a D-D fusion product of the D(LR)2 and D(XP)2 germ line DH genes and the germ line JH3 gene. HuMAb 22-13 Vkappa belongs to the kappa light chain variable subgroup IIIb family and appears to be derived from the Humkv325 germ line gene and is rearranged with a germ line Jkappa5 gene. The results reveal that production of a HuMAb 22-13 is achieved by rearrangement of the 51P1/Humkv325 germ line variable region gene combination, associated with the autoimmune repertoire and that HuMAb 22-13 has a striking sequence homology to rheumatoid factors (RFs) of the Wa idiotypic family. HuMAb 22-13 and Wa RFs have in common V(H)Ia and VkappaIIIb gene segments, but use different DH, JH and Jkappa gene segments. However, in spite of this structural similarity, HuMAb 22-13 does not display rheumatoid factor activity. Taken together with the reported findings, these data indicate the representation of the shared usage of highly homologous variable region genes in entirely different humoral immune responses in the human system.