Yasuo Koishihara

The humanized anti-HM1.24 antibody effectively kills multiple myeloma cells by human effector cell-mediated cytotoxicity

A mouse monoclonal antibody, anti-HM1.24 (IgG2a/kappa), binds to a surface antigen preferentially overexpressed on multiple myeloma (MM) cells, and exhibits potent antitumor cell activity against MM cells by antibody-dependent cell-mediated cytotoxicity (ADCC). To develop an antibody-based immunotherapy against MM, a humanized anti-HM1.24 antibody, in which all FRs correspond to naturally processed human FRs, has been successfully constructed with the aid of both the hybrid variable region and two-step design methods. This humanized anti-HM1.24 antibody (IgG1/kappa) is able to effectively induce ADCC against human myeloma KPMM2 and ARH77 cells in the presence of human PBMCs as effectively as a chimeric anti-HM1.24 antibody. The humanized anti-HM1.24 antibody, therefore, could be expected as a potent immunotherapeutic agent for MM patients.

Humanization of a mouse anti-human interleukin-6 receptor antibody comparing two methods for selecting human framework regions

Mouse monoclonal antibody AUK12-20 binds to human IL-6 receptor and inhibits IL-6 functions. It has been humanized by CDR-grafting for therapeutic use. In the design of reshaped human AUK12-20 VL region, the human framework regions (FRs) from the human Bence-Jones protein REI were used. The reshaped human AUK12-20 light chain, in combination with chimeric AUK12-20 heavy chain, bound to antigen as well as chimeric AUK12-20 antibody. In the design of reshaped human AUK12-20 VH region, two sets of the human FRs were chosen and compared. One set was from the consensus amino acid sequence for human VH regions subgroup (HSG)-I and the other set was from human antibody HAX, the most similar human VH region found in a database of human immunoglobulin sequences. The HSG-I-based and the HAX-based reshaped human AUK12-20 heavy chains in combination with the reshaped human AUK12-20 light chain, showed approximately 90 and 100% antigen-binding and competition-binding activities as compared to the chimeric or mouse AUK12-20 heavy chains. Most importantly, these humanized antibodies inhibited the IL-6-dependent tumor cell growth as well as the original mouse antibody suggesting that these humanized antibodies could be efficacious in human patients. Our results show that both approaches for the design of reshaped human antibodies can be used for successful humanization. The approach based on FRs from the most similar individual human antibody, however, seemed to be best for designing a reshaped human antibody that mimicked as closely as possible the original mouse antibody.

Generation of potent antitumor CTL from patients with multiple myeloma directed against HM1.24.

Purpose: The purpose of this work was to test the suitability of the HM1.24 antigen as a CTL target for immunotherapy of patients with multiple myeloma. Experimental Design: Antigen-specific T cells were generated from patients with multiple myeloma using stimulation with protein-pulsed dendritic cells and tested in ELISPOT and CTL assays. Results: HM1.24-primed T cells responded selectively to HM1.24-loaded autologous peripheral blood mononuclear cells (PBMC) in an IFN-γ ELISPOT assay (median, 342; range, 198-495 IFN-γ–producing cells/105 cf. unloaded PBMC median, 98; range, 7-137; P < 0.05, n = 5) and also to autologous malignant plasma cells (MPC; median, 227; range, 153-335; P < 0.05 when compared with the response to allogeneic MPC median, 57; range, 22-158; n = 5). HM1.24-primed T cells lysed autologous MPC (at 20:1 E/T ratio: median, 48% specific killing; range, 23-88%; at 10:1 E/T ratio: median, 43%; range, 15-80%; n = 12) but not allogeneic MPC. Lysis of autologous MPC was inhibited by anti–MHC class I but not anti–MHC class II antibodies and was blocked by Concanamycin A. Lysis of autologous MPC was blocked by competition with autologous HM1.24-transfected dendritic cells (10:1 ratio with autologous MPC). Unmanipulated, or control plasmid–transfected dendritic cells had no effect on lysis of autologous MPC. Conclusion: Our results indicate that HM1.24 is a promising target for immunotherapy of multiple myeloma.

Humanized anti-interleukin-6 receptor monoclonal antibody induced apoptosis of fresh and cloned human myeloma cells in vitro

We investigated the effect of anti-IL-6 receptor monoclonal antibody (hPM1) on the in vitro proliferation of cloned and freshly isolated myeloma cells from 20 patients with advanced stage multiple myeloma (MM). Humanized PM1 significantly inhibited the growth of a myeloma cell line in a dose-dependent manner and inhibited more than 30% of the proliferation of fresh myeloma cells in 10 of the 19 cases. Flow cytometric analysis using annexin V and 7AAD showed that hPM1 induced apoptosis of myeloma cells. These observations suggest the possibility of using hPM1 for treating some patients with MM whose growth depends on IL-6.

Production of inteleukin 6 by human spleen cells stimulated with streptococcal preparation OK-432

The streptococcal preparation OK-432 was tested for the ability to stimulate human spleen leukocytes (SPL) for generation of interleukin 6 (IL-6). When SPL were cultured with OK-432 for 24 h in serum-free T medium, the cell-free supernatant induced production of IgM in the SKW6.CL-4 and IgG in the CESS human B cell line, while no such activity was detected in unstimulated SPL culture. The activity was neutralized by treatment with antiserum directed against B cell stimulatory factor 2 (BSF-2). An optimum production of BSF-2 was observed when SPL were stimulated with 10 micrograms/ml of OK-432. The culture supernatant also induced proliferation of IL-6-dependent murine hybridoma MH-60.BSF2 (hybridoma growth factor; HGF). It is thus evident that the molecule produced by OK-432-activated human SPL is BSF-2/HGF/IL-6. These results indicate that the antitumor agent OK-432 stimulates human spleen cells to produce IL-6.

Humanization of an anti-human IL-6 mouse monoclonal antibody glycosylated in its heavy chain variable region

Interleukin-6 (IL-6) inhibitors are good potential therapeutic agents in human patients, and anti-IL-6 antibodies are among the best candidates. Here, we have successfully humanized mouse monoclonal antibody SK2, which specifically binds to IL-6 and strongly inhibits IL-6 functions. Since this antibody possesses N-linked carbohydrates on Asn-30 of VH region, which seems to be very close to an antigen-binding site, influence of these carbohydrates on antigen-binding was investigated. A biosensor study showed that the mouse SK2 Fab and its deglycosylated fragments had almost equal Kd (Kon/Koff), 26.8 nM (1.05 x 10(6)/2.81 x 10(-2)) and 24.7 nM (1.28 x 10(6)/3.15 x 10(-2)), respectively. Furthermore, a mutant chimeric SK2 antibody, in which the N-glycosylation site was removed from the VH region, showed a Kd of 11 nM, almost similar to that of the original chimeric SK2 antibody, determined by Scatchard analysis with 125I-IL-6. These data indicate the carbohydrates of mouse SK2 VH region do not significantly influence antigen-binding activity. In the next step, two versions of each humanized SK2 VL and VH regions were carefully designed based on the amino acid sequences of human REI and DAW, respectively. Only one alteration, Tyr to Phe, was made at position 71 in the two light chains, according to the canonical residue for LI. A N-glycosylation site was introduced on the two heavy chains, by changing Ser to Asn at position 30. All four combinations of humanized light and heavy chains could bind to IL-6 as well as the chimeric SK2 antibody. The light chain first version, however, could not efficiently inhibit IL-6 binding to its receptor, indicating the importance of the LI loop conformation for the inhibitory activity of SK2 antibody. In contrast, both versions of the heavy chains were comparable, in yielding good humanized SK2 antibodies, suggesting that the glycosylation of the SK2 VH region has no influence in recreating a functional antigen-binding site in this humanization.

Immunologic abnormality in NZB/W F1 mice. Thymus-independent expansion of B cells responding to interleukin-6.

We have previously reported that B cell abnormality in NZB/W Fl mice developed independently of thymus. Here we examined further whether B cells from NZB/W F1 mice respond to interleukin‐6 (IL‐6), a factor for terminal differentiation of B cells. When freshly isolated splenic B cells were incubated for 5 days in the presence of human IL‐6, an increased production of both IgM and IgG, including anti‐DNA antibody, was evident in NZB/W Fl mice; there was no increase in BALB/c mice. A magnitude of augmentation in IgG but not IgM production by IL‐6 became more apparent in older NZB/W F1 mice. The increased immunoglobulin production seen with IL‐6 was neutralized by treatment with rabbit anti‐recombinant human IL‐6 antibody. As B cells from athymic NZB/W Fl nude mice also responded to IL‐6, it was suggested that B cells in NZB/W Fl mice differentiated into the IL‐6‐responding state in a thymus‐independent manner. This B cell abnormality may be associated with the pathogenesis of autoimmune disease in NZB/W Fl mice.

Mouse anti-human interleukin-6 receptor monoclonal antibody inhibits proliferation of fresh human myeloma cells in vitro.

Interleukin‐6 (IL‐6) is a major growth factor in multiple myeloma. We investigated the effect of mouse anti‐human IL‐6 receptor monoclonal antibody (anti‐IL‐6R mAb) on the in vitro proliferation of freshly isolated myeloma cells from 21 patients to evaluate the therapeutic potential. The addition of anti‐IL‐6R mAb inhibited more than 30% of the spontaneous proliferation of myeloma cells in 9 of 21 cases in a dose‐ (0.1 to 20 /μ/ml) and time‐dependent manner. The inhibitory effects of anti‐IL‐6R mAb did not differ significantly from that of anti‐IL‐6 mAb, and were correlated with the extent of the response of myeloma cells to IL‐6. Flow cytometric analysis showed that all myeloma cells expressed IL‐6R, whose intensity was not correlated with either the extent of response of myeloma cells to IL‐6 or the inhibitory effects of anti‐IL‐6R mAb on proliferation of myeloma cells. Although our study showed heterogeneity in the proliferative responses of myeloma cells to IL‐6 and anti‐IL‐6R mAb, these observations suggest the possibility of using anti‐IL‐6R mAbs for treating some patients with multiple myeloma whose growth depends on IL‐6.

Estimation of interleukin 6 production by reverse transcriptase-polymerase chain reaction in four human myeloma cell lines

Although an autocrine growth mechanism through interleukin 6 has been advocated in human myeloma cells, reports of IL-6 production by cells from established myeloma cell lines are rare. In the present study, we examined whether or not a minute amount of interleukin 6 is produced in 4 human myeloma cell lines. IL-6 production was not detected in any of the 4 lines by enzyme immunoassay, bioassay with two interleukin 6-dependent murine hybridoma cell lines and Northern hybridization. However, we detected interleukin 6 mRNA in one (U266) of the 4 lines by the reverse transcriptase-polymerase chain reaction. Nevertheless, the proliferation of all 4 lines was not inhibited by an anti-interleukin 6 antibody. These results suggest that autocrine stimulation by interleukin 6 is not involved in the majority of human myeloma cell lines.