So Ohta

Engineered antibodies of IgG1/IgG3 mixed isotype with enhanced cytotoxic activities.

Enhancement of multiple effector functions of an antibody may be a promising approach for antibody therapy. We have previously reported that fucose removal from Fc-linked oligosaccharides greatly enhances antibody-dependent cellular cytotoxicity (ADCC) of therapeutic antibodies. Here, we report a unique approach to enhance complement-dependent cytotoxicity (CDC), another important effector function of antitumor antibodies, by using engineered constant region of human IgG1/IgG3 chimeric isotypes. We systematically shuffled constant domains of IgG1 and IgG3 to generate a comprehensive set of mixed chimeric isotypes of anti-CD20 antibodies. Among these, the variant 1133, consisting of the CH1 and the hinge each from IgG1 and the Fc from IgG3, was unexpectedly found to exhibit markedly enhanced CDC that exceeded wild-type levels. However, it lacked protein A-binding capacity, an important feature for the industrial production. To eliminate this deficiency, a portion in COOH-terminal CH3 domain of 1133 was substituted with IgG1, resulting in full recovery of protein A binding without compromising the enhanced CDC and ADCC activities. The CDC-enhancing effect using a chimeric isotype was also shown in CD52 antigen/antibody system. The ADCC activity of the variants was also maximized by the absence of fucose from its carbohydrate structure, a phenomenon that has previously been observed for wild-type antibodies. Enhanced cytotoxicity of a variant was confirmed in a cynomolgus monkey model. These findings suggest that the variant antibodies with IgG1/IgG3 chimeric constant regions and nonfucosylated oligosaccharides that possess dual-enhanced cytotoxic functions may be an improvement for the next generation of therapeutic antitumor antibodies.

Enhanced tumor cell selectivity of adriamycin-monoclonal antibody conjugate via a poly(ethylene glycol)-based cleavable linker

A novel linker consisting of poly(ethylene glycol) (PEG) and dipeptide was used for conjugation of adriamycin with tumor-specific monoclonal antibody, NL-1, to confirm that the linker can be cleaved selectively with the tumor specific enzyme to express cytotoxicity of the anti-tumor agent. Initially, adriamycin-conjugated PEG linkers through different amino acid compositions, alanyl-valine (Ala-Val), alanyl-proline (Ala-Pro), and glycyl-proline (Gly-Pro) sequences, were prepared to confirm selective digestion with model enzymes. Adriamycin was released by particular model endoproteases, thermolysin and proline endopeptidase, from the linkers with different efficiency. When conjugates were prepared using these adriamycin-bound linkers, conjugates had no loss of binding affinity and specificity for common acute lymphoblastic leukemia antigen (CALLA) expressed on the Daudi cell surfaces as the target of NL-1 antibody. In addition, adriamycin release from the conjugates was also confirmed by incubating them with specific proteases. Tumor cell growth was inhibited dose-dependently for the conjugates carrying Ala-Val and Gly-Pro linkers, whereas significant inhibitory effect was abolished for the conjugate carrying Ala-Pro linker, indicating that cytotoxic effect can be controlled by specificity of antibody and composition of linker peptide. IC(50) for Ala-Val linked conjugate was approximately 3.5 microg/ml and that for Gly-Pro linked conjugate was 5.2 microg/ml. PEG-dipeptidyl linker demonstrated here will be an effective tool for the preparation of immunoconjugate, especially specific activation of anti-tumor agents at desired tumor tissues.

Synthesis of a novel duocarmycin derivative DU-257 and its application to immunoconjugate using poly(ethylene glycol)-dipeptidyl linker capable of tumor specific activation.

Novel anti-tumor agent, duocarmycin derivative DU-257, was designed and synthesized to prepare immunoconjugate in order to confirm the feasibility of enzymatically cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine. Oxyethylamine arm was introduced at 4-methoxy position of segment B of DU-86 to form DU-257 and evaluated its property. DU-257 retained similar stability and potency with DU-86 while enhanced hydrophilicity suggested. DU-257 was condensed to the PEG-dipeptidyl linker through carboxyl terminal of dipeptide, and enzymatic release of DU-257 using a model enzyme, thermolysin, similar enzyme of which was shown to be overexpressed at various tumor sites, was evaluated by HPLC analysis. Cleavage between the linker amino acids by the model protease and release of DU-257 as valine conjugated form was confirmed. The enzymatically released form of DU-257 expressed its cytotoxicity without loss of the potency for HeLaS3 and SW1116 tumor cell lines, although the efficacy was different in individual cells. DU-257 was then conjugated through the linker to KM231 monoclonal antibody specifically reactive to GD3 antigen which was shown to be expressed on the surface of many malignant tumors such as SW1116. The conjugate retained its binding specificity for SW1116 cell with a similar activity with KM231. Furthermore, the conjugate showed significant growth inhibition on SW1116 cell at a concentration of 75 microg/mL while no effect on antigen negative cell, HeLaS3. These results suggest that the conjugate retained its anti-tumor effect only when it bound on and was activated at the target cell, simultaneously. DU-257 will be one of the candidate of anti-tumor agent for application to immunoconjugate and its conjugate with KM231 via PEG-dipeptidyl linker will be a useful entity for cancer therapy related to sLe(a) expression.

A mouse/human chimeric anti-(ganglioside GD3) antibody with enhanced antitumor activities

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin has been focused on as a target molecule for passive immunotherapy. We have cloned the cDNA encoding the immunoglobulin light and heavy chains of an anti-GD3 monoclonal antibody KM641 (murine IgG3, κ), and constructed the chimeric genes by linking the cDNA fragments of the murine light and heavy variable regions to cDNA fragments of the human κ and γ1 constant regions, respectively. The transfer of these cDNA constructs into SP2/0 mouse myeloma cells resulted in the production of the chimeric antibody, designated KM871, that retained specific binding activity to GD3. Indirect immunofluorescence revealed the same staining pattern for chimeric KM871 and the mouse counterpart KM641 on GD3-expressing melanoma cells. When human serum and human peripheral blood mononuclear cells were used as effectors in complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity respectively, the chimeric KM871 was more effective in killing GD3-expressing tumor cells than was the mouse counterpart KM641. Intravenous injection of chimeric KM871 markedly suppressed tumor growth in nude mice. The chimeric KM871, having enhanced antitumor activities and less immunogenicity than the mouse counterpart, would be a useful agent for passive immunotherapy of human cancer.

Synthesis and HPLC analysis of enzymatically cleavable linker consisting of poly(ethylene glycol) and dipeptide for the development of immunoconjugate.

A model compound of anti-tumor agent, segment B of duocarmycin derivative DU-86, was conjugated to tumor-specific antibody via a cleavable linker consisting of poly(ethylene glycol) (PEG) and dipeptide, L-alanyl-L-valine (Ala-Val), to confirm the feasibility of the linker for application to immunoconjugate. The release of segment B from the linker was evaluated by HPLC analysis. When segment B was derivatized to have an amino residue and then linked to PEG through a dipeptide, segment B was cleaved at the peptide bond by a particular enzyme, thermolysin (EC 3.4.24.4), but not by plasmin (EC 3.4.2 1.7.), indicating that certain protease specifically expressed at the tumor site would be capable of peptide-specific digestion and release of anti-tumor agent since a thermolysin-like enzyme has been reported to be expressed at many tumor cells. Furthermore, the results showing that cell extract from G361 human melanoma had an ability to digest the linker peptide while the linker was stable in normal human serum suggested the tumor-specific activation of the conjugated agent. Segment B was conjugated via the linker to murine monoclonal antibody KM641 reactive to GD3 ganglioside to form immunoconjugate and the quantitative release of segment B under the treatment with the enzyme was also confirmed. These results indicate the possibility of double targeting based on both the recognition ability of tumor specific antibody and tumor specific activation of the anti-tumor agents to enhance tumor treatment efficacy and to decrease unwanted side effects.

Antitumor effects of a novel monoclonal antibody with high binding affinity to ganglioside GD3

Ganglioside GD3, which is one of the major gangliosides expressed on the cell surface human tumors of neuroectodermal origin, has been studied as a target molecule for passive immunotherapy. We established ten kinds of anti-GD3 monoclonal antibodies (mAb) of the mouse IgG3 subclass by immunization with purified GD3 and melanoma cells. One of the established mAb, KM641, showed major reactivity with GD3 and minor reactivity with GQ1b out of 11 common gangliosides in an enzymelinked immunosorbent assay. Immunostaining of gangliosides, separated on thin-layer chromatography plates, using KM641 revealed that most of the melanoma cell lines contained immunoreactive GD3 and GD3-lactone at a high level, but only the adrenal gland and the urinary bladder out of 21 human normal tissues had immunoreactive GD3. In immunofluorescence, KM641 bound to a variety of living tumor cell lines especially melanoma cells, including some cell lines to which another anti-GD3 mAb R24, established previously, failed to bind. High-affinity binding of KM641 to a tumor cell line was quantified by Scatchard analysis (Kd = 1.9×10−8 M). KM641 exerted tumor-killing activity in the presence of effector cells or complement against melanoma cells expressing GD3 at a high level. Not only natural killer cells but also polymorphonuclear cells were effective as the effector cells in antibody-dependent cellular cytotoxicity. Intravenous injection of KM641 markedly suppressed the tumor growth of a slightly positive cell line, C24.22 (7.2×105 binding sites/cell), as well as a very GD3-positive cell line, G361 (1.9×107 binding sites/cell), inoculated intradermally in nude mice. KM641, characterized by a high binding affinity for GD3, has the potential to be a useful agent for passive immunotherapy of human cancer.

The role of lymphokine-activated cell-associated antigen: III. Inhibition of T-cell activation by monoclonal killer-blocking antibody

The addition of monoclonal killer blocking antibodies (KBA MAb) to cultured T cells resulted in significant inhibition of T-cell responses to concanavalin A (Con A), class I antigen and class II antigen, whereas T-cell responses to phytohemagglutinin are insensitive to KBA MAb. The inhibitory effect of KBA MAb is observed only when KBA MAb is added to the culture at an early time. This indicates that the lymphokine-activated cell-associated antigen (LAA) defined by KBA MAb plays an important role in the early stages of T-cell activation. Con A-induced interleukin 2 (IL-2) receptor acquisition and IL-2 production, both of which are required for the early steps of T-cell activation, were greatly inhibited by KBA MAb. However, KBA MAb did not inhibit the action of IL-2, which is required for later stages of T-cell activation.

Immunoglobulin class switch of anti-ganglioside monoclonal antibody from IgM to IgG.

Ganglioside GM2, which is one of the major gangliosides expressed on cell surface of neuroectodermal-origin human tumors, has been focused on as a target molecule for passive immunotherapy. One of the problems in this area was that monoclonal antibodies (mAbs) raised against GM2 were of IgM class even if donors of B cells were varied in mouse, rat or human. We stimulated two kinds of mice hybridomas having membrane-bound anti-GM2 IgM on their surface with GM2 incorporated in synthetic liposomes in the presence of the mouse thymocytes to accelerate the class switch of immunoglobulins (Igs). After the stimulation, protein A-reactive clones were sorted out using a cell sorter. We finally isolated two class switch variants generating mouse IgG3, designated KM796 and KM750, from original hybridomas producing mouse IgM anti-GM2 mAbs, KM696 and KM697, respectively after over 20-time repetitions of the sorting. ELISA with 11 common gangliosides revealed that one of the variant, KM750, retained the same binding specificity to N-acetyl GM2 as that of the parental IgM, KM697. By ELISA using panel of anti-idiotype (Id) mAbs to anti-GM2 mAbs, KM750 was shown to retain the parental KM697 Id. Another variant KM796 almost lost its activity in purification process in acidic condition and changes in Id were suggested. In immunofluorescence assay, KM750 was confirmed to bind to GM2-expressing tumor cell lines. The class switch hybridoma has been stably cultured with the production of the IgG3-class mAb for more than 22 months.

Therapeutic potential of chimeric anti-(ganglioside GD3) antibody KM871: antitumor activity in xenograft model of melanoma and effector function analysis

Abstract KM871 is a chimeric antibody recognizing ganglioside GD3, which is one of the major gangliosides expressed on the cell surface of human tumors of neuroectodermal origin. This study demonstrates the antitumor activity of KM871 against human melanoma xenografts in nude mice, and analyzes the effector function operating in mice. In a well-established tumor model, KM871 showed antitumor activity against H-15 and SK-MEL-28 human melanoma but not against H-187 and G361 human melanoma when administered intravenously 5 days/week for 2 weeks. The G361 tumor became sensitive when KM871 was first administered on the day of tumor inoculation. In this assay, it was observed that almost all the mice were tumor-free, but a few mice developed tumors. Therefore, we examined the amount and expression pattern of GD3 antigen on G361 tumors escaping from KM871 treatment, but no change was observed. Next we examined the optimal administration schedule for KM871 in mice, using H-15 melanoma. KM871 showed antitumor activity when administered intravenously either 5 days/week for 2 weeks or three biweekly doses. However, the effect of the former schedule was stronger than three biweekly doses. To compare the effector function in humans and mice, we studied the complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity and antibody-dependent macrophage-mediated cytotoxicity of KM871 using complement or effector cells prepared from humans and mice. It was found that the antibody-dependent cell-mediated cytotoxicity exerted by polymorphonuclear cells and antibody-dependent macrophage-mediated cytotoxicity were the only antitumor mechanism of KM871 in mice. However their action was very weak compared with that in humans, and complement-mediated cytotoxicity, which was strong in humans, was not observed in mice. Therefore, the antitumor activity of KM871 against human melanomas evaluated by the nude mouse model might be underestimated. These results indicate that KM871 shows good antitumor activity against GD3-positive human melanoma and the antitumor activity expected in humans might be superior to that of the nude mouse model.

Effect of a chimeric anti-ganglioside GM2 antibody on ganglioside GM2-expressing human solid tumors in vivo

Ganglioside GM2 is expressed on the surface of neuroblastoma and glioblastoma cells, and may also be detected on lung cancer cells. We reported previously that anti‐ganglioside GM2 antibody exhibited strong in vitro anti‐tumor activity against adriamycin‐resistant cancer cells, which overexpressed ganglioside GM2. In the present study, we examined the in vivo anti‐tumor effect of the chimeric anti‐ganglioside GM2 antibody, KM966, against human lung and breast carcinoma cells, SBC‐3 and MCF‐7, and respective adriamycin‐resistant clones, SBC‐3/ADM and AdrR MCF‐7 in BALB/c nu/nu mice. Ratios of tumor volume (T/C) between KM966‐treated group and control group were 0.01 for SBC‐3, 0.00 for SBC‐3/ADM, 0.85 for MCF‐7 and 0.34 for AdrR MCF‐7 cells, respectively. Nude mice, which were pretreated with anti‐asialo GM1 antibody to remove natural killer cells, were transplanted with 4 × 107 of SBC‐3 and SBC‐3/ADM subcutaneously. Seven days later, when tumors had grown to a diameter of over 8 mm, mice began to receive intravenous treatment of 120 μg/mouse KM966 daily. Fourteen daily treatments induced regression to less than 4‐mm diameter in 4/5 SBC‐3 tumors and 5/5 of SBC‐3/ADM tumors. All SBC‐3/ADM tumors disappeared completely, suggesting that KM966 exerts a strong in vivo anti‐tumor effect on ganglioside GM2‐expressing cancer cells. In KM966‐treated mice, the surface of the tumor cells stained positive with anti‐human IgG. In addition, numerous leukocytes had infiltrated into the tumor mass. Antibody‐dependent cell‐mediated cytotoxicity (ADCC) of KM966 against tumor cells was examined in vitro by 51Cr‐release assay and revealed that KM966 induces ADCC activity against ganglioside GM2‐expressing tumors. Our results suggest that immunotherapy using KM966 may be useful for the treatment of ganglioside GM2‐expressing solid tumors. Int. J. Cancer 82:759–764, 1999.