Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2)

Losman, Michele J.; Hansen, Hans J.; Dworak, Heather; Krishnan, Indira; Qu, Zhengxing; Shih, Lisa B.; Li, Zeng; Goldenberg, David M.; Leung, Shui-on

doi:10.1002/(sici)1097-0142(19971215)80:12+<2660::aid-cncr43>3.0.co;2-7

Cited by 13 publications

(11 citation statements)

References 25 publications

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“…Development of hLL2, the humanized anti-CD22 MAb, now referred to as epratuzumab, has been described previously (20,21). Similar procedures were adopted to develop the humanized anti-CD20 MAb, designated as IMMU-106, or hA20.…”

Section: Antibodiesmentioning

confidence: 99%

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Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma

Stein

Qu²,

Chen³

et al. 2004

Clinical Cancer Research

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153

124

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Purpose: A new humanized anti-CD20 monoclonal antibody (MAb), IMMU-106, was evaluated to elucidate its action as an antilymphoma therapeutic, as a single agent, and in combination with the anti-CD22 MAb, epratuzumab.Experimental Design: Antiproliferative effects, apoptotic effects, and the ability of IMMU-106 to mediate complement-mediated cytotoxicity and antibody-dependent cellular cytotoxicity on a panel of non-Hodgkin's lymphoma (NHL) cell lines were compared with the chimeric anti-CD20 MAb, rituximab, and evaluated in light of the various levels of antigen expression by the cell lines. In vivo therapy studies were performed in SCID mice bearing disseminated Raji lymphoma.Results: The mechanisms of cytotoxicity of IMMU-106 were found to be similar to rituximab, and include direct apoptosis, antibody-dependent cellular cytotoxicity, and complement-mediated cytotoxicity. IMMU-106 was also found to be very similar to rituximab in terms of antigenbinding specificity, binding avidity, and dissociation constant. Treatment of Raji-bearing SCID mice with IMMU-106 yielded median survival increases of up to 4.2-fold compared with control mice. Survival in mice treated with IMMU-106 plus epratuzumab was compared with IMMU-106 treatment alone. Although the combined treatment did not improve median survival, an increased proportion of long-term survivors was observed. An enhanced antiproliferative effect was also observed in vitro in SU-DHL-6 cells when IMMU-106 was combined with epratuzumab. These findings are consistent with the up-regulation of CD22 expression observed after pretreatment of NHL cells in vitro with CD20 MAb (IMMU-106).Conclusions: It is expected that in humans IMMU-106 should be at least as effective as rituximab and, due to its human framework construction, it may exhibit different pharmacokinetic, toxicity, and therapy profiles. In addition, it may be possible to enhance efficacy by combination therapy comprised of anti-CD20 and other B-cell lineage targeting MAbs, such as epratuzumab. The current results emphasize that in vitro as well as in vivo studies with many of the NHL cell lines were generally predictive of the known activity of anti-CD20 MAbs in NHL patients, as well as the enhanced efficacy of epratuzumab combined with rituximab observed in early clinical trials.

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Section: Antibodiesmentioning

confidence: 99%

“…IMMU-106 was expressed in Sp2/0-Ag14 cells (American Type Culture Collection). A high-level IMMU-106-producing clone was developed as described (21). Both epratuzumab and IMMU-106 were produced in bioreactors and purified by a combination of affinity chromatography on Protein A columns and gel filtration on SE columns under GMP compliance.…”

Section: Antibodiesmentioning

confidence: 99%

Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma

Stein

Qu²,

Chen³

et al. 2004

Clinical Cancer Research

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153

124

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“…Some subcloning procedures used pGem3Z vector (Promega, Madison, WI). The genes encoding the hBS14 polypeptides were ultimately engineered into the mammalian expression vector pdHL2, which permits the amplification of antibody production (15,16). The pdHL2 vector contains the genes for immunoglobulin G (IgG) constant regions (C H and C K ) and was originally designed to accept variable domain cassettes and direct the synthesis of whole IgG.…”

Section: Methodsmentioning

confidence: 99%

“…Sp2/0-Ag14 mouse myeloma cells have been used previously in conjunction with the pdHL2 expression vector for highlevel expression of recombinant IgG (16). The hBS14-pdHL2 DNA vector was linearized by digestion with EcoRI restriction endonuclease and successfully transfected into Sp2/0-Ag14 (4 Â 10 6 cells) by electroporation (450 V, 25 AF).…”

Section: Methodsmentioning

confidence: 99%

Pretargeting of Carcinoembryonic Antigen–Expressing Cancers with a Trivalent Bispecific Fusion Protein Produced in Myeloma Cells

Rossi

Chang

Losman

et al. 2005

Clinical Cancer Research

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Purpose:To characterize a novel trivalent bispecific fusion protein and evaluate its potential utility for pretargeted delivery of radionuclides to tumors. Experimental Design: hBS14, a recombinant fusion protein that binds bispecifically to carcinoembryonic antigen (CEA) and the hapten, histamine-succinyl-glycine (HSG), was produced by transgenic myeloma cells and purified to near homogeneity in a single step using a novel HSG-based affinity chromatography system. Biochemical characterization included sizeexclusion high-performance liquid chromatography (SE-HPLC), SDS-PAGE, and isoelectric focusing. Functional characterization was provided by BIAcore and SE-HPLC.The efficacy of hBS14 for tumor pretargeting was evaluated in CEA-expressing GW-39 human colon tumor^bearing nude mice using a bivalent HSG hapten (IMP-241) labeled with 111 In.Results: Biochemical analysis showed that single-step affinity chromatography provided highly purified material. SE-HPLC shows a single protein peak consistent with the predicted molecular size of hBS14. SDS-PAGE analysis shows only two polypeptide bands, which are consistent with the calculated molecular weights of the hBS14 polypeptides. BIAcore showed the bispecific binding properties and suggested that hBS14 possesses two functional CEA-binding sites. This was supported by SE-HPLC immunoreactivity experiments. All of the data suggest that the structure of hBS14 is an 80 kDa heterodimer with one HSG and two CEA binding sites. Pretargeting experiments in the mouse model showed high uptake of radiopeptide in the tumor, with favorable tumorto-nontumor ratios as early as 3 hours postinjection. Conclusions:The results indicate that hBS14 is an attractive candidate for use in a variety of pretargeting applications, particularly tumor therapy with radionuclides and drugs.

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“…The amplified PCR product was cloned in the TOPO-TA cloning vector (Invitrogen) and confirmed by DNA sequencing. SacII-EagI fragment containing the heavy chain constant region of IgG 1 in the expression vector hLL2-pdHL2 31 was replaced with SacII-EagI of TOPO-TA-IgG 4 plasmid to obtain the vector pdHL2-hLL2-␥4. The mutation (S228P) was then introduced into pdHL2-hLL2-␥4 by replacing PstI-StuI fragment with a synthetic PstI-StuI fragment (56 bp) containing the mutated sequence, resulting in the vector pdHL2-hLL2-␥4P.…”

Section: Construction Of Igg 4 Constant Region Containing a Proline Mmentioning

confidence: 99%

Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin

Chang

Sapra

Vanama

et al. 2005

Blood

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Ranpirnase (Rap) is a cytotoxic ribonuclease (RNase) isolated from frog oocytes. Here we describe high antitumor activity of a novel immunotoxin, 2L-Rap-hLL1-␥4P, composed of 2 Rap molecules, each fused to the N terminus of the light chain of hLL1, an internalizing anti-CD74 humanized antibody. To reduce unwanted side effects, the constant region of hLL1 was changed from ␥1 to ␥4 and further to ␥4P by replacing serine 228 to proline to prevent the formation of a half immunoglobulin G (IgG) common for IgG 4 . In vitro, 2L-Rap-hLL1-␥4P retained RNase activity, specific binding to CD74, and was significantly more potent against CD74 ؉ cell lines (Daudi, Raji, and MC/CAR) than naked hLL1. In vivo, the pharmacokinetic profile of 2L-Rap-hLL1-␥4P was similar to that of naked hLL1. The maximum tolerated dose of 2L-Rap-hLL1-␥4P in severe combined immunodeficient mice (SCID) or BALB/c mice was 50 g per mouse. In Raji and Daudi Burkitt lymphoma xenograft models, treatment with a single 5 to 50 g dose of 2L-Rap-hLL1-␥4P, given as early or delayed treatment, resulted in cures of most animals. Treatment with 2L-Rap-hLL1-␥4P was significantly better than all controls, including saline, naked hLL1, and nonspecific immunotoxin. IntroductionRanpirnase (Rap), a monomeric protein (Mr, 11 817; 104 amino acids), is an amphibian ribonuclease (RNase) belonging to the RNase A superfamily. 1 Native Rap, isolated from Rana pipiens eggs, 2 demonstrated significant cytostatic and cytotoxic effects on a variety of tumor cell lines in vitro 3 and in vivo. 4 Rap has a low affinity (more than 1 M) for RNase inhibitor (RI), which constitutes about 0.01% of the cytosolic protein in mammalian cells, 1 and therefore could evade inactivation by RI. As indicated by its resistance to both protease degradation and denaturation at elevated temperatures, 5 Rap is highly stable. Its enzymatic activity requires an N-terminal pyroglutamyl residue. 6 Recombinant Rap (rRap) expressed in Escherichia coli with methionine (Met) at the N terminus (Met [Ϫ1]) displays much reduced activity, 7 but glycosylation of rRap (expressed in Pichia pastoris) increases its conformational stability and toxicity to cancer cells. 8 Rap enters cells via receptor-mediated endocytosis. 9 Once internalized into the cytosol, it selectively degrades tRNA, 10,11 resulting in inhibition of protein synthesis and induction of apoptosis. 10 Immunotoxins consisting of Rap and LL2, an internalizing anti-CD22 murine monoclonal antibody (mAb), 12 have been prepared by chemical conjugation and shown to have potent and specific antitumor effects against CD22 ϩ cells both in vitro and in vivo. 13 Rap was studied in the treatment of patients with unresectable malignant mesothelioma, 14 where the reversible dose-limiting toxicity was renal and where there appeared to be no immunogenicity. 15 These encouraging results prompted us to develop a series of novel recombinant immunotoxins involving Rap and other internalizing mAbs. 16,17 CD74 (invariant chain, Ii) is a type II transmembrane glycoprotein...

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Generation of a high-producing clone of a humanized anti-B-cell lymphoma monoclonal antibody (hLL2)

Cited by 13 publications

References 25 publications

Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma

Characterization of a New Humanized Anti-CD20 Monoclonal Antibody, IMMU-106, and Its Use in Combination with the Humanized Anti-CD22 Antibody, Epratuzumab, for the Therapy of Non-Hodgkin’s Lymphoma

Pretargeting of Carcinoembryonic Antigen–Expressing Cancers with a Trivalent Bispecific Fusion Protein Produced in Myeloma Cells

Effective therapy of human lymphoma xenografts with a novel recombinant ribonuclease/anti-CD74 humanized IgG4 antibody immunotoxin

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