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Blood Oct 2010We have generated hexavalent antibodies (HexAbs) comprising 6 Fabs tethered to one Fc of human IgG1. Three such constructs, 20-20, a monospecific HexAb comprising 6 Fabs...
Multiple signaling pathways induced by hexavalent, monospecific, anti-CD20 and hexavalent, bispecific, anti-CD20/CD22 humanized antibodies correlate with enhanced toxicity to B-cell lymphomas and leukemias.
We have generated hexavalent antibodies (HexAbs) comprising 6 Fabs tethered to one Fc of human IgG1. Three such constructs, 20-20, a monospecific HexAb comprising 6 Fabs of veltuzumab (humanized anti-CD20 immunoglobulin G1κ [IgG1κ]), 20-22, a bispecific HexAb comprising veltuzumab and 4 Fabs of epratuzumab (humanized anti-CD22 IgG1κ), and 22-20, a bispecific HexAb comprising epratuzumab and 4 Fabs of veltuzumab, were previously shown to inhibit pro-liferation of several lymphoma cell lines at nanomolar concentrations in the absence of a crosslinking antibody. We now report an in-depth analysis of the apoptotic and survival signals induced by the 3 HexAbs in Burkitt lymphomas and provide in vitro cytotoxicity data for additional lymphoma cell lines and also chronic lymphocytic leukemia patient specimens. Among the key findings are the significant increase in the levels of phosphorylated p38 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) by all 3 HexAbs and the notable differences in the signaling events triggered by the HexAbs from those incurred by crosslinking veltuzumab or rituximab with a secondary antibody. Thus, the greatly enhanced direct toxicity of these HexAbs correlates with their ability to alter the basal expression of various intracellular proteins involved in regulating cell growth, survival, and apoptosis, with the net outcome leading to cell death.
Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Humans; Leukemia; Lymphoma, B-Cell; MAP Kinase Signaling System; Mitochondrial Membranes; NF-kappa B; PTEN Phosphohydrolase; Proto-Oncogene Proteins c-bcl-2; Rituximab; Sialic Acid Binding Ig-like Lectin 2; Signal Transduction
PubMed: 20628151
DOI: 10.1182/blood-2010-03-276857 -
Cancer Feb 2010Radioimmunotherapy of non-Hodgkin lymphoma comprises a (90)Y- or (131)I-labeled murine anti-CD20 IgG, but both agents also include a substantial dose of unlabeled...
Radioimmunotherapy of non-Hodgkin lymphoma comprises a (90)Y- or (131)I-labeled murine anti-CD20 IgG, but both agents also include a substantial dose of unlabeled anti-CD20 IgG given immediately before the radioconjugate to reduce its uptake in the spleen (primary normal B-cell antigen sink); this extends its plasma half-life and improves tumor visualization. Thus, these treatments combine an effective anti-CD20 radioconjugate with an unconjugated anti-CD20 antibody that is also therapeutically active, but the large anti-CD20 IgG predose ( approximately 900 mg) may diminish the tumor localization of the radioimmunoconjugate (eg, 10-35 mg). We have examined alternative approaches that enhance radionuclide targeting and improve antitumor responses. One uses a (90)Y-labeled anti-CD22 IgG (epratuzumab) combined with an antibody therapy regimen of a humanized anti-CD20 IgG (veltuzumab). Pretargeted radionuclide therapy using a trivalent, humanized, recombinant bispecific anti-CD20 antibody with a (90)Y-hapten-peptide is another highly effective method that is also less toxic than directly radiolabeled IgG. Finally, all approaches benefit from the addition of a consolidation-dosing regimen of the anti-CD20 IgG antibody. This article reviews these various options and discusses how some fundamental changes could potentially enhance the response and duration from radionuclide-targeted therapy.
Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, CD20; Humans; Immunoconjugates; Indium Radioisotopes; Lymphoma, Non-Hodgkin; Mice; Mice, Nude; Radioimmunotherapy
PubMed: 20127947
DOI: 10.1002/cncr.24802 -
Blood Oct 2009Interferon-alpha (IFN-alpha) has direct inhibitory effects on some tumors and is a potent stimulator of both the innate and adaptive immune systems. A tumor-targeting...
Interferon-alpha (IFN-alpha) has direct inhibitory effects on some tumors and is a potent stimulator of both the innate and adaptive immune systems. A tumor-targeting antibody-IFN-alpha conjugate (mAb-IFN-alpha) could kill by direct actions of the monoclonal antibody (mAb) and IFN-alpha on tumor cells and also potentiate a tumor-directed immune response. The modular Dock-and-Lock method (DNL) was used to generate 20-2b, the first immunocytokine having 4 cytokine (IFN-alpha2b) groups that are fused to the humanized anti-CD20 mAb, veltuzumab. Additional mAb-IFN-alpha constructs, each retaining potent IFN-alpha2b biologic activity, also were produced by DNL. The 20-2b shows enhanced antibody-dependent cellular cytotoxicity compared with veltuzumab but lacks complement-dependent cytotoxicity. The 20-2b inhibits in vitro proliferation of lymphoma cells and depletes them from whole human blood more potently than the combination of veltuzumab and a nontargeting, irrelevant, mAb-IFN-alpha. The 20-2b demonstrated superior therapeutic efficacy compared with veltuzumab or nontargeting mAb-IFN-alpha in 3 human lymphoma xenograft models, even though mouse immune cells respond poorly to human IFN-alpha2b. Targeting IFN-alpha with an anti-CD20 mAb makes the immunocytokine more potent than either agent alone. These findings suggest that 20-2b merits clinical evaluation as a new candidate antilymphoma therapeutic.
Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antineoplastic Agents; Humans; Interferon alpha-2; Interferon-alpha; Lymphoma, B-Cell; Male; Mice; Recombinant Fusion Proteins; Recombinant Proteins; Xenograft Model Antitumor Assays
PubMed: 19710501
DOI: 10.1182/blood-2009-06-228890 -
Blood Jun 2009The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a...
The dock and lock (DNL) method is a new technology for generating multivalent antibodies. Here, we report in vitro and in vivo characterizations of 20-22 and 22-20, a pair of humanized hexavalent anti-CD20/22 bispecific antibodies (bsAbs) derived from veltuzumab (v-mab) and epratuzumab (e-mab). The 22-20 was made by site-specific conjugation of e-mab to 4 Fabs of v-mab; 20-22 is of the opposite configuration, composing v-mab and 4 Fabs of e-mab. Each bsAb translocates both CD22 and CD20 into lipid rafts, induces apoptosis and growth inhibition without second-antibody crosslinking, and is significantly more potent in killing lymphoma cells in vitro than their parental antibodies. Although both bsAbs triggered antibody-dependent cellular toxicity, neither displayed complement-dependent cytotoxicity. Intriguingly, 22-20 and 20-22 killed human lymphoma cells in preference to normal B cells ex vivo, whereas the parental v-mab depleted malignant and normal B cells equally. In vivo studies in Daudi tumors revealed 20-22, despite having a shorter serum half-life, had antitumor efficacy comparable with equimolar v-mab; 22-20 was less potent than 20-22 but more effective than e-mab and control bsAbs. These results indicate multiple advantages of hexavalent anti-CD20/22 bsAbs over the individual parental antibodies and suggest that these may represent a new class of cancer therapeutics.
Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antibody-Dependent Cell Cytotoxicity; Antigens, CD20; Apoptosis; Calcium; Caspases; Cell Proliferation; Cytotoxicity, Immunologic; Female; Humans; Immunotherapy; Lymphoma, B-Cell; Mice; Mice, SCID; Sialic Acid Binding Ig-like Lectin 2; Survival Rate
PubMed: 19372261
DOI: 10.1182/blood-2008-10-187138 -
Journal of Nuclear Medicine : Official... Mar 2009We determined whether therapeutic responses using a bispecific antibody that pretargeted (90)Y-hapten-peptide radioimmunotherapy or a directly radiolabeled, humanized,... (Comparative Study)
Comparative Study
UNLABELLED
We determined whether therapeutic responses using a bispecific antibody that pretargeted (90)Y-hapten-peptide radioimmunotherapy or a directly radiolabeled, humanized, (90)Y-anti-CD20 IgG (veltuzumab) could be improved by combining these treatments with unlabeled humanized antibodies against CD22 (epratuzumab), CD74 (milatuzumab), or veltuzumab.
METHODS
Nude mice bearing established subcutaneous Ramos human Burkitt lymphoma were treated with antibodies alone or in combination with pretargeted radioimmunotherapy (PT-RAIT) or radioimmunotherapy, and tumor growth was monitored. Biodistribution studies examined the effect that predosing with unlabeled veltuzumab had on radioimmunotherapy and PT-RAIT targeting.
RESULTS
None of the unconjugated antibodies was effective against established and rapidly growing xenografts, but PT-RAIT, at approximately 30% of its maximum tolerated dose, and radioimmunotherapy alone, at its maximum tolerated dose, were able to arrest growth and even entirely ablate tumors in some animals. Only combinations with veltuzumab improved therapeutic responses, most significantly when a veltuzumab regimen (weekly, 1.0 mg followed by 3 x 0.5 mg) was initiated 1 wk after PT-RAIT or (90)Y-veltuzumab. Biodistribution data indicated that when unlabeled veltuzumab (1.0 or 0.25 mg) was administered in advance of the radiolabeled veltuzumab or bispecific antibody injection, tumor uptake was significantly reduced ((111)In-veltuzumab, 47% and 25%, respectively; (111)In-hapten-peptide, 74% and 49%, respectively). Despite an approximately 50% decrease in radioactivity uptake in the tumor, antitumor responses were not diminished significantly for (90)Y-veltuzumab, and in the case of PT-RAIT responses were improved. However, higher amounts of predosed veltuzumab reduced the effects of PT-RAIT.
CONCLUSION
These studies suggest that administering unlabeled anti-CD20 IgG therapy after the radioactivity dose provides the best efficacy and that the amount of unlabeled anti-CD20 IgG administered as a predose to anti-CD20-targeted radionuclide therapy should be minimized.
Topics: Animals; Antibodies, Bispecific; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens, CD20; Antigens, Differentiation, B-Lymphocyte; Burkitt Lymphoma; Drug Therapy, Combination; Histocompatibility Antigens Class II; Humans; Indium Radioisotopes; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Transplantation; Radioimmunotherapy; Radiopharmaceuticals; Sialic Acid Binding Ig-like Lectin 2; Transplantation, Heterologous; Yttrium Radioisotopes
PubMed: 19223402
DOI: 10.2967/jnumed.108.058602 -
Blood Jan 2009Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st... (Comparative Study)
Comparative Study
Veltuzumab is a humanized anti-CD20 monoclonal antibody with complementarity-determining regions (CDRs) identical to rituximab, except for one residue at the 101st position (Kabat numbering) in CDR3 of the variable heavy chain (V(H)), having aspartic acid (Asp) instead of asparagine (Asn), with framework regions of epratuzumab, a humanized anti-CD22 antibody. When compared with rituximab, veltuzumab has significantly reduced off-rates in 3 human lymphoma cell lines tested, as well as increased complement-dependent cytotoxicity in 1 of 3 cell lines, but no other in vitro differences. Mutation studies confirmed that the differentiation of the off-rate between veltuzumab and rituximab is related to the single amino acid change in CDR3-V(H). Studies of intraperitoneal and subcutaneous doses in mouse models of human lymphoma and in normal cynomolgus monkeys disclosed that low doses of veltuzumab control tumor growth or deplete circulating or sessile B cells. Low- and high-dose veltuzumab were significantly more effective in vivo than rituximab in 3 lymphoma models. These findings are consistent with activity in patients with non-Hodgkin lymphoma given low intravenous or subcutaneous doses of veltuzumab. Thus, changing Asn(101) to Asp(101) in CDR3-V(H) of rituximab is responsible for veltuzumab's lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.
Topics: Amino Acid Substitution; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antineoplastic Agents; B-Lymphocytes; Cell Line, Tumor; Chlorocebus aethiops; Complement System Proteins; Complementarity Determining Regions; Disease Models, Animal; Drug Screening Assays, Antitumor; Humans; Lymphoma; Mice; Mutation, Missense; Rituximab; Structure-Activity Relationship
PubMed: 18941114
DOI: 10.1182/blood-2008-07-168146 -
Blood Feb 2008Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly...
Combination immunotherapy with anti-CD20 and anti-CD22 mAbs shows promising activity in non-Hodgkin lymphoma. Therefore, bispecific mAbs (bsAbs) were recombinantly constructed from veltuzumab (humanized anti-CD20) and epratuzumab (humanized anti-CD22) and evaluated in vitro and in vivo. While none of the parental mAbs alone or mixed had notable antiproliferative activity against Burkitt lymphoma cells when not cross-linked, the bsAbs [eg, anti-CD20 IgG-anti-CD22 (scFv)(2)] were inhibitory without cross-linking and synergistic with B-cell antigen (BCR)-mediated inhibition. The bsAbs demonstrated higher antibody-dependent cellulary cytoxicity (ADCC) activity than the parental mAbs, but not complement-dependent cytoxicity (CDC) of the parental CD20 mAb. Cross-linking both CD20 and CD22 with the bsAbs resulted in the prominent redistribution of not only CD20 but also CD22 and BCR into lipid rafts. Surprisingly, appreciable translocation of CD22 into lipid rafts was also observed after treatment with epratuzumab. Finally, the bsAbs inhibited Daudi lymphoma transplant growth, but showed a significant advantage over the parental anti-CD20 mAb only at the highest dose tested. These results suggest that recombinantly fused, complementary, bispecific, anti-CD20/22 antibodies exhibit functional features distinct from their parental antibodies, perhaps representing new candidate therapeutic molecules.
Topics: Antibodies, Bispecific; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Burkitt Lymphoma; Cell Division; Cell Line; Humans; Immunotherapy; Lymphoma, B-Cell; Rituximab; Sialic Acid Binding Ig-like Lectin 2
PubMed: 18025153
DOI: 10.1182/blood-2007-08-110072 -
Cancer Oct 2005Intestinal metaplasia (IM) is often a component of Barrett esophagus (BE) and is characterized by a distinctive type of epithelium. Because IM has the potential to...
BACKGROUND
Intestinal metaplasia (IM) is often a component of Barrett esophagus (BE) and is characterized by a distinctive type of epithelium. Because IM has the potential to progress to dysplasia or adenocarcinoma, an accurate identification of its presence is important clinically. A cytopathologic diagnosis of IM on esophageal brushings by morphology alone can be difficult. The authors investigated the role of hepatocyte paraffin 1 (Heppar-1) immunostaining as a possible marker of IM in cytologic samples of BE.
METHODS
Eleven samples each of BE with and without IM diagnosed on esophageal brushings were retrieved from the cytopathology files of The Johns Hopkins Hospital over an 8-year period (1993-2001). All 22 specimens were confirmed histologically on subsequent tissue biopsies. Slides initially were prepared by cytospin and stained with Papanicolaou stain. After destaining, the slides were immunostained with hepatocyte paraffin 1 (Heppar-1) antibody at 1:100 dilution employing conventional methodology.
RESULTS
Among the samples of BE with IM, 9 of 11 samples (82%) were immunoreactive for Heppar-1, compared with 0 of 11 specimens of BE with only cardiac-type metaplasia. The immunoexpression was cytoplasmic and granular and was seen only focally in the glandular fragments.
CONCLUSIONS
Heppar-1 was a moderately sensitive (82%) and highly specific (100%) immunomarker for IM in BE. It was easy to use in limited cytologic samples that originally were stained with Papanicolaou stain and is recommended for inclusion in morphologically difficult samples of BE with questionable IM. The immunostaining pattern predominantly was focal, necessitating a careful evaluation of positive reactions on cytologic samples.
Topics: Adenocarcinoma; Aged; Aged, 80 and over; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Antigens; Barrett Esophagus; Biomarkers; Cytological Techniques; Esophageal Neoplasms; Hepatocytes; Humans; Immunoassay; Metaplasia; Middle Aged; Retrospective Studies; Sensitivity and Specificity
PubMed: 15918175
DOI: 10.1002/cncr.21099