Improving the treatment of non-Hodgkin lymphoma with antibody-targeted radionuclides.

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.

Therapy of B-cell malignancies by anti-HLA-DR humanized monoclonal antibody, IMMU-114, is mediated through hyperactivation of ERK and JNK MAP kinase signaling pathways.

A humanized IgG4 anti-HLA-DR monoclonal antibody (IMMU-114), engineered to avoid side effects associated with complement activation, was examined for binding and cytotoxicity on leukemia, lymphoma, and multiple myeloma cell lines and chronic lymphocytic leukemia (CLL) patient specimens, followed by evaluation of the effects of IMMU-114 on extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signaling pathways. HLA-DR was expressed on the majority of these cells at markedly higher levels than CD20, CD22, and CD74. IMMU-114 was toxic to mantle cell lymphoma, CLL, acute lymphoblastic leukemia, hairy cell leukemia, non-Hodgkin lymphoma (including rituximab-resistant), and multiple myeloma cell lines, and also patient CLL cells.

Pretargeted radioimmunotherapy of pancreatic cancer xenografts: TF10-90Y-IMP-288 alone and combined with gemcitabine.

Unlabelled: Pancreatic cancer is a silent disease that most commonly presents in an already metastatic form. Current treatment options provide little survival benefit. Radiolabeled PAM4 IgG, a monoclonal antibody that recognizes a unique epitope associated with a mucin found almost exclusively in pancreatic cancer, has shown encouraging therapeutic effects in animal models and in early clinical testing ((90)Y-humanized PAM4 IgG, (90)Y-clivatuzumab tetraxetan).

Ceramide regulates gemcitabine-induced senescence and apoptosis in human pancreatic cancer cell lines.

Bioactive sphingolipids are potent intracellular signaling molecules having profound effects on cell death, growth, and differentiation. Pharmacologic manipulation of sphingolipid levels could have a significant effect on the induction of apoptosis by anticancer agents, and thus, improve treatment efficacy. We observed that gemcitabine cannot completely kill AsPc1 and Panc1 human pancreatic cancer cells in culture; even at high concentrations of gemcitabine, 30% to 40% of the cells remain viable.

Induction of apoptosis by cross-linking antibodies bound to human B-lymphoma cells: expression of Annexin V binding sites on the antibody cap.

Unlabelled: There are many reports that cross-linking antibodies (Abs) bound to the surface of B-lymphoma cells can induce apoptosis and/or cell death, especially with anti-CD20 Abs. This study was intended to extend our understanding of these effects. To determine if CD20 is a unique target in this respect, or whether Abs to other antigens would have similar effects, six Abs were tested, with and without cross-linking with a secondary Ab, on three target cell lines.

Combining milatuzumab with bortezomib, doxorubicin, or dexamethasone improves responses in multiple myeloma cell lines.

Purpose: The humanized anti-CD74 monoclonal antibody, milatuzumab, is in clinical evaluation for the therapy of multiple myeloma (MM). The ability of milatuzumab to increase the efficacy of bortezomib, doxorubicin, and dexamethasone was examined in three human CD74+ MM cell lines, CAG, KMS11, KMS12-PE, and one CD74-MM cell line, OPM-2.

Experimental Design: Activity of milatuzumab as a monotherapy and combined with the drugs was evaluated by studying in vitro cytotoxicity, signaling and apoptotic pathways, and in vivo therapeutic activity in severe combined immunodeficient (SCID) mouse models of MM.

Pretargeted versus directly targeted radioimmunotherapy combined with anti-CD20 antibody consolidation therapy of non-Hodgkin lymphoma.

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.

A re-examination of radioimmunotherapy in the treatment of non-Hodgkin lymphoma: prospects for dual-targeted antibody/radioantibody therapy.

Antibody-based therapies, both unconjugated antibodies and radioimmunotherapy, have had a significant impact on the treatment of non-Hodgkin lymphoma. Single-agent rituximab is an effective therapy, but it is being increasingly used with combination chemotherapy to improve the objective response and its duration. The approved anti-CD20 radioimmunoconjugates ((90)Y-ibritumomab tiuxetan or (131)I-tositumomab) have had encouraging results, with trials now seeking to incorporate a radioimmunoconjugate in various settings.

Novel radioimmunopharmaceuticals for cancer imaging and therapy.

As interest in targeted therapies for cancer increases, radionuclides stand out not only for their ability to be detected by external scintigraphy, but also for their therapeutic capacity. Radionuclides need to be directed to the invading cancer however, and in this context antibodies have been at the forefront of targeting radionuclides for more than 50 years. Antibodies have been dissected and reconstituted in many different forms to fit a multitude of functions.

Properties and structure-function relationships of veltuzumab (hA20), a humanized anti-CD20 monoclonal antibody.

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).

Therapy of advanced B-lymphoma xenografts with a combination of 90Y-anti-CD22 IgG (epratuzumab) and unlabeled anti-CD20 IgG (veltuzumab).

Purpose: Antibodies are effective therapeutic agents in cancer, but cures are rarely if ever obtained. Combination therapies are likely to be more effective than a single agent. In this study, the combination of a new unconjugated humanized anti-CD20 IgG, veltuzumab, with a (90)Y-conjugated humanized antibody to CD22 (epratuzumab) was evaluated for the treatment of B-cell lymphoma in a nude mouse model system.

Improved therapeutic results by pretargeted radioimmunotherapy of non-Hodgkin's lymphoma with a new recombinant, trivalent, anti-CD20, bispecific antibody.

We examined whether a pretargeting method using a new recombinant anti-CD20 bispecific antibody (bsMAb) followed by (90)Y-1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid ((90)Y-DOTA)-peptide could reduce hematologic toxicity yet improve therapeutic responses compared with conventional (90)Y-anti-CD20 IgG and a chemically conjugated bsMAb. TF4, a humanized, tri-Fab bsMAb with two Fabs binding CD20 and one Fab binding histamine-succinyl-glycine (HSG), developed by the dock and lock (DNL) method, was tested in nude mice with Ramos B-cell lymphomas. Optimal pretargeting required a 29-h interval between TF4 and (90)Y-DOTA-HSG, and 20-fold more moles of TF4.

A novel bispecific, trivalent antibody construct for targeting pancreatic carcinoma.

Preclinical and clinical studies have demonstrated the application of radiolabeled mAb-PAM4 for nuclear imaging and radioimmunotherapy of pancreatic carcinoma. We have now examined the ability of a novel PAM4-based, bispecific monoclonal antibody (mAb) construct, TF10, to pretarget a radiolabeled peptide for improved imaging and therapy. TF10 is a humanized, bispecific mAb, divalent for mAb-PAM4 and monovalent for mAb-679, reactive against the histamine-succinyl-glycine hapten.

Use of antibodies and immunoconjugates for the therapy of more accessible cancers.

There are currently 6 unconjugated antibodies and 3 immunoconjugates approved for use in the United States in a variety of cancers, with a considerable number of new agents in clinical testing and preclinical development. Unconjugated antibodies alone can be effective, but more often, antibodies need to be combined with chemotherapy, which enhances the efficacy of the standard treatment. Immunoconjugates tend to be more effective than their unconjugated counterparts, but their increased toxicity often restricts when and how they are used.

The mechanism of killing of B-lymphoma cells by 111In-conjugated antibodies.

Purpose: To determine the mode of killing of B-lymphoma cell lines by 111In-conjugated antibodies (Ab).

Materials And Methods: Cells were treated with a range of radiolabeled Ab concentrations to produce a level of killing from 80 - 99.9%, as determined by a long-term proliferation assay.

Cancer Imaging and Therapy with Bispecific Antibody Pretargeting.

This article reviews recent preclinical and clinical advances in the use of pretargeting methods for the radioimmunodetection and radioimmunotherapy of cancer. Whereas directly-labeled antibodies, fragments, and subfragments (minibodies and other constructs) have shown promise in both imaging and therapy applications over the past 25 years, their clinical adoption has not fulfilled the original expectations due to either poor image resolution and contrast in scanning or insufficient radiation doses delivered selectively to tumors for therapy. Pretargeting involves the separation of the localization of tumor with an anticancer antibody from the subsequent delivery of the imaging or therapeutic radionuclide.

Therapy of human carcinoma xenografts with antibodies to EGFr and HER-2 conjugated to radionuclides emitting low-energy electrons.

Purpose: Low-energy electrons (10-50 keV) can be effective and specific cytotoxic agents when delivered to the cell surface by antibodies, because their path length in tissue is comparable to a cell diameter. In this study, we have begun to evaluate the therapeutic potential of antibodies (Abs) conjugated to (111)In against carcinoma xenografts in nude mice.

Methods: Abs to EGFr or HER-2 were labeled with (111)In to a high specific activity of approximately 1.

Metastatic human colonic carcinoma: molecular imaging with pretargeted SPECT and PET in a mouse model.

Purpose: To prospectively determine if a bispecific monoclonal antibody (MoAb) pretargeting method with a radiolabeled hapten peptide can depict small (<0.3 mm in diameter) microdisseminated human colon cancer colonies in the lungs of nude mice.

Materials And Methods: Animal studies were approved in advance by animal care and use committees.

PAM4-reactive MUC1 is a biomarker for early pancreatic adenocarcinoma.

Purpose: The anti-MUC1 monoclonal antibody (MAb), PAM4, has a high specificity for pancreatic adenocarcinoma compared with other cancers, normal tissues, or pancreatitis. In order to assess its role in early pancreatic cancer development, we examined the expression of the PAM4-reactive MUC1 in the noninvasive precursor lesions, pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN).

Experimental Design: Tissue microarrays prepared from formalin-fixed, paraffin-embedded specimens were assessed by immunohistology for expression of the PAM4-reactive, non-variable number of tandem repeats (VNTR), MUC1 epitope, and the VNTR epitope bound by the MA5 MAb.

Multifunctional antibodies by the Dock-and-Lock method for improved cancer imaging and therapy by pretargeting.

The Dock-and-Lock (DNL) method, which makes bioactive molecules with multivalency and multifunctionality, is a new approach to develop targeting molecules for improved cancer imaging and therapy. It involves the use of a pair of distinct protein domains involved in the natural association between cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) and A-kinase anchoring proteins (AKAPs). The dimerization and docking domain found in the regulatory subunit of PKA and the anchoring domain (AD) of an interactive AKAP are each attached to a biologic entity, and the resulting derivatives, when combined, readily form a stably tethered complex of a defined composition that fully retains the functions of the individual constituents.

Bispecific antibody pretargeting of radionuclides for immuno single-photon emission computed tomography and immuno positron emission tomography molecular imaging: an update.

Molecular imaging is intended to localize disease based on distinct molecular/functional characteristics. Much of today's interest in molecular imaging is attributed to the increased acceptance and role of 18F-flurodeoxyglucose (18F-FDG) imaging in a variety of tumors. The clinical acceptance of 18F-FDG has stimulated research for other positron emission tomography (PET) agents with improved specificity to aid in tumor detection and assessment.

A divalent hapten-peptide induces apoptosis in human non hodgkin lymphoma cell lines targeted by anti-CD20xanti-hapten bispecific antibodies.

Purpose: Bispecific antibody (bsMAb) pretargeting procedures use divalent hapten-peptides to stabilize the binding of the hapten-peptide on tumor cells by a process known as the affinity enhancement system. The goal of this study was to determine if a divalent hapten-peptide could induce apoptosis by cross-linking bsMAb bound to CD20.

Methods: Three forms of bsMAbs were prepared by coupling the IgG, F(ab')2, or Fab' of a humanized anti-CD20 antibody to a Fab' of a murine antibody directed against the hapten histamine-succinyl-glycine (HSG).

CD74: a new candidate target for the immunotherapy of B-cell neoplasms.

CD74 is an integral membrane protein that functions as a MHC class II chaperone. Moreover, it has recently been shown to have a role as an accessory-signaling molecule and has been implicated in malignant B-cell proliferation and survival. These biological functions combined with expression of CD74 on malignant B cells and limited expression on normal tissues implicate CD74 as a potential therapeutic target.

Novel radiolabeled antibody conjugates.

This article reviews the development of radioimmunoconjugates as a new class of cancer therapeutics. Numerous conjugates involving different antigen targets, antibody forms, radionuclides and methods of radiochemistry have been studied in the half-century since radioactive antibodies were first used in model systems to selectively target radiation to tumors. Whereas directly conjugated antibodies, fragments and subfragments have shown promise preclinically, the same approaches have not gained success in patients except in radiosensitive hematological neoplasms, or in settings involving minimal or locoregional disease.

Peroxisome proliferator-activated receptor-gamma antagonists exhibit potent antiproliferative effects versus many hematopoietic and epithelial cancer cell lines.

Peroxisome proliferator-activated receptor-gamma ligands have preclinical and clinical anticancer activity. Most studies in this area address agonists, with relatively few reports on anticancer effects of peroxisome proliferator-activated receptor-gamma antagonists. Thus, we evaluated the two pure peroxisome proliferator-activated receptor-gamma antagonists, T0070907 and GW9662, on a panel of hematopoietic and epithelial cell lines.