Unconjugated Antibody Research Articles

Publication of optimized multicolor immunofluorescence panels

Publication of optimized multicolor immunofluorescence panels

GPNMB expression in uveal melanoma: a potential for targeted therapy

Uveal melanoma is an aggressive disease without effective adjuvant therapy for metastases. Despite genomic differences between cutaneous and uveal melanomas, therapies based on shared biological factors could be effective against both tumor types. High expression of glycoprotein-NMB (GPNMB) in cutaneous melanomas led to the development of CDX-011 (glembatumumab vedotin), a fully human monoclonal antibody against the extracellular domain of GPNMB conjugated to the cytotoxic microtubule toxin monomethylauristatin E. Ongoing phase II trials suggest that CDX-011 has activity against advanced cutaneous melanomas. To determine the potential role of CDX-011 in uveal melanomas, we studied their GPNMB expression. Paraffin-embedded tissues from 22 uveal melanomas treated by enucleation from 2004-2007 at one institution were evaluated immunohistochemically for expression of GPNMB using biotinylated CDX-011 (unconjugated) antibody. Melanoma cells were evaluated for percentage and intensity of expression. Spectral imaging was used in one case with high melanin content. Clinical data were reviewed. Twelve women and 10 men with a median age of 58.7 years (range: 28-83 years) were included. Eighteen of 21 tumors evaluated immunohistochemically (85.7%) expressed GPNMB in 10-90% of tumor cells with variable intensity (5 tumors, 1+; 11, 2+; and 2, 3+). Eleven of 18 tumors (61.1%) expressed GPNMB in >or=50% of cells. Spectral imaging showed diffuse CDX-011 (unconjugated) reactivity in the remaining case. Uveal melanoma, like cutaneous melanoma, commonly expresses GPNMB. Ongoing clinical trials of CDX-011 should be extended to patients with metastatic uveal melanoma to determine potential efficacy in this subset of patients with melanoma.

Gemtuzumab ozogamicin (Mylotarg®) upregulates tissue factor expression but not secretion of inflammatory cytokines TNF-α or IL-8

Gemtuzumab ozogamicin (GO; Mylotarg ®) is an antibody-targeted chemotherapeutic agent approved for the treatment of CD33+ acute myelogenous leukemia (AML). GO is more commonly associated with the development of Sinusoidal Obstructive Syndrome (SOS) than any other chemotherapeutic agent in this patient group. Previous investigations have shown that SOS is a pro-coagulant and pro-inflammatory syndrome. Treatment of THP1 cells (CD33+ AML cell line) with GO alone, or in combination with unconjugated CD33 antibody (hP67.6), resulted in a statistically significant ( P < 0.01) increase in tissue factor (TF) expression, but HepG2 cells (CD33 hepatocyte cell line) were unaffected. Cytokine secretion was not affected by GO treatment in either cell line. These results indicate that GO exerts a pro-coagulant response in certain cell types, which may predispose individuals to developing SOS.

Biological activities of cytokine-neutralizing hyaluronic acid-antibody conjugates

Wound healing represents a highly regulated, orchestrated response of cells recruited to sites of injury. High molecular weight hyaluronic acid was conjugated with monoclonal antibodies to the cytokine interleukin-1beta to create a matrix-forming polymer capable of modifying healing. Using gel electrophoresis and fluorescence immunosorbent assays, we determined a degree of antibody functionalization per hyaluronic acid chain of 13.6+/-1.6%. The biological activity of the conjugate in vitro, measured using a nuclear factor-kappaB translocation assay in activated THP-1 monocytes, was comparable in inhibiting cytokine signaling to a similar level as the unconjugated antibody. Incisional wound studies in Sprague-Dawley rats indicates that viscous hyaluronic acid solutions were immunologically active, but covalent functionalization with antibodies against tumor necrosis factor-alpha and interleukin-1beta resulted in significant reductions in the inflammatory response. Covalent attachment of cytokine-neutralizing antibodies to matrix-forming polymers could lead to the development of materials capable of locally regulating wound healing and inflammatory responses in the setting of tissue regeneration.

Abstract 2577: In vitro and in vivo efficacy of the anti-MN immunoconjugate BAY 79-4620, MN-IC, in MN (CAIX) expressing preclinical tumor models

Abstract BAY 79-4620 is a novel immunoconjugate consisting of a fully human monoclonal antibody directed against MN (carbonic anhydrase IX; CAIX) conjugated with the auristatin derivative MMAE currently in Phase I testing. CAIX is overexpressed in a range of tumor types, such as gastric cancer, non-small cell lung cancer, pancreatic cancer or colorectal cancer. CAIX expression is regulated by HIF-1α, making this protein a marker associated with tumor hypoxia. Expression of this protein has been linked to higher aggressiveness of tumors and is predictive of poor prognosis in several cancers. We report on the pharmacological profile of a novel immunoconjugate directed against MN (CAIX) conjugated with the auristatin derivative MMAE to a fully human anti-MN (CAIX) monoclonal antibody (BAY 79-4620). In vitro studies showed the specific binding and internalization of BAY 79-4620 into CAIX-expressing tumor cells. Intracellular release of the tubulin-inhibiting toxophore MMAE led to mitotic arrest and specific cell kill of tumor cells, with EC50s in the low nanomolar range in most CAIX-positive tumor cell lines tested. In CAIX-negative tumor cell lines, a cytotoxic effect of BAY 79-4620 was seen at only very high doses. Cytotoxicity of this immunoconjugate depends on both CAIX expression and sensitivity of tumor cells to tubulin inhibitors. In vivo activity of the CAIX targeted Mab-MMAE conjugate resulted in higher efficacy (minimum effective dose (MED) of 0.625mg/kg in the HeLa-MaTu model, Q4Dx3 dosing schedule) compared to the systemic administration of either free MMAE toxophore (MED not achieved) or unconjugated anti-CAIX antibody which lacked efficacy in all models tested. Tumor regressions (in 80% of the animals) were achieved at doses of immunoconjugate as low as 1.25mg/kg while higher doses up to 10 mg/kg resulted in complete tumor eradication (in 90% of the animals treated). Efficacy in the HT29 model tested was schedule independent when comparing single dose, Q7Dx2 and Q4Dx3 schedules. The in vivo mode of action of the immunoconjugate was confirmed by tubulin staining in the tumor sections. Treatment with the immunoconjugate was found to be less toxic than that with free MMAE. The maximum tolerated dose of BAY 79-4620 in mice was 60mg/kg, which in terms of toxophore delivered exceeds the LD50 of MMAE (1mg/kg). In summary, these results demonstrate that targeted delivery of MMAE resulted in higher efficacy of BAY 79-4620 in tumor models with high CAIX expression. The immunoconjugate BAY 79-4620 currently in Phase I testing is a promising novel agent for the treatment of gastric, non-small cell lung, pancreatic, colorectal and other MN (CAIX)-positive tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2577.

Generation and Accumulation of Immunosuppressive Adenosine by Human CD4+CD25highFOXP3+ Regulatory T Cells

Naturally occurring regulatory T cells (nTreg) are crucial for maintaining tolerance to self and thus preventing autoimmune diseases and allograft rejections. In cancer, Treg down-regulate antitumor responses by several distinct mechanisms. This study analyzes the role the adenosinergic pathway plays in suppressive activities of human nTreg. Human CD4(+)CD25(high)FOXP3(+) Treg overexpress CD39 and CD73, ectonucleotidases sequentially converting ATP into AMP and adenosine, which then binds to A(2a) receptors on effector T cells, suppressing their functions. CD4(+)CD39(+) and CD4(+)CD25(high) T cells express low levels of adenosine deaminase (ADA), the enzyme responsible for adenosine breakdown, and of CD26, a surface-bound glycoprotein associated with ADA. In contrast, T effector cells are enriched in CD26/ADA but express low levels of CD39 and CD73. Inhibitors of ectonucleotidase activity (e.g. ARL67156) and antagonists of the A(2a) receptor (e.g. ZM241385) blocked Treg-mediated immunosuppression. The inhibition of ADA activity on effector T cells enhanced Treg-mediated immunosuppression. Thus, human nTreg characterized by the presence of CD39 and the low expression of CD26/ADA are responsible for the generation of adenosine, which plays a major role in Treg-mediated immunosuppression. The data suggest that the adenosinergic pathway represents a potential therapeutic target for regulation of immunosuppression in a broad variety of human diseases.

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. 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.

Monitoring the Structural Changes of Conjugated Antibodies by High-Resolution Electron Microscopy and Individual-Particle Electron Tomography

Peptides have promise as potent and selective drug candidates but have not succeeded primarily because of poor pharmacokinetics. The fusion of these peptides to our scaffold antibody has produced molecules, CovX-Bodies, which protect the peptide from renal elimination and/or enzymatic degradation. However, it is possible that the fusion process may in itself affect the intrinsic properties of the antibody scaffold (eg. effector function). Changes in function may relate to changes in structure. However, the structural study of highly dynamic and structurally heterogenous antibodies by X-ray crystallization and NMR is extremely difficult. These studies were designed to examine structural changes that may have occurred during the conjugation process.Here, we studied the structural changes of antibodies with the high-resolution electron microscopy and individual-particle electron tomography (IPET, for details, see the abstract presented by Lei Zhang and Gang Ren). We found 1) the average angle between the Fab regions was 55 degrees +-15 in unconjugated antibodies, but 40 degrees +-10 in unconjugated antibodies and 2) the average sizes of the Fc domain were similar regardless of conjugation. However, 3) the Fc domain shape differed significantly depending on conjugation. The Fc domains of conjugated antibodies were significantly elongated (>30%). Using IPET, we reconstructed a dozen of three-dimensional density maps of individual conjugated and unconjugated antibodies. Comparing the maps, it showed that all three domains of conjugated antibodies were elongated after fusing with the drug. This example demonstrates that this method could be a useful tool for monitoring the structural changes of antibodies.

Binding and Internalization of Iron Oxide Nanoparticles Targeted To Nuclear Oncoprotein

A targeted nanoconjugate is being developed for non-invasive detection of gene expression in cells expressing the JC virus oncoprotein, T-antigen, which has been associated with medulloblastoma and other cancers. JC virus T-antigen localizes predominantly to the nucleus via a classical monopartite nuclear localization signal (NLS). An antibody fragment which recognizes JC virus T-antigen was attached to cross-linked dextran coated iron oxide nanoparticles. Radiolabeled conjugates were added to mouse medulloblastoma cells expressing the target T-antigen to test their ability to bind to tumor cells and be internalized by the cells. All conjugates containing targeting antibody bound to cells and were internalized, with increasing levels over time. There was no difference in cell binding or internalization among conjugates containing 2, 4, 6 or 8 antibody fragments per nanoparticle. Conjugates with only nonspecific antibody on nanoparticles, or unconjugated nonspecific antibody, had significantly lower total binding and internalization than conjugates with targeting antibody. Unconjugated targeting antibody had equivalent or lower cell uptake compared with targeted nanoparticle conjugates. Specificity of uptake was demonstrated by >80% reduction of nanoconjugate uptake in the presence of 100 fold excess of unconjugated antibody. The presence of a membrane translocation peptide (Tat) on the nanoparticles in addition to targeting antibody did not improve nanoconjugate internalization over the internalization caused by the antibody alone. This antibody nanoconjugate demonstrates feasibility of targeting a nuclear protein and suggests that a minimum number of antibody fragments per nanoparticle are sufficient for achieving binding specificity and efficient uptake into living cells.

Protective effects of anti-ricin A-chain antibodies delivered intracellularly against ricin-induced cytotoxicity

To evaluate the ability of anti-ricin A-chain antibodies, delivered intracellularly, to protect against ricin-induced cytotoxicity in RAW264.7 cells. Anti-deglycosylated ricin A-chain antibody and RAC18 anti-ricin A-chain monoclonal antibody were delivered intracellularly by encapsulating in liposomes or via conjugation with the cell-penetrating MTS-transport peptide. RAW264.7 cells were incubated with these antibodies either before or after ricin exposure. The changes in cytotoxicity were estimated by MTT assay. Co-localization of internalized antibody and ricin was evaluated by fluorescence microscopy. Internalized antibodies significantly increased cell viability either before or after ricin exposure compared to the unconjugated antibodies. Fluorescence microscopy confirmed the co-localization of internalized antibodies and ricin inside the cells. Intracellular delivery of antibodies to neutralize the ricin toxin after cellular uptake supports the potential use of cell-permeable antibodies for post-exposure treatment of ricin intoxication.

Therapy of Murine Pulmonary Aspergillosis with Antibody-Alliinase Conjugates and Alliin

Aspergillus fumigatus is an opportunistic fungal pathogen responsible for invasive aspergillosis in immunocompromised individuals. The high morbidity and mortality rates as well as the poor efficacy of antifungal agents remain major clinical concerns. Allicin (diallyl-dithiosulfinate), which is produced by the garlic enzyme alliinase from the harmless substrate alliin, has been shown to have wide-range antifungal specificity. A monoclonal antibody (MAb) against A. fumigatus was produced and chemically ligated to the enzyme alliinase. The purified antibody-alliinase conjugate bound to conidia and hyphae of A. fumigatus at nanomolar concentrations. In the presence of alliin, the conjugate produced cytotoxic allicin molecules, which killed the fungus. In vivo testing of the therapeutical potential of the conjugate was carried out in immunosuppressed mice infected intranasally with conidia of A. fumigatus. Intratracheal (i.t.) instillation of the conjugate and alliin (four treatments) resulted in 80 to 85% animal survival (36 days), with almost complete fungal clearance. Repetitive intratracheal administration of the conjugate and alliin was also effective when treatments were initiated at a more advanced stage of infection (50 h). The fungi were killed specifically without causing damage to the lung tissue or overt discomfort to the animals. Intratracheal instillation of the conjugate without alliin or of the unconjugated monoclonal antibody significantly delayed the death of the infected mice, but only 20% of the animals survived. A limitation of this study is that the demonstration was achieved in a constrained setting. Other routes of drug delivery will be investigated for the treatment of pulmonary and extrapulmonary aspergillosis.

Mechanisms of Discrepancy Between CD33 Expression and Gemtuzumab Ozogamicin-Induced Cytotoxicity in Leukemia Cells.

Abstract 4812Gemtuzumab ozogamicin (GO) is an immunoconjugate of an anti-CD33 antibody and a toxic calicheamicin-γ1 derivative that facilitates cellular uptake of the latter drug. CD33 is selectively expressed on myeloid cells, especially on acute myeloid leukemia (AML) cells, but usually not on non-hematopoietic cells, and therefore the clinical use is confined to AML. Despite its predominant expression on AML cells, GO induces complete remission only in 30% of recurrent AML. The major side-effects of GO include not only myelosuppression but also hepatotoxicity, although CD33 is not expressed on hepatobiliary cells. It suggests that the efficacy of GO is not strictly regulated by CD33 expression. We investigated relationship between CD33 expression and GO cytotoxicity by using leukemia cell lines, and classified them into four groups; group A defined as CD33(+) and cytotoxicity (+), group B as CD33(+) and cytotoxicity (-), group C as CD33(-) and cytotoxicity (+), and group D as CD33(-) and cytotoxicity (-). We selected four cell lines, HL-60, MEG-01, MOLT-3 and K562 which belong to group A, B, C and D, respectively, and were used for further experiment. CD33 was expressed on HL-60 but not on MOLT-3. However, GO-induced cytotoxicity was seen in both cells. We therefore compared effects of GO in HL-60 and MOLT-3. When incubated with GO for 72 hours, viability of HL-60 significantly decreased at 10 ng/mL of GO, while cytotoxic effect of GO against MOLT-3 became apparent when GO concentration was raised to 100 ng/mL. Viability of HL-60 markedly decreased when treated with GO for 30 minutes and then cultured in medium without containing GO for 72 hours, but no cytotoxic effect was observed in MOLT-3 unless culture with GO was prolonged to more than 6 hours. GO-induced apoptosis was detected in both HL-60 and MOLT-3 by Annexin V assay and TUNEL assay. The pretreatment with unconjugated anti-CD33 antibody (hP67.6) resulted in reduced cytotoxic effects of GO in HL-60, but not in MOLT-3. It indicates that GO induces apoptosis in CD33-non-expressing MOLT-3. Higher doses and longer exposure of GO treatment are necessary to induce cytotoxicity against MOLT-3 when compared with HL-60. GO might cause cytotoxicity against MOLT-3 through drug absorption pathway other than CD33-mediated endocytosis. Although HL-60 and MEG-01 expressed CD33, GO induced cytotoxicity on HL-60, but not on MEG-01. We then investigated internalization of antibody-bound CD33. After incubated with hP67.6 on ice, cells were cultured in antibody-free medium, and remaining cell surface-associated CD33 was measured. The loss of cell surface CD33 was observed in MEG-01 as well as HL-60, showing that internalization of CD33 following antibody binding occurred in MEG-01. It indicates that resistance to GO-induced cytotoxicity in MEG-01 is not associated with internalization property. Drug transport pathway other than receptor-mediated endocytosis was investigated. Cells were cultured in rhodamine 123 (Rh123)-containing medium for 30 minutes and the influx of Rh123 was assessed by the fluorescence activity of Rh123-containing cells. Cells were then cultured in medium without containing Rh123 for further 3 hours, and the efflux of Rh123 was analyzed by measuring the reduction curve of fluorescence activity. Rh123 accumulated in MEG-01 twice higher than HL-60. However, fluorescence activity of Rh123 decreased to 20% after 3 hours, while it remained unchanged in HL-60, indicating that drug efflux was activated in MEG-01. Multidrug resistance (MDR)-1 protein was strongly expressed in MEG-01. When cyclosporin-A, a modulator of MDR-1, was given with GO to MEG-01, GO-induced cytotoxicity was significantly increased, while no such effect was observed in HL-60. It suggests that p-glycoprotein-mediated efflux of GO is one of the resistance mechanisms in MEG-01. Disclosures:No relevant conflicts of interest to declare.

CD19: a promising B cell target for rheumatoid arthritis

B-cell depletion with unconjugated CD20 monoclonal antibody (mAb) is used to treat rheumatoid arthritis and other autoimmune diseases. CD20-targeted immunotherapy depletes mature B cells through monocyte-mediated antibody-dependent cellular cytotoxicity, but does not effectively deplete pre-B or immature B cells, certain peripheral B cell subpopulations, most antibody-producing cells, or their malignant counterparts. As immature B cells expressing autoreactive antigen receptors are not depleted by anti-CD20 mAb, a new strategy for eliminating autoantigen-selected B cells and for treating early lymphoblastic leukemias and/or lymphomas was developed using CD19-specific mAbs that induce Fcgamma receptor-dependent and monocyte-dependent B-cell depletion. Preclinical studies using transgenic mice expressing human CD19 have shown that pre-B cells and their malignant counterparts, as well as pre-existing antibody-producing and autoantibody-producing cells, are depleted. Therefore, CD19-directed immunotherapy is expected to treat diverse pre-B-cell-related and plasmablast-related malignancies, and humoral transplant rejection. Moreover, in contrast to CD20-directed immunotherapies, CD19 mAbs could purge the B cell repertoire of autoreactive clones and reset the developmental clock to a point that curtails the extent of emerging self-reactivity, in addition to reducing autoreactive T-cell activation through the elimination of mature B cells. Humanized CD19 mAbs are expected to enter clinical trials in 2009, offering a new approach for the treatment of autoimmune disease that removes both immature B cells and antibodies with autoreactive specificities. CD19-directed immunotherapy could, therefore, offer a new horizon in B-cell depletion for the treatment of multiple autoimmune diseases.

Recognition Versus Adaptive Up-regulation and Degradation of CC Chemokines by the Chemokine Decoy Receptor D6 Are Determined by Their N-terminal Sequence

The chemokine decoy receptor D6 controls inflammatory responses by selective recognition and degradation of most CCR1 to CCR5 agonistic ligands. CCL14 is a homeostatic chemokine present at high concentrations in the serum with a weak agonist activity on CCR1. Under inflammatory conditions, plasmin and UPA-mediated truncation of 8 amino acids generates the potent CCR1/CCR3/CCR5 isoform CCL14(9-74), which is further processed and inactivated by dipeptidyl peptidase IV/CD26 that generates CCL14(11-74). Here we report that D6 efficiently binds both CCL14 and its truncated isoforms. Like other D6 ligands, the biologically active CCL14(9-74) induces adaptive up-regulation of D6 expression on the cell membrane and is rapidly and efficiently degraded. In contrast, the D6-mediated degradation of the biologically inactive isoforms CCL14(1-74) and CCL14(11-74) is very inefficient. Thus, D6 cooperates with CD26 in the negative regulation of CCL14 by the selective degradation of its biologically active isoform. Analysis of a panel of CC chemokines and their truncated isoforms revealed that D6-mediated chemokine degradation does not correlate with binding affinity. Conversely, degradation efficiency is positively correlated with D6 adaptive up-regulation. Sequence analysis indicated that a proline residue in position 2 of D6 ligands is dispensable for binding but crucial for D6 adaptive up-regulation and efficient degradation.

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

AbstractAntibody-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 (90Y-ibritumomab tiuxetan or 131I-tositumomab) have had encouraging results, with trials now seeking to incorporate a radioimmunoconjugate in various settings. However, new preclinical data raise important questions concerning current radioimmunoconjugate treatment regimens and ways to improve them. In radioconjugate therapy, nearly 900 mg of the unlabeled anti-CD20 IgG antibody is predosed to the patient before the anti-CD20 antibody conjugated to either 90Y or 131I is given. Combining an unconjugated anti-CD20 antibody therapy with a radioimmunoconjugate binding to a noncompeting antigen might improve responses by allowing optimal uptake of each agent. Preclinical models have indicated that careful consideration should be given to predosing when using competing antibodies, but that consolidation anti-CD20 therapy enhances the efficacy of radioimmunoconjugate therapy. New technologies, such as pretargeted radioimmunotherapy, also hold promise by reducing toxicity without sacrificing efficacy, and consideration should be given to fractionating or giving multiple radioimmunoconjugate treatments. This perspective discusses how these issues could affect current and future clinical trials.

Pharmacodynamics, Pharmacokinetics, and Tolerability of a B-Cell Specific Antibody-Targeted Chemotherapeutic Agent, Anti-CD79b-MCC-DM1, in Cynomolgus Monkeys (41.12)

Abstract Antibody-based therapeutic approaches have revolutionized the treatment of non-Hodgkin's lymphomas (NHL) as well as other hematological malignancies. However, a large variability in clinical response has been observed and patients with refractory or relapsed lymphomas have limited therapeutic options. Antibody drug conjugates (ADC) may prove valuable in this respect. The B cell surface antigen, CD79b, is an attractive target for this approach as it is rapidly internalized upon antibody engagement and has an appropriate expression pattern, being expressed only on normal and malignant B-cells. Anti-CD79b-MCC-DM1 is an ADC consisting of a chimeric monoclonal anti-CD79b antibody, 10D10, conjugated via a thioether linker to a cytotoxic drug, DM1 (a potent tubulin polymerization inhibitor). To investigate the pharmacodynamics (PD), pharmacokinetics (PK), and tolerability of 10D10-MCC-DM1 in nonhuman primates, we administered 10D10-MCC-DM1 or unconjugated antibody 10D10 to cynomolgus monkeys. 10D10-MCC-DM1 and 10D10 resulted in sustained depletion of B-cells and B cell subsets in blood, with more substantial depletion in the group given 10D10-MCC-DM1. These findings correlated well with the depletion of B-cells observed in lymphoid tissues (bone marrow, lymph nodes, and spleen). Both 10D10-MCC-DM1 and 10D10 were well tolerated. Taken together, these preclinical data suggest that targeting CD79b with ADCs may provide a safe and effective therapy for B-cell malignancies in humans.

Pretargeted Versus Directly Targeted Radioimmunotherapy Combined with Anti-CD20 Antibody Consolidation Therapy of Non-Hodgkin Lymphoma

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. 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. 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. 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.

The Immunotoxin HM1.24-ETA′ Is a Potent Inducer of Apoptosis in Malignant Plasma Cells

Despite new treatment modalities, the clinical outcome of at least a subgroup of patients with multiple myeloma (MM) still needs improvement. Antibody-based targeted therapies are increasingly used for tumor therapy, and may represent interesting options for MM patients. HM1.24 is a surface molecule that is over expressed on malignant plasma cells and efficiently internalized from the cell surface. It may represent a promising target for the development of myeloma-directed immunoconstructs. Here, the development and characterization of a novel single-chain immunotoxin, HM1.24-ETA′, is described. HM1.24-ETA′ was generated by genetic fusion of an HM1.24-specific single-chain Fv (scFv) antibody and a truncated variant of Pseudomonas aeruginosa exotoxin A (ETA′). The immunotoxin was expressed in E. coli and purified to homogeneity by affinity chromatography. Specific binding to HM1.24 was demonstrated by immunofluorescence staining and flow cytometry using antigen positive and negative cells. HM1.24-ETA′ efficiently inhibited growth of IL-6 dependent and IL-6 independent myeloma cell lines (INA-6, RPMI8226, U266). Half maximal growth inhibition was observed at low nanomolar concentrations. Further analyses demonstrated that target cell killing occurred via induction of apoptosis, as evidenced by Annexin V/propidium iodide staining and detection of PARP cleavage. The cytotoxic effect was completely blocked by adding excess of unconjugated parental antibody, demonstrating that the effect was antigen-specific and not mediated by non-specific uptake of the immunotoxin. Importantly, HM1.24-ETA′ efficiently triggered apoptosis (>80% Annexin V positive cells) of freshly isolated plasma cell leukemia cells within 48h. In conclusion, HM1.24-ETA′ efficiently triggered apoptosis of multiple myeloma cell lines as well as freshly isolated tumor cells. These results indicate that HM1.24 may represent a promising target structure for efficient antigen-specific delivery of cytotoxic compounds to plasma cell tumors.

Pharmacodynamics, Pharmacokinetics, and Tolerability of a B-Cell Specific Antibody-Targeted Chemotherapeutic Agent, Anti-CD79b-MCC-DM1, in Cynomolgus Monkeys

Antibody-based therapeutic approaches have revolutionized the treatment of non- Hodgkin's lymphomas (NHL) as well as other hematological malignancies. However, given the biological and clinical heterogeneity of the diseases, a large variability in clinical response has been observed. Currently, patients with refractory or relapsed lymphomas have limited therapeutic options, and therefore the need to develop effective new treatments remains urgent.Antibody drug conjugates (ADC) may prove valuable in this respect. The B cell surface antigen, CD79b, is an attractive target for this approach as it is rapidly internalized upon antibody engagement and has an appropriate expression pattern, being expressed only on normal and malignant B-cells. Anti-CD79b-MCC-DM1 is an ADC consisting of a chimeric monoclonal anti-CD79b antibody, 10D10, conjugated via a thioether linker to a cytotoxic drug, DM1. DM1, a maytansinoid derivative, is a potent tubulin polymerization inhibitor. Anti-CD79b ADCs have demonstrated strong therapeutic activity against B-cell lymphoma xenografts (Polson, 2007). To investigate the pharmacodynamics (PD), pharmacokinetics (PK), and tolerability of 10D10-MCC-DM1 in nonhuman primates, we administered 2 intravenous ~30 mg/kg doses (q 3 weeks) of 10D10-MCC-DM1 or unconjugated antibody 10D10 to cynomolgus monkeys, along with vehicle controls. 10D10-MCC-DM1 and 10D10 resulted in sustained depletion of B-cells and B cell subsets in peripheral blood, with more substantial depletion in the group given 10D10- MCC-DM1. These findings correlated well with the depletion of B-cells observed in lymphoid tissues (bone marrow, lymph nodes, and spleen). We observed an initial rapid depletion of B-cells, likely attributed to probable antibody-mediated clearance by the reticuloendothelial system. This was followed by a greater sustained B-cell depletion by the ADC, attributed to the anti-mitotic pharmacology expected following B-cell receptorbound ADC internalization. In addition, unconjugated 10D10 showed substantially less depletion than 10D10-MCC-DM1 of the CD20+CD21− B cell subset, a subgroup of B cells which is phenotypically similar to human germinal center cells. There was no substantial difference in T cells or NK cells. The PD profiles are particularly encouraging from a therapeutic perspective, as the ADC may be expected to target proliferating NHL cells in a manner similar to dividing B-cells in germinal centers.The pharmacokinetics are described by the plasma measures of total antibody, conjugate, and DM1. 10D10-MCC-DM1 was cleared slowly from the plasma with a terminal half-life of 8.98 ± 1.7 days. The apparent central volume of distribution for 10D10-MCC-DM1 was 47.7 mL/kg, which approximated the physiological plasma volume in monkeys. Modest and expected accumulation was observed following the second dose. At the similar dose level of 10D10 and 10D10-MCC-DM1, the exposure of the total antibody was similar. These PK data, when compared alongside the PD results, point to the increased effectiveness of the drug conjugate in depleting B-cells. Free DM1 concentration in plasma following administration of 10D10-MCC-DM1 exhibited a profile similar to that of 10D10-MCC-DM1 but concentrations were much lower than the corresponding 10D10-MCC-DM1 with a Cmax of 189 ± 23 ng/mL. Based on the species-invariant method, it is projected that the half-life in humans for the conjugate is approximately 19.3 ± 3.4 days. Thus the pharmacokinetic profiles of the conjugate appear to support a dosing schedule of every three weeks in clinical studies.Both 10D10-MCC-DM1 and 10D10 were well tolerated. In monkeys administered 10D10-MCC-DM1, in addition to changes in the lymphoid and hematopoietic systems (B-cell depletion), minimal axonal degeneration of the sciatic nerve was observed. Trastuzumab (Herceptin)-MCC-DM1, an ADC which has shown promising safety and efficacy profiles in clinical trials, displayed similar non-B-cell related effects in cynomolgus monkeys with dose-limiting thrombocytopenia in humans. Thus it appears that targeting B-cells by an anti-CD79b ADC does not have any additional target-independent toxicity. Taken together, these preclinical data suggest that targeting CD79b with ADCs may provide a safe and effective therapy for B-cell malignancies in humans.

306 POSTER Targeted suicide gene therapy for small cell lung cancer

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. In order to improve efficacy, a number of immunoconjugates are being examined in settings where the disease is more easily accessible, such as leukemias, or within compartments that allow easier and more direct access to the tumor, such as in the peritoneal cavity or brain, or both locally and systemically, in adjuvant situations, where the disease burden has been reduced by some other means, and with the main goal of these treatments being to kill residual disease.