Susan Chen

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

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 antigen-binding 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.

Preclinical Therapy of Breast Cancer with a Radioiodinated Humanized Anti-EGP-1 Monoclonal Antibody: Advantage of a Residualizing Iodine Radiolabel

AbstractBackground. A humanized monoclonal antibody (MAb), hRS7, labeled with 131I-IMP-R4, was evaluated for the preclinical radioimmunotherapy (RAIT) of breast cancer. 131I-IMP-R4 is an improved residualizing form of 131I that overcomes the short tumor residence time associated with conventionally radioiodinated MAbs. RS7, an internalizing MAb, recognizes epithelial glycoprotein-1, which is highly expressed in the carcinomas of breast, lung, ovary, and prostate. Methods. A humanized version of RS7 was generated by CDR-grafting and transfection. In vivo experiments were carried out in nude mice bearing subcutaneous MDA-MB-468 human breast cancer xenografts. Therapy experiments were performed using established tumors with mean tumor volume (MTV) of 0.3 cm3, and single administrations, at ∼70% of the estimated maximum tolerated doses (MTD), of the residualizing 131I-IMP-R4-hRS7 and 131I-hRS7 prepared by the conventional chloramine-T method [131I-hRS7 (CT)]. Therapeutic specificity was determined by comparison with untreated and non-specific MAb controls. Results. hRS7 was functionally very similar to murine and chimeric RS7. A biodistribution study using 125I-IMP-R4-hRS7 and 131I-hRS7 (CT) indicated a dosimetric advantage for the former. The MTVs 8 weeks post-treatment were 20, 163, and 280% of the starting MTVs of 131I-IMP-R4-hRS7-treated, 131I-hRS7 (CT)-treated, and untreated groups, respectively. Complete remissions were seen in 5 of 11 [and 6 of 8] mice treated with 131I-IMP-R4-hRS7, and in 1 of 11 mice treated with 131I-hRS7(CT). 131I-IMP-R4-hRS7 was significantly more efficacious than 131I-hRS7 (CT) [P = 0.01 for AUC] and the control 131I-IMP-R4-MAb. Conclusion. 131I-IMP-R4-hRS7 is a promising new agent for RAIT, providing significant therapeutic advantage in comparison to the conventionally 131I-labeled antibody.

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. Results: Given as a monotherapy, cross-linked milatuzumab, but not milatuzumab alone, yielded significant antiproliferative effects in CD74+ cells. The combination of cross-linked milatuzumab with bortezomib, doxorubicin, or dexamethasone caused more growth inhibition than either cross-linked milatuzumab or drug alone, producing significant reductions in the IC50 of the drugs when combined. Efficacy of combined treatments was accompanied by increased levels of apoptosis measured by increases of activated caspase-3 and hypodiploid DNA. Both milatuzumab and bortezomib affect the nuclear factor-κB pathway in CAG MM cells. In CAG- or KMS11-SCID xenograft models of disseminated MM, milatuzumab more than doubled median survival time, compared with up to a 33% increase in median survival with bortezomib but no significant benefit with doxorubicin. Moreover, combining milatuzumab and bortezomib increased survival significantly compared with either treatment alone. Conclusions: The therapeutic efficacies of bortezomib, doxorubicin, and dexamethasone are enhanced in MM cell lines when given in combination with milatuzumab, suggesting testing these combinations clinically.

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. IMMU-114 induced disease-free survival in tumor-bearing SCID mice with early-stage disease and in models that are relatively resistant to anti-CD20 monoclonal antibodies. Despite positive staining, acute myelogenous leukemic cells were not killed by IMMU-114. The ability of IMMU-114 to induce activation of ERK and JNK signaling correlated with cytotoxicity and differentiates the mechanism of action of IMMU-114 from monoclonal antibodies against CD20 and CD74. Thus, antigen expression is not sufficient for cytotoxicity; antibody-induced hyperactivation of ERK and JNK mitogen activated protein kinase signaling pathways are also required.

Combining radioimmunotherapy and chemotherapy for treatment of medullary thyroid carcinoma: Effectiveness of dacarbazine

To enhance the efficacy of chemotherapy for medullary thyroid carcinoma (MTC), we evaluated the effect of combining radioimmunotherapy (RAIT) with 90Y‐anticarcinoembryonic antigen (CEA) monoclonal antibody MN‐14 and chemotherapy in nude mice bearing human MTC xenografts. A preliminary study evaluated doxorubicin, dacarbazine (DTIC), cyclophosphamide, and vincristine, singly and in combination, for their effect on the growth of MTC xenografts (TT) in nude mice. Given individually, DTIC yielded the most effective tumor growth inhibition, delaying the mean time to doubling from 1 week for untreated tumor‐bearing mice to 7.5 weeks. Administering either the 4 drugs in combination or a 2‐drug combination comprised of doxorubicin and DTIC significantly improved the efficacy compared with any single drug alone, increasing the mean doubling time to 10–12 weeks.

Advantage of yttrium-90-labeled over iodine-131-labeled monoclonal antibodies in the treatment of a human lung carcinoma xenograft†

The purpose of this investigation was to compare the therapeutic effectiveness of yttrium‐90 (90Y)‐labeled monoclonal antibodies (MoAbs) with iodine‐131 (131I)‐labeled MoAbs delivered to human tumor xenografts at their maximum tolerated doses (MTD).

Evaluation of anti-human leukocyte antigen-DR monoclonal antibody therapy in spontaneous canine lymphoma

A pilot study of anti-human leukocyte antigen (HLA)-DR monoclonal antibody (mAb) in dogs with lymphoma was undertaken to verify the suitability of a canine model to address therapeutically relevant endpoints prior to a full trial in dogs, and ultimately human investigation. In vitro studies demonstrated that L243, a murine IgG1 anti-HLA-DR, binds to normal and malignant canine lymphocytes and induces apoptosis in canine lymphoma cells. Moreover, L243 was administered safely to normal dogs and dogs with lymphoma, and bound to malignant cells in nodal tissue. Preliminary evidence of transient disease stabilization was observed in a subset of dogs with advanced-stage lymphoma following L243 immunotherapy. hL243γ4P (IMMU-114), a humanized IgG4 anti-HLA-DR, currently under evaluation preclinically for human trials, was also shown to bind malignant canine lymphocytes, and safety and pharmacokinetic data from the administration of IMMU-114 to normal dogs indicate similar behavior to L243 in these assessments. These findings provide a rationale for the use of dogs with lymphoma in safety and efficacy evaluations of anti-HLA-DR mAbs for both veterinary and human applications.

Successful therapy of a human lung cancer xenograft using MAb RS7 labeled with residualizing radioiodine

We have recently reported that a radioiodinated, DTPA-appended peptide, designated IMP-R1, is a residualizing iodine label that overcomes many of the limitations that have impeded the development of residualizing iodine for clinical use. In this study the potential of 131I-IMP-R1-RS7, an internalizing anti-EGP-1 monoclonal antibody, was evaluated by performing preclinical therapy studies in nude mice bearing Calu-3 human non-small cell carcinoma of the lung xenografis. Elimination of 6 of 9 established tumors (mean tumor volume=0.3 cm(3)) was observed using a single dose of 350 microCi/mouse of 131I-IMP-R1-RS7, with all animals tolerating the dose. At the same dose and specific activity of 131I-RS7, labeled using the conventional chloramine-T method, there were four deaths, and one complete remission in nine treated mice. At the maximum tolerated dose of conventionally 131I-labeled RS7, 275 microCi, mean stable disease for approximately 5 weeks was observed, with no complete responses. Specificity of the therapeutic effect was shown in an isotype-matched control experiment, where 131I-IMP-R1-RS7 was markedly more effective than the (131)I-IMP-R1-labeled control antibody. These studies demonstrate that (131)I-IMP-R1-RS7 provides a therapeutic advantage in comparison to conventional 131I-labeled RS7, as predicted by the increased tumor accretion observed previously in targeting studies. A direct comparison of the maximum tolerated doses of (131)I-IMP-R1-RS7 (350 microCi) and 90Y-DOTA-RS7 (105 microCi) was performed in this tumor model using large established tumors (mean tumor volume=0.85 cm(3)). Anti-tumor efficacy and toxicity of the two treatments were comparable.

Abstract 5341: Sensitivity of NHL to killing by anti-HLA-DR and anti-CD74 mAbs is increased by interferon-gamma

Background: HLA-DR and CD74 are similarly, but not identically, expressed and induced by interferons on a variety of cells. Expression of both antigens on hematological malignancies led to their development as targets for antibody-based therapy. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab (Immunomedics Inc, Morris Plains, NJ), is in clinical evaluation for therapy of NHL, multiple myeloma (MM), and CLL after preclinical evidence of activity in these tumor types. A humanized anti-HLA-DR mAb, hL243γ4P (IMMU-114, Immunomedics) has demonstrated anti-tumor activity in vitro and in vivo, and clinical evaluation is planned. In addition to expression in hematologic cancers, these antigens are expressed on the surface of other tumor types, including melanoma and renal cell carcinoma, and in the cytoplasm of others, including pancreatic and colonic carcinomas, and glioblastomas (GBM). Methods: We examined whether the ability of anti-HLA-DR and anti-CD74 mAbs to kill cancer cells can be increased by using IFNγ as an inducer of antigen expression. Using a panel of diverse cancer cell lines (including NHL, MM, GBM, and pancreatic and colonic carcinomas), we examined IFNγ-induced changes in surface and cytoplasmic HLA-DR and CD74 expression. Sensitivity of malignant cells to milatuzumab and hL243γ4P was assessed with and without INFγ by cytotoxicity assays. Results: Without IFNγ surface expression of HLA-DR and CD74 were present on 2/2 NHL, 2/2 MM, and only weakly positive on 2/2 GBM cell lines. Surface CD74 and HLA-DR were weak or undetectable on 4/4 colon and 4/4 pancreatic carcinomas. Cytoplasmic CD74 and HLA-DR were seen in NHL, MM, GBM, and 1/4 colon and 1/4 pancreatic (CD74 only) carcinomas. Two-day incubation with IFNγ increased surface and cytoplasmic expression of both HLA-DR and CD74 in all the NHL and GBM, and 3/4 pancreatic cancer lines, but not MM cell lines. In all 4 colon lines, IFNγ increased cytoplasmic expression of both antigens, and surface expression of HLA-DR in 3/4 and CD74 in 2/4. Upregulation of HLA-DR and CD74 ranged from 23-3700%. Increased killing by both hL243γ4P (58%) and milatuzumab (33%) was seen in vitro after INFγ exposure in WSU-FSCCL NHL cells. No cell killing was observed using these mAbs in vitro on U118 (GBM), Capan-1 (pancreatic carcinoma), or LoVo (colon carcinoma), despite upregulation of the antigens in these cell lines. A CD74-transfected version of the U118 GBM cell line has been prepared for comparison of milatuzumab sensitivity based on antigen density only. Conclusions: Cell surface and cytoplasmic expression of CD74 and HLA-DR are increased on cell lines from a variety of cancer types after INFγ exposure. This increased expression correlates with increased toxicity of anti-HLA-DR and anti-CD74 mAbs in a NHL cell line, and is under evaluation in other cancer types. These studies could prove useful in predicting the potential benefit of combined INFγ and mAb therapies. 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 5341.