The emergence of trophoblast cell-surface antigen 2 (TROP-2) as a novel cancer target.

TROP-2 is a glycoprotein first described as a surface marker of trophoblast cells, but subsequently shown to be increased in many solid cancers, with lower expression in certain normal tissues. It regulates cancer growth, invasion and spread by several signaling pathways, and has a role in stem cell biology and other diseases. This review summarizes TROP-2's properties, especially in cancer, and particularly its role as a target for antibody-drug conjugates (ADC) or immunotherapy. When the irinotecan metabolite, SN-38, is conjugated to a humanized anti-TROP-2 antibody (sacituzumab govitecan), it shows potent broad anticancer activity in human cancer xenografts and in patients with advanced triple-negative breast, non-small cell and small-cell lung, as well as urothelial cancers.

Prevention of acute graft-versus-host disease in a xenogeneic SCID mouse model by the humanized anti-CD74 antagonistic antibody milatuzumab.

Prevention and treatment of graft-versus-host disease (GVHD) remains a major challenge, given that current T-cell depletion and mainstay immunosuppressive therapies compromise preexisting T-cell immunity, often leading to severe infections and disease relapse. Thus, there is a critical need for novel anti-GVHD agents that can spare protective T-cell memory. Here we show that milatuzumab (hLL1), a humanized anti-CD74 antagonist monoclonal antibody, can moderately reduce the numbers of CD74-expressing B cells and myeloid dendritic cells, but has no effect on the survival of T cells that are CD74(-). Consequently, milatuzumab inhibits allogeneic T-cell proliferation in mixed leukocyte reactions. In a human/mouse xenogeneic SCID mouse model in which GVHD is induced and mediated by engrafted human CD4(+) T cells and dendritic cells, milatuzumab effectively prevents the onset and manifestations of acute GVHD, suppresses serum levels of human IFN-γ and IL-5, eliminates the infiltration of human lymphocytes into GVHD target organs (ie, lung, liver, and spleen), and significantly promotes survival (90% versus 20% for controls; P = .0012). Importantly, exposure to milatuzumab does not affect the number of cytomegalovirus-specific, IFN-γ-producing human CD8(+) T cells in allogeneic mixed leukocyte reactions. These encouraging results warrant further exploration of milatuzumab as a possible new therapeutic agent for GVHD.

Preclinical studies on targeted delivery of multiple IFNα2b to HLA-DR in diverse hematologic cancers.

The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting mAb (mAb-IFNα) can enhance potency because of increased tumor localization and improved pharmacokinetics. We used the Dock-and-Lock method to generate C2-2b-2b, a mAb-IFNα comprising tetrameric IFNα2b site-specifically linked to hL243 (humanized anti-HLA-DR). In vitro, C2-2b-2b inhibited various B-cell lymphoma leukemia and myeloma cell lines. In most cases, this immunocytokine was more effective than CD20-targeted mAb-IFNα or a mixture comprising the parental mAb and IFNα. Our findings indicate that responsiveness depends on HLA-DR expression/density and sensitivity to IFNα and hL243. C2-2b-2b induced more potent and longer-lasting IFNα signaling compared with nontargeted IFNα. Phosphorylation of STAT1 was more robust and persistent than that of STAT3, which may promote apoptosis. C2-2b-2b efficiently depleted lymphoma and myeloma cells from whole human blood but also exhibited some toxicity to B cells, monocytes, and dendritic cells. C2-2b-2b showed superior efficacy compared with nontargeting mAb-IFNα, peginterferonalfa-2a, or a combination of hL243 and IFNα, using human lymphoma and myeloma xenografts. These results suggest that C2-2b-2b should be useful in the treatment of various hematopoietic malignancies.

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.

A bispecific antibody-IFNalpha2b immunocytokine targeting CD20 and HLA-DR is highly toxic to human lymphoma and multiple myeloma cells.

The short circulating half-life and side effects of IFNα affect its dosing schedule and efficacy. Fusion of IFNα to a tumor-targeting monoclonal antibody (MAb-IFNα) can enhance potency due to increased tumor localization and improved pharmacokinetics. We report the generation and characterization of the first bispecific MAb-IFNα, designated 20-C2-2b, which comprises two copies of IFNα2b and a stabilized F(ab)(2) of hL243 (humanized anti-HLA-DR; IMMU-114) site-specifically linked to veltuzumab (humanized anti-CD20). In vitro, 20-C2-2b inhibited each of four lymphoma and eight myeloma cell lines, and was more effective than monospecific CD20-targeted MAb-IFNα or a mixture comprising the parental antibodies and IFNα in all but one (HLA-DR(-)/CD20(-)) myeloma line, suggesting that 20-C2-2b should be useful in the treatment of various hematopoietic malignancies. 20-C2-2b displayed greater cytotoxicity against KMS12-BM (CD20(+)/HLA-DR(+) myeloma) compared with monospecific MAb-IFNα, which targets only HLA-DR or CD20, indicating that all three components in 20-C2-2b could contribute to toxicity. Our findings indicate that a given cell's responsiveness to MAb-IFNα depends on its sensitivity to IFNα and the specific antibodies, as well as the expression and density of the targeted antigens.

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.

Enhanced expression of CD74 in gastrointestinal cancers and benign tissues.

CD74, a transmembrane glycoprotein that associates with MHC II, is an important chaperone that regulates antigen presentation for immune response. In addition, CD74 is the receptor for macrophage migration-inhibitory factor which, when bound to CD74, initiates survival pathways and cell proliferation. Formalin fixed, paraffin embedded clinical specimens were evaluated by immunohistochemical procedures for expression of CD74. Overall, expression of CD74 within gastrointestinal carcinomas showed a statistically greater expression than in the normal tissue counterparts (P<0.001 or better). CD74 expression was observed in 95% of pancreatic carcinomas with the majority of cases presenting a mostly intense, diffuse labeling pattern. The results suggested a trend towards greater expression within the higher grade carcinomas (P=0.06). Colorectal and gastric carcinomas gave similar results with 60% and 86%, respectively, positive for CD74 with an intense, diffuse staining pattern. We hypothesized that precursor lesions would express levels of CD74 as high, or higher, than their respective carcinomas, since activation of survival pathways would be of particular importance at the early stages of neoplastic development. For PanIN lesions there was greater expression of CD74 within higher grade, PanIN-3 lesions, whereas the colonic adenomas showed no such trend, but overall, a higher frequency and intensity of CD74 labeling than was observed within the colon carcinomas. These findings are supportive of a role for CD74 in the development and maintenance of gastrointestinal neo-plasia, and provide a rationale for development of therapeutic agents that are able to block CD74 function, specifically within the tumor cell.

CD20-targeted tetrameric interferon-alpha, a novel and potent immunocytokine for the therapy of B-cell lymphomas.

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.

Hexavalent bispecific antibodies represent a new class of anticancer therapeutics: 1. Properties of anti-CD20/CD22 antibodies in lymphoma.

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.

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 IC(50) 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-kappaB 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.

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). 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.

Novel designs of multivalent anti-CD20 humanized antibodies as improved lymphoma therapeutics.

Multivalent antibodies, either monospecific or bispecific, may improve the efficacy of current therapeutic interventions involving a single monoclonal antibody (mAb). We have applied the Dock-and-Lock (DNL) method, a new platform technology for the site-specific and covalent assembly of modular components into stably tethered complexes of defined composition, to prepare a hexavalent, anti-CD20 antibody, designated Hex-hA20, which comprises six Fabs with one Fc. We show that Hex-hA20 retains the binding activity of all six Fabs, associates with CD20 in lipid rafts, affects antibody-dependent cell-mediated cytotoxicity, but not complement-dependent cytotoxicity, and inhibits proliferation of Daudi, Raji, and Ramos cells in vitro at subnanomolar concentrations without the need for a cross-linking antibody. In addition, Hex-hA20 induces strong homotypical adhesion and is inefficient in stimulating calcium mobilization. Thus, Hex-hA20 exhibits biological properties attributable to both type I and type II anti-CD20 mAbs, as exemplified by rituximab and tositumomab, respectively. Although Hex-hA20 has a short serum half-life, it shows antitumor efficacy in tumor-bearing mice comparable with veltuzumab at equivalent doses. The versatile DNL method was also applied to generate two other multivalent anti-CD20 antibodies without the Fc region, Tri-hA20 and Tetra-hA20, comprising three and four Fabs of veltuzumab, respectively. Similar to Hex-hA20, these were purified to near homogeneity and shown to have potent antiproliferative activity in vitro, thus indicating the need for clustering three or more CD20 molecules on the cell surface to induce growth inhibition.

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. The anti-CD74 monoclonal antibody LL1 has been humanized (hLL1 milatuzumab or IMMU-115) and can provide the basis for novel therapeutic approaches to B-cell malignancies, particularly because this antibody shows rapid internalization into CD74+ malignant cells. This article reviews the preclinical evaluations of LL1, its humanized form, and isotope, drug, and toxin conjugates. These studies show that unconjugated hLL1 and conjugates of hLL1 constructs with radioisotopes, doxorubicin, and frog RNase have high antitumor activity in non-Hodgkin's lymphoma and multiple myeloma in vitro and in tumor xenograft models. Single-dose studies of hLL1 in monkeys showed no adverse effects but did decrease circulating B and T lymphocytes and natural killer cells. When evaluated in combination with rituximab, either equivalent or improved efficacy, compared with either antibody alone, was observed. CD74 is a new candidate target for the immunotherapy of neoplasms expressing this antigen, which can be exploited using either a naked antibody or conjugated to isotopes, drugs, or toxins.

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). A recombinant bsMAb with divalent binding to CD20 and monovalent binding to HSG was also examined. Induction of apoptosis on SU-DHL-6, RL, and Ramos cells was examined by propidium iodide staining, caspase-3 activation, and mitochondrial membrane potential collapse, and compared with induction by cross-linking an anti-CD20 IgG with an antispecies antibody. RESULTS: The various forms of bsMAb had differing baseline levels of apoptosis in the absence of the divalent HSG peptide. The addition of the divalent HSG peptide significantly increased the level of apoptosis seen with the Fab'xFab' bsMAb by 2.2- to 3.9-fold, as well as the F(ab')2xFab', IgGxFab', and the recombinant bsMAbs by approximately 1.5-fold. CONCLUSIONS: The addition of a divalent HSG peptide to various forms of bispecific anti-CD20 MAbs could enhance apoptotic signaling in several lymphoma cells. This effect was more consistently measured when the orientation of the anti-hapten-binding arm of the bsMAb was well defined, such as in the Fab'xFab' and recombinant forms of bsMAb.

Epratuzumab, a CD22-targeting recombinant humanized antibody with a different mode of action from rituximab.

Epratuzumab is a humanized anti-CD22 monoclonal antibody currently in clinical trials for treatment of non-Hodgkin lymphoma (NHL) and certain autoimmune diseases. Here we report the results of investigations of epratuzumab's mode of action in comparison to and in combination with the anti-CD20 mAb, rituximab. In vitro cell growth inhibition, induction of apoptosis, and the ability of the mAbs to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were evaluated. We also investigated the potential activity of epratuzumab in the regulation of B-cell antigen receptor (BCR) activation. Epratuzumab and rituximab displayed very distinct modes of action; epratuzumab acts as an immunomodulatory agent, while rituximab is an acutely cytotoxic therapeutic antibody. Epratuzumab has distinct effects on cell growth from rituximab. For example, rituximab+anti-human IgG Fcgamma yielded marked inhibition of proliferation in human NHL cell lines, while epratuzumab had little or no effect in this assay. However, when cells were immobilized and stimulated with anti-IgM, epratuzumab, but not rituximab, caused a significant antiproliferative effect. Unlike rituximab, no CDC could be detected, and ADCC was modest but significant with epratuzumab. Importantly, combining rituximab and epratuzumab did not decrease rituximab's ability to induce apoptosis, CDC, and ADCC. In fact, the combination is more effective than rituximab alone in inhibiting proliferation of Daudi Burkitt lymphoma cells in the presence of second antibody, and at least equally effective to rituximab in the absence of crosslinking. These observations suggest that it may be possible to enhance clinical efficacy by combination therapy comprised of anti-CD20 and anti-CD22 mAbs.

Characterization of a humanized IgG4 anti-HLA-DR monoclonal antibody that lacks effector cell functions but retains direct antilymphoma activity and increases the potency of rituximab.

HLA-DR is under investigation as a target for monoclonal antibody (mAb) therapy of malignancies. Here we describe a humanized IgG4 form of the anti-HLA-DR mAb L243, hL243gamma4P (IMMU-114), generated to provide an agent with selectivity toward neoplastic cells that can kill without complement-dependent cytotoxicity (CDC) or antibody-dependent cellular-cytotoxicity (ADCC), so as to reduce reliance on intact immunologic systems in the patient and effector mechanism-related toxicity. In vitro studies show that replacing the Fc region of hL243gamma1, a humanized IgG1 anti-HLA-DR mAb, with the IgG4 isotype abrogates the effector cell functions of the antibody (ADCC and CDC) while retaining its antigen-binding properties, antiproliferative capacity (in vitro and in vivo), and the ability to induce apoptosis concurrent with activation of the AKT survival pathway. Growth inhibition was evaluated compared with and in combination with the anti-CD20 mAb rituximab, with the combination being more effective than rituximab alone in inhibiting proliferation. Thus, hL243gamma4P is indistinguishable from hL243gamma1 and the parental murine mAb in assays dependent on antigen recognition. The abrogation of ADCC and CDC, which are believed to play a major role in side effects of mAb therapy, may make this antibody an attractive clinical agent. In addition, combination of hL243gamma4P with rituximab offers the prospect for improved patient outcome.

Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys.

PURPOSE: IMMU-110 is a drug immunoconjugate composed of doxorubicin conjugated to the humanized anti-CD74 monoclonal antibody, hLL1, at a doxorubicin/monoclonal antibody ratio of approximately 8:1 (mol/mol). CD74 is a rapidly internalizing molecule associated with HLA-DR, which has high expression by several tumor types. Here, we describe safety evaluations of IMMU-110 in mice and monkeys as well as efficacy studies in a xenograft model of the human multiple myeloma cell line, MC/CAR. EXPERIMENTAL DESIGN: In vitro binding of IMMU-110 was determined by a cell-based ELISA and cytotoxicity of IMMU-110 assayed with a tetrazolium assay. Pharmacokinetics and biodistribution of radiolabeled IMMU-110 were examined in tumor-free BALB/c mice, and the therapeutic effectiveness was evaluated in severe combined immunodeficient mice bearing MC/CAR cells. Acute toxicity of IMMU-110 was studied in CD74-positive cynomolgus monkeys (Macaca fascicularis). RESULTS: In vitro, IMMU-110 specifically binds to CD74 and is cytotoxic against MC/CAR cells. In vivo, IMMU-110 displayed a pharmacokinetic and biodistribution profile identical to that of unconjugated hLL1 monoclonal antibody, except for higher kidney uptake. Treatment with a single dose of IMMU-110 as low as 50 microg antibody/mouse (or 1.4 microg doxorubicin/mouse), 5 days postinjection of the multiple myeloma cells, resulted in cure of most mice. In mice, no host toxicity of IMMU-110 was observed at the highest protein dose tested (125 mg/kg). In cynomolgus monkeys, bone marrow toxicity was observed at 30 and 90 mg/kg doses. CONCLUSIONS: The excellent safety and efficacy profile of IMMU-110 supports clinical testing of this immunoconjugate in the treatment of CD74-positive B-cell malignancies.

Advantage of a residualizing iodine radiolabel in the therapy of a colon cancer xenograft targeted with an anticarcinoembryonic antigen monoclonal antibody.

PURPOSE: A disadvantage of conventionally radioiodinated monoclonal antibodies (mAb) for cancer therapy is the short retention time of the radionuclide within target cells. To address this issue, we recently developed a method in which radioiodine is introduced onto antibodies using an adduct consisting of a nonmetabolizable peptide attached to the aminopolycarboxylate diethylenetriaminepentaacetic acid, designated IMP-R4. This adduct causes the radioiodine to become trapped in lysosomes following antibody catabolism. Clinical-scale production of 131I-IMP-R4-labeled antibodies is possible using a recently developed facile method. EXPERIMENTAL DESIGN: The properties of 131I-IMP-R4-labeled anticarcinoembryonic antigen (CEA) humanized mAb hMN-14 were compared with the directly radioiodinated hMN-14 (131I-hMN-14) in CEA-expressing human colon cancer cell lines, LoVo and LS174T, and in nude mice bearing established LoVo tumor xenografts. RESULTS: 125I-IMP-R4-hMN-14 retention in the cell lines was significantly increased (61.5% after 3 days) compared with 125I-hMN-14. In vivo, a significant improvement in tumor accretion of radiolabel was obtained using 131I-IMP-R4-hMN-14, which led to a marked improvement in therapeutic efficacy. Eight weeks post-treatment, mean tumor volumes were 0.16 +/- 0.19 and 1.99 +/- 1.35 cm3 in mice treated with 131I-IMP-R4-hMN-14 and 131I-hMN-14, respectively, with complete remissions observed in 27% of mice treated with 131I-IMP-R4-hMN-14 and none using 131I-hMN-14. CONCLUSION: 131I-IMP-R4-hMN-14 provides a significant therapeutic advantage in comparison to the conventionally 131I-labeled antibody. The ability of this labeling method to lend itself to clinical-scale labeling, the broad applicability of a humanized anti-CEA mAb for CEA-expressing cancers, and the clinical benefits of radioimmunotherapy with anti-CEA mAb shown recently for small-volume and minimal residual disease combine to make 131I-IMP-R4-hMN-14 a promising new agent for radioimmunotherapy.