Rhona Stein

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.

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–Hodgkins 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.

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 veltuzumabs lower off-rate and apparent improved potency in preclinical models that could translate into advantages in patients.

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 ∼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 μg antibody/mouse (or 1.4 μg 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.

CD74 is expressed by multiple myeloma and is a promising target for therapy

Purpose: CD74 (HLA-DR-associated invariant chain) plays a role in antigen presentation. In addition to its expression on antigen-presenting cells, it is expressed by carcinomas of renal, lung, gastric, and thymic origin and by certain sarcomas. The restricted expression of CD74 by normal tissues and its very rapid internalization make CD74 an attractive therapeutic target for both cancer and immunologic diseases. Preclinical efficacy of anti-CD74 monoclonal antibody (mAb) therapy has been demonstrated in B-lymphoma models. Because there are few validated antigenic targets in multiple myeloma, CD74 expression was examined. Experimental Design: CD74 expression was assessed by immunohistochemistry in bone marrow biopsies of known multiple myeloma cases. Its expression was measured by flow cytometry in multiple myeloma lines, and CD74 mRNA expression was determined by reverse transcription-PCR. In addition, the in vitro antiproliferative effect of LL1 mAb was evaluated on a CD74+ multiple myeloma cell line using a [3H]thymidine incorporation assay. Results: CD74 expression was observed in 19 of 22 cases of multiple myeloma, with most expressing moderate to high levels in the majority of malignant plasma cells. CD74 was expressed by most multiple myeloma cell lines, as was CD74 mRNA, at levels mirroring CD74 protein. Also, unlabeled LL1 mAb mediated in vitro growth inhibition of a CD74+ multiple myeloma cell line. Conclusions: CD74 expression is frequent in multiple myeloma, with predominant expression by the malignant plasma cells. Because anti-CD74 mAbs internalize very rapidly and LL1 mAb has shown efficacy in B-lymphoma models, CD74 represents a novel and promising target for treatment of multiple myeloma. Therefore, LL1 mAb is well suited as a carrier of radionuclides, drugs, or toxins, and also has activity as an unlabeled mAb, thereby supporting its development for this unmet need in cancer therapy.

Epitope specificity of the anti-(B cell lymphoma) monoclonal antibody, LL2

LL2 is a murine monoclonal antibody IgG2a reactive with B cells and non-Hodgkins B-cell lymphoma, which, in a radioiodinated form, induces responses in lymphoma patients [Goldenberg et al. (1991) J Clin Oncol 9:548–564]. In this report we identify LL2 as a member of the CD22 cluster. The molecular size of the antigen, its expression profile, and competitive blocking studies were used to establish this identification. By Western blot analysis and immunoprecipitation studies using the Raji Burkitts lymphoma cell line metabolically labelled with [3H]leucine, the LL2 antigen was determined to correspond to a molecular mass of 140 kDa. The molecular mass of the LL2 antigen, and the B-cell-restricted reactivity of the LL2 antibody, were consistent with both the CD21 and CD22 clusters. To assess additional similarities and differences between LL2 and anti-CD22 and anti-CD21, the binding of these mAb to cultured cell lines. Nalm-6 and Molt-4, was compared by flow cytometry. The binding profile of LL2 on these cell lines was consistent with anti-CD22, but not anti-CD21. Sequential immunoprecipitation and cross-blocking studies with anti-CD22 monoclonal antibodies recognizing established CD22 epitopes were performed to confirm that LL2 reacts with CD22 and to determine which epitope LL2 recognizes. Binding of131I-LL2 to Raji cells is inhibited over 90% by prior incubation of the target cells with unlabelled RFB4, indicating that LL2 belongs to the same epitope group as RFB4, i.e., epitope B.

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.

CD20-targeted tetrameric interferon-α, 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.

Advantage of residualizing radiolabels for an internalizing antibody against the B-cell lymphoma antigen, CD22

Abstract LL2 is an anti-CD22 pan-B-cell monoclonal antibody which, when radiolabeled, has a high sensitivity for detecting B-cell, non-Hodgkin’s lymphoma (NHL), as well as an antitumor efficacy in therapeutic applications. The aim of this study was to determine whether intracellularly retained radiolabels have an advantage in the diagnosis and therapy of lymphoma with LL2. In vitro studies showed that iodinated LL2 is intracellularly catabolized, with a rapid release of the radioiodine from the cell. In contrast, residualizing radiolabels, such as radioactive metals, are retained intracellularly for substantially longer. In vivo studies were performed using LL2-labeled with radioiodine by a non-residualizing (chloramine-T) or a residualizing method (dilactitol-tyramine, DLT), or with a radioactive metal (111In). The biodistribution of a mixture of 125I (non-residualizing chloramine-T compared to residualizing DLT), 111In-labeled LL2 murine IgG2a or its fragments [F(ab′)2, Fab′], as well as its humanized, CDR-grafted form, was studied in nude mice bearing the RL human B-cell NHL cell line. Radiation doses were calculated from the biodistribution data according to the Medical International Radiation Dose scheme to assess the potential advantage for therapeutic applications. At all assay times, tumor uptake was higher with the residualizing labels (i.e., 111In and DLT-125I) than with the non-residualizing iodine label. For example, tumor/blood ratios of 111In-labeled IgG were 3.2-, 3.5- and 2.8-fold higher than for non-residualizing iodinated IgG on days 3, 7 and 14, respectively. Similar results were obtained for DLT-labeled IgG and fragments with residualized radiolabels. Tumor/organ ratios also were higher with residualizing labels. No significant differences in tumor, blood and organ uptake were observed between murine and humanized LL2. The conventionally iodinated anti-CD20 antibody, 1F5, had tumor uptake values comparable to those of iodinated LL2, the uptake of both antibodies being strongly dependent on tumor size. These data suggest that, with internalizing antibodies such as LL2, labeling with intracellularly retained isotopes has an advantage over released ones, which justifies further clinical trials with residualizing 111In-labeled LL2 for diagnosis, and residualizing 131I and 90Y labels for therapy.

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.