First Bispecific Antibody Immunocytokine (Anti-CD20/HLA-DR-Interferon-á2b) Is Highly Toxic for Human Lymphoma Cells in Vitro.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1695-1695
Author(s):  
Diane L Nordstrom ◽  
Edmund A Rossi ◽  
David M. Goldenberg ◽  
Chien-Hsing Chang
Keyword(s):  
Bispecific Antibody ◽  
Size Exclusion ◽  
Biological Entity ◽  
Lymphoma Cells ◽  
Hla Dr ◽  
Human Lymphoma ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 1695 Poster Board I-721 Background IFN-á2 is indicated for the therapy of a variety of hematopoietic tumors. As with most cytokines, the short serum half-life and severe side effects of IFN-á2 are major factors affecting its dosing schedule and efficacy. Fusion or conjugation of IFN-á2 to a tumor-targeting IgG has the potential to enhance in vivo potency due to increased tumor localization and more favorable pharmacokinetics. We have recently demonstrated that 20-2b, a monospecific immunocytokine generated by the dock-and-lock (DNL) method to comprise tetrameric IFN-á2b covalently linked to veltuzumab, a humanized anti-CD20 mAb, exhibited very potent anti-tumor activity in vitro and in human lymphoma xenografts (Rossi et al., Blood, in press). However, lymphomas and leukemias that express little or no CD20 are expected to be resistant to therapy with 20-2b. HLA-DR is expressed on many hematopoietic tumors and some solid cancers. A bispecific immunocytokine that could target IFN-á to both CD20 and HLA-DR might be a more effective therapeutic against a wide variety of hematopoietic malignancies, including those that express CD20, HLA-DR, or both. Since each component of the multifunctional complex (veltuzumab, anti-HLA-DR F(ab)2, and IFN-á2b) has anti-tumor activity independently, we evaluated if the bispecific immunocytokine can potentially be even more potent than the monospecific immunocytokine, 20-2b. Methods One strategy of the modular DNL method is to fuse either the dimerization-and-docking domain (DDD) derived from protein kinase A, or the anchoring domain (AD) of a cognate A-kinase anchoring protein, to a biological entity, resulting in respective DDD- and AD-modules that are readily combined to quantitatively generate stably-tethered structures of defined composition with retained bioactivity. We have selectively combined recombinant DDD-modules of both IFN-á2b and anti-HLA-DR Fab (derived from humanized L243) together with a recombinant AD-module of anti-CD20 IgG (veltuzumab) to generate the first bispecific antibody-based immunocytokine, designated 20-C2-2b, which comprises two copies of IFN-á2b and a stabilized F(ab)2 of hL243 site-specifically linked to veltuzumab. Results Each of the three modules, veltuzumab-AD, hL243-Fab-DDD, and IFN-á2b-DDD, was produced recombinantly in separate myeloma cell cultures. Combining equimolar amounts of the three modules under mild redox conditions resulted in the formation of 20-C2-2b, which was purified by sequential chromatographic processes involving Protein A, IMAC and anion exchange chromatography to remove potential side-products such as 20-2b and 20-C2 (the hexavalent bispecific antibody comprising veltuzumab and four Fabs of hL243). Size-exclusion HPLC analysis indicated a major peak of a retention time consistent with a ∼310 kDa protein. Reducing SDS-PAGE of 20-C2-2b revealed the presence of all three constituents. The complex was immunoreactive with an anti-IFN-á2b, an anti-idiotype to hL243, as well as an anti-idiotype to veltuzumab, and showed increased binding to Raji lymphoma cells compared to either veltuzumab or hL243. More importantly, 20-C2-2b was found to be extremely cytotoxic to Daudi, an IFNá-sensitive Burkitt lymphoma cell line, having an IC50 = 0.035 pM, which was 100,000-fold more potent than hL243 IgG, 100-fold more potent than a combination of veltuzumab, hL243 IgG and a structural analog comprising an irrelevant IgG and IFN-á2b, and 5-fold more potent than 20-2b. In the same assay, we have also determined that 20-C2-2b was about 2-fold more potent than C2-2b, which comprises hL243 IgG linked to four molecules of IFN-á2b. Conclusions The DNL method provides a modular approach to enable the creation of novel multifunctional complexes. Based on our experience with 20-2b, the bispecific immunocytokine 20-C2-2b is expected to have greater in vivo potency than IFN-á due to improved pharmacokinetics and endowed targeting specificity, and may potentially be useful for therapy of a variety of hematopoietic tumors that express either CD20 or HLA-DR. Disclosures Nordstrom: Immunomedics, Inc.: Employment. Rossi:Immunomedics, Inc: Employment. Goldenberg:Immunomedics Inc.: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Chang:Immunomedics Inc.: Employment.

Lenalidomide (Revlimid®) Enhances Monoclonal Antibody-Associated Anti-Tumor Activity Against Rituximab-Sensitive and Rituximab-Resistant B-Cell Lymphoma Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2522-2522 ◽  
Author(s):  
Nishitha Reddy ◽  
Raymond Cruz ◽  
Francisco Hernandez-Ilizaliturri ◽  
Joy Knight ◽  
Myron S. Czuczman
Keyword(s):  
B Cell ◽  
Cell Lines ◽  
Scid Mouse ◽  
In Vitro Exposure ◽  
Human Lymphoma ◽  
Scid Mouse Model ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Background: Lenalidomide is a potent thalidomide analogue shown to activate both the innate and adoptive immune system, inhibit angiogenesis, and modify the tumor microenvironment. While lenalidomide has received approval by the U.S. Federal Drug Administration (FDA) for the treatment of various hematological conditions, ongoing clinical trials are addressing its role in the treatment of B-cell lymphomas. There is a dire need to develop novel well-tolerated, therapies which combine various target-specific agents such as lenalidomide and monoclonal antibodies (mAbs). We previously demonstrated that lenalidomide is capable of expanding natural killer (NK) cells in a human-lymphoma-bearing SCID mouse model and improve rituximab anti-tumor activity in vivo. Methods: In our current work we studied the effects of lenalidomide on the biological activity of a panel of mAbs against various B-cell lymphomas, utilizing various rituximab-sensitive (RSCL) and rituximab-resistant cell lines (RRCL) generated in our laboratory from Raji and RL cell lines. Functional assays including antibody-dependant cellular cytotoxicity (ADCC) and complement-mediated cytotoxicity (CMC) were performed to demonstrate changes in sensitivity to rituximab. RSCL and RRCL (1′105 cells/well) were exposed to either lenalidomide (5 μg/ml) or vehicle with or without mAb at a final concentration of 10μg/ml. The mAb panel consisted of two anti-CD20 mAbs: rituximab (Biogen IDEC, Inc.) and hA20, a humanized anti-CD20 mAb (Immunomedics, Inc.); an anti-CD80 mAb (galixumab, Biogen IDEC Inc.), and an anti-CD52 antibody (Alemtuzumab, Berlex Inc.). Changes in DNA synthesis and cell proliferation were determined at 24 and 48 hrs by [3H]-thymidine uptake. For ADCC/CMC studies, NHL cells were exposed to lenalidomide or vehicle for 24 hrs and then labeled with 51Cr prior to treatment with one of various mAbs (10 mg/ml) and peripheral blood mononuclear cells (Effector: Target ratio, 40:1) or human serum, respectively. 51Cr-release was measured and the percentage of lysis was calculated. Changes in antigen (CD20, CD80, and CD52) expression following in vitro exposure to lenalidomide were studied by multicolor flow cytometric analysis. Results: Concomitant in vitro exposure of various RSCL and RRCL cells to lenalidomide and either galixumab, hA20 or alemtuzumab for 24 hrs resulted in improved anti-tumor activity when compared to controls. In addition, pre-incubation of both RSCL and RRCL with lenalidomide rendered cells more susceptible to alemtuzumab-, hA20- and galixumab-mediated ADCC and CMC. No antigen modulation (i.e., upregulation) was observed following in vitro exposure of lenalidomide to NHL cell lines, suggesting an alternative mechanism involved in the improvement antitumor activity observed. Conclusions: Our data suggest that the augmented antitumor effect of lenalidomide is not limited to its combination with rituximab, but also that it augments the antiproliferative and biological activity of alemtuzumab, hA20 and galixumab. Furthermore, these interactions are observed even in our RRCL. Future studies will be directed towards evaluating whether similar activity will be seen in vivo using a human lymphoma-bearing SCID mouse model. (Supported by USPHS grant PO1-CA103985 from the National Cancer Institute.)

Multivalent Anti-CD20/Anti-CD22 Bispecific Antibody Fusion Proteins Made by the DNL Method Show Potent Lymphoma Cytotoxicity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2495-2495 ◽  
Author(s):  
Edmund A. Rossi ◽  
Michele J. Losman ◽  
Diane L. Nordstrom ◽  
Thomas Cardillo ◽  
Meiyu Loo ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Bispecific Antibody ◽  
Experimental Conditions ◽  
Fab Fragments ◽  
Lymphoma Treatment ◽  
Daudi Cells ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Purpose: Multivalent and either monospecific or bispecific antibodies have shown the potential for improved efficacy in a number of preclinical studies. A panel of such agents based on hLL2 (epratuzumab, anti-CD22) and hA20 (humanized anti-CD20) was made and evaluated for cytotoxicity in vitro and anti-tumor activity in vivo. Methods: The Dock and Lock (DNL) method (Rossi et al., Proc. Natl. Acad. Sci. USA, 2006,103:6841) is a novel platform technology for the site-specific and covalent assembly of modular components. DNL was utilized to generate two hexavalent monospecific (hex-hA20 and hex-hLL2), and two hexavalent bispecific (DNL-1 and DNL-2) stably tethered complexes, each composed of four Fab fragments conjugated to an IgG at the latter’s carboxyl termini of the heavy chain. DNL-1 consists of four hA20 Fab fragments tethered to an hLL2 IgG. DNL-2 consists of four hLL2 Fab fragments tethered to an hA20 IgG. Results: For each construct, the DNL method produced a single, defined, homogeneous structure that is stable in serum. All of the constituent Fab fragments are functional, with binding affinities similar to the parental MAbs. In vitro analyses using human Burkitt lymphoma cell lines demonstrate that both the monospecific and bispecific constructs induce strong cell adhesion and potently inhibit cell growth without the requirements for further cross-linking. DNL-1, DNL-2 and hex-hA20 each completely inhibited the growth of cultured Daudi cells at concentrations as low as 1 nM and exhibited two to three logs greater potency than that of hA20 IgG or rituximab under the same experimental conditions. Anti-tumor efficacy was demonstrated in SCID mice bearing disseminated Daudi lymphoma. Treatment with 4 μg of hex-hA20 or DNL-2 at 1, 4 and 7 days post intravenous injection of 1 × 107 Daudi cells resulted in significantly increased survival of tumor-bearing mice compared to controls treated with saline. Conclusion: These findings suggest that the DNL method can be used to make multivalent proteins with potent anti-tumor activity, as shown here with anti-CD20/anti-CD22 bispecific antibody fusion proteins.

scholarly journals Treatment with 177 lu-HH1 Increases CD20 Expression in Non-Hodgkin Lymphoma Cells in Vitro and In Vivo

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3245-3245
Author(s):  
Ada H.V. Repetto-Llamazares ◽  
Roy Hartvig Larsen ◽  
Landsverk Kirsti ◽  
Trond Stokke ◽  
Bergthora Eiriksdottir ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Burkitt’S Lymphoma ◽  
Burkitt's Lymphoma ◽  
Lymphoma Cells ◽  
Employment Equity ◽  
Cd20 Expression ◽  
Equity Ownership ◽  
Anti Cd20

Abstract Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-DOTA-HH1 (177Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents. The in vitro expression of CD20 in Burkitt's Lymphoma, Daudi, cells 1-7 days after treatment with 177Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb, while Ramos (Burkitt's Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation. Treatment of nude mice with Ramos xenografts with 177Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05). SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg 177Lu-HH1 or with the combination of 40 MBq/kg 177Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of 177Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with 177Lu-HH1 alone (Table 1). In conclusion, 177Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future. Table 1. Therapy experiment groups and result Group Median ± SD Surviving fraction at the end of the study % Increase in symptom free survival compared to control NaCl + NaCl 64 ± 2 0.1 ---- NaCl + Rituximab 75 ± 10 0.3 15.4 177 Lu-HH1 + NaCl 92 ± 14 * 0.3 43.8 177 Lu-HH1 + Rituximab > 132 * 0.7 > 106.3 *Significantly different from NaCl + NaCl group (p < 0.01) Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stokke:Nordic nanovector ASA: Equity Ownership. Generalov:Nordic Nanovector ASA: Employment. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.

A new anti-idiotype antibody capable of binding rituximab on the surface of lymphoma cells

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2540-2542 ◽  
Author(s):  
Mark S. Cragg ◽  
Mike B. Bayne ◽  
Alison L. Tutt ◽  
Ruth R. French ◽  
Stephen Beers ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Cell Surface ◽  
Immunoglobulin G ◽  
Human Immunoglobulin ◽  
Lymphoma Cells ◽  
Fine Specificity ◽  
High Affinity ◽  
Anti Cd20

Abstract The chimeric anti-CD20 monoclonal antibody (mAb), rituximab, is an established part of the management of many non-Hodgkin lymphomas. The in vivo action of rituximab remains elusive, and this partially reflects a lack of highly specific reagents to detect rituximab binding at the cell surface. Here we report a new high-affinity mAb (MB2A4) with fine specificity for the idiotype of rituximab. It is able to detect rituximab in vitro, in the presence of high levels of human immunoglobulin G (IgG), in the serum of patients receiving rituximab therapy, and, surprisingly, when rituximab is bound to CD20 on the cell surface. We propose that the anti–idiotype (Id) binds to rituximab molecules bound univalently at the cell surface, facilitated by the relatively high off-rate of rituximab. This reagent provides new insights into the binding of rituximab at the cell surface and demonstrates a mode of binding that could be exploited for the surface detection of other mAbs with clinical and biologic applications.

scholarly journals 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

Blood ◽  
2006 ◽  
Vol 108 (8) ◽  
pp. 2736-2744 ◽  
Author(s):  
Rhona Stein ◽  
Zhengxing Qu ◽  
Susan Chen ◽  
David Solis ◽  
Hans J. Hansen ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Effector Cell ◽  
Cell Functions ◽  
Hla Dr ◽  
Complement Dependent Cytotoxicity ◽  
Survival Pathway ◽  
Anti Cd20

AbstractHLA-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, hL243γ4P (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 hL243γ1, 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, hL243γ4P is indistinguishable from hL243γ1 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 hL243γ4P with rituximab offers the prospect for improved patient outcome.

Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis

Blood ◽  
2000 ◽  
Vol 95 (12) ◽  
pp. 3900-3908 ◽  
Author(s):  
Josée Golay ◽  
Luisella Zaffaroni ◽  
Thomas Vaccari ◽  
Manuela Lazzari ◽  
Gian-Maria Borleri ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Low Grade ◽  
Lymphoma Cells ◽  
B Lymphoma Cells ◽  
Biologic Response ◽  
Anti Cd20 ◽  
Hodgkin's Lymphomas

Abstract The chimeric anti-CD20 MAb rituximab has recently become a treatment of choice for low-grade or follicular non-Hodgkin's lymphomas (FL) with a response rate of about 50%. In this report, we have investigated the mechanism of action of rituximab on 4 FL and 1 Burkitt's lymphoma (BL) cell lines, 3 fresh FL samples and normal B cells in vitro. Rituximab efficiently blocks the proliferation of normal B cells, but not that of the lymphoma lines. We did not detect significant apoptosis of the cell lines in response to rituximab alone. All cell lines were targets of antibody-dependent cellular cytotoxicity (ADCC). On the other hand, human complement-mediated lysis was highly variable between cell lines, ranging from 100% lysis to complete resistance. Investigation of the role of the complement inhibitors CD35, CD46, CD55, and CD59 showed that CD55, and to a lesser extent CD59, are important regulators of complement-mediated cytotoxicity (CDC) in FL cell lines as well as in fresh cases of FL: Blocking CD55 and/or CD59 function with specific antibodies significantly increased CDC in FL cells. We conclude that CDC and ADCC are major mechanisms of action of rituximab on B-cell lymphomas and that a heterogeneous susceptibility of different lymphoma cells to complement may be at least in part responsible for the heterogeneity of the response of different patients to rituximab in vivo. Furthermore, we suggest that the relative levels of CD55 and CD59 may become useful markers to predict the clinical response.

scholarly journals Rac1 Inhibits Apoptosis in Human Lymphoma Cells by Stimulating Bad Phosphorylation on Ser-75

2004 ◽  
Vol 24 (14) ◽  
pp. 6205-6214 ◽  
Author(s):  
Baolin Zhang ◽  
Yaqin Zhang ◽  
Emily Shacter
Keyword(s):  
Cell Survival ◽  
Phosphorylation Site ◽  
Small Gtpase ◽  
Lymphoma Cells ◽  
Drug Induced ◽  
Chemotherapy Drugs ◽  
Human Lymphoma ◽  
Induced Apoptosis

ABSTRACT The small GTPase Rac1 has emerged as an important regulator of cell survival and apoptosis, but the mechanisms involved are not completely understood. In this report, constitutively active Rac1 is shown to stimulate the phosphorylation of the Bcl-2 family member Bad, thereby suppressing drug-induced caspase activation and apoptosis in human lymphoma cells. Rac1 activation leads to human Bad phosphorylation specifically at serine-75 (corresponding to murine serine-112) both in vivo and in vitro. Inhibition of constitutive and activated Rac1-induced Bad phosphorylation by a cell-permeable competitive peptide inhibitor representing this Bad phosphorylation site sensitizes lymphoma cells to drug-induced apoptosis. The data show further that endogenous protein kinase A is a primary catalyst of cellular Bad phosphorylation in response to Rac activation, while Akt is not involved. These findings define a mechanism by which active Rac1 promotes lymphoma cell survival and inhibits apoptosis in response to cancer chemotherapy drugs.

Comparison of Biodistributions and Therapeutic Efficacies of Pretargeted Radioimmunoconjugates Targeting the CD20, CD22, and DR Molecules on Human B Cell Lymphomas.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 611-611
Author(s):  
Anastasia Pantelias ◽  
John M. Pagel ◽  
Nathan Hedin ◽  
Yukang Lin ◽  
Donald Axworthy ◽  
...  
Keyword(s):  
Cell Lines ◽  
Half Life ◽  
Lymphoma Cell ◽  
In Vivo Studies ◽  
Cell Binding ◽  
Normal Tissues ◽  
Human Lymphoma ◽  
Anti Cd20

Abstract Radioimmunotherapy (RIT) using anti-CD20 monoclonal antibodies (Ab) produces response rates of 60–95% in relapsed non-Hodgkin’s lymphoma (NHL) patients; however, tumor-to-normal organ ratios of absorbed radiation are low and many patients relapse. The efficacy of RIT is limited by non-specific delivery of radiation to normal tissues due to the long circulating half-life of radiolabeled antibodies. Pretargeted RIT (PRIT) using streptavidin (SA)-Ab conjugates followed by a clearing agent and radiolabeled biotin can augment the efficacy of RIT and decrease toxicity compared with conventional RIT. Although PRIT using anti-CD20-SA Abs have been studied with promising results, targeting multiple antigens may increase efficacy. Since successful clinical trials have been conducted with directly radiolabeled anti-DR and anti-CD22 Abs, we initiated in vitro and in vivo studies comparing pretargeted anti-CD20 Ab-SA conjugate (1F5/SA) with pretargeted anti-CD22-SA (HD39/SA) and anti-HLA-DR-SA (Lym-1/SA) conjugates in three different human B-lymphoma cell lines, RAMOS (Burkitt), RAJI (Burkitt) and FL-18 (transformed follicular). Using standard flow cytometry techniques all three Ab-SA conjugates bound to ≥ 97% of FL18 cells. Cell binding for 1F5/SA, Lym-1/SA, and HD39/SA was 99%, 99%, and 83% to RAJI cells, respectively, and 99%, 22%, and 85% to RAMOS cells. The blood half-life of each conjugate in vivo was measured by injecting groups of 4 mice i.v. with 0.7nmol (150μg) of 125I labeled Ab-SA conjugate and drawing 10μl of blood at various time points to determine the percent injected dose per gram (% ID/g). The half-lives of 1F5/SA, Lym-1/SA and HD39/SA were 18.38, 14.92 and 16.23 hours, respectively. When 5.8nmol (50μg) of a clearing agent (synthetic biotin-N-acetyl-galactosamine) was given 24 hours post 125I-Ab-SA injection, the % ID/g in blood fell by more than 80% of the initial dose within a half-hour. Blood, tumor and non-specific organ uptake was determined by biodistribution experiments in mice (Balb/c nu/nu) bearing human lymphoma xenografts. Athymic mice with s.c. RAMOS, RAJI, or FL-18 xenografts received 1.4nmol (300μg) of either 1F5/SA, HD39/SA, or Lym-1/SA i.v. followed 24 hours later by 5.8nmol (50μg) clearing agent to remove non-localized conjugate from circulation, and 3 hours later by an 111In labeled DOTA-biotin ligand (1μg). The biodistributions of each conjugate were evaluated by sacrificing mice at 24 and 48 hours after 111In-DOTA-biotin. At 24 hours, the ID/g was 18.2±13.6% in FL18 xenografts for pretargeted Lym-1/SA, 18.2±17.4% ID/g for 1F5/SA and 3.3±0.7% ID/g for HD39/SA. Conversely, at 24 hours pretargeted Lym-1/SA uptake in RAJI tumors was 10.8±2.1% ID/g, and 1F5/SA and HD39/SA RAJI tumor localization was 5.2±1.9% ID/g and 2.2±0.5% ID/g. respectively. 1F5/SA had superior uptake (7.1±3.3% ID/g) in RAMOS xenografts compared with Lym-1/SA (3.5±1.5% ID/g) and HD39/SA (2.7±1.0% ID/g). These data suggest a strong correlation between in vitro cell binding results and in vivo biodistributions for all three Ab-SA conjugates in all three human lymphoma cell lines. Using these agents in combination may result in a synergistic effect that has the potential to increase the efficacy of PRIT over using any one of the agents alone. Biodistribution and therapy studies using the Ab-SA conjugates in combination in tumored mice are on-going.

Ublituximab (TGTX-1101), A Novel Anti-CD20 Monoclonal Antibody (mAb), Demonstrates Activity in Rituximab-Sensitive and Rituximab–resistant B Non-Hodgkin Lymphoma (B-NHL) Pre-Clinical In vitro and in Vivo models.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2756-2756 ◽  
Author(s):  
John Miller ◽  
Matthew J. Barth ◽  
Cory Mavis ◽  
Ping-Chiao Tsai ◽  
Pavel Klener ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Primary Tumor ◽  
Scid Mice ◽  
In Vivo Experiments ◽  
Non Hodgkin Lymphoma ◽  
Tail Vein Injection ◽  
Anti Cd20 ◽  
Anti Tumor Activity

Abstract Abstract 2756 The addition of rituximab to chemotherapy regimens utilized in treating B-cell non-Hodgkin lymphoma (B-NHL) has resulted in significant improvement in treatment response and clinical outcomes. On the other hand, the use of rituximab is changing the biology and response to second-line therapy in patients with relapsed/refractory disease. Novel anti-CD20 mAbs continue to be developed that may offer additional treatment options for relapsed/refractory rituximab-pre-treated patients. Ublituximab (TGTX-1101) is a novel, chimeric mAb targeting a unique epitope on the CD20 antigen. Ublituximab has been glycoengineered to enhance affinity for all variants of FcγRIIIa receptors. To further characterize the activity of ublituximab, we evaluated its anti-tumor activity in a panel of rituximab-sensitive (RSCL), rituximab–resistant (RRCL) cell lines, primary tumor cells isolated from patients with B-NHL by negative selection using magnetic beads, and in lymphoma SCID mice xenograft models. RSCL (Raji, RL, U2932, Granta, HBL-2, Jeko-1, Mino, Rec1 and Z-138), RRCL (Raji-2R, Raji 4RH, RL-4RH, and U2932-4RH); and cytarabine-resistant (AraCR) mantle cell lymphoma cell (MCL) lines (Granta-AraCR, HBL-2-AraCR, Jeko-AraCR, Mino-AraCR and Rec1-AraCR) were labeled with 51Cr. Subsequently, cells were exposed to ublituximab, rituximab or isotope control and human serum (25%) for complement dependent cytotoxicity (CDC) assays or to effector cells isolated from healthy volunteers (effector:target ratio 40:1) for antibody dependent cellular cytotoxicity (ADCC) assays, respectively. Antibody-induced direct anti-proliferative effects and induction of apoptosis were determined by alamar blue reduction assay and Annexin-V and propidium iodide staining, respectively. Primary tumor cells (n=11) were exposed to ublituximab, rituximab or isotype control +/− pooled human serum for 48 hr. Changes in ATP content were determined using the CellTiterGlo assay. For in vivo studies, 6–8 week old SCID mice were inoculated via tail vein injection with 1×106 Raji cells on day 0 and assigned to rituximab (10mg/kg/dose), ublituximab (10mg/kg/dose) or control group. MAb was given via tail vein injection on days +3, +7, +10 and +14. Differences in survival were analyzed by Kaplan-Meier curves and p values calculated using log rank test. Ublituximab induced significantly higher ADCC when compared to rituximab in 13 out of 17 cell lines tested (including all RRCL and cytarabine resistant MCL cells): (Raji 44.4% vs. 19.8%; Raji 4RH 17.5% vs. 8.3%; Raji 2R 28.2% vs. 12%; RL 40.9% vs. 17.8%; RL-4RH 33.5% vs. 17.2%; U2932 46.9% vs. 28.8%; U2932-4RH 40.2% vs. 22.1%; HBL-2AraCR 30.7% vs. 16.6%; Jeko 34.8% vs. 18.4; Jeko-AraCR 23.8% vs. 9.6; Mino 47.4% vs. 11.6%; Mino-AraCR 32.5% vs. 15.5; Rec1 30.9% vs. 0%; p-values <0.05). There was no significant difference between ublituximab and rituximab in terms of CMC (including studies performed in primary tumor cells) or direct signaling (i.e. apoptosis or cell proliferation). While ublituximab therapy prolonged the survival of lymphoma-bearing SCID mice when compared to controls, the anti-tumor activity in vivo was similar to rituximab. Our results suggest that ublituximab exhibits higher ADCC than rituximab in vitro, including in RRCL and elicits similar CDC and direct anti-tumor effects. Despite this enhanced ADCC activity, initial in vivo experiments did not result in improved survival compared to rituximab, however additional in vivo experiments investigating the activity of ublituximab in RRCL and MCL mouse models, testing alternative dose/schedule regimens and/or in combination with other anti-lymphoma agents are planned. Updated research results will be presented at the annual meeting. A Phase I/II trial of ublituximab in patients with relapsed/refractory NHL is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

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