No abstract

CD20 is an important target for the treatment of B-cell malignancies, including non-Hodgkin lymphoma as well as autoimmune disorders. B-cell depletion therapy using monoclonal antibodies against CD20, such as rituximab, has revolutionized the treatment of these disorders, greatly improving overall survival in patients. Here, we report the development of GA101 as the first Fc-engineered, type II humanized IgG1 antibody against CD20. Relative to rituximab, GA101 has increased direct and immune effector cellmediated cytotoxicity and exhibits superior activity in cellular assays and whole blood B-cell depletion assays. In human lymphoma xenograft models, GA101 exhibits superior antitumor activity, resulting in the induction of complete tumor remission and increased overall survival. IntroductionRituximab, a type I chimeric IgG1 anti-CD20 antibody, has revolutionized the management and treatment of B-cell malignancies, increasing the median overall survival of patients with many of these diseases. 1 In combination with chemotherapy, it has significantly improved response rates and progression-free and overall survival of patients with diffuse large B-cell lymphoma (DLBCL) or follicular lymphoma. 1,2 Rituximab treatment has also benefited patients with other diseases amenable to B-cell depletion therapy, including B-cell chronic lymphocytic leukemia (B-CLL) and rheumatoid arthritis. 2,3 Nevertheless, relapse is a common occurrence, for example, in B-CLL, and there remains a need for treatments that delay the onset of relapse without increasing toxicity. 1 To this end, various therapeutic approaches are being explored, including new chemotherapies, small molecules, antibodydrug conjugates, and the use of alternative B-cell targets. However, in contrast to the situation with rituximab, the clinical benefit of these therapies remains to be demonstrated. In addition, many of these agents exhibit poor safety and tolerability profiles or necessitate the use of more complex treatment regimens.Thus far, CD20 has been the most effective unconjugated antibody target for the treatment of B-cell malignancies. An alternative and complementary approach is to generate new unconjugated CD20 antibodies with enhanced functional activities that may lead to superior efficacy. Three types of functional activities of anti-CD20 antibodies have been described: signaling in target cells on CD20 binding leading to growth inhibition and (nonclassic) apoptosis (referred to as "direct cell death"), complement-dependent cytotoxicity (CDC), and antibodydependent cellular cytotoxicity (ADCC) mediated by cells displaying Fc␥ receptors (Fc␥Rs), such as Fc␥RIIIa-expressing NK cells and macrophages. 4,5 Anti-CD20 antibodies with different functions may be generated either (1) by selecting antibodies that bind to a different CD20 epitope, which bind in an alternative mode or with changed affinity, resulting in altered intensity or type of functional mechanism; or (2) by engineering the Fc region of the antibody to enhance immune effector functions. The ...

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cellmediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation. Mol Cancer Ther; 12(10); 2031-42. Ó2013 AACR.

Purpose: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. Experimental Design: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. Results: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA–expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1–positive tumors. Conclusions: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286–97. ©2016 AACR.

Cubilin is the intestinal receptor for the endocytosis of intrinsic factor-vitamin B12. However, several lines of evidence, including a high expression in kidney and yolk sac, indicate it may have additional functions. We isolated apolipoprotein A-I (apoA-I), the main protein of high-density lipoprotein (HDL), using cubilin affinity chromatography. Surface plasmon resonance analysis demonstrated a high-affinity binding of apoA-I and HDL to cubilin, and cubilin-expressing yolk sac cells showed efficient 125I-HDL endocytosis that could be inhibited by IgG antibodies against apoA-I and cubilin. The physiological relevance of the cubilin-apoA-I interaction was further emphasized by urinary apoA-I loss in some known cases of functional cubilin deficiency. Therefore, cubilin is a receptor in epithelial apoA-I/HDL metabolism.

We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies.

Background-Carriers of the ⑀4 allele of the apolipoprotein E gene are at a higher risk of coronary heart disease than individuals with other genotypes. We examined whether the risk of death or a major coronary event in survivors of myocardial infarction depended on apolipoprotein E genotype and whether the benefits of treatment with simvastatin differed between genotypes. Methods and Results-Cox proportional hazards models were used to analyze 5.5 years of follow-up data from 966 Danish and Finnish myocardial infarction survivors enrolled in the Scandinavian Simvastatin Survival Study. A total of 16% of the 166 ⑀4 carriers in the placebo group died compared with 9% of the 312 patients without the allele, which corresponds to a mortality risk ratio of 1.8 (95% confidence interval, 1.1 to 3.1). The risk ratio was unaffected by considerations of sex, age, concurrent angina, diabetes, smoking, and serum lipids in multivariate analyses. Simvastatin treatment reduced the mortality risk to 0.33 (95% confidence interval, 0.16 to 0.69) in ⑀4 carriers and to 0.66 (95% confidence interval, 0.35 to 1.24) in other patients (Pϭ0.23 for treatment by genotype interaction). Apolipoprotein E genotype did not predict the risk of a major coronary event.Baseline serum levels of lipoprotein(a) also predicted mortality risk and could be combined with ⑀4-carrier status to define 3 groups of patients with different prognoses and benefits from treatment. Conclusions-Myocardial infarction survivors with the ⑀4 allele have a nearly 2-fold increased risk of dying compared with other patients, and the excess mortality can be abolished by treatment with simvastatin. (Circulation.

Therapeutic Abs possess several clinically relevant mechanisms of action including perturbation of tumor cell signaling, activation of complement-dependent cytotoxicity, Ab-dependent cellular cytotoxicity (ADCC), Ab-dependent cellular phagocytosis (ADCP), and induction of adaptive immunity. In view of the important role of phagocytic lineage cells in the mechanism of action of therapeutic Abs, we analyzed FcγR receptor-dependent effector functions of monocytes and macrophages triggered by glycoengineered (GE) Abs (having enhanced FcγRIIIa [CD16a] binding affinity) versus their wild-type (WT) counterparts under different experimental conditions. We first defined the precise FcγR repertoire on classical and nonclassical intermediate monocytes—M1 and M2c macrophage populations. We further show that WT and GE Abs display comparable binding and induce similar effector functions (ADCC and ADCP) in the absence of nonspecific, endogenous IgGs. However, in the presence of these IgGs (i.e., in a situation that more closely mimics physiologic conditions), GE Abs display significantly superior binding and promote stronger monocyte and macrophage activity. These data show that in addition to enhancing CD16a-dependent NK cell cytotoxicity, glycoengineering also enhances monocyte and macrophage phagocytic and cytotoxic activities through enhanced binding to CD16a under conditions that more closely resemble the physiologic setting.