Pharmacokinetics and pharmacodynamics of pozelimab alone or in combination with cemdisiran in non-human primates.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease caused by uncontrolled complement activation; effective and approved treatments include terminal complement inhibition. This study assessed whether combination cemdisiran (an investigational N-acetylgalactosamine-conjugated RNAi therapeutic that suppresses liver production of complement component C5) and pozelimab (an investigational fully human monoclonal antibody against C5) results in more effective and durable complement activity inhibition than the individual agents alone in non-human primates. Cynomolgus monkeys received a single subcutaneous injection of cemdisiran (5 or 25 mg/kg), pozelimab (5 or 10 mg/kg), or combination cemdisiran and pozelimab (5+5 mg/kg, 5+10 mg/kg, or 25+10 mg/kg, respectively). When given in combination, pozelimab was administered 2 weeks after cemdisiran dosing. Pharmacokinetics and ex vivo pharmacodynamic properties were assessed. The half-life of pozelimab alone was 12.9-13.3 days; this increased to 19.6-21.1 days for pozelimab administered in combination with cemdisiran. In ex vivo classical pathway hemolysis assays (CH50), pozelimab + cemdisiran combinations achieved durable and more complete suppression of complement activity (8-13 weeks) vs monotherapy of either agent. Cemdisiran monotherapy demonstrated dose-dependent suppression of total C5 concentrations, with the higher dose (25 mg/kg) achieving >90% maximum suppression. Total C5 concentrations after administration of pozelimab + cemdisiran combinations were similar compared with administration of cemdisiran alone. The combination of pozelimab + cemdisiran mediates complement activity inhibition more efficiently than either pozelimab or cemdisiran administered alone. The pharmacokinetic/pharmacodynamic profile of combination pozelimab + cemdisiran in non-human primates appears suitable for further clinical investigation as a potential long-acting treatment for PNH and other complement-mediated diseases.

Translational findings for odronextamab: From preclinical research to a first-in-human study in patients with CD20+ B-cell malignancies.

Odronextamab is a fully-human IgG4-based CD20xCD3 bispecific antibody that binds to CD3 on T cells and CD20 on B cells, triggering T-cell-mediated cytotoxicity independent of T-cell-receptor recognition. Adequate safety, tolerability, and encouraging durable complete responses have been observed in an ongoing first-in-human (FIH) study of odronextamab in patients with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (B-NHL; NCT02290951). We retrospectively evaluated the pharmacokinetic, pharmacodynamic, and antitumor characteristics of odronextamab in a series of in vitro/in vivo preclinical experiments, to assess their translational value to inform dose escalation for the FIH study. Half-maximal effective concentration values from in vitro cytokine release assays (range: 0.05-0.08 mg/L) provided a reasonable estimate of odronextamab concentrations in patients associated with cytokine release at a 0.5 mg dose (maximum serum concentration: 0.081 mg/L) on week 1/day 1, which could therefore be used to determine the week 1 clinical dose. Odronextamab concentrations resulting in 100% inhibition of tumor growth in a Raji xenograft tumor mouse model (1-10 mg/L) were useful to predict efficacious concentrations in patients and inform dose-escalation strategy. Although predicted human pharmacokinetic parameters derived from monkey data overestimated projected odronextamab exposure, they provided a conservative estimate for FIH starting doses. With step-up dosing, the highest-tested weekly odronextamab dose in patients (320 mg) exceeded the 1 mg/kg single dose in monkeys without step-up dosing. In conclusion, combination of odronextamab in vitro cytokine data, efficacious concentration data from mouse tumor models, and pharmacokinetic evaluations in monkeys has translational value to inform odronextamab FIH study design in patients with R/R B-NHL.

A Biparatopic Antibody-Drug Conjugate to Treat MET-Expressing Cancers, Including Those that Are Unresponsive to MET Pathway Blockade.

Lung cancers harboring mesenchymal-to-epithelial transition factor (MET) genetic alterations, such as exon 14 skipping mutations or high-level gene amplification, respond well to MET-selective tyrosine kinase inhibitors (TKI). However, these agents benefit a relatively small group of patients (4%-5% of lung cancers), and acquired resistance limits response durability. An antibody-drug conjugate (ADC) targeting MET might enable effective treatment of MET-overexpressing tumors (approximately 25% of lung cancers) that do not respond to MET targeted therapies. Using a protease-cleavable linker, we conjugated a biparatopic METxMET antibody to a maytansinoid payload to generate a MET ADC (METxMET-M114). METxMET-M114 promotes substantial and durable tumor regression in xenografts with moderate to high MET expression, including models that exhibit innate or acquired resistance to MET blockers. Positron emission tomography (PET) studies show that tumor uptake of radiolabeled METxMET antibody correlates with MET expression levels and METxMET-M114 efficacy. In a cynomolgus monkey toxicology study, METxMET-M114 was well tolerated at a dose that provides circulating drug concentrations that are sufficient for maximal antitumor activity in mouse models. Our findings suggest that METxMET-M114, which takes advantage of the unique trafficking properties of our METxMET antibody, is a promising candidate for the treatment of MET-overexpressing tumors, with the potential to address some of the limitations faced by the MET function blockers currently in clinical use.

Generation of T-cell-redirecting bispecific antibodies with differentiated profiles of cytokine release and biodistribution by CD3 affinity tuning.

T-cell-redirecting bispecific antibodies have emerged as a new class of therapeutic agents designed to simultaneously bind to T cells via CD3 and to tumor cells via tumor-cell-specific antigens (TSA), inducing T-cell-mediated killing of tumor cells. The promising preclinical and clinical efficacy of TSAxCD3 antibodies is often accompanied by toxicities such as cytokine release syndrome due to T-cell activation. How the efficacy and toxicity profile of the TSAxCD3 bispecific antibodies depends on the binding affinity to CD3 remains unclear. Here, we evaluate bispecific antibodies that were engineered to have a range of CD3 affinities, while retaining the same binding affinity for the selected tumor antigen. These agents were tested for their ability to kill tumor cells in vitro, and their biodistribution, serum half-life, and anti-tumor activity in vivo. Remarkably, by altering the binding affinity for CD3 alone, we can generate bispecific antibodies that maintain potent killing of TSA + tumor cells but display differential patterns of cytokine release, pharmacokinetics, and biodistribution. Therefore, tuning CD3 affinity is a promising method to improve the therapeutic index of T-cell-engaging bispecific antibodies.

A BCMAxCD3 bispecific T cell-engaging antibody demonstrates robust antitumor efficacy similar to that of anti-BCMA CAR T cells.

CD3-engaging bispecific antibodies (bsAbs) and chimeric antigen receptor (CAR) T cells are potent therapeutic approaches for redirecting patient T cells to recognize and kill tumors. Here we describe a fully human bsAb (REGN5458) that binds to B-cell maturation antigen (BCMA) and CD3, and compare its antitumor activities vs those of anti-BCMA CAR T cells to identify differences in efficacy and mechanism of action. In vitro, BCMAxCD3 bsAb efficiently induced polyclonal T-cell killing of primary human plasma cells and multiple myeloma (MM) cell lines expressing a range of BCMA cell surface densities. In vivo, BCMAxCD3 bsAb suppressed the growth of human MM tumors in murine xenogeneic models and showed potent combinatorial efficacy with programmed cell death protein 1 blockade. BCMAxCD3 bsAb administration to cynomolgus monkeys was well tolerated, resulting in the depletion of BCMA+ cells and mild inflammatory responses characterized by transient increases in C-reactive protein and serum cytokines. The antitumor efficacy of BCMAxCD3 bsAb was compared with BCMA-specific CAR T cells containing a BCMA-binding single-chain variable fragment derived from REGN5458. Both BCMAxCD3 bsAb and anti-BCMA CAR T cells showed similar targeted cytotoxicity of MM cell lines and primary MM cells in vitro. In head-to-head in vivo studies, BCMAxCD3 bsAb rapidly cleared established systemic MM tumors, whereas CAR T cells cleared tumors with slower kinetics. Thus, using the same BCMA-binding domain, these results suggest that BCMAxCD3 bsAb rapidly exerts its therapeutic effects by engaging T cells already in place at the tumor site, whereas anti-BCMA CAR T cells require time to traffic to the tumor site, activate, and numerically expand before exerting antitumor effects.

Preclinical PET imaging with the novel human antibody 89Zr-DFO-REGN3504 sensitively detects PD-L1 expression in tumors and normal tissues.

BACKGROUND: Programmed cell death protein 1/programmed death-ligand 1 (PD-1/PD-L1) blocking antibodies including cemiplimab have generated profound clinical activity across diverse cancer types. Tumorous PD-L1 expression, as assessed by immunohistochemistry (IHC), is an accepted predictive marker of response to therapy in some cancers. However, expression is often dynamic and heterogeneous, and therefore not reliably captured by IHC from tumor biopsies or archival samples. Thus, there is significant need for accurate whole-body quantification of PD-L1 levels. METHODS: We radiolabeled the novel human anti-PD-L1 antibody REGN3504 with zirconium-89 (89Zr) using the chelator p-SCN-Bn-Deferoxamine to enable non-invasive immuno-positron emission tomography (immuno-PET) of PD-L1 expression. PET imaging assessed the localization of 89Zr-REGN3504 to multiple human tumor xenografts. Mice genetically humanized for PD-1 and PD-L1 were used to assess the biodistribution of 89Zr-REGN3504 to normal tissues and the estimated human radiation dosimetry of 89Zr-REGN3504 was also determined. Pharmacokinetics of REGN3504 was assessed in monkeys. RESULTS: Clear localization of 89Zr-REGN3504 to human tumor xenografts was observed via PET imaging and ex vivo biodistribution studies demonstrated high (fourfold to sixfold) tumor:blood ratios. 89Zr-REGN3504 specifically localized to spleen and lymph nodes in the PD-1/PD-L1 humanized mice. 89Zr-REGN3504 immuno-PET accurately detected a significant reduction in splenic PD-L1 positive cells following systemic treatment with clodronate liposomes. Radiation dosimetry suggested absorbed doses would be within guidelines for other 89Zr radiolabeled, clinically used antibodies. Pharmacokinetics of REGN3504 was linear. CONCLUSION: This work supports the clinical translation of 89Zr-REGN3504 immuno-PET for the assessment of PD-L1 expression. Future clinical studies will aim to investigate the utility of 89Zr-REGN3504 immuno-PET for predicting and monitoring response to anti-PD-1 therapy.

Inhibition of complement pathway activation with Pozelimab, a fully human antibody to complement component C5.

Complement is a key component of the innate immune system. Inappropriate complement activation underlies the pathophysiology of a variety of diseases. Complement component 5 (C5) is a validated therapeutic target for complement-mediated diseases, but the development of new therapeutics has been limited by a paucity of preclinical models to evaluate the pharmacokinetic (PK) and pharmacodynamic (PD) properties of candidate therapies. The present report describes a novel humanized C5 mouse and its utility in evaluating a panel of fully human anti-C5 antibodies. Surprisingly, humanized C5 mice revealed marked differences in clearance rates amongst a panel of anti-C5 antibodies. One antibody, pozelimab (REGN3918), bound C5 and C5 variants with high affinity and potently blocked complement-mediated hemolysis in vitro. In studies conducted in both humanized C5 mice and cynomolgus monkeys, pozelimab demonstrated prolonged PK and durable suppression of hemolytic activity ex vivo. In humanized C5 mice, a switch in dosing from in-house eculizumab to pozelimab was associated with normalization of serum C5 concentrations, sustained suppression of hemolytic activity ex vivo, and no overt toxicity. Our findings demonstrate the value of humanized C5 mice in identifying new therapeutic candidates and treatment options for complement-mediated diseases.

A Mucin 16 bispecific T cell-engaging antibody for the treatment of ovarian cancer.

Advanced ovarian cancer is frequently treated with combination chemotherapy, but high recurrence rates show the need for therapies that can produce durable responses and extend overall survival. Bispecific antibodies that interact with tumor antigens on cancer cells and activating receptors on immune cells offer an innovative immunotherapy approach. Here, we describe a human bispecific antibody (REGN4018) that binds both Mucin 16 (MUC16), a glycoprotein that is highly expressed on ovarian cancer cells, and CD3, thus bridging MUC16-expressing cells with CD3+ T cells. REGN4018 induced T cell activation and killing of MUC16-expressing tumor cells in vitro. Binding and cytotoxicity of REGN4018 in vitro were minimally affected by high concentrations of CA-125, the shed form of MUC16, which is present in patients. In preclinical studies with human ovarian cancer cells and human T cells in immunodeficient mice, REGN4018 potently inhibited growth of intraperitoneal ovarian tumors. Moreover, in a genetically engineered immunocompetent mouse expressing human CD3 and human MUC16 [humanized target (HuT) mice], REGN4018 inhibited growth of murine tumors expressing human MUC16, and combination with an anti-PD-1 antibody enhanced this efficacy. Immuno-PET imaging demonstrated localization of REGN4018 in MUC16-expressing tumors and in T cell-rich organs such as the spleen and lymph nodes. Toxicology studies in cynomolgus monkeys showed minimal and transient increases in serum cytokines and C-reactive protein after REGN4018 administration, with no overt toxicity. Collectively, these data demonstrate potent antitumor activity and good tolerability of REGN4018, supporting clinical evaluation of REGN4018 in patients with MUC16-expressing advanced ovarian cancer.

Anti-PCSK9 antibody pharmacokinetics and low-density lipoprotein-cholesterol pharmacodynamics in nonhuman primates are antigen affinity-dependent and exhibit limited sensitivity to neonatal Fc receptor-binding enhancement.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as an attractive therapeutic target for cardiovascular disease. Monoclonal antibodies (mAbs) that bind PCSK9 and prevent PCSK9:low-density lipoprotein receptor complex formation reduce serum low-density lipoprotein-cholesterol (LDL-C) in vivo. PCSK9-mediated lysosomal degradation of bound mAb, however, dramatically reduces mAb exposure and limits duration of effect. Administration of high-affinity mAb1:PCSK9 complex (1:2) to mice resulted in significantly lower mAb1 exposure compared with mAb1 dosed alone in normal mice or in PCSK9 knockout mice lacking antigen. To identify mAb-binding characteristics that minimize lysosomal disposition, the pharmacokinetic behavior of four mAbs representing a diverse range of PCSK9-binding affinities at neutral (serum) and acidic (endosomal) pH was evaluated in cynomolgus monkeys. Results revealed an inverse correlation between affinity and both mAb exposure and duration of LDL-C lowering. High-affinity mAb1 exhibited the lowest exposure and shortest duration of action (6 days), whereas mAb2 displayed prolonged exposure and LDL-C reduction (51 days) as a consequence of lower affinity and pH-sensitive PCSK9 binding. mAbs with shorter endosomal PCSK9:mAb complex dissociation half-lives (<20 seconds) produced optimal exposure-response profiles. Interestingly, incorporation of previously reported Fc-region amino acid substitutions or novel loop-insertion peptides that enhance in vitro neonatal Fc receptor binding, led to only modest pharmacokinetic improvements for mAbs with pH-dependent PCSK9 binding, with only limited augmentation of pharmacodynamic activity relative to native mAbs. A pivotal role for PCSK9 in mAb clearance was demonstrated, more broadly suggesting that therapeutic mAb-binding characteristics require optimization based on target pharmacology.

Impact of glycation on antibody clearance.

Glycation of therapeutic proteins occurs during mammalian cell culture expression and upon administration to patients. Since the chemical attachment of mannose or other sugars via a chemical linker has been shown to increase a protein's clearance rate in mice through the mannose receptor, we explored the effect of mannose glycation on the clearance of an IgG in mice. An IgG decorated with high levels of mannose (~18 mol/mol protein) through glycation did not clear faster in mice than the underivatized protein, whereas the same IgG decorated with mannose attached in a way to maintain the normal glycosidic bond (2-imino-2-methoxyethyl-1-thiomannoside, or IMT-mannose) at similar derivatization levels cleared significantly faster. Surface plasmon resonance studies revealed that the IgG derivatized with IMT-mannose bound tightly to the mannose receptor (KD = 20 nM) but the IgG glycated with mannose did not bind. These results indicate that glycation, even at unnaturally elevated levels, does not appear to be a clearance concern for therapeutic proteins.

Evaluation of matrix microsampling methods for therapeutic drug candidate quantification in discovery-stage rodent pharmacokinetic studies.

BACKGROUND: AMG 517 or 1-aminobenzotriazole were quantified by LC-MS/MS from low blood/plasma volumes for rat pharmacokinetic (PK) characterization in order to qualify manual/automated dried blood spot (DBS) sampling and plasma separation capillary sampling. In addition, mouse serial automated blood sampling was compared with standard composite sampling. MATERIALS & METHODS: AMG 517 or 1-aminobenzotriazole was administered to rats or mice and multiple microsampling techniques were used to obtain blood or plasma. RESULTS: PK parameters derived from DBS and whole blood-obtained drug concentrations were within 7% for manual DBS and 20% for automated DBS. Plasma PK parameters derived from capillary or standard plasma-obtained drug concentrations differed by 6%. Plasma PK parameters obtained from serial automated blood sampling or manual composite sampling were within 20%. CONCLUSION: Collectively, these results suggest that the microsampling applications that were investigated are attractive approaches for quantifying drug candidates in low matrix volumes that can be successfully employed within discovery-stage rodent PK studies.

Investigation of maternal and fetal exposure to an IgG2 monoclonal antibody following biweekly intravaginal administration to cynomolgus monkeys throughout pregnancy.

To assess the potential for male-mediated drug transfer to their female partner and/or developing conceptus, vaginal uptake of a monoclonal antibody (mAb) biotherapeutic was assessed in cynomolgus monkeys. A human IgG2 mAb (IgG2X; bound human and cynomolgus monkey neonatal Fc-receptor, FcRn, with similar high affinity) was administered intravaginally (IvG; 100mg/dose) to 5 pregnant cynomolgus monkeys biweekly from gestation day (gd) 21 to gd133. In all maternal samples collected before gd119, IgG2X plasma concentrations were below the limit of quantification (BLQ; <25ng/mL). After dosing on gd119 and 133, maternal IgG2X plasma concentrations remained BLQ in 3/5 monkeys and were very low in 2/5 (up to 116ng/mL; ∼0.01% of the IvG dose). IgG2X was BLQ in all fetal plasma samples. These data indicate that male-mediated mAb drug transfer via seminal fluid does not present a health risk to the female partner and is not bioavailable to the developing conceptus.

Placental transfer of a fully human IgG2 monoclonal antibody in the cynomolgus monkey, rat, and rabbit: a comparative assessment from during organogenesis to late gestation.

Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo-fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6-fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3-fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10-fold early in the second trimester (gd50-70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6-fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high-dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans.

Homeostasis of human NK cells is not IL-15 dependent.

IL-15 is a proinflammatory cytokine that plays an important role in the development and activation of NK cells and is a potential target for inflammatory disease therapy. Studies conducted in IL-15- and IL-15R knockout mice identified IL-15 as an important cytokine for NK cell homeostasis. Consistent with this information derived from genetically modified mice, we demonstrated that neutralizing IL-15 with a mouse anti-mouse IL-15 mAb (M96) depletes C57BL/6 mouse NK cells. An mAb directed against macaque IL-15 (Hu714MuXHu) was manufactured and demonstrated to block IL-15-induced activation of nonhuman primate (NHP) NK cells in vitro. Neutralization of macaque IL-15 by parenteral administration of Hu714MuXHu reduces (>95%) circulating NK cell counts in NHPs. A blocking mAb directed against human IL-15 (huIL-15; AMG 714) was manufactured. Unexpectedly, when human subjects were treated with the blocking anti-IL-15 Ab AMG 714 in clinical trials, no reductions in circulating NK cell counts were observed despite achieving significantly higher exposures than the levels of Hu714MuXHu needed to cause NK cell count reductions in NHPs in vivo. Both AMG 714 and Hu714MuXHu are able to block huIL-15 activity in a human T cell blast proliferation and IFN-γ production assay. Both Abs block huIL-15-mediated Stat5 activation and CD69 expression in human NK cells. Collectively, these results demonstrate that NK cell homeostasis is obligatorily dependent upon IL-15 in both mice and NHPs, but that IL-15 is dispensable for maintenance of circulating human NK cells.

Measurement of in vivo therapeutic mAb concentrations: comparison of conventional serum/plasma collection and analysis to dried blood spot sampling.

BACKGROUND: Development of an alternative sampling method that uses small amounts of whole blood, such as dried blood spots (DBS), would be an advance in the quantitative assay field. Previously, we assessed the ability to quantitate therapeutic monoclonal antibodies present in DBS compared with a typical serum sample-based method, and concluded that measurements in DBS were reproducible and yielded methods that met requirements for precision, accuracy and sensitivity. The goal herein was to assess the measurement of therapeutic antibodies in DBS compared with serum and plasma in vivo. RESULTS: Comparison of DBS versus serum in Sprague-Dawley rats and DBS versus plasma in cynomolgus monkeys for measurement of antibody concentrations revealed a two- to three-fold difference in exposure between the samples. CONCLUSION: Overall, there was good correlation between DBS versus serum and DBS versus plasma, but there was a discrepancy in DBS exposures, presumably attributable to hematocrit and recovery effects.

Effects of AMG 145 on low-density lipoprotein cholesterol levels: results from 2 randomized, double-blind, placebo-controlled, ascending-dose phase 1 studies in healthy volunteers and hypercholesterolemic subjects on statins.

OBJECTIVES: The aim of this study was to evaluate the safety, tolerability, and effects of AMG 145 on low-density lipoprotein cholesterol (LDL-C) in healthy and hypercholesterolemic subjects on statin therapy. BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) down-regulates surface expression of the low-density lipoprotein receptor (LDL-R), increasing serum LDL-C. AMG 145, a fully human monoclonal antibody to PCSK9, prevents PCSK9/LDL-R interaction, restoring LDL-R recycling. METHODS: Healthy adults (phase 1a) were randomized to 1 dose of AMG 145: 7, 21, 70, 210, or 420 mg SC; 21 or 420 mg IV; or matching placebo. Hypercholesterolemic adults (phase 1b) receiving low- to moderate-dose statins were randomized to multiple SC doses of AMG 145: 14 or 35 mg once weekly (QW) ×6, 140 or 280 mg every 2 weeks (Q2W) ×3, 420 mg every 4 weeks ×2, or matching placebo. Eleven subjects receiving high-dose statins and 6 subjects with heterozygous familial hypercholesterolemia were randomized to SC AMG 145 140 mg or placebo Q2W ×3. RESULTS: In the trials (AMG 145 n = 85, placebo n = 28), AMG 145 reduced LDL-C up to 64% (p < 0.0001) versus placebo after 1 dose ≥21 mg and up to 81% (p < 0.001) with repeated doses ≥35 mg QW. No serious adverse events (AEs) occurred. Overall incidence of treatment-emergent AEs was similar in AMG 145 versus placebo groups: 69% versus 71% (phase 1a); 65% versus 64% (phase 1b). CONCLUSIONS: In phase 1 studies, AMG 145 significantly reduced serum LDL-C in healthy and hypercholesterolemic statin-treated subjects, including those with heterozygous familial hypercholesterolemia or taking the highest doses of atorvastatin or rosuvastatin, with an overall AE profile similar to placebo.

Enhancing antibody Fc heterodimer formation through electrostatic steering effects: applications to bispecific molecules and monovalent IgG.

Naturally occurring IgG antibodies are bivalent and monospecific. Bispecific antibodies having binding specificities for two different antigens can be produced using recombinant technologies and are projected to have broad clinical applications. However, co-expression of multiple light and heavy chains often leads to contaminants and pose purification challenges. In this work, we have modified the CH3 domain interface of the antibody Fc region with selected mutations so that the engineered Fc proteins preferentially form heterodimers. These novel mutations create altered charge polarity across the Fc dimer interface such that coexpression of electrostatically matched Fc chains support favorable attractive interactions thereby promoting desired Fc heterodimer formation, whereas unfavorable repulsive charge interactions suppress unwanted Fc homodimer formation. This new Fc heterodimer format was used to produce bispecific single chain antibody fusions and monovalent IgGs with minimal homodimer contaminants. The strategy proposed here demonstrates the feasibility of robust production of novel Fc-based heterodimeric molecules and hence broadens the scope of bispecific molecules for therapeutic applications.

A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduces serum cholesterol in mice and nonhuman primates.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.

Endowing self-binding feature restores the activities of a loss-of-function chimerized anti-GM2 antibody.

Our previous studies have described a rare type of antibody that spontaneously binds to itself, or homodimerizes. This self-binding, or autophilic antibody provides stronger protection against bacterial infection than a non-self-binding antibody with identical specificity and affinity, due to an increase of polymeric avidity. Furthermore, we have shown that a peptide derived from the self-binding domain of the autophilic T15 antibody can be crosslinked to the Fc carbohydrate of monoclonal antibodies specific for the B-cell receptor of B-cell tumors. These peptide-crosslinked antibodies can exert self-binding properties, leading to an increase in binding efficiency to the target cells as well as an increase in potential to induce apoptosis. Herein, we report a novel finding that crosslinking of the autophilic T15 peptide rescues a loss-of-function chimerized (ch) anti-GM2 antibody. The parental antibody demonstrates in vivo anti-tumor activity against melanoma xenografts. The T15 peptide-conjugated antibody shows the ability to bind to itself, as well as an increased binding to its antigen, ganglioside GM2. Moreover, the peptide-conjugated antibody also demonstrates an increased ability to bind to two GM2-positive tumor cell lines and notably important, restores its ability to induce apoptosis in two types of tumor cells. These results provide strong support for the clinical potential of the autophilic technology.

Characterization of a proapoptotic antiganglioside GM2 monoclonal antibody and evaluation of its therapeutic effect on melanoma and small cell lung carcinoma xenografts.

Monoclonal antibodies have begun to show great clinical promise for the treatment of cancer. Antibodies that can directly affect a tumor cell's growth and/or survival are of particular interest for immunotherapy. Previously, we described monoclonal antibody DMF10.62.3 that had antiproliferative and proapoptotic effects when it bound an antigen of unknown identity on tumor cells in vitro. In this report, we determined that DMF10.62.3 and a clonally related antibody DMF10.167.4 recognize the ganglioside GM2. These antibodies react with a GM2 epitope that is expressed on a large number of tumor cell lines, including human melanoma and small cell lung carcinoma, but not on normal primary lines or most normal tissues. Interestingly, this pattern of cellular reactivity is distinct from that reported for other previously described GM2 antibodies, a difference that is presumably due to DMF10.167.4's binding to a unique GM2-associated epitope. Additional characterization of DMF10.167.4 revealed that this antibody was able to induce apoptosis and/or block cellular proliferation when cultured in vitro with the human Jurkat T lymphoma, CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines. In vivo, DMF10.167.4 antibody was well tolerated in mice and did not detectably bind to or damage normal tissues. However, this antibody was able to prevent murine E710.2.3 lymphoma, human CHL-1 melanoma, and SBC-3 small cell lung carcinoma lines from establishing tumors in vivo and blocked progression of established CHL-1 and SBC-3 tumors in vivo. Therefore, monoclonal antibody DMF10.167.4 has immunotherapeutic potential.