Table of Contents

Volume 4, Issue 1

  January/February 2012

  Pages 1 - 131

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EDITORIAL

Open Access Article

Which are the antibodies to watch in 2012?

Janice M. Reichert
Pages 1 - 3
http://dx.doi.org/10.4161/mabs.4.1.18719
Abstract | PDF

As we enter the new year of 2012, the realm of antibody therapeutics development seems full of possibilities for antibodies to watch. The commercial pipeline of antibody-based therapeutics continues to grow and now totals nearly 350 candidates. The molecular diversity of the candidates, especially those for cancer, is remarkable. Modified antibodies such as antibody-drug conjugates (ADCs), bispecific antibodies, Fc or glyco-engineered antibodies and antibody fragments/domains now comprise more than half of the anticancer antibodies at Phase 1, and ~40% of those at Phase 2 and Phase 3. These types of modified antibodies have not been developed as frequently for other disorders–by comparison, ~90% of antibodies developed for non-cancer indications are unmodified IgG–but development of the new formats for inflammatory and autoimmune disorders is expected to grow. In addition, antibody mixtures and antibodies with indirect mechanisms of action (e.g., agonism of immune activation receptors or antagonism of immune inhibitory receptors) are entering clinical study more frequently.




MEETING REPORTS

Open Access Article

World Bispecific Antibody Summit, September 27–28, 2011, Boston, MA

Eugen Dhimolea and Janice M. Reichert
Pages 4 - 13
http://dx.doi.org/10.4161/mabs.4.1.18821
Abstract | PDF

With more than 30 therapeutic monoclonal antibodies (mAbs) approved and annual global sales of the products at ~$50 billion in 2010, these products have proven to be successful in many ways. Nevertheless, there is room for improvement in performance, and substantial unmet medical needs remain. As a consequence, numerous organizations are devoting resources to engineering novel mAbs such as bispecific antibodies that have increased functionality compared with unmodified IgG molecules. The World Bispecific Antibody Summit, organized by Hanson Wade, drew over 100 participants to Boston to discuss engineering novel bispecific antibodies, generating lead candidates and clinical study and commercialization of the molecules. Approaches such as the trifunctional antibody (TRION), dual variable domain-Ig (Abbott), two-in-one (Genentech), dual affinity retargeting (MacroGenics), kappa-lambda body (NovImmune), bispecific T-cell engager (Micromet) and chemical generation (CovX/Pfizer) were discussed in detail. In addition, posters describing bispecific Affibody® molecules for targeting of EGFR and HER2 (Affibody), T-cell receptor based bi-specifics that target HLA-peptides (Immunocore), a novel mAb-Fv bispecific antibody format utilizing Fc region (Xencore), generation of a tetravalent bispecific antibody against IL4 and IL13 for the treatment of idiopathic pulmonary fibrosis (Sanofi), Combining Affibody® molecules and the Albumod™ technology to create long acting multispecific protein therapeutics (Royal Institute of Technology, Affibody) and COVA301 as a highly potent bispecific inhibitor of IL-17A and TNF-α (Covagen) were presented.

Open Access Article

Poster Sessions: September 27-28, 2011

Eugen Dhimolea and Janice M. Reichert
Pages 14 - 16
http://dx.doi.org/10.4161/mabs.19908
PDF




REVIEW

Disulfide bond structures of IgG molecules: Structural variations, chemical modifications and possible impacts to stability and biological function

Hongcheng Liu and Kimberly May
Pages 17 - 23
http://dx.doi.org/10.4161/mabs.4.1.18347
Abstract | Full Text | PDF

The disulfide bond structures established decades ago for immunoglobulins have been challenged by findings from extensive characterization of recombinant and human monoclonal IgG antibodies. Non-classical disulfide bond structure was first identified in IgG4 and later in IgG2 antibodies. Although, cysteine residues should be in the disulfide bonded states, free sulfhydryls have been detected in all subclasses of IgG antibodies. In addition, disulfide bonds are susceptible to chemical modifications, which can further generate structural variants such as IgG antibodies with trisulfide bond or thioether linkages. Trisulfide bond formation has also been observed for IgG of all subclasses. Degradation of disulfide bond through β-elimination generates free sulfhydryls disulfide and dehydroalanine. Further reaction between free sulfhydryl and dehydroalanine leads to the formation of a non-reducible cross-linked species. Hydrolysis of the dehydroalanine residue contributes substantially to antibody hinge region fragmentation. The effect of these disulfide bond variations on antibody structure, stability and biological function are discussed in this review.




REPORTS

Open Access Article

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

Catalin Doneanu, Alex Xenopoulos, Keith Fadgen, Jim Murphy, St. John Skilton, Holly Prentice, Martha Stapels and Weibin Chen
Pages 24 - 44
http://dx.doi.org/10.4161/mabs.4.1.18748
Abstract | Full Text | PDF

Assays for identification and quantification of host-cell proteins (HCPs) in biotherapeutic proteins over 5 orders of magnitude in concentration are presented. The HCP assays consist of two types: HCP identification using comprehensive online two-dimensional liquid chromatography coupled with high resolution mass spectrometry (2D-LC/MS), followed by high-throughput HCP quantification by liquid chromatography, multiple reaction monitoring (LC-MRM). The former is described as a “discovery” assay, the latter as a “monitoring” assay. Purified biotherapeutic proteins (e.g., monoclonal antibodies) were digested with trypsin after reduction and alkylation, and the digests were fractionated using reversed-phase (RP) chromatography at high pH (pH 10) by a step gradient in the first dimension, followed by a high-resolution separation at low pH (pH 2.5) in the second dimension. As peptides eluted from the second dimension, a quadrupole time-of-flight mass spectrometer was used to detect the peptides and their fragments simultaneously by alternating the collision cell energy between a low and an elevated energy (MSE methodology). The MSE data was used to identify and quantify the proteins in the mixture using a proven label-free quantification technique (“Hi3” method). The same data set was mined to subsequently develop target peptides and transitions for monitoring the concentration of selected HCPs on a triple quadrupole mass spectrometer in a high-throughput manner (20 min LC-MRM analysis). This analytical methodology was applied to the identification and quantification of low-abundance HCPs in six samples of PTG1, a recombinant chimeric anti-phosphotyrosine monoclonal antibody (mAb). Thirty three HCPs were identified in total from the PTG1 samples among which 21 HCP isoforms were selected for MRM monitoring. The absolute quantification of three selected HCPs was undertaken on two different LC-MRM platforms after spiking isotopically labeled peptides in the samples. Finally, the MRM quantitation results were compared with TOF-based quantification based on the Hi3 peptides, and the TOF and MRM data sets correlated reasonably well. The results show that the assays provide detailed valuable information to understand the relative contributions of purification schemes to the nature and concentrations of HCP impurities in biopharmaceutical samples, and the assays can be used as generic methods for HCP analysis in the biopharmaceutical industry.

A recombinant triplebody with specificity for CD19 and HLA-DR mediates 
preferential binding to antigen double-positive cells by dual-targeting

Ingo Schubert, Christian Kellner, Christoph Stein, Markus Kügler, Michael Schwenkert, Domenica Saul, Bernhard Stockmeyer, Christian Berens, Fuat S. Oduncu, Andreas Mackensen and Georg H. Fey
Pages 45 - 56
http://dx.doi.org/10.4161/mabs.4.1.18498
Abstract | PDF

To test the hypothesis that dual-targeting confers the novel ability of selective binding to antigen double-positive over antigen single-positive cells, a single-chain triplebody (sctb), HLA-ds16-hu19, was produced and characterized. The molecule carries three single-chain Fv (scFv) antibody fragments in a single polypeptide chain, the two distal ones specific for the human histocompatibility protein HLA-DR and the B-lymphoid cell surface protein CD19, the central one for CD16, the human low affinity Fc-receptor FcγRIII. For comparison, the bispecific scFvs (bsscFv) hu19-ds16 and HLA-ds16 were also produced. All CD16 binding modules are disulfide-stabilized (ds). The sctb bound simultaneously to both CD19 and HLA-DR on the same cancer cell and, thus, showed functional dual-targeting. In a mixing-experiment with HLA-DR single-positive HUT-78 cells and (HLA-DR plus CD19) double-positive SEM cells, the triplebody showed preferential binding to the double-positive cells, even when the single-positive cells were present in a numerical excess of up to 20-fold. In antibody-dependent cellular cytotoxicity experiments with mononuclear cells as effector cells, the sctb promoted equal lysis of Raji cells, an antigen double-positive cell line, at 130-fold lower concentrations than the bsscFv hu19-ds16, indicating that both distal scFvs of the sctb contributed to tumor cell lysis. A panel of stably-transfected HEK293 cell lines was generated that included CD19- and HLA-DR single-positive and (HLA-DR plus CD19) double-positive lines with antigen-surface densities varying over a broad range. Using a pair of cell lines with matching densities, the sctb eliminated double-positive target cells preferentially single-positive cells. This ability of preferential or selective targeting of antigen double-positive over single-positive cells opens attractive new perspectives for the use of dual-targeting sctbs in cancer therapy.

Immunotoxins constructed with chimeric, short-lived anti-CD22 monoclonal antibodies induce less vascular leak without loss of cytotoxicity

Xiao-yun Liu, Laurentiu M. Pop, John Schindler and Ellen S. Vitetta
Pages 57 - 68
http://dx.doi.org/10.4161/mabs.4.1.18348
Abstract | Full Text | PDF

An immunotoxin (IT) constructed with RFB4, a murine anti-CD22 monoclonal antibody, and the “deglycosylated” A chain of ricin has shown activity at safe doses in patients with non-Hodgkin lymphoma and in children with acute lymphoblastic leukemia. The dose limiting toxicity is vascular leak syndrome (VLS), which appears to be due to a unique amino acid motif in the ricin toxin A (RTA) chain that damages vascular endothelial cells. We mutated recombinant (r) RTA to disable this site, but await testing of the IT prepared with this mutant RTA in humans. Another possible approach to reducing IT-induced VLS is to shorten the half-life of the IT in vivo. We previously constructed a mouse-human chimeric RFB4 by grafting the variable genes of RFB4 onto the human IgG1k constant regions. Here, we report the expansion of our panel of mutant chimeric RFB4s (mcRFB4s) that lack the ability to bind to the neonatal Fc receptor (FcRn). In comparison with cRFB4, which had a T1/2 of 263 h, the mcRFB4s had T1/2s ranging from 39 to 106 h. ITs were constructed with these mcRFB4s and rRTA. The mcRFB4-RTA ITs retained their cytotoxicity in vitro and had shorter half lives than the parental cRFB4-RTA IT. In addition, the mcRFB4 IT with the shortest T1/2 induced less pulmonary vascular leak in mice, which we have postulated is a surrogate marker for VLS in humans.

Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies

Mark F. Maurer, Ursula Garrigues, Stephen R. Jaspers, Brent Meengs, Mark W. Rixon, Brenda L. Stevens, Kenneth B. Lewis, Susan H. Julien, Thomas R. Bukowski, Anitra C. Wolf, Nels B. Hamacher, Mark Snavely and Stacey R. Dillon
Pages 69 - 83
http://dx.doi.org/10.4161/mabs.4.1.18713
Abstract | Full Text | PDF

Interleukin-21 (IL-21) is a type I four-helical bundle cytokine that exerts a variety of significant effects on many hematopoietic cells, including T and B lymphocytes and natural killer cells. IL-21 is produced predominantly by CD4+ T cells and natural killer T cells and, when aberrantly overexpressed, appears to play important roles in a wide variety of autoimmune disorders. To generate potential therapeutic reagents capable of inhibiting IL-21 for clinical use, we immunized human immunoglobulin transgenic mice with IL-21 and then identified and cloned a panel of human anti-human IL-21 binding monoclonal antibodies. IL-21 neutralizing and IL-21-binding, non-neutralizing antibodies were assigned to distinct epitope “bins” based on surface plasmon resonance competition studies. The most potent neutralizing antibodies had extremely high (sub pM) affinity for IL-21 and were able to block IL-21 activity in various biological assays using either an IL-21R-transfected pre-B-cell line or primary human B cells, and their neutralizing activity was, in some cases, superior to that of a soluble form of the high affinity heterodimeric IL-21 receptor. Characterization of this panel of IL-21 antibodies provided the basis for the selection of a therapeutic candidate antibody capable of inhibiting IL-21 activity for the treatment of autoimmune and inflammatory diseases.

Analytical characterization of ch14.18: A mouse-human chimeric disialoganglioside-specific therapeutic antibody

Gopalan Soman, Abraham T. Kallarakal, Dennis Michiel, Xiaoyi Yang, Nirmala Saptharish, Hengguang Jiang, Steve Giardina, John Gilly and George Mitra
Pages 84 - 100
http://dx.doi.org/10.4161/mabs.4.1.18566
Abstract | Full Text | PDF

Ch14.18 is a mouse-human chimeric monoclonal antibody to the disialoganglioside (GD2) glycolipid. In the clinic, this antibody has been shown to be effective in the treatment of children with high-risk neuroblastoma, either alone or in combination therapy. Extensive product characterization is a prerequisite to addressing the potential issues of product variability associated with process changes and manufacturing scale-up. Charge heterogeneity, glycosylation profile, molecular state and aggregation, interaction (affinity) with Fcγ receptors and functional or biological activities are a few of the critical characterization assays for assessing product comparability for this antibody. In this article, we describe the in-house development and qualification of imaged capillary isoelectric focusing to assess charge heterogeneity, analytical size exclusion chromatography with online static and dynamic light scattering (DLS), batch mode DLS for aggregate detection, biosensor (surface plasmon resonance)-based Fcγ receptor antibody interaction kinetics, N-glycoprofiling with PNGase F digestion, 2-aminobenzoic acid labeling and high performance liquid chromatography and N-glycan analysis using capillary electrophoresis. In addition, we studied selected biological activity assays, such as complement-dependent cytotoxicity. The consistency and reproducibility of the assays are established by comparing the intra-day and inter-day assay results. Applications of the methodologies to address stability or changes in product characteristics are also reported. The study results reveal that the ch14.18 clinical product formulated in phosphate-buffered saline at a concentration of 5 mg/ml and stored at 2–8°C is stable for more than five years.

Subcutaneous bioavailability of therapeutic antibodies as a function 
of FcRn binding affinity in mice

Rong Deng, Y. Gloria Meng, Kwame Hoyte, Jeff Lutman, Yanmei Lu, Suhasini Iyer, Laura DeForge, Frank-Peter Theil, Paul J. Fielder and Saileta Prabhu
Pages 101 - 109
http://dx.doi.org/10.4161/mabs.4.1.18543
Abstract | Full Text | PDF

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.

Tolerability, pharmacokinetics and pharmacodynamics of CMAB007, a humanized anti-immunoglobulin E monoclonal antibody, in healthy Chinese subjects

Bo Zhou, Birong Lin, Jing Li, Weizhu Qian, Sheng Hou, Dapeng Zhang, Geng Kou, Bohua Li, Hao Wang, Yongchuan Chen and Yajun Guo
Pages 110 - 119
http://dx.doi.org/10.4161/mabs.4.1.18349
Abstract | Full Text | PDF

The goal of the studies presented here was to determine the tolerability, pharmacokinetic and pharmacodynamic profiles of CMAB007, a biosimilar of omalizumab (Xolair; a humanized anti-immunoglobulin E monoclonal antibody), in healthy, male Chinese subjects. Thirty-six healthy Chinese men participated in two open-label, dose-escalation studies: 27 in a single-dose study (150, 300 or 600 mg) and 9 in a multiple-dose study (150 or 300 mg every 4 weeks for 20 weeks). The safety profiles of both studies were generally unremarkable. No drug-related adverse event was observed. CMAB007 exhibited a linear PK profile over the dose range of 150–600 mg. In the single-dose study, maximum concentration (Cmax) was reached within 6–8 d, and Cmax and area under concentration-time curve (AUC) increased linearly with the dose. In the multiple-dose study, steady-state appeared to have been achieved after the third dose. Css-max and AUCτ also showed dose-linearity. A dose-dependent suppression of free IgE was observed during treatment, as a median percentage change from baseline, 91.9–98.8%, in the three single-dose groups. No anti-CMAB007 antibodies were detected after dosing in any subject. Subcutaneous administration of CMAB007 was well-tolerated and seemed to be effective in reducing free IgE in healthy Chinese volunteers, which provides important information for further clinical studies.

New developments in malaria diagnostics: Monoclonal antibodies against Plasmodium dihydrofolate reductase–thymidylate synthase, heme detoxification protein and glutamate rich protein

Johanna H. Kattenberg, Inge Versteeg, Stephanie J. Migchelsen, Iveth J. González, Mark D. Perkins, Petra F. Mens and Henk D.F.H. Schallig
Pages 120 - 126
http://dx.doi.org/10.4161/mabs.4.1.18529
Abstract | Full Text | PDF

Currently available rapid diagnostic tests (RDTs) for malaria show large variation in sensitivity and specificity, and there are concerns about their stability under field conditions. To improve current RDTs, monoclonal antibodies (mAbs) for novel malaria antigens have been developed and screened for their possible use in new diagnostic tests. Three antigens, glutamate rich protein (GLURP), dihydrofolate reductase-thymidylate synthase (DHFR-TS) and heme detoxification protein (HDP), were selected based on literature searches. Recombinant antigens were produced and used to immunize mice. Antibody-producing cell lines were subsequently selected and the resulting antibodies were screened for specificity against Plasmodium falciparum and Plasmodium vivax. The most optimal antibody couples were selected based on antibody affinity (expressed as dissociation constants, KD) and detection limit of crude antigen extract from P. falciparum 3D7 culture. The highest affinity antibodies have KD values of 0.10 nM ± 0.014 (D5) and 0.068 ± 0.015 nM (D6) for DHFR-TS mAbs, 0.10 ± 0.022 nM (H16) and 0.21 ± 0.022 nM (H18) for HDP mAbs and 0.11 ± 0.028 nM (G23) and 0.33 ± 0.093 nM (G22) for GLURP mAbs. The newly developed antibodies performed at least as well as commercially available histidine rich protein antibodies (KD of 0.16 ± 0.13 nM for PTL3 and 1.0 ± 0.049 nM for C1–13), making them promising reagents for further test development.




EDITOR'S CORNER

Open Access Article

2012 upcoming meetings

Janice M. Reichert
Pages 127 - 128
http://dx.doi.org/10.4161/mabs.4.1.18718
PDF

Open Access Article

Note of appreciation to reviewers

Janice M. Reichert
Pages 129 - 131
http://dx.doi.org/10.4161/mabs.4.1.18717
PDF