Humanized antibody neutralizing 2009 pandemic H1N1 virus.

Biotechnology Journal

Biotechnol. J. 2014, 9, 1594–1603

DOI 10.1002/biot.201400083

www.biotechnology-journal.com

Research Article

Humanized antibody neutralizing 2009 pandemic H1N1 virus Nachiket Shembekar1, Vamsee V Aditya Mallajosyula2, Piyush Chaudhary1, Vaibhav Upadhyay3, Raghavan Varadarajan2 and Satish Kumar Gupta1 1 Reproductive

Cell Biology Laboratory, National Institute of Immunology, New Delhi, India Biophysics Unit, Indian Institute of Science, Bangalore, India 3 Product Development Cell, National Institute of Immunology, New Delhi, India 2 Molecular

The 2009 pandemic H1N1 S-OIV (swine origin influenza A virus) caused noticeable morbidity and mortality worldwide. In addition to vaccine and antiviral drug therapy, the use of influenza virus neutralizing monoclonal antibodies (MAbs) for treatment purposes is a viable alternative. We previously reported the isolation of a high affinity, potently neutralizing murine MAb MA2077 against 2009 pandemic H1N1 virus. We describe here the humanization of MA2077 and its expression in a mammalian cell line. Six complementarity-determining regions (CDRs) of MA2077 were grafted onto the human germline variable regions; along with six and eight back mutations in the framework of heavy and light chains, respectively, pertaining to the vernier zone and interchain packing residues to promote favorable CDR conformation and facilitate antigen binding. The full length humanized antibody, 2077Hu2, expressed in CHO-K1 cells, showed high affinity to hemagglutinin protein (KD = 0.75 ± 0.32 nM) and potent neutralization of pandemic H1N1 virus (IC50 = 0.17 μg/mL), with marginally higher IC50 as compared to MA2077 (0.08 μg/mL). In addition, 2077Hu2 also retained the epitope specificity for the “Sa” antigenic site on pandemic HA. To the best of our knowledge, this is the first report of a humanized neutralizing antibody against pandemic H1N1 virus.

Received Revised Accepted Accepted article online

24 MAR 2014 02 JUN 2014 02 JUL 2014 07 JUL 2014

Supporting information available online

Keywords: Antibody humanization · CDR grafting · Epitope mapping · 2009 pandemic H1N1 · Neutralizing monoclonal antibodies See accompanying commentary by Reingard Grabherr DOI 10.1002/biot.201400409

1 Introduction Influenza virus is among the most common causes of human respiratory infections and is responsible for recurrent epidemics and occasional/sporadic pandemic outbreaks. The 2009 pandemic caused by H1N1 swine origin influenza A virus [S-OIV, hereafter referred to as pandemic H1N1 (pH1N1)] resulted in more than 5 million infections with about 200 000 deaths worldwide, since April 2009 [1].

Correspondence: Dr. Satish Kumar Gupta, National Institute of Immunology, Reproductive Cell Biology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India E-mail: [email protected] Abbreviations: CDRs, complementarity-determining regions; HA, hemagglutinin; HI, hemagglutination Inhibition; HRP, horseradish peroxidase; MAbs, monoclonal antibodies; pH1N1, pandemic H1N1; TMB, 3,3′,5,5′tetramethylbenzidine; VH, heavy chain variable region; VL, light chain variable region; WAM, web based antibody modeling

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Influenza virus infection poses a serious risk for people with underlying medical conditions such as diabetes, asthma, and kidney or heart problems (WHO fact sheet; http://www.who.int/mediacentre/factsheets/2003/ fs211/en/). Annually updated trivalent vaccine and antiviral drugs are the primary modes of prophylaxis and treatment for influenza infection. However, in addition, newer alternate treatment modalities, especially in a pandemic situation, can be very useful. Neutralizing antibodies against the major surface glycoprotein of the influenza virus, hemagglutinin (HA), is the primary correlate of protection in humans [2]. It is evident from previous studies carried out in animal models, regarding usage of influenza virus neutralizing monoclonal antibodies (MAbs) as well as polyclonal antibodies for treatment purposes that such an alternate treatment option is feasible and effective [3–6]. Passive parenteral immunization with antibodies is available for prophylaxis and therapy of infectious diseases including hepatitis A and B, rabies, tick-borne encephalitis, varicella, respiratory syncytial virus (RSV)

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infection, and Kawasaki syndrome [7]. In addition, the identification of protective B-cell epitopes recognized by these antibodies, can aid in vaccine design and may reveal novel pathogenic and evolutionary mechanisms [8]. Murine MAbs have certain limitations for clinical use in humans as murine MAbs do not trigger appropriate effector functions of complement and Fc receptors. Further, murine antibodies are recognized as foreign by the patient’s immune system evoking human anti-murine-antibody immune response (HAMA) thus cutting short the therapeutic window [9]. Hence, murine antibodies need to be humanized for clinical administration in humans [10]. Antibody humanization involves designing an antibody molecule with minimal immunogenicity for humans while retaining the affinity and specificity of the parental non-human antibody. Several approaches for humanization have been described in recent years, such as complementarity-determining region (CDR) grafting, resurfacing, and generation of framework libraries [11]. In spite of recent progress, antibody engineering, in this case “humanization” remains challenging and success varies from case to case, and depends upon several factors such as choice of method, selection of human template, regions from the parental antibody incorporated during humanization, and compatibility of human template to accept the mutations. We have earlier reported the isolation of a high affinity murine MAb MA2077, that potently neutralized pandemic H1N1 virus [12]. We report here the humanization of murine MAb MA2077 by CDR-grafting with rational modifications in the framework region (FR). The humanized antibody was expressed in mammalian cells and characterized for its ability to neutralize H1N1 virus and epitope mapping.

2 Materials and methods 2.1 Cell lines and virus Madine Darby Canine Kidney (MDCK; CCL-34) and CHO-K1 cell line (CCL-61) were obtained from American Type Culture Collection, Manassas, VA, USA and cultured in Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium, respectively (Sigma–Aldrich, Inc., St. Louis, MO, USA). Medium was supplemented with 10% fetal bovine serum (FBS) and an antibiotic–antimycotic cocktail [penicillin (100 units/mL), streptomycin (100 μg/mL), and amphotericin B (0.25 μg/mL); Biological Industries, Kibbutz beit, Haemek, Israel]. Cell lines were cultured at 37°C under humidified conditions with 5% CO2. Pandemic H1N1 NYMCX-179A virus (A/California/07/2009, NIBSC Code 09/124) was received from NIBSC, UK, passage in MDCK cells in presence of TPCK-trypsin (2 μg/mL; Sigma–Aldrich, Inc.), and kindly provided to us by Serum Institute of India Limited, Pune,


India. Titer of the virus stock was calculated using Reed and Muench method [13].

2.2 Amplification of antibody variable (V) regions of MA2077 The murine monoclonal antibody MA2077 was generated as described elsewhere [12]. Total RNA was isolated from approximately 2 million hybridoma cells secreting the murine MA2077, using the Tri reagent (Sigma–Aldrich, Inc.) with protocol using the chloroform–isopropanol–ethanol steps for its purification. Reverse transcription into cDNA was carried out using Superscript III reverse transcriptase enzyme (Life Technologies, NY, USA) and amplification of heavy and light chain V regions (VH and VL) was carried out using high fidelity Taq polymerase (Life Technologies), as per the protocols described elsewhere [14]. Amplified VH and VL regions (~400 bp) were then sequenced commercially (Biolinkk, New Delhi, India).

2.3 Humanization of MA2077 Humanization of MA2077 was carried out using CDRgrafting method with some modifications. The humanized variant was designated as 2077Hu2. The CDRs were identified as per Kabat definitions [15]. Human germline V sequences, to be used as FRs for CDR-grafting, with highest identity to murine VH and VL regions were identified independently using NCBI IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/). The “J” region for the VH and VL was selected from the most identical human consensus sequence [15]. Canonical structure class for the CDRs of murine and human template was identified as per Chothia definitions [16, 17]. Certain FR residues that contribute significantly to a favorable conformation of CDRs, such as vernier zone and interchain packing residues were studied [18–20] (Humanization by design http://people.cryst.bbk.ac.uk/~ubcg07s/). These important FR residues were identified, analyzed in a homology model of MA2077, and a decision of whether to retain the murine or human residue was made. Finally, the sequence composition for V region of 2077Hu2 was assembled and synthetic genes separately for the VH and VL were procured commercially (Genscript, NJ, USA).

2.4 Molecular modeling Molecular modeling of V region of MA2077 was performed by Web Based Antibody Modeling Software (WAM; http://antibody.bath.ac.uk/) [21]. The 3D structures were viewed using PDB molecular visualization programs, Deepview and PyMOL (http://www.pymol.org/). The interaction of each residue belonging to the vernier zone and interchain packing group, within 5 Å of CDRs and VH/VL interface was analyzed. The compatibility of the

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crucial murine residue substitutions in human FR was analyzed.

muir interaction model using BIA EVALUATION 3.1 software.

2.5 Cloning and mammalian expression of full length 2077Hu2

2.6.3 In vitro microneutralization assay

The 2077Hu2 VH was digested and cloned into a mammalian expression vector with human IgG1 constant region, pFUSEss-CHIg-hG1 (InvivoGen, CA, USA) at EcoRI/NheI (New England Biolabs, MA, USA) restriction sites. The VL was digested and cloned into mammalian expression vector with human kappa chain constant region, pFUSE2ss-CLIg-hk (InvivoGen) at EcoRI/BsiWI (New England Biolabs) restriction sites. The VH and VL expression vectors for 2077Hu2, were then co-transfected into CHO-K1 cells using calcium-phosphate transfection method [22]. The cells were further grown for 4 weeks in the presence of Zeocin (250 μg/mL) and Blasticidin (5 μg/mL; Life Technologies) and single clones were selected. The large-scale antibody expression was carried out in CD-CHO medium supplemented with 8 mM L-glutamine (Life Technologies). The antibodies from culture supernatant were purified using protein-G sepharose (GE Healthcare Biosciences AB, Uppsala, Sweden).

2.6 Characterization of 2077Hu2 The purified 2077Hu2 was characterized as follows:

2.6.1 ELISA ELISA was carried out using microtiter plates coated with HA protein of pandemic H1N1 virus (A/California/07/2009; Sino Biological, Inc., Beijing, China; 160 ng/well) and employing varying concentrations (2.5–0.075 μg/mL) of purified MA2077 and 2077Hu2, as described previously [12]. For detecting 2077Hu2 binding, HRP conjugated goatanti-human antibody (Pierce Biotechnology, Inc., Rockford, IL, USA) at 1:5000 dilution was used.

2.6.2 Binding affinity studies using surface plasmon resonance (SPR) The binding affinity of 2077Hu2 to pH1N1 (A/California/ 04/2009) mammalian-expressed recombinant HA (rHA) protein (Sino Biological, Inc.) was determined using SPR experiments performed with a Biacore 2000 optical biosensor (Biacore, Uppsala, Sweden) as described previously [12]. Seven hundred and fifty resonance units (RU) of purified 2077Hu2 were immobilized on an activated surface of a CM5 chip (GE HealthCare) by standard amine coupling. A sensor channel immobilized with ovalbumin served as the negative control for each binding interaction. Different concentrations of pH1N1 rHA or H1N1 (A/Puerto Rico/8/34) rHA trimer (Sino Biological, Inc.) were passed over each channel. Each binding curve was corrected for non-specific binding. The kinetic parameters were obtained by fitting the data to a 1:1 Lang-

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Microneutralization assay was performed using MDCK cell line as per the WHO guidelines as described previously [12] (WHO manual. WHO Manual on Animal Influenza Diagnosis and Surveillance WHO/CDS/CSR/NCS/ 2002). Briefly, equal volume of antibody and 100 TCID50 units of the virus were incubated for 1 h at room temperature and added onto the MDCK cells (15 000/well), preseeded in a 96-well cell culture plate (Greiner Bio-One, GmbH, Frickenhausen, Germany). After incubation for 24 h at 32°C, cells were fixed with 80% acetone and probed with 1:4000 dilution of anti-influenza A-nucleoprotein antibody (Millipore, Billerica, MA, USA). The binding of nucleoprotein antibody was further detected by ELISA as described elsewhere [12]. The 50% inhibitory concentration (IC50) of the neutralizing antibody was calculated using the nonlinear regression program of GraphPad Prism software (http://www.graphpad.com/ scientific-software/prism/).

2.6.4 Hemagglutination inhibition (HI) assay The HI assay was carried out in V-bottom 96-well plates (Greiner Bio-One), as described elsewhere, using varying concentrations of the antibodies and 4 HA units of the pH1N1 virus with 0.5% guinea pig RBCs [12]. The HI titer of the antibody represents the lowest concentration of the antibody showing HI activity.

2.6.5 Thermal denaturation using tryptophan fluorescence Fluorescence emission spectra of purified MA2077 and 2077Hu2 were recorded using the Cary Eclipse spectrofluorimeter equipped with a temperature controller (Varian, California, USA). Purified protein (100 μg/mL) was taken in a 1 cm path length cuvette and incubated at different temperatures ranging from 20 to 95°C. After incubation for 20 min at each temperature, the sample was excited at 280 nm and emission spectra were collected from 300 to 400 nm with excitation and emission slit width set at 5 nm. Each spectrum was scanned three times and the average spectrum was plotted.

2.7 Epitope mapping 2.7.1 Competition ELISA Briefly, a 96-well plate was coated with 160 ng of pH1N1 HA protein at 37ºC for 1 h followed by overnight incubation at 4ºC. The following day, the plate was blocked with 1% milk protein in phosphate buffer saline (PBS) (pH 7.4) for 2 h at 37ºC. After washing the plate with PBS, 80 μL mixture containing equal volumes of varying concentrations of MA2077 (5–0.04 μg/mL) and fixed concentration of 2077Hu2 (0.5 μg/mL) was added in quadruplicates and





plate was incubated for 1 h at 37ºC. After 1 h incubation, the plate was washed three times with PBS containing 0.05% Tween-20 (PBST) and either HRP conjugated goatanti-mouse antibody (1:10 000 dilution) or goat-anti-human antibody (1:5000 dilution) were added in duplicate wells. After incubation for 1 h at 37ºC, the plate was again washed three times with PBST. The reaction was developed using TMB substrate (Sigma–Aldrich, Inc.). The reaction was stopped with 50 μL 5 N H2SO4 and optical density was measured at 450 nm with a reference wavelength of 630 nm.

2.7.2 Yeast surface display of HA1 fragments The construction of pPNLS yeast cell-surface display plasmids (pPNLS-H1pHA9, H1pHA9-Sa, and H1pHA9Sb) has been described previously [12]. Briefly, a stable HA1 fragment, H1pHA9, from the pandemic H1N1 strain A/California/07/2009 was designed, which retained the ability to bind conformation sensitive neutralizing antibodies binding within the head of HA. We further introduced mutations to disrupt either the “Sa” or “Sb” antigenic sites to map the epitope of HA head-specific antibodies. The yeast cells (EBY100 strain) were transformed with the yeast display plasmids using the LiAc/SS carrier DNA/PEG method as described previously [23]. Yeast cell-surface expression of the displayed proteins was performed as previously described [24]. Briefly, a primary culture (3 mL) in synthetic dextrose casamino acids (SDCAA) medium (pH 4.5) was grown at 30°C under shaking conditions until an OD600 of 2–3 was reached. The yeast cells were subsequently induced for protein expression by transferring to SGCAA minimal media (similar to SDCAA medium except dextrose replaced by galactose) and incubated with shaking at 20°C for 24 h. After induction, 1 million yeast cells were processed for individual analysis. The cells were washed with PBS containing 1% BSA (PBSB). Surface expression of the displayed proteins with a C-terminal c-Myc tag was detected using anti-c-Myc chicken antibody (Life Technologies) at a pre-determined dilution (1:400 in PBSB) incubated for 1 h at 4°C and subsequently stained with Alexa Fluor-488 goat anti-chicken antibody (Life Technologies) at a pre-determined dilution (1:300 in PBSB) for 1 h at 4°C, protected from light. Binding titration of 2077Hu2 with the displayed HA1 fragments was performed to determine the antibody epitope. The induced cells were incubated with varying concentrations of 2077Hu2 for 1 h at 4°C, and subsequently stained with Alexa Fluor-488 goat anti-human antibody (Life Technologies) at a pre-determined dilution (1:400 in PBSB), as mentioned above. After washing, the fluorescence from the stained cells was detected by flow cytometric analysis (BD FACS-Aria, BD FACS-Diva Cytometry Software). The binding titration data were fit as described previously using the following equation [25]:


Fractional binding 

[MAb] ([MAb]  K D )

[MAb] is the concentration of 2077Hu2 antibody and KD is the apparent dissociation constant.

2.8 Preliminary assessment of “humanness” The preliminary analysis of “degree of humanness” of the humanized antibody was performed bioinformatically. The web based tool provides a Z score that defines closeness of the humanized sequence to a typical human sequence [26] (http://www.bioinf.org.uk/abs/shab/). Using this program, Z scores for murine and human VH and VL were compared separately.

2.9 Ethics statement All the experiments were performed after due approval from Institutional Animal Ethical Committee, National Institute of Immunology, New Delhi (IAEC#247/10) and following its guidelines.

3 Results 3.1 Humanization of MA2077 Using NCBI IgBLAST, the human germline V region from IGHV1-46*03 group showed the highest identity (60.2%) to MA2077 VH and IGKV6D-41*01 showed the highest identity (59.6%) to MA2077 VL. The identity was considered only with respect to the FR residues. The sequence alignment of the mouse and human templates showed that there were 21 non-identical residues in VH and 23 non-identical residues in VL in FR (Fig. 1 and 2). For the “J” region, an identical “J” region from the human consensus sequence was selected, which showed two mismatched residues each for VH and VL (Fig. 1 and 2). Comparison of canonical structure class between the murine and human templates revealed, class matching for 4 CDRs as: H1:1, H2:2, L1:1, and L2:1. The mismatching canonical structure class for L3 was determined as similar to class 1 for MA2077 and was indeterminate for human template due to absence of canonical of the same loop length. The vernier zone residues were identified and studied using a homology model of MA2077 (described in Section 3.2). Sequence comparison between the murine and human FR residues at vernier zone positions showed that 6 out of 12 such residues for VH and 7 out of 14 residues for VL were non-identical (Supporting information, Table S1). Similarly, interchain packing residues were identified using the compiled information given in the website program, “Humanization by design” (http://people.cryst.bbk.

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Figure 1. Amino acid sequence alignment of VH. Sequence alignment of MA2077 VH and human germline (IGHV1-46*03) V region has been shown. Identical amino acids have been marked as “*.” The crucial murine FR residues that were retained in the humanized construct, 2077Hu2, have been colored in red. The final sequence of 2077Hu2 VH has also been aligned.

Figure 2. Amino acid sequence alignment of VL. Sequence alignment of MA2077 VL and human germline (IGKV6D-41*01) V region has been shown. Identical amino acids have been marked as “*.” The crucial murine FR residues that were retained in the humanized construct, 2077Hu2, have been colored in red. The final sequence of 2077Hu2 VL has also been aligned.

ac.uk/~ubcg07s/), which was in agreement with analysis on a homology model of MA2077. Sequence comparison of the FR packing residues between the murine and human templates showed that one out of seven residues for VH, and two out of six residues for VL were mismatched (Supporting information, Table S2). These mismatched FR residues were then analyzed in the homology model of MA2077.

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3.2 Molecular modeling of MA2077 Molecular modeling of MA2077 was performed using WAM [21] and the model was viewed as mentioned in Section 2.4. In MA2077, five residues of VH pertaining to vernier zone (H48:I, H67:A, H69:L, H71:V, and H78:A) and one residue belonging to both vernier zone as well as interchain packing (H93:T) were analyzed for interaction with CDRs, since these were non-identical in the human





Table 1. Kinetic parameters for binding of 2077Hu2 to rHA by surface plasmon resonance

Analytea) A/California/04/2009 HA (H1N1) A/Puerto Rico/8/34 HA(H1N1)

kon (1/Ms) 2.44 × –c)

106

koff (1/s) 1.84 × –c)

10–3

KD (M) 0.75 ± 0.32 × 10–9 b) –c)

a) 2077Hu2 was immobilized on the surface of a CM5-chip and analytes were passed over this surface at different concentrations. b) Reported values are the mean ± SD of kinetic parameters obtained at different concentrations. The experiment was repeated thrice. c) No detectable binding.

template. Representative analysis of interaction of H48:I (I156) and H93:T (T205) with other residues within a distance cut-off of 5 Å´ has been shown (Supporting information, Fig. S1A and S1B). The isoleucine at H48 (I156) was predicted to interact with six other residues, out of which, two residues H169 and F172 belong to CDR2. Moreover, I156 interacts with W155 and G157, which were both predicted to be in the vernier zone and common to the human template. In addition, I156 also interacted with the CDR1 terminating residue W144, which is conserved across the murine and human templates (Supporting information, Fig. S1A). Similarly, threonine at H93 (T205) was predicted to interact with six other residues, three of which G207, F213, and Y215 belong to CDR3. T205 also interacted with W216 at the boundary of CDR3 and the vernier zone residue R206, both of which are conserved across the murine and human sequences (Supporting information, Fig. S1B). In case of MA2077 VL, seven residues pertaining to the vernier zone (L2:I, L4:L, L46:R, L47:Y, L49:Y, L64:A, and L71:Y) and one belonging to the interchain packing residues (L87:F), which mismatched with the human template were analyzed. All the eight residues interacted with the residues influencing CDR conformation. Representative analysis of interaction of L49:Y (Y48) and L87:F (F86) with other residues within 5 Å´ distance has been shown (Supporting information, Fig. S1C and S1D). The tyrosine at L49 (Y48) interacted with seven residues, three of which N49, T50, and E52 were part of CDR2. Two of the residues M32 and H33 belonged to CDR1 whereas the remaining two residues R45 and I47 were predicted to be in the vernier zone (Supporting information, Fig. S1C). Phenylalanine at L87 (F86) also showed interaction with seven other residues, two of which were vernier zone residues (W34 and Y35), one residue (Q88) belonged to CDR3 and another (H33), belonged to CDR1 (Supporting information, Fig. S1D). Hence, we decided to retain six murine FR residues in VH and eight murine FR residues in VL of 2077Hu2, in addition to grafting of CDRs (Fig. 1 and 2). The back-mutations were subsequently evaluated in a model of 2077Hu2. None of these mutations result to unfavorable interactions in 2077Hu2 (data not shown).

3.3 Mammalian expression and characterization of 2077Hu2 The sequences for VH and VL regions of 2077Hu2 were assembled; genes were separately synthesized and cloned.


Mammalian expression was carried out in CHO-K1 cells as described in Section 2.5. The antibody was purified from the culture supernatant using protein-G column. The yield of the humanized antibody was around 0.3–0.6 mg/L after purification. The purified humanized antibody was characterized further. The purified 2077Hu2 showed sig-

Figure 3. Characterization of humanized antibody 2077Hu2. (A) ELISA reactivity profile: Purified 2077Hu2 and MA2077 were tested at varying concentrations in ELISA against rHA protein of pandemic H1N1 virus (160 ng/well). The Y-axis represents the absorbance at 490 nm. Values are represented as mean ± SEM of three independent experiments performed in duplicates. (B) In vitro microneutralization assay: Purified 2077Hu2 and MA2077 were tested at varying concentrations against 100 TCID50 dose of the pandemic H1N1 virus in an in vitro microneutralization assay using MDCK cell line. Data are represented as mean ± SEM of four independent experiments performed in duplicates.

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nificant binding to pandemic H1N1 HA protein in ELISA comparable to MA2077, upto 0.075 μg/mL (Fig. 3A). Binding affinity studies showed that 2077Hu2 bound pH1N1 rHA (A/Cal/04/2009) with high affinity with an equilibrium dissociation constant (KD) of 0.75 ± 0.32 nM (Table 1, Supporting information, Fig. S2). The binding affinity of the MAb to pH1N1 rHA upon humanization decreased by ~350-fold. The higher dissociation rate (koff) between pH1N1 rHA and 2077Hu2 (1.84 × 10–3 s–1) when compared to MA2077 (9.27 × 10–6 s–1) results in the decreased affinity (KD) to rHA upon humanization. Despite the reduced affinity upon humanization, 2077Hu2 had retained the specificity, as binding to H1N1 (A/Puerto Rico/8/34) rHA could not be detected even at the highest concentrations (100 nM) of analyte (Table 1, Supporting information, Fig. S2). Further, 2077Hu2 showed potent neutralization of the pandemic H1N1 virus in an in vitro microneutralization assay with an IC50 of 0.17 μg/mL, which is marginally more than the IC50 of murine MAb MA2077 (0.08 μg/mL) (Fig. 3B). The reduced affinity of 2077Hu2 to pH1N1 rHA possibly explains the difference in potency of neutralization between the murine and the humanized antibody. Stability of MA2077 and 2077Hu2 antibodies under thermal stress was monitored using intrinsic tryptophan fluorescence. MA2077 and 2077Hu2 appear to be equally stable to thermal stress. Both the proteins showed a significant red-shift in the emission maxima upon heat denaturation with an apparent transition mid-point at ~70 ºC indicating that the proteins were well folded (Supporting information, Fig. S3). Additionally, the 2077Hu2 antibody showed HI activity against the pandemic H1N1 virus with HI titre of 2.5 μg/mL (Supporting information, Fig. S4).

3.4 Mapping of the 2077Hu2 epitope The competition ELISA was carried out using varying concentrations of the MA2077, (5–0.04 μg/mL), and a fixed concentration of the 2077Hu2 (0.5 μg/mL; equivalent to absorbance of 1.0) to check whether the humanized antibody still recognizes a similar epitope on HA. Competitive ELISA observations revealed that 2077Hu2 indeed competed with MAb MA2077 for binding to pandemic HA protein as evident by increase in absorbance from 0.26 to 1.2 for binding of the 2077Hu2 as a function of decreasing concentration of MA2077 (Fig. 4A). In order to corroborate the results of competition ELISA, the established yeast surface display methodology that we have previously reported for epitope mapping of HA head-domain specific antibodies was used [12]. The probing of yeast display constructs, H1pHA9, H1pHA9-Sa, and H1pHA9-Sb with anti-c-Myc antibody showed that all the fragments were well expressed on the yeast cell-surface (data not shown). Further, the dissociation constants (KD) for all the constructs with 2077Hu2 were determined to ascertain its binding specificity. The

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Figure 4. Epitope mapping of 2077Hu2. (A) Competition ELISA: Competition ELISA using varying concentrations of MA2077 (5–0.04 μg/mL) and fixed concentration of 2077Hu2 (0.5 μg/mL, equivalent to absorbance of 1.0) was carried out with pH1N1 HA protein (160 ng/well). (B) Yeast surface display: Binding titration curve of varying concentrations of 2077Hu2 with yeast cell-surface displaying HA1 fragments, H1pHA9, H1pHA9-Sa, and H1pHA9-Sb has been shown. Both in panels (A and B), a representative data of three independent experiments have been shown. The symbols indicate data points while the solid lines show the fit.

H1pHA9 construct bound 2077Hu2 with high affinity [KD of 9.04 ± 1.2 nM (mean ± SD)]. The construct H1pHA9-Sb, wherein residues of the “Sb” antigenic site were mutated bound 2077Hu2 with a KD of 55.8 ± 3.2 nM (mean ± SD). The “Sa” antigenic site mutant, H1pHA9-Sa, bound 2077Hu2 with extremely low affinity (KD of >2.5 μM) (Fig. 4B). The binding data clearly indicates that perturbation of the “Sa” antigenic site of HA leads to a drastic reduction in affinity to 2077Hu2. Therefore, residues lining the “Sa” antigenic site form the epitope of 2077Hu2, similar to the parent MA2077 antibody.

3.5 Analysis of “degree of humanness” The Z-scores for the VH and VL of MA2077 and 2077Hu2 were compared independently using the web-based tool



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described in Section 2.8. Z-scores for both VH and VL of 2077Hu2 showed significant shift toward “zero” (VH: from −1.868 for MA2077 to –1.026 for 2077Hu2; VL: from –1.709 for MA2077 to –1.299 for 2077Hu2), which suggested that the 2077Hu2 sequence has increased “human” content.

4 Discussion The use of neutralizing MAbs for treatment of influenza virus infections is currently being explored worldwide. Several pandemic H1N1 neutralizing MAbs have been isolated recently, from either murine/human B cells or phage-display libraries; specific as well as broadly crossreactive [3–5, 27–31]. However, MAb MA2077 can also be considered as valuable tool against the 2009 pandemic H1N1 virus in view of its neutralization efficacy, affinity, and specificity [12]. The 2009 pandemic H1N1 virus has shown remarkable antigenic stability since entering into the human population in 2009. The 99.9% of the viruses isolated so far have been characterized as, A/California/07/2009-like, the vaccine strain that has been used in the current study (Weekly Influenza Surveillance Report, CDC, USA, 2013–2014 Influenza Season Week 9 ending March 1, 2014. http://www.cdc.gov/flu/weekly/). In addition, the H1N1 component of the 2013–2014 seasonal vaccine also contains the identical pandemic strain, also indicative of the fact that this strain is likely to be the prevalent strain for the next season as well. Hence, despite high specificity of MA2077, we decided to humanize this antibody for its potential therapeutic usage. The methodology of generating high affinity murine MAbs followed by their humanization for therapeutic purposes, is cost effective and hence, widely used. Numerous humanized antibodies are currently being used therapeutically for various indications including cancer, transplant rejection, and even for infectious disease such as RSV [32]. Some studies have reported generation of chimeric and humanized neutralizing MAbs against H5 subtype influenza viruses [33, 34]. However, to the best of our knowledge, there are no reports of humanized neutralizing MAbs against H1-subtype influenza viruses, especially the 2009 pandemic H1N1 virus. For humanizing the murine MAb MA2077, we adopted a rational approach involving CDR-grafting. It has been predicted that CDR-grafted humanized antibodies against extracellular antigen, such as influenza virus in this case, may be less immunogenic than against cell surface antigens due to the later being more effectively internalized and presented to the humoral immune system [35]. Moreover, many MAbs that are being used clinically in humans, such as alemtuzumab and daclizumab, have been humanized by CDR-grafting method [36, 37]. For grafting the CDRs of MA2077, we first identified homologous human germline V regions. The choice of other human templates such as fixed framework or human con-



sensus sequences was not considered, since the fixed framework of a mature antibody may already contain somatic mutations and consensus sequences being bioinformatically derived are artificial and hence may cause undesirable immunogenicity. Further, the canonical structure class of the CDRs between murine and human template was matched in order to determine the compatibility for CDR-grafting. Restricting the “humanization” to only CDR-grafting may lead to a loss in affinity and/or in extreme cases, specificity of the parental antibody [38, 39]. The loss in affinity/specificity has been attributed to an unfavourable CDR conformation. Several reports suggest that successful humanization leading to restoration of binding affinity, depends upon careful analysis of back mutations of important FR residues, which drive the CDR to a productive conformation. The number of back-mutations is case-specific and has to be analyzed either structurally or in a homology model [40]. One such group of FR residues called vernier zone residues, play a significant role in CDR conformation and make enthalpic contributions to antigen binding and hence have been implicated for retention of immunological reactivity [20, 41, 42]. Another such group is interchain packing residues, which play a crucial role in CDR conformation as well as interactions at the VH/VL interface and their importance has been evaluated in humanization of antibody 1B4 [18, 42]. The analysis of such residues using a homology model of MA2077 identified six vernier zone residues in VH, seven vernier zone residues in VL, and one interchain packing residue in VL of MA2077, which were non-identical with the human template as CDR-interacting residues, and hence all were retained in the humanized antibody. Mammalian expression and characterization of 2077Hu2 showed reduced affinity as compared to MA2077. However, 2077Hu2 showed potent neutralization of pandemic H1N1 virus and retained epitope specificity of “Sa” antigenic site. Further, both 2077Hu2 and MA2077 showed similar thermal stability. Though, the binding affinity and the potency of neutralization of the parental antibody have been reduced, they are still higher than many of the previously reported neutralizing MAbs against the pandemic H1N1 virus [27, 29, 31, 43]. Moreover, nanomolar affinity is considered as a cut-off for therapeutic antibody candidates [44]. Further, the statistical assessment of “humanness” showed that the humanized antibody has increased “human” content. Moreover, the Z-scores obtained for the 2077Hu2, were comparable and even superior as compared to some of the humanized antibodies being used clinically [26]. However, these data provide only a preliminary assessment of antigenicity of the molecule and this issue would require further study. The HI test principally detects anti-HA antibodies, which are responsible for neutralization of influenza virus

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physiologically. Hence, HI antibody titer is commonly used as immune correlate for protection in infections as well as vaccine efficacy studies [2]. The 2077Hu2 antibody showing HI titer of 2.5 μg/mL may thus be protective physiologically. In conclusion, we have successfully humanized a murine MAb, which potently neutralizes pandemic H1N1 virus. Its therapeutic usage can be enhanced by synergistic administration with other antiviral drugs or neutralizing MAbs. Moreover, our strategy for humanization of murine MAbs can be further fine tuned and will be useful for generating therapeutic MAbs against influenza viruses as well as other relevant indications.

This work was funded by the Department of Biotechnology, Government of India (BT/BIPP/0213/04/09). The funding agency had no role in study design, collection, analysis, or interpretation of the data. The authors also thank Aparna Asok for maintaining the FACS facility at the Molecular Biophysics Unit, Indian Institute of Science, Bangalore. The authors declare no financial or commercial conflict of interest.

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ISSN 1860-6768 · BJIOAM 9 (12) 1459–1623 (2014) · Vol. 9 · December 2014

Systems & Synthetic Biology · Nanobiotech · Medicine

12/2014 Asian Congress of Biotechnology 2013


This Special issue covers contributions from the Asian Congress of Biotechnology 2013 (New Delhi, India, December 2013) in collaboration with the Asian Federation of Biotechnology (AFOB). The issue is edited by Virendra Bisaria and Akihiko Kondo and includes articles on stem cells, algae biotechnology and recombinant protein. The cover image shows the cell-penetrating mechanism of the 30Kc19 protein derived from the silkworm hemolymph. The silkworm image also symbolizes the Asian region. Image designed by Helpdesign. See the article by Park et al. http://dx.doi.org/10.1002/biot.201400253

Biotechnology Journal – list of articles published in the December 2014 issue. Editorial: Asian Congress on Biotechnology 2013 Akihiko Kondo and Virendra S. Bisaria http://dx.doi.org/10.1002/biot.201400774 Editorial: Building on the BTJ experience to foster scientific and research communication Judy Peng http://dx.doi.org/10.1002/biot.201400785 Commentary The passive strategy: Increasing the force in the battle against influenza Reingard Grabherr

http://dx.doi.org/10.1002/biot.201400409 Commentary Production of vitamin B12 in recombinant Escherichia coli: An important step for heterologous production of structurally complex small molecules Yin Li

http://dx.doi.org/10.1002/biot.201400472 Review Expanding frontiers in plant transcriptomics in aid of functional genomics and molecular breeding Pinky Agarwal, Swarup K. Parida, Arunima Mahto, Sweta Das, Iny Elizebeth Mathew, Naveen Malik and Akhilesh K. Tyagi

http://dx.doi.org/10.1002/biot.201400063 Review Aquatic proteins with repetitive motifs provide insights to bioengineering of novel biomaterials Yun Jung Yang, Dooyup Jung, Byeongseon Yang, Byeong Hee Hwang and Hyung Joon Cha

http://dx.doi.org/10.1002/biot.201400070 Review Open and continuous fermentation: Products, conditions and bioprocess economy Teng Li, Xiang-bin Chen, Jin-chun Chen, Qiong Wu and Guo-Qiang Chen

http://dx.doi.org/10.1002/biot.201400084

Technical Report Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae Yuetong Ji, Yuehui He, Yanbin Cui, Tingting Wang, Yun Wang, Yuanguang Li, Wei E. Huang, and Jian Xu

http://dx.doi.org/10.1002/biot.201400165 Research Article Zinc, magnesium, and calcium ion supplementation confers tolerance to acetic acid stress in industrial Saccharomyces cerevisiae utilizing xylose Ku Syahidah Ku Ismail, Takatoshi Sakamoto, Tomohisa Hasunuma, Xin-Qing Zhao and Akihiko Kondo

http://dx.doi.org/10.1002/biot.201300553 Research Article Coenzyme B12 can be produced by engineered Escherichia coli under both anaerobic and aerobic conditions Yeounjoo Ko, Somasundar Ashok, Satish Kumar Ainala, Mugesh Sankaranarayanan, Ah Yeong Chun, Gyoo Yeol Jung and Sunghoon Park

http://dx.doi.org/10.1002/biot.201400221 Research Article Volatile fatty acids derived from waste organics provide an economical carbon source for microbial lipids/biodiesel production Gwon Woo Park, Qiang Fei, Kwonsu Jung, Ho Nam Chang, Yeu-Chun Kim, Nag-jong Kim, Jin-dal-rae Choi, Sangyong Kim and Jaehoon Cho

http://dx.doi.org/10.1002/biot.201400266 Research Article Photon up-conversion increases biomass yield in Chlorella vulgaris Kavya R. Menon, Steffi Jose and Gadi K. Suraishkumar

http://dx.doi.org/10.1002/biot.201400216 Research Article Chromosomal integration of hyaluronic acid synthesis (has) genes enhances the molecular weight of hyaluronan produced in Lactococcus lactis Rothangmawi Victoria Hmar, Shashi Bala Prasad, Guhan Jayaraman and Kadathur B. Ramachandran

http://dx.doi.org/10.1002/biot.201400215 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


Research Article Ionic liquids as novel solvents for the synthesis of sugar fatty acid ester

Research Article Humanized antibody neutralizing 2009 pandemic H1N1 virus

Ngoc Lan Mai, Kihun Ahn, Sang Woo Bae, Dong Woo Shin, Vivek Kumar Morya and Yoon-Mo Koo

Nachiket Shembekar, Vamsee V Aditya Mallajosyula, Piyush Chaudhary, Vaibhav Upadhyay, Raghavan Varadarajan and Satish Kumar Gupta



Research Article Electron donation to an archaeal cytochrome P450 is enhanced by PCNA-mediated selective complex formation with foreign redox proteins

Research Article Efficient myogenic commitment of human mesenchymal stem cells on biomimetic materials replicating myoblast topography

Risa Suzuki, Hidehiko Hirakawa and Teruyuki Nagamune

Eunjee A. Lee, Sung-Gap Im and Nathaniel S. Hwang



Research Article Dimerization of 30Kc19 protein in the presence of amphiphilic moiety and importance of Cys-57 during cell penetration

Research Article Modulation of the stemness and osteogenic differentiation of human mesenchymal stem cells by controlling RGD concentrations of poly(carboxybetaine) hydrogel

Hee Ho Park, Youngsoo Sohn, Ji Woo Yeo, Ju Hyun Park, Hong Jai Lee, Jina Ryu, Won Jong Rhee and Tai Hyun Park


Hsiu-Wen Chien, Szu-Wei Fu, Ai-Yun Shih and Wei-Bor Tsai




Comparison of the Protective Efficacy of Neutralizing Epitopes of 2009 Pandemic H1N1 Influenza Hemagglutinin.

Hospitalised children with pandemic (H1N1) 2009 influenza.

The 2009 H1N1 Pandemic Influenza in Korea.

Similarity of currently circulating H1N1 virus with the 2009 pandemic clone: viability of an imminent pandemic.

Prevalence of Oseltamivir-Resistant 2009 H1N1 Influenza Virus among Patients with Pandemic 2009 H1N1 Influenza infection in NRITLD, Tehran, Iran.

Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine.

Changes in heterosubtypic antibody responses during the first year of the 2009 A(H1N1) influenza pandemic.

A monoclonal antibody-based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines.

H1N1 Pandemic Influenza Virus Infection and Vaccination in Norway, 2009-2010.

Neuraminidase Activity and Resistance of 2009 Pandemic H1N1 Influenza Virus to Antiviral Activity in Bronchoalveolar Fluid.

Pulmonary immune responses to 2009 pandemic influenza A (H1N1) virus in mice.

2009 pandemic influenza A (H1N1) infection in Saurashtra region, India.

Post-pandemic influenza a (H1N1) 2009 virus infection in pregnant women in Ceará, Brazil.

An NA-deficient 2009 pandemic H1N1 influenza virus mutant can efficiently replicate in cultured cells.

Mammalian pathogenesis of oseltamivir-resistant pandemic (H1N1) 2009 influenza virus isolated in South Korea.

H1N1 virus in commercial piggery, Lagos Nigeria.

Specific Inhibitory Effect of κ-Carrageenan Polysaccharide on Swine Pandemic 2009 H1N1 Influenza Virus.

H1N1 Virus Infection during the 2009 Pandemic.

Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin.

Return of pandemic H1N1 influenza virus.

Quantifying the mortality caused by the H1N1 influenza virus during the 2009 pandemic in Mexico.

Maternal mortality due to pandemic influenza A H1N1 2009 virus in Colombia.

Maternal mortality due to pandemic influenza A H1N1 2009 virus in Colombia.

Prophylaxis and therapy of pandemic H1N1 virus infection using egg yolk antibody.