DEVELOPMENT OF TETRAVALENT, BI-SPECIFIC CCR5 ANTIBODIES WITH ANTIVIRAL ...

43 Pages·2011·0.67 MB·English

AAC Accepts, published online ahead of print on 7 February 2011
Antimicrob. Agents Chemother. doi:10.1128/AAC.00215-10
Copyright © 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
DEVELOPMENT OF TETRAVALENT, BI-SPECIFIC CCR5 ANTIBODIES WITH
ANTIVIRAL ACTIVITY AGAINST CCR5 MAB-RESISTANT HIV-1 STRAINS
Jürgen Schanzera#*, Andreas Jekleb#%, Junichi Nezua^, Adriane Lochnerb $,Rebecca
Croasdalea, Marianna Dioszegib, Jun Zhangb°, Eike Hoffmanna, Wilma Dormeyera,
Jan Strackea, Wolfgang Schäfera, Changhua Jib°, Gabrielle Heilekb, Nick Cammackb,
Michael Brandta, Pablo Umanaa and Ulrich Brinkmanna
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Roche Pharma Research (a) TPI Penzberg/Schlieren, Nonnenwald 2, D-82372 o
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Penzberg, Germany, and (b) Roche Palo Alto, Department of Virology, 3431 Hillview n
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Ave, Palo Alto, CA 94304, United States. a
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* to whom correspondence should be sent: Roche Diagnostics GmbH, Nonnenwald 2, fr
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D-82372 Penzberg, Germany, [email protected]

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% current address: Novabay Pharmaceuticals, Emeryville, California //a
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$ current address: Biosciences Division and BioEnergy Science Center, Oak Ridge s
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National Laboratory, Oak Ridge, Tennessee .o
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^ current address: Chugai Pharmaceuticals, Tokyo, Japan
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° current address: Hofmann La Roche, Nutley, New Jersey
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# contributed equally to this work 4
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running title: High Antiviral Activity of Tetravalent CCR5 Antibodies
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KEYWORDS: Antibody Engineering; Bispecific Antibodies, Disulfide stabilization, g
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single chain Fv, HIV, CCR5, CD4, Entry Inhibitor. s
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Schanzer et al. 2
1 ABSTRACT
2 In this study, we describe novel tetravalent-bispecific antibody derivatives that bind
3 two different epitopes on the HIV co- receptor CCR5. The basic protein formats that
4 we have applied were derived from ‘Morrison-Type’ bispecific antibodies: whole IgG’s
5 to which we connected single chain antibodies (scFvs) via (Gly Ser) sequences at D
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6 either C- or N-termini of the light chain or heavy chain. By design optimization n
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7 including disulfide-stabilization of scFvs or introduction of 30 amino acid linkers, d
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8 stable molecules could be obtained in amounts that are within the same range or no fr
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9 less than four-fold lower as observed with monoclonal antibodies in transient h
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10 expression. In contrast to monospecific CCR5 antibodies, bispecific antibody :/
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11 derivatives block two alternative docking sites of CCR5-tropic HIV strains on the c
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12 CCR5 co-receptor. Consequently, these molecules showed 18 – 57 fold increased m
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13 antiviral activities compared to the parent antibodies. Most importantly, one prototypic g/

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14 tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the J
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15 single parental antibodies. In summary, physical linkage of two CCR5 antibodies u
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16 targeting different epitopes on the HIV co-receptor CCR5 resulted in tetravalent, 4
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17 bispecific antibodies with enhanced antiviral potency against wildtype and CCR5- 0
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18 antibody resistant HIV-1 strains. by

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Schanzer et al. 3
1 INTRODUCTION
2 HIV is a retrovirus that infects components of the human immune system such as
3 CD4+ T cells, macrophages and dendritic cells (6, 28), contributing to the loss of
4 cellular immunity (5, 11). HIV infects host cells by the binding of the viral glycoprotein
5 gp120 to the cellular receptor CD4, followed by association to co-receptors such as D
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6 CCR5 or CXCR4 (3, 20, 31). The CCR5 and CXCR4 co-receptors are members of n
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7 the G protein-coupled receptors, characterized by seven transmembrane helices, d
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8 three intracellular loops, an amino-terminal domain (NTD) and a intracellular domain fr
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9 (2). Individuals who are homozygous for the CCR5 ∆32 mutation have a natural h
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10 resistance to HIV infection (2, 25, 35). Interference of HIV binding to the CD4 :/
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11 receptor, or one of the co-receptors can reduce or prevent HIV infection of cells. c
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12 Therefore, antibodies which bind viral entry receptors may provide an attractive m
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13 addition to standard HIV therapy. Current therapeutics under development such as g/

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14 the anti-CD4 antibody Ibalizumab (formerly TNX-355) (10, 12, 14, 22), and the anti- J
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15 CCR5 antibodies Pro-140 and HGS004 (14, 23) have shown efficacy against viral u
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16 infections in vitro and in clinical trials (11, 15, 16, 24, 25). We recently described two 4
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17 CCR5 antibodies with high antiviral activity in vitro (16, 18, 40): RoAb13, which binds 0
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18 to the N-terminal domain (NTD) of CCR5 and MAb3952 which recognizes the by

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19 extracellular domain 2 (ECL-2) of CCR5 (16). ue
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20 HIV adapts to therapy regimens and frequently evades treatment by developing
21 resistance mutations (36). Several mechanisms of resistance to CCR5 inhibitors have
22 been described, including increased affinity of the viral envelope to CCR5, binding of
23 the virus to the inhibitor-occupied receptors and a change in co-receptor utilization
24 from CCR5 to CXCR4 or other co-receptors (27, 32, 39). Recently, we proposed
Schanzer et al. 4
1 CCR5 epitope switching as an additional mechanism of resistance to epitope-
2 specific CCR5 antibodies. We showed that after in vitro resistance selection to the
3 ECL-2-specific CCR5 antibody MAb3952, two CCR5-tropic virus strains preferentially
4 bound to the NTD of CCR5, rendering them more susceptible to the NTD-specific
5 antibody RoAb13 (16).
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6 Here, we describe the generation and characterization of tetravalent-bispecific ow
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7 antibody derivatives composed of whole IgGs with N- and C-terminal scFvs which lo
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8 bind two different epitopes on CCR5. In contrast to monospecific antibodies, these d

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9 molecules can block both potential HIV docking sites on CCR5 leading to highly m

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10 improved antiviral potency in peripheral blood mononuclear cells (PBMC) antiviral ttp
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11 assays against wildtype HIV strains as well as virus variants resistant to epitope- aa
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12 specific CCR5 antibodies. s
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Schanzer et al. 5
1 METHODS
2 Construction of bispecific antibodies
3 All DNA sequences encoding bispecific antibodies were prepared by automated
4 gene synthesis (Geneart AG, Regensburg, Germany) from synthetic oligonucleotides
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5 and PCR products. Gene Segments coding the MAb3952 antibody heavy chain with ow
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6 a C-terminal (G4S)2 (two repeats of four glycine and one serine residues) unit linked loa
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7 to the VH and VL regions of the RoAb13 antibodies joined by a (G S) linker (= d
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8 CCR5-2320 heavy chain), were synthesized with flanking BamHI and XbaI restriction m

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9 sites. DNA sequences encoding heavy chain constructs with cysteine residues for ttp
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10 disulfide stabilization in the VH (Kabat position 44) region of the attached scFv or aa
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11 increased connector (G S) and linker (G S) length were prepared by gene s
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12 synthesis with flanking BamHI/EcoNI (N-terminal scFv) or EcoNI/XbaI (C-terminal o
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13 scFv) restriction sites and the corresponding overlapping region of the MAb3952 o
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14 heavy chain. In a similar manner, gene segments encoding the MAb3952 antibody an
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15 light chain with a C-terminal (G4S)2 unit linked to the VH and VL region of the RoAb13 ry

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16 antibody joined by a (G4S)3 linker (= CCR5-4320 light chain), were synthesized with , 2
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17 flanking BamHI and XbaI restriction sites. In addition, DNA sequences coding for the 9

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18 unmodified and N-terminal scFv modified MAb3952 light chain (= CCR5-2320 light g
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19 chain) were prepared. DNA sequences coding light chain constructs with cysteine st
20 residues for disulfide stabilization in the VL (Kabat position 100) region of the
21 attached scFv or increased connector (G S) and linker (G S) length were
4 2-5 4 4-6
22 prepared by gene synthesis with flanking BamHI/BsgI (N-terminal scFv) or BsgI/XbaI
23 (C-terminal scFv) restriction sites and the corresponding overlapping region of the
24 MAb3952 light chain. All constructs were designed with a 5’-end DNA sequence
Schanzer et al. 6
1 coding for a leader peptide, which targets proteins for secretion in eukaryotic cells. A
2 pUC18 derived eukaryotic expression vector (Roche Diagnostics, Penzberg) was
3 used for the construction of all heavy and light chain scFv fusion protein expression
4 plasmids. The constructed heavy and light chain DNA segments as well as the
5 expression vector were digested with BamHI and XbaI restriction enzymes (Roche
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6 Molecular Biochemicals) and subjected to agarose gel electrophoresis. Purified ow
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7 heavy and light chain coding DNA fragments were then ligated to the restriction lo
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8 enzyme digested vector resulting in the final expression plasmids. For the cysteine, d

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9 connector and linker modified constructs, gene synthesized fragments were digested m

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10 with BamHI/EcoNI, BamHI/BsgI, EcoNI/XbaI or BsgI/XbaI restriction enzymes and ttp
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11 ligated to the analogous treated expression vector fragment. The final expression aa
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12 vectors were transformed into E. coli cells, expression plasmid DNA was isolated and s
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13 subjected to restriction enzyme analysis and DNA sequencing. Correct clones were o
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14 grown in 150 ml LB-Amp medium, again plasmid DNA was isolated and sequence o
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15 integrity confirmed by DNA sequencing. an
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17 Expression and purification 0
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18 Bispecific antibodies were expressed by transient transfection of human embryonic g
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19 kidney cells. Briefly, in suspension growing HEK cells were cultivated at 37°C/8 % st
20 CO and seeded in fresh medium at a density of 1-2 x 106 viable cells/ml prior to
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21 transfection. Then, DNA/293fectin™ complexes were prepared in Opti-MEM I
22 medium (Invitrogen, Germany) using 325 µl of 293fectin™ (Invitrogen, Germany) and
23 250 µg of heavy and light chain plasmid DNA in a 1:1 molar ratio and added to 250
24 ml cell suspension. Bispecific antibody containing cell culture supernatants were
Schanzer et al. 7
1 harvested 7 days after transfection by centrifugation at 14000 g for 30 minutes and
2 filtered through a sterile filter (0.22 µm). Supernatants were stored at -20° C until
3 purification. Bispecific antibodies were purified in two steps by affinity
4 chromatography using Protein A-SepharoseTM (GE Healthcare, Sweden) and
5 Superdex 200 size exclusion chromatography. Briefly, the harvested cell culture
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6 supernatants were applied on a HiTrap ProteinA HP (5 ml) column equilibrated with ow
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7 PBS buffer (10 mM Na HPO , 1 mM KH PO , 137 mM NaCl and 2.7 mM KCl, pH 7.4). lo
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8 Unbound proteins were washed out with equilibration buffer. Bound bispecific d

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9 antibodies were eluted with 0.1 M citrate buffer, pH 2.8, and the protein containing m

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10 fractions were neutralized with 0.1 ml 1 M Tris, pH 8.5. Then, the eluted protein ttp
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11 fractions were pooled, concentrated with an Amicon Ultra centrifugal filter device aa
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12 (MWCO: 30 K, Millipore) to a volume of 3 ml and loaded on a Superdex 200 HiLoad s
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13 120 ml 16/60 gel filtration column (GE Healthcare, Sweden) equilibrated with 20 mM o
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14 histidine, 140 mM NaCl, pH 6.0. Fractions containing purified bispecific antibodies o
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15 with less than 5 % high molecular weight aggregates were pooled and stored in 1.0 an
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16 mg/ml and 0.1 mg/ml aliquots at -80°C. ry

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18 Biochemical and biophysical analysis of purified recombinant proteins by

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19 The protein concentration of purified protein samples was determined by measuring st
20 the optical density (OD) at 280 nm. Purity and molecular weight of bispecific
21 antibodies were analyzed by SDS-PAGE in the presence and absence of a reducing
22 agent (5 mM 1,4-dithiotreitol). Aggregation of bispecific antibody samples was
23 analyzed by high-performance size exclusion chromatography using a Superdex 200
24 analytical size-exclusion column (GE Healthcare, Sweden) in 200 mM KH PO , 250
2 4
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1 mM KCl, pH 7.0 running buffer at 25°C. 25 µg protein were injected on the column
2 at a flow rate of 0.5 ml/min and eluted isocratic over 50 minutes. For aggregation
3 analysis, concentrations of 0.1 mg/ml, 1 mg/ml and 5 mg/ml of purified proteins were
4 prepared and incubated at 4°C and 40°C for 7 days and then evaluated by high-
5 performance SEC. The integrity of the amino acid backbone and the molecular
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6 weight of reduced bispecific antibody light and heavy chains was verified by ow
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7 NanoElectrospray Q-TOF mass spectrometry after removal of N-glycans by lo
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8 enzymatic treatment with Peptide-N-Glycosidase F (Roche Molecular Biochemicals). d

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9 m

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10 CCR5 cell-based ELISA :/
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11 Twenty thousand CHO-CCR5, CHO-CCR5-dN8 or CHO-CCR5-K171A cells per well s
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12 were seeded into 96-well tissue culture plates and incubated overnight at 37°C as o
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13 described earlier (Zhang et al, 2007). Thereafter, the cell culture medium was o
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14 aspirated, 50 µl new medium containing serially diluted bispecific or control an
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15 antibodies was added and plates were incubated for 2 hours at 4°C. Then, cells were ry

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16 fixed in PBS containing 0.05% (vol/vol) glutaraldehyde for 10 min. After washing three , 2
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17 times with assay medium, 50 µl per well of horseradish peroxidase (HRP)-conjugated 9

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18 sheep anti-mouse immunoglobulin G (IgG) antibody (GE Healthcare, USA) diluted g
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19 1:2,000 was added to the plates and incubated at room temperature for 2 h. After st
20 extensive washing with PBS, 50 µl of tetramethylbenzidine substrate (Roche Applied
21 Science, Germany) was added to each well. The reaction was stopped after 5
22 minutes by adding 25 µl of 1 M sulfuric acid and measured at 450 to 620 nm on an
23 Envision plate reader (Perkin-Elmer, Shelton, CT).
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Schanzer et al. 9
1 RANTES radioligand binding assay
2 125I-RANTES was purchased from PerkinElmer Life Sciences Inc. (USA). Binding
3 assays were performed on CHO cells expressing recombinant human CCR5
4 receptors. Cells were seeded in 96-well culture plates at 1.5 × 105 cells/well in ice
5 cold binding buffer (phenol red-free F12 medium supplemented with freshly made D
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6 0.1% BSA and 0.1% NaN ). Serially diluted CCR5 bispecific and control antibodies n
3 lo
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7 were added to the cells, followed by addition of 100 pM 125I-labeled RANTES. After a d
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8 2 hour incubation at room temperature with gentle shaking, cells were harvested onto fr
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9 GF/C UniFilter plates (PerkinElmer Life Sciences Inc., USA) using a cell harvester. h
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10 UniFilter plates were pretreated with 0.3% PEI/0.2% BSA for 30 min prior to harvest. :/
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11 Filter plates were washed five times with 25 mM pH 7.1 HEPES buffer containing 500 c
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12 mM NaCl, 1 mM CaCl and 5 mM MgCl . Plates were dried in 70 °C oven for 20 min m
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13 and 40 µl scintillation fluid was added and radioactivity was measured using g/

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14 TopCount NXT (PerkinElmer, USA). In all experiments, each data point was assayed J
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15 in duplicate. Data are presented as the percentage of counts obtained in absence of u
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16 cold competing ligand. Curve fitting and subsequent data analysis were carried out 4
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17 using GraphPad PRIZM software (Intuitive Software for Science, San Diego, CA) and 0
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18 IC50 values were calculated using non-linear regression analysis. by

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20 Antiviral assay using peripheral blood mononuclear cells (PBMC)
21 Human PBMC were isolated from buffy-coats (obtained from the Stanford
22 Blood Center) by a Ficoll-Paque (Amersham, USA) density gradient centrifugation
23 according to manufacturer’s protocol. Briefly, blood was transferred from the buffy
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1 coats in 50 ml conical tubes and diluted with sterile Dulbecco’s phosphate buffered
2 saline (Invitrogen/Gibco) to a final volume of 50 ml. Twenty-five ml of the diluted
3 blood was transferred to two 50 ml conical tubes, carefully underlayed with 12.5 ml of
4 Ficoll-Paque Plus (Amersham Biosciences) and centrifuged at room temperature for
5 20 min at 450 x g without brakes. The white cell layer was carefully transferred to a
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6 new 50 ml conical tube and washed twice with PBS. To remove remaining red blood ow
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7 cells, cells were incubated for 5 minutes at room temperature with ACK lysis buffer lo
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8 (Biosource, Rockville, MD)) and washed one more time with PBS. PBMC were d

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9 counted and incubated at a concentration of 2–4 x 106 cells/ml in RPMI1640 m

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10 containing 10% FCS (Invitrogen/Gibco), 1% penicillin/streptomycin, 2 mM L- ttp
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11 glutamine, 1 mM sodium-pyruvate, and 2 µg/ml Phytohemagglutinin (Invitrogen) for a
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12 24h at 37°C. Cells were incubated with 5 Units/ml human IL-2 (Roche Molecular s
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13 Biochemicals) for a minimum of 48h prior to the assay. In each well of a 96 well round r
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14 bottom plate, 1 x 105 PBMC were infected with HIV-1 NLBal (NL4.3 strain (1) with the n

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15 env of BaL (gp120)) , RU570, 301567, CC1/85_NDC or CC1/85_3952res (16) in the n
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16 presence of serially diluted bispecific and control antibodies. The amount of virus ry

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17 used was equivalent to 0.8 to 1.2 ng HIV-1 p24 antigen/well. Infections were set up in 2
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18 quadruplicates. Plates were incubated for 6 days at 37°C. Virus production was
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19 measured at the end of infection by using a p24 ELISA (Perkin-Elmer, USA) gu
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20 according to the manufacturer's instructions. For all antiviral assays, the 50% t
21 inhibitory concentration (IC ) was calculated by using the sigmoidal dose-response
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22 model with one binding site in Microsoft XLFit.