MODIFIED FC REGION
20220340684 · 2022-10-27
Inventors
- Cheng-I Wang (Singapore, SG)
- Patricia Ng (Singapore, SG)
- John Connolly (Singapore, SG)
- Chia Yin Lee (Singapore, SG)
Cpc classification
C07K16/2851
CHEMISTRY; METALLURGY
C07K2317/72
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
C07K2317/24
CHEMISTRY; METALLURGY
C07K16/283
CHEMISTRY; METALLURGY
C07K2317/76
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
International classification
Abstract
Fc regions comprising modification to increase the affinity of association between the Fc region and TRIM21 are disclosed. Also disclosed are constituent polypeptides of such Fc regions, antigen-binding molecules and immunogens comprising such Fc regions, and nucleic acids encoding and methods using such Fc regions, antigen-binding molecules and immunogens.
Claims
1. An Fc region, optionally isolated, comprising modification to increase the affinity of association between the Fc region and TRIM21.
2. The Fc region according to claim 1, wherein the modification comprises one or more substitutions to the amino acid sequence of a polypeptide of the Fc region.
3. The Fc region according to claim 2, wherein the polypeptide comprises substitution at one or more positions corresponding to the following positions of IGHG1 (EU numbering): 252, 253, 254, 256, 309, 310, 311, 314, 315, 345, 428, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439 or 440; or wherein the Fc region comprises a polypeptide comprising substitution at one or more positions corresponding to the following positions of IGHG1 (EU numbering): 253, 256, 433, 434, 435, 436 or 440.
4. (canceled)
5. The Fc region according to claim 1, wherein the Fc region comprises a polypeptide comprising: I or L at the position corresponding to position 253; P, A, T, V, G, I, K, N or S at the position corresponding to position 256; S, V, H, A, D, P, Q, T or I at the position corresponding to position 433; H, R, N, I, L or M at the position corresponding to position 434; H or L at the position corresponding to position 435; Y, F, T, L, W or S at the position corresponding to position 436; and S, G, I, R, Y, N, V, T, P, D, W, M, A, K, F, E or C at the position corresponding to position 440.
6. The Fc region according to claim 1, wherein the Fc region comprises a polypeptide comprising an amino acid sequence having at least 60% sequence identity to one of SEQ ID NOs:167 to 175; or wherein the Fc region comprises a polypeptide comprising an amino acid sequence having at least 60% sequence identity to one of SEQ ID NOs:32 to 166.
7. (canceled)
8. A polypeptide, optionally isolated, comprising: an amino acid sequence having at least 60% sequence identity to SEQ ID NO:10, wherein the polypeptide comprises the following amino acid residues at the specified positions numbered relative to SEQ ID NO:10: I or L at the position corresponding to position 26; P, A, T, V, G, I, K, N or S at the position corresponding to position 29; S, V, H, A, D, P, Q, T or I at the position corresponding to position 206; H, R, N, I, L or M at the position corresponding to position 207; H or L at the position corresponding to position 208; Y, F, T, L, W or S at the position corresponding to position 209; and S, G, I, R, Y, N, V, T, P, D, W, M, A, K, F, E or C at the position corresponding to position 213; and wherein the amino acid sequence of the polypeptide is not identical to the amino acid sequence of a constituent polypeptide of the Fc region of a wildtype immunoglobulin.
9. The polypeptide according to claim 8, wherein the polypeptide comprises an amino acid sequence having at least 60% sequence identity to one of SEQ ID NOs:167 to 175; or wherein the polypeptide comprises an amino acid sequence having at least 60% sequence identity to one of SEQ ID NOs:32 to 166.
10. (canceled)
11. (canceled)
12. The polypeptide according to claim 8 wherein the polypeptide is a constituent polypeptide of an Fc region.
13. (canceled)
14. The Fc region according to claim 1, wherein the Fc region is comprised in an antigen-binding molecule; and wherein the antigen-binding molecule further comprises an antigen-binding domain capable of specific binding to a target antigen.
15. The Fc region according to claim 14, wherein the target antigen is an antigen of a pathogen, a cancer-associated antigen or an autoimmune disease-associated antigen.
16. The Fc region according to claim 14, wherein the antigen-binding molecule comprises an antigen-binding domain capable of specific binding to an endocytosis receptor.
17. The Fc region according to claim 14, wherein the antigen-binding molecule is a multispecific antigen-binding molecule.
18. The Fc region according to claim 14, wherein the antigen-binding molecule further comprises an antigenic sequence of a target antigen.
19. The Fc region according to claim 1, wherein the Fc region is comprised in an immunogen and wherein the immunogen further comprises an antigenic sequence of a target.
20. The Fc region according to claim 19, wherein the target antigen is an antigen of a pathogen, a cancer-associated antigen or an autoimmune disease-associated antigen.
21. The Fc region according to claim 19, wherein the immunogen additionally comprises an antigen-presenting cell (APC)-targeting region.
22. The Fc region according to claim 21, wherein the APC-targeting region comprises or consists of a moiety capable of specific binding to an endocytosis receptor.
23. The Fc region according to claim 19, wherein the immunogen further comprises an antigen-binding domain capable of specific binding to a target antigen.
24.-33. (canceled)
34. A method of treating or preventing an infectious disease, a cancer or an autoimmune disease, comprising administering to a subject a therapeutically or prophylactically effective amount of: an antigen-binding molecule comprising an antigen-binding domain capable of specific binding to a target antigen and an Fc region, wherein the Fc region comprises modification to increase the affinity of association between the Fc region and TRIM21; or an immunogen comprising an antigenic sequence of a target antigen and an Fc region, wherein the Fc region comprises modification to increase the affinity of association between the Fc region and TRIM21.
35.-39. (canceled)
Description
BRIEF DESCRIPTION OF THE FIGURES
[0316] Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.
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EXAMPLES
[0333] In the following Examples, the inventors describe the engineering of Immunoglobulin Fc to increase its affinity for TRIM21, and analysis of the effect of this improved affinity for TRIM21-mediated functions.
[0334] The entire contents of Ng et al., J Immunol. (2019) 202(8):2307-2319 is specifically incorporated by reference in its entirety.
Example 1: Materials and Methods
[0335] 1.1 PBMCs
[0336] Peripheral blood mononuclear cells (PBMCs) were isolated from apheresis blood of healthy donors using Ficoll-Paque Premium (GE Healthcare).
[0337] 1.2 MoDCs
[0338] Monocytes were isolated from PBMCs using CD14 Microbeads (Miltenyi). Monocytes were differentiated into moDC by culture in the presence of 100 ng/ml GM-CSF (premium grade, Miltenyi) and 100 ng/ml IL-4 (premium grade, Miltenyi) in RPMI-1640 with 25 mM HEPES and L-Glutamine (Hyclone) further supplemented with 10% FBS (South American origin, Gibco) and antibiotics comprising 100 U/ml Penicillin and 100 ug/ml Streptomycin (Gibco). On the fourth day of the culture, one third of the culture medium was replaced with fresh culture medium with the same supplements. After 7 days, moDC (floating cells) from the cell culture were harvested for use in experiments.
[0339] 1.3 Cell Lines
[0340] HEK293T cells and HeLa cells were maintained in 1 g/L glucose DMEM (Hyclone) supplemented with 10% FBS (South American origin, Gibco) and antibiotics (100 U/ml Penicillin, 100 ug/ml Streptomycin, Gibco).
[0341] 1.4 Viruses
[0342] Purified E1- and E3-deleted replication-deficient adenovirus type 5 with the eGFP reporter gene (VQAdCMV eGFP, ViraQuest) was used for the antibody-dependent intracellular neutralization (ADIN) assays. The same strain of adenovirus with no eGFP gene (VQAd EMPTY, ViraQuest) was used for all other assays. To titer the virus, HEK293T cells were seeded onto 0.01% poly-L-lysine (Sigma) coated 24-well plates at a cell density of 2.5×10.sup.5 cells/ml. Once the cells had adhered, 10-fold serial dilutions of virus were added to the cell culture and incubated for 2 days. Viral titer was calculated from the number of infected cells as detected by the Adeno-X Rapid Titer Kit (Clontech).
[0343] 1.5 Human TRIM21 (PRYSPRY Domain) Protein.
[0344] The human recombinant TRIM21 PRYSPRY domain sequence shown in SEQ ID NO:184 was cloned into the 3′-end of a His-tag in the pQE-2 bacteria expression vector, and expressed in E. coli BL21 (DE3). A 20 ml overnight culture was inoculated into 380 ml fresh growth medium and grown at 37° C. until the OD.sub.600 reached 1.0. Protein expression was induced with 1 mM IPTG for 4.5 hours at room temperature. The expressed protein was then purified from cell lysates using Ni-NTA resin (Qiagen), followed by gel filtration in PBS using a Superdex 75 chromatography column (GE Healthcare).
[0345] 1.6 Antibodies
[0346] The variable domains of the chimeric anti-adenovirus antibody were cloned from hybridoma 9C12 (TC31-9C12.C9)(Varghese et al., 2004b), obtained from the Developmental Studies Hybridoma bank (NICHD/University of Iowa). The variable domains of the human anti-DEC205 antibody were synthesized based on the published sequence of clone 3G9-2D2 (U.S. Pat. No. 8,236,318 B2). The variable domains were cloned into the N-terminal of the constant domains of the human IgG1 in the pTT5 vector. For the Fc-modified antibodies, the 5 amino acid modifications in the constant regions were modified using the Quikchange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies). Antibodies were expressed in HEK293-6E cells or in CHO cells, and purified from their supernatants using Protein G beads. The purified antibodies were buffer-exchanged into 20 mM His, 150 mM NaCl, pH 6.0, then filtered and assayed for endotoxin content using the Limulus Amoebocyte Lysate-QCL1000 (Lonza). Only antibody preparations with an endotoxin content of <1 EU/μg were used for cell-based assays.
[0347] 1.7 Peptides
[0348] Peptides were synthesized according to the following sequences: TYFSLNNKF (SEQ ID NO:176; HLA-A*2401-Ad5 epitope), YVLFEVFDVV (SEQ ID NO:177; HLA-A*0201-Ad5 epitope), LAVFEDYVAF (SEQ ID NO:178; scrambled peptide) and SLYNTVATL (SEQ ID NO:179; HLA-A2-HIV epitope).
[0349] 1.8 Phage Display Library Construction
[0350] The PN04-44AD phagemid has the human IgG1 Fc gene encoding amino acid positions 239 to 447 (EU numbering) fused to an amber stop (TAG) followed by a truncated gill. PN04-44AD was used as the template for making the phage display library using a Kunkel reaction-based site-directed mutagenesis strategy previously described (Tonikian et al., 2007): briefly, a single-stranded template was used for annealing of oligonucleotides carrying the library sequences; double-stranded DNA was then produced and electroporated into E. coli TG-1 cells that were pre-infected with helper phage. The electroporated TG-1 cells were enumerated by plating dilutions of an aliquot onto 2YT plates with ampicillin and kanamycin. The remaining electroporated TG-1 were cultured at 37° C., overnight, in 2YT medium with ampicillin and kanamycin for the production of bacteriophages. The next day, bacteriophages were harvested from the cell culture supernatant and stored in PBS with 20% glycerol until use.
[0351] 1.9 Biopanning of the Phage Display Library
[0352] Recombinant human TRIM21 PRYSPRY domain was biotinylated and then used as bait for biopanning. In the first round of biopanning, 2×10.sup.12 bacteriophages were incubated with 5 nM of bait protein for 1 hour to allow for interaction. The bait was then captured using DynaBeads M-280 Streptavidin (Invitrogen). The beads were then washed five times with casein, incubated for 30 min with 1 μM of soluble Fc and eluted using trypsin. In the second round of biopanning, the eluted bacteriophages were amplified and incubated first with empty streptavidin beads, and followed by incubation with 0.5 nM of bait protein. The bait was then captured using streptavidin beads. The beads were then washed ten times with casein and incubated with 1 μM soluble Fc for two rounds of 30 mins, before elution using trypsin. In the third round of biopanning, the eluted phages were amplified, incubated with empty streptavidin beads, and then incubated with 0.05 nM of bait protein. The bait was then captured using streptavidin beads. The beads were washed ten times, then incubated for 5 hours with 1 μM of soluble Fc, before elution using trypsin. 50 clones from each of rounds two and three were sequenced.
[0353] 1.10 Surface Plasmon Resonance (SPR) Analysis
[0354] The affinity constants were measured using ProteON XPR36 (Bio-Rad). Antibodies were immobilized onto a GLC sensor chip via amine coupling, and PBS with 0.05% Tween-20 was used as the running buffer. Curves were fitted with the ProteON Manager software using five concentrations of TRIM21 and based on a Langmuir 1:1 model.
[0355] 1.11 Antibody-Dependent Intracellular Neutralization (ADIN) Assays
[0356] For ADIN assays, 1.25×10.sup.5 HeLa cells were seeded into 24-well plates and 1×10.sup.5 moDC were seeded into 96-well plates for infection with immune complexes, virus alone, antibodies alone or medium alone for 48 h. As HeLa cells are highly permissive to adenovirus infection (Fechner et al., 2000), an MOI of 1.2 was used, while for moDCs, an MOI of 400 was used. Infected cells were detected by analysis of expression of eGFP by flow cytometry. For HeLa cells, immune complexes were pre-formed by incubation of virus with antibodies at concentrations ranging from 0.3 pM to 35 nM for 1 h at room temperature prior to use. Thereafter, the incubation mixture was diluted 55-fold into the HeLa cell culture to give a final concentration ranging from 0.006 pM to 627 pM. For moDCs, immune complexes were pre-formed by incubation of virus with antibodies at concentrations ranging from 0.17 nM to 690 nM for 1 h at room temperature prior to use. Thereafter, the incubation mixture was diluted by 12.5-fold into the moDC culture to give a final concentration ranging from 0.01 nM to 55 nM.
[0357] Coimmunoprecipitation: Ad5 was preincubated with Abs for 1 h at room temperature in PBS to form immune complexes. Immature moDC were then treated with the immune complexes in RPMI 1640 supplemented with 10% FBS at an MOI of 200 and incubated at 37° C. with 5% CO.sub.2 for 4 h. Cells were lysed on ice with RIPA buffer containing 1% Nonidet P-40 substitute (Sigma-Aldrich), 50 mM Tris-Cl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1% Phosphatase Inhibitor Cocktail 3 (Sigma-Aldrich), and 10% glycerol, supplemented with Protease Inhibitor Mini Tablets (Thermo Fisher Scientific), 1 mM PMSF (Roche), 10 mM MG132 (Sigma-Aldrich), and 20 mM N-ethylmaleimide (Sigma-Aldrich). Goat Fab anti-human Fab (Jackson ImmunoResearch) was coupled onto M-270 Epoxy Dynabeads (Thermo Fisher Scientific), following the manufacturer's instructions, in a Dynabeads Antibody Coupling Kit. Immunoprecipitations were performed by incubating cell lysates from 1.5 million cells with 1 mg of Dynabeads (prebound with anti-human Fab) overnight at 4° C. Beads were then washed with RIPA buffer, and immunoprecipitates were eluted with 0.1 M Glycine buffer (pH 2.7).
[0358] Western blot: Cell extracts and immunoprecipitates were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using Trans-Blot SD semi-dry transfer cell (Bio-Rad Laboratories), according to the manufacturer's protocols. After incubation with 5% nonfat milk in TBST (25 mM Tris [pH 7.2], 140 mM NaCl, and 3 mM KCl, 0.2% Tween 20) for 1 h, the membrane was incubated with Abs against IgG Fab (1:5000; Jackson ImmunoResearch), Ro52/TRIM21 (1:500, D-12; Santa Cruz Biotechnology), and GAPDH (1:10000, VPA00187; Bio-Rad Laboratories) overnight at 4° C. Membranes were washed with TBST four times, then incubated with HRP-conjugated anti-mouse (1:10000; Dako) or anti-rabbit (1:20000; Thermo Fisher Scientific) Abs for 1 h. Blots were washed with TBST four times and developed with ECL Plus Western Blotting Substrate (Thermo Fisher Scientific) according to the manufacturer's protocols. Blot images were acquired using the ChemiDoc imaging system (Bio-Rad Laboratories).
[0359] 1.12 MoDC Maturation Assay
[0360] 1×10.sup.5 moDCs were incubated in 200 μl of cell culture medium in 96-well plates with either PBS, 4×10.sup.7 Ad5 virus, 1.6 μg antibodies or immune complexes made from 4×10.sup.7 Ad5 virus and 1.6 μg antibodies that were pre-incubated for 1 h at room temperature. All treatments were administered to the cell culture in a volume of between 8-80 μl, and medium was added accordingly to make the final cell culture volume up to 200 μl. For a positive control, 1 μg/ml LPS was used. The cells were analyzed by FACS after 24 h.
[0361] 1.13 MoDC: Autologous CD14.sup.− PBMC Co-Culture Assay
[0362] MoDCs were prepared and treated in the same way as in the moDC maturation for 4 h.
[0363] Treatment of moDC with MG132 is done by incubating moDC in a six-well plate with 10 μM of MG132 (Sigma-Aldrich) in medium for 1 h at 37° C., 5% CO2. After 1 h, the cells were centrifuged to remove MG132. MG132-treated or -untreated moDC were resuspended in fresh medium and treated in the same way as in the moDC maturation for 4 h.
[0364] After 4 h, the cells were co-cultured in fresh medium with 5×10.sup.5 autologous CD14.sup.− PBMCs that had been labeled with 10 μM CFSE using the Vybrant™ CFDA SE Cell Tracer kit (Life technologies): briefly, 100 million cells were labeled in 1 ml of 10 μM CFSE in FBS-supplemented medium for 5 mins at 20° C. Excess dye was then removed by centrifugation at 10,000×g for 1 min and the cells were washed three times with fresh medium. The co-culture was maintained for up to 13 days with replacement of one third of old medium with fresh medium on days 4 and 7. For positive controls, either 1 μg/ml LPS was added to the moDCs, or 2 μl T Cell TransACT™ (Miltenyi) was used.
[0365] 1.14 Peptide Re-Stimulation Assays
[0366] To prepare moDCs for pulsing of peptides, autologous moDCs were generated using the same method as above from frozen monocyte stock. After 6 days, moDCs were pulsed for a day with either 10 μg/ml of sterile-filtered peptides, or 200 μg peptide libraries (Miltenyi, PepTivator AdV5 Hexon or PepTivator NY-ESO-1) in medium containing 100 ng/ml GM-CSF (Miltenyi), 100 ng/ml IL-4 (Miltenyi) and 50 ng/ml TNF-α (Miltenyi). Thereafter, 1×10.sup.5 peptide-pulsed moDCs were used to re-stimulate autologous 11 day old moDC:CD14.sup.− PBMC co-culture, at a ratio of 1:5 peptide-pulsed moDCs to 11 day old co-culture. This was done by harvesting the 11-d coculture, resuspending the cells to a concentration of 5×10.sup.6 cells/ml in fresh medium (with or without brefeldin A), and adding 100 μl of cells (i.e., 5×10.sup.5 cells) to the peptide-pulsed moDC in the 96-well, round-bottom plate. For donor LCY02, cells were re-stimulated for 16 hours and the supernatant of the re-stimulated cells was harvested for analysis by ELISA. The cells were then treated with fresh medium containing 1 μg/ml Brefeldin A. After 5 h, cells were harvested and labelled for analysis by flow cytometry. For donors LCY10, PAT35 and LCY25 cells were re-stimulated for 16 hours in the presence of 1.5 μg/ml Brefeldin A and then harvested for analysis by flow cytometry. For the 11 day old moDC:CD14.sup.− PBMC co-cultures, replacement of one third old medium with fresh medium was performed at days 4 and 7 for LCY02, PAT35 and LCY25; and for LCY10, feeding was performed on day 4 followed by dilution of the co-culture into an equal volume of fresh medium containing 10 ng/mL IL-7 and IL-15 on days 7 and day 10.
[0367] 1.15 Flow Cytometry
[0368] In all assays, LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used to exclude dead cells. In the moDC maturation assay, cells were incubated with anti-FcR-blocking antibody (eBioscience, San Diego, Calif., USA) and then labelled using combinations of the following antibodies: Pacific-Blue-anti-CD14 (M5E2), APC-anti-CD11c (S-HCL-3), Alexa Fluor700-anti-CD80 (L307.4), FITC-anti-CD83 (HB15e), PE-Cy7-anti-CD86 (FUN-1), APC-Cy7-anti-HLA-DR (L243), BV650-anti-CCR7 (G043H7) and PE-anti-CD206 (19.2). In the co-culture assay, cells were incubated with human FcR blocking reagent (Miltenyi) and then labelled using the following antibodies: Alexa Fluor647-anti-CD3 (SK1), PE-Cy7-anti-CD4 (OKT4), Pacific Blue-anti-CD8 (SK7), and PE-anti-CD56 (AF12-7H3). In the haplotyping of donors, the dyes used were Alexa Fluor 647-anti-HLA-A24 (17A10) and PE-anti-HLA-A2 (BB7.2). For haplotype controls, a HLA-A24+ cell line HT29 and HLA-A2+ cell line MDA-MB-231 were also labelled and included in the analysis. In the re-stimulation assays, the dyes used were Alexa Fluor647-anti-CD3 (SK7), PE-Cy7-anti-IFN-γ (4S.B3), Pacific Blue-anti-CD8 (SK1), and PE-anti-CD56 (AF12-7H3). After the surface markers are labelled, cells were fixed and permeabilized using BD Cytofix/Cytoperm solution followed by PE-Cy7-anti-IFN-γ. For absolute cell counts 10 μl of CountBright™ Absolute Counting Beads were added to cells. Samples were acquired using BD FACSDiva software on the LSRFortessa cell analyzer and FACSCanto II (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Ash-land, OR, USA).
[0369] 1.16 Cytokine and Chemokine Analysis
[0370] Supernatants from the moDC maturation assays were analyzed by a multiplex analysis using human cytokine/chemokine bead panel 1 and 2, which measure a total of 64 targets (Milliplex MAP kits, Millipore) on a Flexmap 3D system (Luminex Corp, Texas, USA). Supernatants from the co-culture assays were analyzed by ELISA for the level of IFN-γ using Human IFN-γ ELISA MAX™ Standard (Biolegend).
[0371] 1.17 Statistical Analysis
[0372] Statistical analyses were performed using GraphPad Prism 7.01 software using repeated measure ANOVA with Dunnett's multiple comparison testing, or Friedman with Dunn's multiple comparison testing. The adjusted p-values are indicated in the Figures by asterisks.
Example 2: Results
[0373] 2.1 A Modified Fc Exhibits Increased Affinity for TRIM21
[0374] The inventors first identified the amino acids in Fc region that are in contact with TRIM21, based on the crystal structure of the human IgG Fc-TRIM21 complex (
TABLE-US-00003 Amino acid CH Secondary (EU numbering) Domain Structure 252(MET) CH2 Loop 253(ILE) Loop 254(SER) Loop 309(LEU) Helix 310(HIS) Helix 311(GLN) Helix 314(LEU) Helix 315(ASN) Helix 345(GLU) CH3 Loop 428(MET) Sheet 430(GLU) Loop 431(ALA) Loop 432(LEU) Loop 433(HIS) Loop 434(ASN) Loop 435(HIS) Loop 436(TYR) Sheet 437(THR) Sheet 438(GLN) Sheet 439(LYS) Sheet 440(SER) Sheet
[0375] The majority of the amino acids interacting with TRIM21 in the CH2 domain were in the α-helix, and the inventors reasoned that this region could be destabilised by modification. The inventors therefore instead focused on modification of the amino acids in the CH3 domain, and selected 7 amino acids whose side groups were proximal to and facing towards TRIM21 for modification (see
[0376] A phage library of 2 billion bacteriophages displaying the human IgG1 Fc was generated with randomly-substituted amino acids in the selected 7 positions. Recombinant human TRIM21 PRYSPRY domain was used as bait for biopanning. After three sequential rounds of biopanning, approximately 50 clones from the output of Rounds 2 and 3 were sequenced. One sequence was found in ˜10% of the clones analysed in Round 2; and in 94% of the clones analysed in Round 3 (
[0377] The inventors next investigated whether antibodies bearing the modified Fc bound to TRIM21 with higher affinity than antibodies comprising unmodified Fc. Two sets of antibodies were constructed: the first set comprises the variable domains of human antibody, 3G9-2D2 (Cheong et al., 2010) which recognizes human DEC-205, joined to human IgG1 constant regions that have the modified or unmodified Fc; the second set comprises variable domains of the mouse antibody, 9C12 (Varghese et al., 2004a) which recognizes the Adenovirus Type 5 (Ad5) hexon, joined to human IgG1 constant regions that have the modified or unmodified Fc. The first set was designated ‘human IgG1’, and the second set was designated ‘chimeric IgG1’ (
[0378] The affinity of the unmodified and Fc-modified antibodies for TRIM21 was measured by surface plasmon resonance analysis. Modifying the Fc region of human IgG1 increased its affinity for the PRYSPRY domain of TRIM21 by at least 100 fold: the affinity constants (K.sub.D) were 0.522 nM for the Fc-modified human IgG1 (
[0379] To assess the involvement of each single mutation in the affinity increase for TRIM21, 5 mutants with reversion to wild-type amino-acid in 1 single position were derived from Fc-modified chimeric IgG1 and their affinity for TRIM21 domain PRYSPRY was measured (Table 1). Reversion of mutations in positions 256, 433 and 434 resulted in lower affinity for TRIM21, suggesting that modifications T256P, H433T and N434R are important for improved binding.
TABLE-US-00004 TABLE 1 Affinity of Fc-modified and unmodified chimeric IgG1 for TRIM21. Amino-acid position in IgG1 (EU numbering system) Affinity K.sub.D 256 433 434 436 440 (M) Wildtype T H N Y S 125 chimeric IgG1 PN04-90 P T R F I 4.25 × 10.sup.−1 V1 P H R F I 1.08 × 10.sup.−8 V2 P T N F I 1.62 × 10.sup.−6 V3 P T R Y I 2.11 × 10.sup.−10 V4 P T R F S 2.76 × 10.sup.−10 V5 T T R F I 2.35 × 10.sup.−8
[0380] 2.2 Increasing Fc Affinity for TRIM21 Preserves ADIN
[0381] Antibody-dependent intracellular neutralization (ADIN) was first demonstrated in HeLa cells infected with adenoviruses (Mallery et al., 2010). The hexon protein of adenovirus is recognized by the monoclonal antibody 9C12 (Varghese et al., 2004a), which was used in construction of the chimeric antibodies. 9C12 does not block viral entry, but mediates post-entry neutralization (Varghese et al., 2004b), in a TRIM21-dependent fashion (Mallery et al., 2010). While reducing the affinity of antibody for TRIM21 does not affect ADIN of adenovirus in HeLa cells (Foss et al., 2016), the effects of increasing affinity for TRIM21 have not been investigated. The inventors investigated how increasing Fc affinity for TRIM21 influences ADIN in both HeLa cells (non-immune cells) and monocyte-derived DCs (immune cells).
[0382] Different concentrations of Fc-modified antibodies comprising PN04-90 Fc and unmodified antibodies comprising wildtype human IgG1 Fc were incubated with replication-defective Ad5 which carries the eGFP (enhanced green fluorescent protein) reporter gene to form immune complexes, which were then added to HeLa cells or monocyte-derived DCs (moDCs). The frequency of infection was monitored after 48 h via analysis of eGFP expression (
[0383] To verify that the Fc-modified Abs interact with TRIM21 in moDC, the treated moDC were lysed and beads conjugated with Fab anti-human Fab were used to immunoprecipitate the Fc-modified Abs. Western blot analysis showed that TRIM21 coimmunoprecipitated with modified Ab, suggesting that it binds to the internalized Fc-modified immune complexes (
[0384]
[0385] ADIN mediated by the unmodified Fc and modified Fc remained the same when tested at lower viral MOI (i.e. less than 400).
[0386] 2.3 Increasing Fc Affinity for TRIM21 Promotes moDC Maturation
[0387] Reducing the affinity of antibodies for TRIM21 has previously been shown to impair pro-inflammatory signalling in HEK293T cells (Foss et al., 2016). For moDCs, stimulation of a T cell response requires the simultaneous presentation of cognate antigen, and the provision of activating signals in the form of pro-inflammatory cytokines and co-stimulatory molecules on the DC surface. This process is often subverted by viruses, including adenoviruses (Newton et al., 2008). The inventors therefore investigated whether increasing Fc affinity for TRIM21 promoted moDC expression of co-stimulatory molecules and pro-inflammatory cytokines.
[0388] Incubating moDCs with Ad5 alone for 24 h did not increase expression of maturation markers, even at the highest dose of 400 MOI (
[0389] 2.4 Increasing Fc Affinity for TRIM21 Promotes moDC Production of Th1-Associated Chemokines.
[0390] The inventors next investigated whether increasing Fc affinity for TRIM21 promoted expression of pro-inflammatory cytokines by moDCs. Using a multiplex bead-based assay, the inventors measured the secretion of 64 cytokines and chemokines by moDCs after 24 h of incubation with immune complexes comprising the Fc-modified or unmodified antibodies. For each cytokine/chemokine, fold change in expression level between treatments with PN04-90 Fc (Fc-modified) and wildtype IgG1 Fc (unmodified) immune complexes was calculated and correlated to their dosages (
[0391] As IFN-γ is not a typical cytokine produced by moDCs, the slight increase in the levels of this cytokine most probably came from T cells and/or innate lymphoid cells that could be present in the CD14+ preparations, which had cell purity ranging from 81 to 91% (
[0392] In vivo, early-maturing DCs in peripheral tissues are the main producers of CCL3, CCL4 and CCL5 (Sallusto et al., 1999, 2000), which attract immature DC and T cells that promote a T helper type 1 (Th1) response (Lebre et al., 2005). Taken together, these data show that exposure of moDCs to Ad5 immune complexes comprising antibodies with Fc modified for increased affinity to TRIM21 induced increased co-stimulatory marker expression and greater production of Th1-associated pro-inflammatory cytokines compared to either virus alone, or Fc-unmodified immune complexes.
[0393] 2.5 Increasing Fc Affinity for TRIM21 Increases T Cell Expansion by moDC
[0394] The inventors next assessed how Fc-modified immune complexes influenced CD8 T cell responses in PBMCs. moDCs were incubated either Ad5 alone, or with PN04-90 Fc (Fc-modified) or wildtype IgG1 Fc (unmodified) antibody-Ad5 immune complexes, and then co-cultured with autologous CD14.sup.− PBMCs for 13 days. LPS and TransACT (a CD3/CD28 agonist) were used as positive controls to non-specifically activate DCs and T cells respectively.
[0395] After 13 days of co-culture, the relative proportions of CD4 and CD8 T cells were determined by flow cytometry. Cells were gated for singlets/live/CD3+CD56− cells to include T cells and exclude NKT cells. CD8 T cells were found to be markedly enriched in co-cultures where moDC were treated with PN04-90 Fc (Fc-modified) immune complexes (
[0396] Analyzing results from 7 different donors, the inventors observed that treatment of moDCs with Fc-modified immune complexes significantly increased CD8 T cell counts but not CD4 T cell counts in co-culture experiments, as compared to treatment with Fc-unmodified Ad5 immune complexes (
[0397] Treatment with Fc-modified immune complexes, compared with treatment with unmodified immune complex, resulted in an increase in cell count that was statistically significant for CD8 T cells but not CD4 T cells (
[0398] During T cell expansion, IFN-γ is produced and so the inventors measured secretion of IFN-γ at 4, 7 and 13 days of co-culture for each donor. As in the CD8 T cell response, treating moDCs with Fc-modified immune complexes induced significantly higher IFN-γ production in the moDCs:CD14.sup.− PBMC co-culture than did either virus alone or Fc-unmodified immune complexes at 4, 7 and 13 days post infection, as determined by ELISA of the donors that responded to the modified-Fc immune complexes (
[0399] Analyzing the IFN-γ results from the same 7 donors again revealed a heterogeneity in response that could be grouped into the Type 1 and Type 2 donors (
[0400] Taken together, the data show that through modifying Fc for improved affinity to TRIM21, immune complexes can be generated which increase the ability of moDC to stimulate IFN-γ production by PBMCs, and to stimulate CD4 and CD8 T cell expansion. In the case of CD8 T cell count, this effect is particularly marked in the Type 2 subset of donors that do not respond well to virus alone.
[0401] 2.6 Increasing Fc Affinity for TRIM21 Increases Antigen-Specific CD8 T Cell Proliferation Induced by moDC Incubated with Immune Complexes
[0402] To understand the extent to which the CD8 T cell response was antigen-specific, the inventors further investigated the response of donor LCY02; this donor was HLA-A24- and HLA-A2-positive, and exhibited a 2.7-fold increase in the percentage of CD8 T cells in moDC:CD14.sup.− PBMC co-cultures in response to PN04-90 Fc immune complexes (
[0403] A marked and specific IFN-γ response to the HLA-A24-restricted-Adenovirus epitope was observed after 16 h of re-stimulation (
[0404] In the same experiment, CD14− PBMCs were labelled with CFSE to enable identification of proliferating cell populations. T cells that were cocultured with moDCs pretreated with the virus and Fc-modified Abs showed the most proliferation (
[0405] After 11 days, 43% of the CD8 T cells, and 21% of the CD4 T cells had proliferated (
[0406] In the single peptide experiments, the HLA-A24 adenovirus peptide stimulated 6.57% of the proliferated CD8 T cells to produce IFN-γ, while the response to the HLA-A2 adenovirus peptide was comparable to the negative controls (
[0407] To eliminate the bystander effect of cytokines which could be released during re-stimulation, the experiment was repeated in the presence of Brefeldin A to stop cytokine release right at the start of re-stimulation. Only CD8 but not CD4 T cells were found to display antigen-specific re-stimulation (
[0408] In a separate experiment, toxicity from overnight brefeldin A treatment was directly measured by comparing the number of live cells in CD14− PBMCs in the presence and absence of brefeldin A. LIVE/DEAD staining showed that after 16 h the viability of CD14− PBMCs with brefeldin A treatment was ˜70 and 86% of that without brefeldin A treatment for inactivated and activated cells, respectively (
[0409] Taken together, these results show that Fc-modified immune complexes increase moDC stimulation of antigen-specific IFN-γ production and CD8 T cell proliferation.
[0410] The inventors next investigated whether the CD8 T cell proliferation stimulated by moDCs treated with Fc-modified immune complex were dependent on proteasomal degradation in the moDCs.
[0411] MoDCs were pretreated with MG132 for 1 h, then subjected to treatment with immune complexes or various controls and then cocultured with autologous CFSE-labeled CD14− PBMCs for 11 days.
[0412] The results are shown in
[0413] To ensure that the loss of proliferation was not solely because of MG132-associated toxicity to moDC, moDC viability was analysed 1 d after MG132 treatment. At 22 h posttreatment, the viability of moDC with MG132 treatment was 61% of that without MG132 treatment (
Example 3: Discussion
[0414] The inventors engineered the human IgG Fc region to increase its affinity for TRIM21 by 100-fold, and showed that the resulting antibodies directs viral antigens effectively into the cross-presentation pathway leading to the stimulation of antigen-specific CD8 T cells. This process is mediated by cross-presentation and not the classical MHC class I presentation of viral proteins because most of the endocytosed viruses are neutralized and therefore viral genes are not being expressed. Importantly, the cross-presentation process is remarkably enhanced by Fc-modification for increased TRIM binding, while the host-protective mechanism of ADIN is retained.
[0415] While both the Fcγ receptor (FcγR) and the neonatal Fc receptor (FcRn) have been shown to facilitate cross-presentation (Baker et al. 2011; Regnault et al. 1999), this is the first demonstration that TRIM21 also accesses this pathway. The involvement of TRIM21 with the proteasome has led to speculation that it regulates antigen-processing (signal 1) in DCs, but the inventors also found that TRIM21 ligation by Fc-modified immune complexes induces expression of co-stimulatory molecules (signal 2), and pro-inflammatory cytokine/chemokine release (signal 3) by moDCs. For cross-priming of CD8 T cells, all 3-signals are required. DC-targeted vaccine strategies often rely on TLR-stimulants, such as monophosphorylated lipid A, polyinosinic-polycytidylic acid, and CpG oligonucleotides, or a cocktail of four cytokines, IL1p, IL-6, TNF-α and Prostaglandin E2 (Castiello et al., 2011; de Jong et al., 2002; Han et al., 2009), to provide signals 2 and 3 to DCs. Fc-modified immune complexes could potentially provide all three signals, removing the need to optimize the timing for the adding of antigen (before or after the addition of the maturation cocktail, depending on whether antigen-internalization is required) and dosages of the different components; and also overcomes the issue of ensuring all four components reach the DCs with the correct timing in vivo. In other words, the modified Fc would render in vivo application of DC-targeted vaccines more feasible.
[0416] MoDCs treated with Fc-modified immune complexes consistently upregulated maturation marker expression in five out of six donors, and a sub-population of donors (60%) exhibit markedly increased CD8 T cell expansion. The differences are likely to be due to the level and timing of previous exposure to Ad5 for the donors. Donors with prior exposure to adenovirus may have memory T cells capable of responding to viral antigens in the absence of co-stimulatory molecule expression by DC. In this case, moDCs treated with virus alone would be expected to outperform moDCs treated with immune complexes, due to increased expression of viral proteins: at MOI 400 the replication-deficient Ad5 infects 69% of the moDC, likely leading to expression of viral proteins at a level equivalent to that of a replication-competent adenovirus at an MOI of 1 (Saha and Parks, 2017). Thus in the absence of antibodies (and therefore ADIN), there is a larger population of moDCs (69% versus 4% in the presence of antibody) producing viral proteins, albeit without upregulating their co-stimulatory markers, and able to stimulate memory CD8 T cells in the subpopulation of donors who have them. Importantly, prophylactic vaccines need to stimulate naïve T cells rather than memory T cells, since they are meant to protect individuals not having had prior exposure to the virus while therapeutic vaccines need to revive a virally-damped immune response, which means that the DC might need a ‘boost’ to upregulate their stimulatory status again. In both cases, the ability of the modified-Fc to enhance moDC-cross-priming of CD8 T cells is advantageous.
[0417] Ad5 immune complexes were used to demonstrate the potential of modified-Fc immune complexes to induce and enhance CD8 T cell responses to viral antigen, but these findings are of relevance to other pathogens and diseases. Ad5 immune complexes reach TRIM21 in the cytosol of moDC because adenoviruses release protein VI, which lyses the endosome (Wiethoff et al., 2005; Greber et al., 1993).
[0418] Antibody-antigen fusion proteins targeting DCs such as anti-DEC205-NY-ESO-1 (Dhodapkar et al., 2014) and anti-DEC-HIV gag (Bozzacco et al., 2007) have been shown to stimulate anti-cancer and anti-HIV CD8 T cell responses. Also, antibodies can simply be designed to target antigens of interest to form immune complexes that are taken into the DC via the Fc receptor. Fc receptor-mediated antigen internalization in DCs is known to be channelled to a special transport pathway which allows the antigen efficient access to the cytosol (Amigorena, 2002). HRP-anti-HRP immune complexes were detectable by HRP substrate (DAB) and anti-rabbit IgG Fabs suggesting that both antigen and antibody remains mostly intact in the cytosol (Rodriguez et al., 1999, Gros and Amigorena, 2019). Given that an Fc receptor (TRIM21) and an Ag-processing enzyme (proteasome) both exist in the cytosol, immune complexes may have privileged access to deliver themselves and their cargo completely intact to the cytosol.
[0419] In summary, the inventors have identified a readily-adaptable method of Fc modification for targeting endocytosed antigen in immune complexes to MHC class I cross-presentation pathway via TRIM21, and have shown its potential to provide all the signals necessary for the stimulation of a potent CD8 T cell and cytokine response to specific antigen.
Example 4: Further Fc Variants
[0420] Further variant Fc regions having improved affinity for TRIM21 as compared to wildtype human IgG1 Fc were identified.
[0421] Briefly, an Fc Phage display library was generated, essentially as described in Example 1.8, comprising 200 million Fc variants having amino acids randomised at positions 256, 433, 434, 436 and 440 of human IgG1.
[0422] The Fc Phage display was used in three biopanning experiments performed as described in Example 1.9, as summarised in Table 2 below. Binding to the PRYSPRY domain of TRIM21 was analysed by ELISA.
TABLE-US-00005 TABLE 2 Biopanning with an Fc Phage display library randomised at positions 256, 433, 434, 436 and 440 to identify variants with improved binding to TRIM21 No. clones with improved Host cells binding to Host cells for Fc No. clones TRIM21 as Biopanning for phage expression picked for determined by No. of new Expt conditions amplification for ELISA ELISA ELISA sequences 1 Low TG1 HB2151 94 4 (only 2 have 2 stringency PCR insert) approach (3 rounds of panning) 2 Low TG1 HB2151 24 11 8 stringency approach (repeated pan 3) 3 High XL1-Blue XL1-Blue 372 293 125 stringency approach (2 rounds of panning)
[0423]
[0424]
[0425]
[0426]
[0427] A total of 306 clones were identified as expressing Fc regions which bound to the PRYSPRY domain of TRIM21 with greater affinity to wildtype Fc.
[0428] The 306 clones were sequenced, and identifying 128 new, unique sequences (SNs. 1 to 8, 10 to 39, and 46 to 135 of
[0429] The 135 sequences were divided into 8 subgroups based on their sequence pattern (see
[0430] Fc regions comprising sequences corresponding to 8 of the 129 new sequences were analysed for binding to TRIM21 PRYSPRY domain by Surface Plasmon Resonance, which was performed as described in Example 1.10.
[0431] The results are summarised below:
TABLE-US-00006 SN (of Amino-acid Figure (EU numbering system) Ka Kd KD 13) 256 433 434 436 440 (1/Ms) (1/s) (M) Wildtype T H N Y S 6.47 × 10.sup.5 8.12 × 10.sup.−2 1.26 × 10.sup.−7 IgG1 Fc 1 P S H Y G 7.05 × 10.sup.6 3.88 × 10.sup.−4 5.5 × 10.sup.−11 2 P S H Y S 4.66 × 10.sup.6 7.27 × 10.sup.−4 1.56 × 10.sup.−10 10 P V H Y R 5.15 × 10.sup.5 4.60 × 10.sup.−4 8.92 × 10.sup.−10 9 P V H Y S 5.03 × 10.sup.6 4.81 × 10.sup.−4 9.57 × 10.sup.−11 15 P H H Y S 7.84 × 10.sup.6 1.65 × 10.sup.−2 2.11 × 10.sup.−9 32 P T R Y S 1.06 × 10.sup.6 5.30 × 10.sup.−4 4.98 × 10.sup.−10 46 P V R Y S 7.30 × 10.sup.5 1.24 × 10.sup.−4 1.69 × 10.sup.−10 92 A H N F M 3.94 × 10.sup.6 1.45 × 10.sup.−2 3.68 × 10.sup.−9
[0432] Example 2.3 and
[0433] The inventors next investigated whether the improved ability to stimulate moDC maturation was also possessed by virus-antibody immune complexes comprising variant Fc having a more modest improvement in the affinity of binding to TRIM21 PRYSPRY domain relative to wildtype human IgG1 Fc.
[0434] The inventors therefore investigated the co-stimulatory molecule expression by DCs following co-culture with immune complexes comprising V1 Fc. V1 Fc binds to TRIM21 PRYSPRY domain with an affinity of K.sub.D=1.08×10.sup.−8 M (see Example 2.1 above).
[0435] A moDC maturation assay was performed as described in Example 1.12, using moDCs from PBMCs obtained from three different donors. The following test conditions were investigated: [0436] PBS only (PBS; negative control); [0437] Ad5-specific antibody comprising wildtype IgG1 Fc (WT only) [0438] Ad5-specific antibody comprising V1 Fc (V1 only) [0439] Ad5-specific antibody comprising PN04-90 Fc (PN04-90 only) [0440] Ad5 only (Virus only) [0441] Immunocomplex comprising Ad5-specific antibody comprising wildtype IgG1 Fc+Ad5 (WT+Virus) [0442] Immunocomplex comprising Ad5-specific antibody comprising V1 Fc+Ad5 (V1+Virus) [0443] Immunocomplex comprising Ad5-specific antibody comprising PN04-90 Fc+Ad5 (PN04-90+Virus) [0444] LPS only (LPS; positive control)
[0445] The results are shown in
[0446] Based on these results it can be concluded that even a modest improvement in affinity for TRIM21 provides functional effects relevant to therapeutic and prophylactic applications of molecules and complexes comprising Fc regions.
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