Patent Application
20240010712
The present invention relates to the fields of medical science, immunology and pharmaceutical products. The present invention provides antibodies and compositions capable of affecting the immune system and inhibiting pathogenic infections, such as streptococcal infections. The present invention also provides methods for obtaining such antibodies as well as the uses of such antibodies.
Antibodies are essential components of the immune system used to recognize and neutralize external intruders such as pathogenic bacteria. They are produced by B cells after their B cell receptor reacts with a specific antigen in the lymphoid tissue. B cell maturation and antibody development have evolved to allow for an extraordinary variety enabling the binding of diverse targets. V(D)J recombination events, as well as somatic hypermutation, give rise to a vast repertoire of antibody variable domains. The result of B cell activation, clonal expansion, maturation, and class switching is the generation of IgG antibodies that offer long term protection against infectious agents. An IgG antibody is a Y-shaped molecule composed of two identical Fab fragments and one Fc domain, where the unique specificity is provided by Fab interaction with an antigen. IgG typically binds with either Fab and, dependent on the antigen density and organization, can bind with a higher strength through avidity to two copies of the same antigen (Klein and Bjorkman 2010). We designate this latter form of binding as dual-Fab trans binding. When bound to their target, IgG molecules carry out effector functions by triggering clustering of Fc receptors on immune cells, which induces cell signaling and leads to a variety of downstream effects such as phagocytosis, immune recognition, and activation.
Group A streptococcus (GAS) is a common human pathogen causing significant morbidity and mortality in the human population and is an important causative agent of severe invasive infections. The bacterium has evolved an extensive array of measures to counteract the human immune response, including resistance to phagocytosis, and several immunoglobulin-targeting mechanisms (IdeS, EndoS, protein M/H). The streptococcal M protein, a virulence determinant, has a long coiled-coiled structure with repeating regions (A, B, S, C, and D). These regions are typically associated with distinct protein interactions and bind many components of the humoral immune response such as C4BP and albumin, and forms complexes with fibrinogen that induce vascular leakage and contribute to phagocytosis resistance. The M protein can also reduce phagocytosis by reversing the orientation of IgG by capturing IgG Fc domains. These pathogenic mechanisms deprive the immune system of crucial defenses, allowing GAS to disseminate within a host and across the population.
Although GAS infections generate a humoral immune response, repeated exposures seem to be required to generate protective memory B cell immunity. There are few candidates for anti-bacterial monoclonal antibody therapy in general, and none available for GAS. Much effort has been allocated to developing vaccines against GAS (Dale and Walker 2020), with the prime immunizing antigen being M protein, particularly with M protein-based peptides (Azuar et al. 2019). Yet, no effective vaccine against GAS has been approved to date. It is unclear what makes it so difficult to generate immunity, but potentially formation of antibody subsets is suppressed by immunodominant regions or cryptic epitopes (Ozberk et al. 2018). In severe life-threatening invasive GAS infections, intravenous IgG antibodies (IVIG) from human pooled plasma are used as therapy, even though reports on their efficacy show contradictory results. Also, there remain safety issues with medicaments derived from human plasma. Hence, there is a dire need to find new ways to treat these critically ill patients.
Hence, there is a need for effective and safe new antibodies for combatting diseases originating from external intruders such as pathogenic bacteria and vira. One such pathogenic bacterium is Group A streptococcus (GAS).
The present inventors have discovered that anti-M protein antibodies derived from a healthy donor who had previously undergone a GAS infection, these antibodies when exposed to GAS and M protein bind and exert various effects including a range of protective immune functions, including bacterial agglutination, NFkB activation, phagocytosis, and in vivo protection. The inventors also identified a new type of interaction where the two identical Fabs of one of the monoclonal IgG antibodies simultaneously bind to two distinct epitopes on the same molecule. This form of binding is designated as dual-Fab cis binding.
In a first aspect the present invention provides an antibody binding to streptococcal M protein, wherein said antibody comprises:
In one embodiment the antibody is selected from the group consisting of
In another embodiment the antibody of the present invention binds to streptococcal M protein with a KD of less than 50×10−9 M−1, as determined by binding to Streptococcus pyogenes SF370.
Antibodies of the present invention have been found to have the ability to mediate bacterial agglutination. In further embodiments the antibodies according to the invention have been found to mediate NFkB-activation. In yet further embodiments of the invention the antibodies of the present invention have been found to have the ability to induce phagocytosis.
In yet further embodiments, antibodies of the present invention have the ability to exhibit simultaneous binding to two different epitopes of the streptococcal M protein by way of dual-Fab cis antibody binding.
In a second aspect the present invention provides a pharmaceutical composition comprising the antibody as defined above and at least one pharmaceutically acceptable excipient.
In one embodiment the pharmaceutical composition comprises two or more different antibodies.
In a third aspect the present invention provides the use of the antibody according to the first aspect for the treatment of a streptococcal infection, such as a Group A streptococcus infection.
In a fourth aspect the present invention provides the use of an antibody of the present invention in an application selected from Western blot, Flow Cytometry, ELISA and Immunoflourescence.
In a fifth aspect the present invention provides an antibody exhibiting binding to two different epitopes of a protein by way of dual-Fab cis antibody binding.
In a sixths aspect the present invention provides a method for obtaining an antibody exhibiting simultaneous binding to two different epitopes of a molecule by way of dual-Fab cis antibody binding, comprising the steps:
In one embodiment the method for obtaining an antibody exhibiting dual-Fab cis antibody binding, said two different epitopes of a molecule is two different epitopes of of a protein, such as streptococcal M protein.
In a seventh aspect the present invention provides a method for crosslinking antibody F(ab′)2-fragments bound to a molecule, comprising the steps:
In addition, in comparison with known antibodies and antibody compositions, the antibodies according to the present invention show improved properties for effectively exhibiting range of protective immune functions, including bacterial agglutination, NFkB activation, phagocytosis, and/or in vivo protection.
In a first aspect the present invention provides an antibody binding to streptococcal M protein, wherein said antibody comprises:
In one embodiment the antibody has the sequence A1-A13 to comprises no more than 3 conservative substitutions, such as no more than 1 conservative substitution. In a second embodiment the antibody has the sequence B1-B18 comprises no more than 3 conservative substitutions, such as no more than 1 conservative substitution.
Conservative substitutions as used herein is defined as substitution within the classes of amino acids reflected in one or more of the groups in one or more of the Tables 1-3.
In one embodiment the antibody comprises:
A Complementarity Determining Region (CDR) H3 loop comprising the sequence A1-A18, wherein
The antibody according to any the present invention preferably binds to streptococcal M protein with a KD of less than 50×10−9 M−1, as determined by binding to Streptococcus pyogenes SF370. In one embodiment the antibody binds to streptococcal M protein with a KD less than 15×10−9 M−1, less than 5×10−9 M−1 or less than 1×10−9 M−1.
In a further embodiment the antibody of the invention has said CDR H3 loop selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4.
In a further embodiment, the antibody according to the invention has a H3 chain selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and any variant sequence having less than 20 conservative amino acid substitutions relative to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
In some embodiments, the antibody comprises said variant sequence of H3 chain having less than 10 conservative amino acid substitutions, less than 5 conservative amino acid substitutions, or less than 2 conservative amino acid substitutions.
In another embodiment the antibody comprises said CDR L3 loop being selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
In a further embodiment, the antibody according to the invention has a L3 chain selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and any variant sequence having less than 20 conservative amino acid substitutions relative to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.
In some embodiments, the antibody comprises said variant sequence of L3 chain having less than 10 conservative amino acid substitutions, less than 5 conservative amino acid substitutions, or less than 2 conservative amino acid substitutions.
In a further embodiment the antibody of the invention is selected from the group consisting of
In another embodiment the antibody of the invention is selected from the group consisting of
In another embodiment the antibody of the invention is selected from the group consisting of
In an embodiment, the antibody according to the invention has the ability to mediate bacterial agglutination.
In another embodiment, the antibody according to the invention has the ability to mediate NFkB-activation.
In yet another embodiment the antibody according to the invention has the ability to induce phagocytosis.
In yet another embodiment the antibody has the ability to exhibit simultaneous binding to two different epitopes of the streptococcal M protein by way of dual-Fab cis antibody binding. In an embodiment the antibody has said two different epitopes of the streptococcal M protein being a) in the B repeats and C repeats, b) in the linear sequence, or c) in the 3-dimensional structure.
The term “dual-Fab cis antibody binding” as used herein is intended to mean binding by an antibody where only one binding site binds to two distinct binding sites on the target molecule. Hence, dual-Fab cis antibody binding is different from the binding by known bi-specific antibodies which contain two independent variable sites each binding to one of two sites on the target molecule. Ab25 is capable of dual-Fab cis binding in an intramolecular cis-binding fashion to two distinct, non-identical epitopes.
The requirement of dual-Fab cis binding to separate epitopes that Ab25 exhibits, is an unexpected mode of functional antibody interaction, adding to the already astounding diversity found in antibodies (Kanyavuz et al., 2019). A related phenomenon to the dual-Fab cis binding is the case of the anti-HIV 2G12 antibody (Trkola et al., 1996) which has a mutation in its hinge region leading to Fab dimerization (Gach et al., 2010). The two Fabs of 2G12 bind to high-mannose sugars but due to the fact of their unorthodox dimerization, essentially behave as one large Fab (Calarese et al., 2005). In fact, normal single Fab-based interactions between multiple anti-HIV glycan antibodies gave similar biological outcomes as 2G12 (for a review see Kong et al., 2014). This indicates that while 2G12 has an unorthodox structure, its function is correlated to the specific epitope and not due to a distinct mode of interaction. In the context of unorthodox antibodies, bispecific antibodies (Kontermann and Brinkmann, 2015) or antigen clasping antibodies (Hattori et al., 2016) have been engineered for improved functionality, and the present invention shows that evolution has resulted in similar outcomes. The present invention reveal an, up till now, unknown added value of using F(ab′)2 fragments rather than Fabs when screening for functional antibodies.
In a further aspect the invention provides a pharmaceutical composition comprising the antibody of the invention and at least one pharmaceutically acceptable excipient.
In one embodiment the pharmaceutical composition comprises two or more different antibodies as defined by the invention.
The antibodies and antibody compositions of the invention disclosed herein may be used to combat bacterial or viral infections in individuals. In particular the antibodies may be used to treat infections with Group A streptococcus.
Pharmaceutical compositions for parenteral administration such as intravenous or subcutaneous administration may be liquid or solid formulations for administration. Solid formulations for reconstitution may be delivered by injection or infusion. Such formulations are typically sterile products.
The treatment method may consist of a single administration or a plurality of administrations over a period of time.
Depending upon the particular treatment and the individual to be treated, as well as the route of administration, the compositions may be administered at varying doses and/or frequencies.
The pharmaceutical compositions must be stable under the conditions of manufacture and storage; thus, if necessary they should be preserved against the contaminating action of microorganisms such as bacteria and fungi. In case of liquid formulations such as solutions, the carrier can be a solvent containing, for example, water, ethanol, polyol (e.g. glycerol, propylene glycol and liquid polyethylene glycol), buffer, isotonicity agent and suitable mixtures thereof.
The compositions for use in the treatment methods of the invention comprises at least one pharmaceutically acceptable excipient, such as carriers, solvents, pH-adjusting agents, buffers, stabilizing agents, surfactants, solubilizers, preservatives etc.
It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question. A person skilled in the art will know how to choose a suitable formulation and how to prepare it (see eg Remington's Pharmaceutical Sciences 18 Ed. or later). A person skilled in the art will also know how to choose a suitable administration route and dosage.
In a further aspect the invention provides the use of the antibody of the invention for the treatment of a streptococcal infection, such as Group A streptococcus.
In one embodiment the use of the antibody according to the invention is for the treatment of sepsis.
In a further embodiment the use of said antibody comprises the parenteral administration, such as intravenously, subcutaneously or intramuscularly.
In an embodiment this use is in combination with intravenous immunoglobulin (IVIG). In another embodiment the use of the pharmaceutical composition of the invention is for the treatment of sepsis. In a further embodiment this use of said antibody comprises administration intravenously, subcutaneously or intramuscularly.
In a further aspect the present invention provides the use of an antibody of the invention in an application selected from Western blot, Flow Cytometry, ELISA and Immunoflourescence.
In a further aspect the present invention provides an antibody exhibiting binding to two different epitopes of a molecule by way of dual-Fab cis antibody binding. In an embodiment said two different epitopes of a molecule is two different epitopes of a protein or a carbohydrate. In one embodiment said molecule is a protein. In another embodiment said two different epitopes of a protein is two different epitopes of streptococcal M protein.
In a further aspect the present invention provides a method for obtaining an antibody exhibiting simultaneous binding to two different epitopes of a molecule by way of Dual-Fab cis antibody binding, comprising the steps:
In one embodiment of this method said two different epitopes of a molecule is two different epitopes of streptococcal M protein.
In a further aspect the present invention provides a method for crosslinking antibody F(ab′)2-fragments bound to a molecule, comprising the steps:
In an embodiment of this method said molecule is a protein, such as streptococcal M protein.
The following list of non-limiting embodiments further illustrate the invention:
1. An antibody binding to streptococcal M protein, wherein said antibody comprises:
2. The antibody according to embodiment 1, wherein said sequence A1-A13 comprises no more than 3 conservative substitutions.
3. The antibody according to embodiment 1, wherein said sequence B1-B18 comprises no more than 3 conservative substitutions.
4. The antibody according to embodiment 1, wherein said sequence A1-A13 comprises no more than 1 conservative substitution.
5. The antibody according to embodiment 1, wherein said sequence B1-B18 comprises no more than 1 conservative substitution.
6. The antibody according to any of the preceding embodiments, wherein said antibody comprises:
7. The antibody according to any of the preceding embodiments, which binds to streptococcal M protein with a KD of less than 50×10−9 M−1, as determined by binding to Streptococcus pyogenes SF370.
8. The antibody according to embodiment 7, wherein said KD is less than 15×10−9 M−1.
9. The antibody according to embodiment 7, wherein said KD is less than 5×10−9 M−1.
10. The antibody according to embodiment 7, wherein said KD is less than 1×10−9 M−1.
11. The antibody according to any of the preceding embodiments, wherein said CDR H3 loop is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
12. The antibody according to any of the preceding embodiments, wherein said CDR L3 loop is selected from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
13. The antibody according to any of the preceding embodiments wherein the H3 chain is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12 and any variant sequence having less than 20 conservative amino acid substitutions relative to SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 or SEQ ID NO:12.
14. The antibody according to any of the preceding embodiments, wherein said L3 chain is selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and any variant sequence having less than 20 conservative amino acid substitutions relative to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 or SEQ ID NO:16.
15. The antibody according to any of embodiments 13-14, wherein said variant sequence has less than 10 conservative amino acid substitutions.
16. The antibody according to any of embodiments 13-14, wherein said variant sequence has less than 5 conservative amino acid substitutions.
17. The antibody according to any of embodiments 13-14, wherein said variant sequence has less than 2 conservative amino acid substitutions.
18. The antibody according to any of the preceding embodiments, wherein said H3 chain is selected from SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.
19. The antibody according to any of the preceding embodiments, wherein said L3 chain is selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16.
20. The antibody according to any of the preceding embodiments, which is selected from the group consisting of
21. The antibody according to embodiment 20, which is selected from the group consisting of
22. The antibody according to any of the preceding embodiments, which is selected from the group consisting of
23. The antibody according to any of the preceding embodiments, which has the ability to mediate bacterial agglutination.
24. The antibody according to any of the preceding embodiments, which has the ability to mediate NFkB-activation.
25. The antibody according to any of the preceding embodiments, which has the ability to induce phagocytosis.
26. The antibody according to any of the preceding embodiments, which has the ability to exhibit simultaneous binding to two different epitopes of the streptococcal M protein by way of dual-Fab cis antibody binding.
27. The antibody according to embodiment 26, wherein said two different epitopes of the streptococcal M protein are a) in the B repeats and C repeats, b) in the linear sequence, or c) in the 3-dimensional structure.
28. A pharmaceutical composition comprising the antibody as defined in any of embodiments 1-27 and at least one pharmaceutically acceptable excipient.
29. The pharmaceutical composition according to embodiment 28, which comprises two or more different antibodies as defined in any of embodiments 1-26.
30. The use of the antibody according to any of embodiments 1-27 for the treatment of a streptococcal infection, such as Group A streptococcus.
31. The use of the antibody according to any of embodiments 1-27 for the treatment of sepsis.
32. The use according to any of embodiments 30-31, wherein said antibody is administered parenterally, such as intravenously, subcutaneously or intramuscularly.
33. The use according to any of embodiments 30-32 which is in combination with intravenous immunoglobulin (IVIG).
34. The use of the pharmaceutical composition as defined in any of embodiments 28-29 for the treatment of sepsis.
35. The use according to any of embodiments 30-34, wherein said antibody is administered parenterally, such as intravenously, subcutaneously or intramuscularly.
36. Use of an antibody as defined in any of embodiments 1-27 in an application selected from Western blot, Flow Cytometry, ELISA and Immunoflourescence.
37. An antibody exhibiting binding to two different epitopes of a molecule by way of dual-Fab cis antibody binding.
38. The antibody according to embodiment 37, wherein said two different epitopes of a molecule is two different epitopes of a protein, such as streptococcal M protein.
39. Method for obtaining an antibody exhibiting simultaneous binding to two different epitopes of a molecule by way of Dual-Fab cis antibody binding, comprising the steps:
40. The method according to embodiment 39, wherein said two different epitopes of a molecule is two different epitopes of a protein, such as streptococcal M protein.
41. Method for crosslinking antibody F(ab′)2-fragments bound to a molecule, comprising the steps:
42. The method according to embodiment 41, wherein said molecule is a protein such as streptococcal M protein.
General Methods/Materials
Single B Cell Purification, Baiting, and Isolation
B cell isolation was performed as described previously (Smith et al. 2009), with some modifications. Briefly, 35 ml of blood was drawn (into citrated collection tubes) from a young woman who had recently recovered from a group A streptococcal infection with no post-infection (autoimmune) sequelae. The blood was treated with 2.5 μl/ml Rosettesep B (Stemcell technologies) for 20 mins at room temperature. The blood was then diluted 1:1 in phosphate buffered saline (PBS) and layered onto Lymphoprep gradients. After centrifugation (30 mins at 800×g), the plasma was collected and frozen while the B cell layer (around 7 ml) was removed, diluted with 43 ml of PBS, and centrifuged again. This washing step was repeated twice. The B cells were counted and kept at room temperature for staining (typical yields are 2-5 million cells per 30-40 ml of blood).
B Cell Staining, Baiting, and Sorting
The B cells were concentrated into a final volume of 500 μl in PBS. The cells were then blocked with 5% BSA for 20 minutes before being stained with antibodies against CD19-PE (BD-555413), CD3-BV510 (BD-564713), and IgG-BV421 (BD-562581). The B cells were also labelled with the Sytox-FITC live/dead stain (Thermofischer-S34860). Baiting of the B cells was done using soluble M1 protein isolated from an MC25 group A streptococcus M1 strain. The M1 protein isolation procedure was previously described elsewhere (Collin and Olsen 2000). The M1 protein was directly conjugated to Alexa Fluor 647 using the microscale labeling kit (Invitrogen). In addition to the antibodies and live/dead stains, 0.1 μg/ml of AF694-M1 was added to the cells and the mixture was incubated at 32° C. for 20 minutes (M1 undergoes a conformational change at 4° C. which could obscure important epitopes (Cedervall et al. 1995)). After the incubation, the cells were washed with PBS twice and were kept on ice until further analysis. The gates for sorting were set on a FACSAriaFusion sorter using unstained cells and FMO-1 samples. A total of 100 cells were sorted from 2.5 million B cells directly into 10 μl of water containing RNase inhibitor in 96-well plates and were immediately transferred to a −80° C. freezer. The cells at this point would have been lysed due to osmotic pressure and the RNA stabilized in solution.
Reverse Transcription, Family Identification, and Cloning
The cells previously frozen in plates were thawed on ice and RT-PCR was performed using the OneStep RT-PCR kit (Qiagen) protocol without modification. The primer sequences used in the PCR steps were taken directly from the Smith et al (2009) paper without any modifications. After the RT-PCR, the nested PCR was performed and the bands corresponding to the variable regions of the heavy and light chains were sequenced to identify the antibody families. Family-specific cloning primers were used to clone the variable chains into the plasmids containing the constant regions of the heavy and light chains. The expression plasmids were generously donated by Dr. Patrick Wilson's group.
General Cell Culture and Transfection
THP1 cells (Leukemic monocytes) were maintained in RPMI media supplemented with L-Glutamine and 10% FBS. The cells were kept at a cell density between 5-10×105 cells per ml. THP1-XBlue cells were maintained like regular THP1 cells. HEK293 cells were maintained in DMEM supplemented with L-Glutamine and 10% FBS. The cells were never allowed to grow to 100% confluency. The day before transfection, 8×106 cells were plated in circular 150 mm dishes. This transfection format allowed for the most efficient antibody recovery.
Transfection, Expression, and Purification
In total, 10 antibody construct pairs were successfully generated from 100 starting cells. The Antibody pairs were transformed into Mix'n'go E. coli. Transformant colonies were verified by sequencing and the plasmids were further propagated and DNA was extracted using a Zymoresearch midiprep kit. Plasmid pairs encoding full mature antibodies were co-transfected into HEK293 cells using the PEI transfection method (https://www.addgene.org/protocols/transfection/). Cells were briefly treated with 25 μM Chloroquine for 5 hours. Thereafter, 20 μg of heavy and light chain expression plasmid DNA were diluted in OptiMEM (Life technologies) media containing polyetheleneimine (PEI) at a 1:3 ratio (for 50 μg of DNA, 114 μl of a 1 mg/ml PEI stock was used). The cells were incubated at 37° C. for 18 hours before they were washed 2 times with PBS and the DMEM media was exchanged with OptiMEM. The cells were incubated for a further 72 hours before the supernatants were collected. The antibodies in the supernatants were purified using Protein G beads in a column setup. The antibodies were then titrated by comparing their concentrations on an SDS-PAGE to serial dilutions of a known concentration of Xolair (commercially bought Omalizumab, stored at 150 mg/ml).
Bacterial Strains, Growth, and Transformation
Streptococcus pyogenes strain SF370 (emm1 serotype) and AP1 (emm1 serotype) was grown in Todd-Hewitt Yeast media (THY) at 37° C. The bacteria were maintained on agar plates for 3 weeks before being discarded. We chose to use SF370 in all of our experiments since it is an M1 serotype strain lacking protein H which is a complicating factor (due to its strong Fc binding capacity and extensive homology with M protein (Akesson et al. 1990)). For experiments, overnight cultures were prepared in THY and were diluted 1:20 on the day of the experiments. After dilution, three hours of growth at 37° C. ensured that the bacteria were in mid-log growth. For the generation of GFP-expressing strains, the SF370 and its ΔM isogenic counterpart were grown to mid-log before being washed with ice-cold water. The electrocompetent bacteria were electroporated with 20 μg of the pGFP1 plasmid and plated on Erythromycin supplemented THY plates. The successful transformants were fluorescent when examined under ultraviolet light. Heat killing the bacteria was done by growing the cultures to mid log, washing them once in PBS and incubating them on ice for 5 minutes. The bacteria were then heat shocked at 80° C. for 5 minutes before being placed on ice for 15 minutes. For the phagocytosis assay, the heat killed bacteria were centrifuged at 8000×g for 3 minutes and resuspended in Na-medium (5.6 mM glucose, 127 mM NaCl, 10.8 mM KCl, 2.4 mM KH2PO4, 1.6 mM MgSO4, 10 mM HEPES, 1.8 mM CaCl2; pH adjusted to 7.3 with NaOH). Heat-killed bacteria were stained with 5 μM Oregon Green 488-X succinimidyl ester (Thermofischer) at 37° C. under gentle rotation and protected from light for 30 min. The bacteria were then centrifuged and resuspended in Sodium carbonate buffer (0.1 M, pH 9.0) for an additional staining step with the pH-sensitive dye CypHer5E (Fischer scientific). This was used at a concentration of 7 μg/ml in a volume of 1.5 ml for 2 h at room temperature under gentle rotation, protected from light. The samples were washed once with Na-medium to remove excess dye and stored at 4° C. for later use.
Antibody Screening and Flow Cytometry
For ELISAs: ELISA plates were coated overnight with human fibrinogen (25 μg/ml in 100 μl of PBS) at 4° C. The following day, the coating buffer was washed with PBST and the plates were coated with 10 μg/ml M1, which had been purified from MC25 culture supernatants (Collin and Olsen 2000). After a 1-hour incubation at 37° C., the wells were washed 3 times with PBST and blocked with 2% BSA in 300 μl PBST for 30 minutes. After blocking, 300 μl of antibody containing supernatants were added to the wells, or diluted donor plasma as a control. The samples were incubated for 1 hour at 37° C., washed, and a solution of Protein G-HRP (diluted 1:3000) was added to the wells and incubated at 37° C. for 1 hour. The samples were then washed and developed with 100 μl developing reagent (20 ml Substrate buffer NaCitrate pH 4.5+1 ml ABTS Peroxide substrate+0.4 ml H2O2). Absorbance was read at OD450 following 5-30 minutes of color development at room temperature.
For ELISA using the shorter M1 B1B2B3 and C1C2C3 constructs, open reading frames encoding for the B1B2B3 repeats of the M1 protein (UniProt ID: Q99XV0, emm1; amino acids 132-194) and the C1C2C3 repeats (amino acids 229-348) were cloned at the Lund Protein Production Platform (LP3) (Lund, Sweden). The encoding sequences were ordered as a synthetic construct from Genscript (NJ, USA), and cloned into a pNIC28-Bsa4-based vector incorporating a tandem affinity purification tag (histidine-hemagglutinin-StrepII-tobacco etch virus protease recognition site) at the C-terminus of the construct. The constructs were expressed in Luria-Bertani Broth (Difco) supplemented with 50 μg/ml of kanamycin at 25° C. in E. coli TUNER (DE3) cells. For protein expression the temperature was lowered to 18° C. and the expression induced with 0.1 mM IPTG at OD600 0.6. Expressed cells were harvested and resuspended in phosphate buffer (50 mM NaPO4, 300 mM NaCl, 20 mM imidazole, pH 8.0) supplemented with EDTA-free Complete Protease Inhibitor tablets (Roche). The cells were lysed using a French pressure cell at 18,000 psi. The lysate was cleared via ultracentrifugation (Ti 50.2 rotor, 244,000×g, 60 min, 4° C.) and subsequently passed through a 0.45 μm filter prior to loading on a HisTrap HP column (GE Healthcare). The column was washed with 20 column volumes (CVs) of phosphate buffer, and bound protein was eluted using a gradient of 0-500 mM imidazole in phosphate buffer. Fractions containing the desired protein were pooled, and dialyzed against 1× phosphate buffer saline (PBS; 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl) pH 7.3, and stored at −80° C. These constructs were subsequently used to coat the ELISA wells or as competition for the M antibodies.
For flow cytometric screening: Overnights of SF370-GFP bacteria or its ΔM counterpart were diluted 1:20 into THY and grown until mid-log. 100 μl of the bacteria were distributed into wells of a 96 well plate. Antibodies purified from cell culture supernatants were diluted to 5 μg/ml and were digested with 1 μg/ml of IdeS for 3 hours at 37° C. The digested antibodies were further diluted 1:10 into the bacterial suspension. Reaching a final concentration of 0.5 μg/ml. The bacteria were incubated for 30 minutes at 37° C. before being washed twice with PBS. AF647-conjugated Fab α-Fab antibody fragments were used as secondary antibodies to detect binding of the primary α-M antibodies. After a 30-minute incubation with the Fab α-Fab fragments, the bacteria were washed and analyzed on a Cytoflex flow cytometer (Beckman coulter). The gates for the GFP-expressing bacteria were set using the SF370 parent strain (not expressing the GFP plasmid). GFP-expressing bacteria within the GFP-expressing gate were assessed for antibody staining (APC channel). Antibody staining reflects the presence of surface-bound primary-secondary antibody complexes and is indicative of bound anti-M antibodies.
For western blotting: Antibody reactivity to linear epitopes was assessed by probing the lysates of SF370 and its ΔM mutant using western blotting. Briefly, pellets of logarithmically grown bacteria were incubated with phospholipase C for 30 minutes in PBS until the lysates became clear. The lysates were sonicated and cleared by centrifugation (15,000×g for 3 minutes). We loaded 40 μg of 5 replicate sets of SF370 vs ΔM mutant protein on a gradient SDS-PAGE gel (4-20%). The gel electrophoresis was run for 60 minutes to achieve protein separation. The proteins were transferred from the gel to a PVDF membrane which was blocked for 45 minutes with 5% skimmed milk in PBST. The replicate lanes of the membrane were then cut and probed with 2 or 10 μg/ml of Xolair, Ab25, 32, 49 or IVIgG overnight at 4° C. The membranes were washed 3 times with PBST and probed with the secondary HRP-conjugated goat anti-human IgG secondary (Rockland) antibody for 1 hour at room temperature. The secondary was later washed, and the membrane developed using a chemilumunescence reagent (West-Femto substrate, Thermofischer).
Agglutination Assays
For Agglutination Assays:
Overnight cultures of SF370 and its ΔM strain were diluted 1:5 in RPMI and were treated with 100 μg/ml of the anti-M antibodies, or with 5% donor plasma. It is crucial for this series of experiments that the bacteria are incubated in a cuvette and are not shaken or vortexed during incubation. At indicated time points, the OD600 of the bacteria was measured and at the 3.5 hour mark the cuvettes were photographed.
For Aggregate Dissolution Experiments
SF370 bacteria were grown overnight, diluted 1:20 in THY and left to grow for two hours. The bacteria were then supplemented with 100 μg/ml of the appropriate antibody. Two hours after inoculation, the bacteria were vortexed, imaged (randomly) and the aggregate areas were analyzed using Image J.
SIM Imaging
Logarithmic phase bacteria were sonicated (VialTweeter; Hielscher) for 0.5 minutes to separate any aggregates and incubated fixed in 4% paraformaldehyde for 5 minutes on ice. The bacteria were thereafter washed with PBS twice (10,000×g for 3 min). SF370 was stained with Alexa Fluor 647-conjugated wheat germ agglutinin (WGA). Bacteria were incubated with IdeS-cleaved Xolair, Ab25, Ab32, and Ab49 and stained with fluorescently labelled IgGFab or IgGFc specific F(ab′)2 fragments (DyLight488—conjugated anti-human IgGFc or IgGFab; Jackson ImmunoResearch Laboratory). Samples were mounted on glass slides using Prolong Gold Antifade Mountant with No. 1.5 coverslips. Images of single bacteria were acquired using an N-SIM microscope with LU-NV laser unit, CFI SR HP Apochromat TIRF 100× Oil objective (N.A. 1.49) and an additional 1.5× magnification. The camera used was ORCA-Flash 4.0 sCMOS camera (Hamamatsu Photonics K.K.) and the images were reconstructed with Nikon's SIM software on NIS-Elements Ar (NIS-A 6D and N-SIM Analysis). Images of the bacteria were acquired with 488 and 640 nm lasers. For site localization, single bacteria were manually identified and imaged in time series with 50 frames. The analysis pipeline for site localization was implemented in Julia and is available on GitHub (Kumra Ahnlide et al manuscript 2020). A cut off of initial signal-to-noise ratio (SNR) was set to 0.3 and timeframes included were the ones with at least 70% of the initial SNR.
Binding Curves
SF370 bacteria were grown to mid log, washed and 10 ml of culture were concentrated into 1000 μl of PBS. The bacteria were stained with halving serial dilutions of the anti-M antibodies. 30 μl of bacteria were used per every 100 μl of IdeS treated antibody. The staining was performed at 4° C. for 30 minutes (with shaking) before the bacteria were washed and stained with an excess of AF647-conjugated Fab anti-Fab fragments in a volume of 30 μl for 30 minutes at 4° C. with shaking. The bacteria were then diluted to 250 μl in PBS and analyzed by flow cytometry. Theoretical fit was done in MATLAB using fminsearch for an ideal binding curve with the dissociation constant as an unknown variable.
Crosslinking of Antibody F(Ab′)2 Fragments to the M1 Protein
For the crosslinking of Ab25, Ab32 and Ab49 F(ab′)2 fragments to the M1 protein, we used two different preparations of the M1 protein; one expressed and purified as recombinant in E. coli as described for the B1B2B3 and C1C2C3 constructs above, and one purified from the culture supernatant of the S. pyogenes MC25 strain (Collin and Olsén 2000). The antibody F(ab′)2 fragments were cleaved and purified from the expressed intact antibodies using the FraglT-kit with Fc-capture columns (Genovis) according to the manufacturer's instructions. For crosslinking, 25 μg of the recombinant M1 protein or 8 μg of the MC25 M1 protein were incubated with 5 μg of the respective F(ab′)2 fragments in 1×PBS pH 7.4 at 37° C., 800 rpm, 30 min. Heavy/light disuccinimidylsuberate (DSS; DSS-H12/D12, Creative Molecules Inc.) resuspended in dimethylformamide (DMF) was added to final concentrations 250 and 500 μM and incubated for a further of 60 min at 37° C., 800 rpm. The crosslinking reaction was quenched with a final concentration of 50 mM ammonium bicarbonate at 37° C., 800 rpm, 15 min.
Sample Preparation for MS
The crosslinked samples mixed with 8 M urea and 100 mM ammonium bicarbonate, and the cysteine bonds were reduced with 5 mM TCEP (37° C. for 2 h, 800 rpm) and alkylated with 10 mM iodoacetamide (22° C. for 30 min, in the dark). The proteins were first digested with 1 μg of sequencing grade lysyl endopeptidase (Wako Chemicals) (37° C., 800 rpm, 2 h). The samples were diluted with 100 mM ammonium bicarbonate to a final urea concentration of 1.5 M, and 1 μg sequencing grade trypsin (Promega) was added for further protein digestion (37° C., 800 rpm, 18 h). Samples were acidified (to a final pH 3.0) with 10% formic acid, and the peptides purified with C18 reverse phase spin columns according to the manufacturer's instructions (Macrospin columns, Harvard Apparatus). Peptides were dried in a speedvac and reconstituted in 2% acetonitrile, 0.2% formic acid prior to mass spectrometric analyses.
Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)
All peptide analyses were performed on Q Exactive HF-X mass spectrometer (Thermo Scientific) connected to an EASY-nLC 1200 ultra-high-performance liquid chromatography system (Thermo Scientific). Peptides were loaded onto an Acclaim PepMap 100 (75 μm×2 cm) C18 (3 μm, 100 Å) pre-column and separated on an EASY-Spray column (Thermo Scientific; ID 75 μm×50 cm, column temperature 45° C.) operated at a constant pressure of 800 bar. A linear gradient from 4 to 45% of 80% acetonitrile in aqueous 0.1% formic acid was run for 65 min at a flow rate of 350 nl min-1. One full MS scan (resolution 60000 @ 200 m/z; mass range 390-1 210 m/z) was followed by MS/MS scans (resolution 15000 @ 200 m/z) of the 15 most abundant ion signals. The precursor ions were isolated with 2 m/z isolation width and fragmented using HCD at a normalized collision energy of 30. Charge state screening was enabled, and precursors with an unknown charge state and a charge state of 1 were rejected. The dynamic exclusion window was set to 10 s. The automatic gain control was set to 3e6 and 1e5 for MS and MS/MS with ion accumulation times of 110 ms and 60 ms, respectively. The intensity threshold for precursor ion selection was set to 1.7e4.
Computational Modeling
Several protocols of Rosetta software suit (Koehler Leman et al. 2019) were employed for macromolecular modeling of this study. To model the full-length antibodies, first the antigen-binding domains were characterized using Rosetta antibody protocol (Weitzner et al. 2017). Then, comparative models have been generated for both heavy and light chains using RosettaCM protocol (Song et al. 2013) and aligned on the antigen-binding domains to represent the initial structure of the antibody. HSYMDOCK (Yan et al. 2018), and DaReUS_loop (Karami et al. 2019) web servers were used for symmetric docking of the Fc-domains and to model the hinge regions, respectively. Finally, 4K models were produced for each antibody as the final refinement and the top-scored models were selected based on XLs derived from mass spectrometry combined with rosetta energy scores. Moreover, to characterize the M1 antibody interactions, TX-MS protocol were used (Hauri et al. 2019), through which 2K docking models were generated and filtered out using distance constraints from DDA data. A final round of high-resolution modeling was performed on top models to repack the sidechains using RosettaDock protocol (Gray 2006).
Fluorescent Xolair Competition Experiments
Logarithmically grown SF370 bacteria were heat killed and labelled with Oregon Green (as described previously). The bacteria were mixed with antibodies or plasma/IVIG and incubated for 30 minutes at 37° C. while shaking. Fluorescently conjugated Xolair (conjugated to Alexafluor 647 using the protein labeling kit (Invitrogen) according to the manufacturer's instructions) was then added to the bacteria at a concentration of 100 μg/ml for an additional 30 minutes before being directly analyzed by flow cytometry. For experiments in which Fabs were used, the Fabs were generated using the Fabalactica digestion kit (Genovis) according to manufacturer's instructions.
Phagocytosis Assay
The phagocytosis experiments were performed using persistent association normalization (de Neergaard et al. 2019). Prior to opsonization, the CypHer5E- and Oregon Green-stained SF370 bacteria were sonicated for up to 5 min (VialTweeter; Hielscher) to disperse any large aggregates of bacteria. Sonication was deemed sufficient when clump dispersal was confirmed by microscopy. Staining as well as bacterial count (events/μl in the FITC+ve gate) was assessed by flow cytometry (CytoFLEX, Beckman-Coulter). The pH responsiveness of CypHer5E was tested by measuring the bacterial fluorescent staining in the APC channel before and after the addition of 1 μl of sodium acetate (3 M, pH 5.0) to 100 μl of bacterial suspension. The presence of an acid induced shift in fluorescence indicated successful staining. On the day of experiments, the appropriate number of bacteria were opsonized to suit each experiment. The opsonization with our M-specific antibodies, Xolair or with IVIG was performed at 37° C. for 30 minutes. For experiments with a variable MOP, serial dilutions of the opsonized bacteria were made and used to incubate with the THP-1 cells. By gating on the leukocyte population (Supp.
Flow cytometric acquisition was performed using a CytoFLEX (Beckman-Coulter) with 488 nm and 638 nm lasers and filters 525/40 FITC and 660/10 APC. Threshold was set at FSC-H 70,000 for phagocytosis and for bacteria FSC-H 2000 and SSC-H 2000. Gain was set to 3 for FITC and 265 for APC. Acquisition was set to capture at least 5 000 events of the target population with a velocity of 30 μl/min taking approximately 30 min to assess all samples. Throughout the data acquisition the 96-well plate was kept on an ice-cold insert to inhibit further phagocytosis.
NF-κB Activity Luciferase Assay
THP-XBlue-CD14 (Invivogen) cells were seeded at a density of 200,000 cells per well in 96 well plates. The cells were treated with the appropriate antibodies (at 0.5 μg/ml) with or without M1 protein (2 μg/ml) for 18 hours at 37° C. After the incubation, 20 μl of the cell supernatant were aspirated and mixed with the developing reagent, as described by the assay instructions (QuantiBlue solution, Invivogen). The samples were incubated at 37° C. until development was appropriate and the OD650 measurement of the samples was done using a multi-well spectrophotometer.
Animal Model
All animal use and procedures were approved by the local Malmó/Lund Institutional Animal Care and Use Committee, ethical permit number 03681-2019. Nine-week-old female C57BL/6J mice (Scanbur/Charles River Laboratories) were used. Monoclonal antibody Ab25 (0.4 mg/mouse), or intravenous immunoglobulin (10 mg/mouse) was administered intraperitoneally 6 h pre-infection. S. pyogenes AP1 was grown to logarithmic phase in Todd-Hewitt broth (37° C., 5% C02). Bacteria were washed and resuspended in sterile PBS. 106 CFU of bacteria were injected subcutaneously into the scruff leading to systemic infection within 24 h. Mice were sacrificed 24 h post infection, and organs (blood, livers, spleens, and kidneys) were harvested to determine the degree of bacterial dissemination. The blood cell counts were analyzed by flow cytometry. Cytokines were quantified using a cytometric bead assay (CBA mouse inflammation kit, BD) according to manufacturer instructions.
To understand what constitutes a protective antibody towards GAS infection, we wanted to generate functional human antibodies and analyze their effects on virulence. We choose M protein as a target antigen, with a donor that had successfully cleared a streptococcal infection as a source of M protein-specific antibodies. To identify human antibodies with specificity towards streptococcal M protein, we isolated M-reactive B cells by baiting donor B cells with fluorescently conjugated M protein. We sorted live single CD19+CD3− IgG+ M+ B cells (
To characterize the identified anti-M1 antibodies, we performed a panel of biochemical and immunological assays. We used structured illumination microscopy (SIM) immunofluorescence (IF) to visualize the anti-M binding pattern on the surface of SF370. Interestingly, M protein shows a similar punctate distribution along the surface of the organism with all the monoclonal antibodies, including the Fc-mediated Xolair binding (
Antibody-mediated bacterial agglutination is a well-documented antibody function and has important biological significance such as enchaining bacteria for effective immune clearance (Moor et al. 2017; Mitsi et al. 2017). Another well-known interbacterial, GASs-pecific phenomenon is the formation of M-dependent bacterial aggregates at the bottom of the growth tube (Frick et al. 2000). While it is not possible to grow GAS without having any self-aggregation, the antibodies greatly enhanced bacterial agglutination. Both the triple antibody cocktail as well as individual antibodies led to dose-dependent agglutination, as is also the case with donor serum from the patient from which the M-reactive B cells were obtained (
Phagocytosis is a receptor-mediated process where prey are internalized into phagosomes, followed by their maturation into acidic, hostile compartments. To investigate the ability of the antibodies to trigger phagocytosis, we used persistent association-based normalization (de Neergaard et al. 2019) to study both the antibodies' ability to increase phagocyte association as well as internalization. We incubated phagocytic THP-1 cells with pH-sensitive CypHer5-stained bacteria (
The induction of phagocytosis and NF-κB, as seen with Ab25, are important indicators of immune function. To test the potential protective effects of Ab25 in vivo, we used a mouse model of subcutaneous infection with GAS. The mice were pretreated with intraperitoneal injections of Ab25 or IVIG. High-dose IVIG have been used in mice models of severe GAS infections (Sriskandan et al. 2006) and served as a positive control. Treatment with IVIG or Ab25 reduced the bacterial burden in the spleen, kidney, and liver when compared to untreated controls, with Ab25 exhibiting better protection than IVIG (
Antibodies that bind via their Fabs with high affinity are typically expected to promote an immune response. However, of the tested anti-M antibodies, only Ab25 can promote all tested immune effector functions. To assess structural differences between the antibodies, we present Rosetta-generated molecular models of the Fab fragments of Ab25, Ab32, and Ab49 in which we highlight the complementarity-determining region (CDR) domains (
To determine the epitopes recognized by the respective F(ab′)2-fragments on the M1 protein, we performed targeted in solution cross-linking coupled to mass spectrometry (TX-MS) (Hauri et al. 2019). TX-MS can model accurate quaternary conformations of large protein complexes and support these conformations via identifying several independent cross-links based on many high-accuracy cross-linked peptide fragments. Of the three antibodies analyzed, we identified ten cross-linked peptides between Ab25 and M1-protein. These cross-links are found between the F(ab′)2 and two different regions on the M-protein, indicating that Ab25 has two different binding-sites in the B-S-C domain region (
To validate the observed binding sites through an orthogonal approach, we used site-localization microscopy, where the relative distance between fluorescently labeled cell wall and antibody binding sites is determined by repeated measures of multiple individual bacteria (
Dual-Fab antibody binding, in cis-mode, of two identical Fabs to two different epitopes on a single protein is a novel, previously not observed, mode of antibody interaction. Combined with the fact that dual-Fab cis binding is connected with a clear gain in immunological protective function prompted us to verify this finding and elucidate the particular nature of Ab25s dual-Fab binding capacity. First, we investigated the ability of single Ab25 Fabs to obstruct Fc binding. If either binding site could sustain a steric hindrance of Fc binding, we should see a reduction. However, Fc binding was not affected by single Fabs (
| Number | Date | Country | Kind |
|---|---|---|---|
| 20196885.6 | Sep 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/075789 | 9/20/2021 | WO |
