Patent Application
20210047428
The present application claims priority from Australian Provisional Patent Application No 2018901070 filed on 29 Mar. 2018, the content of which is incorporated herein by reference.
The present disclosure relates to methods of generating antibodies with improved specificity and/or affinity for a target antigen, as well as B-cells and hybridomas expressing the antibodies and compositions comprising antibodies with improved specificity and/or affinity for the target antigen.
In some instances, antibodies can distinguish nearly identical foreign and self-antigens, such as the glycolipids on the cell wall of Campylobacter jejuni and those on human nerve cells, with less than 0.1% of infected people producing cross-reactive antibodies that result in paralysis and Guillain-Barré Syndrome. However, apparent limits to antibody self-foreign discrimination are exploited by Human Immunodeficiency Virus (HIV), Lymphocytic choriomeningitis virus and Lassa fever viruses. They establish persistent infections and evade antibodies by mimicking self and cloaking their foreign envelope proteins with self-glycans.
Furthermore, functionally important vaccine targets are often highly disordered, or surrounded by flexible loops making effective affinity maturation to complex antigens or conformationally-flexible antigens even more challenging.
Immunization of mice or rats with a “non-self” protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Tolerance, the ability of the immune system to prevent responses to self-antigens, makes it difficult to generate a strong immune response in mice with a mouse self-antigen or highly conserved human antigen. Specific knockout mice are often used to overcome the immune tolerance associated with self-antigens.
Accordingly, there remains a need for methods of generating antibodies to self-antigens, highly conserved human antigens and/or conformationally-flexible antigens, and/or methods of generating antibodies with improved specificity and/or affinity for a target antigen.
The method described herein can be used to produce antibodies with desirable specificity and affinity to a target antigen in an animal by introducing the target antigen into an animal expressing a structurally related antigen.
Accordingly, in one aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a target antigen, the method comprising:
whereby the B-cells isolated in iii) express antibodies with improved specificity and/or affinity for the target antigen compared to antibodies expressed by the B-cells prior to affinity maturation.
Accordingly, in another aspect there is provided a method for generating an antibody having improved specificity and/or affinity for a conformationally-flexible target antigen, the method comprising:
whereby the B-cells isolated in iii) express antibodies with improved specificity and/or affinity for the target antigen compared to antibodies expressed by the B-cells prior to affinity maturation.
Accordingly, in another aspect there is provided a method for generating an antibody having improved specificity and/or affinity for a target antigen, the method comprising:
whereby the B-cells isolated in iv) express antibodies with improved specificity and/or affinity for the target antigen compared to antibodies expressed by the B-cells prior to affinity maturation.
Accordingly in another aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a conformationally-flexible target antigen, the method comprising:
whereby the B-cells isolated in iv) express antibodies with improved specificity and/or affinity for the target antigen compared to antibodies expressed by the B-cells prior to affinity maturation.
In one embodiment, the B-cells have been genetically modified to encode the antibody with the basic specificity and/or affinity for the target antigen.
In one embodiment, introducing the B-cell into the animal comprises irradiating the animal and transplanting the B-cells into the animal.
In one embodiment, the basic affinity of the antibody for the target antigen is lower affinity relative to a desired affinity.
In yet another embodiment, the lower affinity for the target antigen is a KD of about 10−6 M to about 10−8 M.
In another embodiment, the improved affinity is an increase in affinity for the target antigen of at least 100-fold.
In yet another embodiment, the improved affinity is an increase in affinity for the target antigen of at least 1000-fold.
In one particular embodiment, the improved specificity for the target antigen is an at least 10-fold increase in specificity.
In one embodiment, the improved specificity for the target antigen is an at least 100-fold increase in specificity.
In yet another embodiment, the improved specificity for the target antigen is an at least 1000-fold increase in specificity.
In one embodiment, the target antigen is a peptide or polypeptide antigen.
In one embodiment, the variant antigen comprises at least one variant amino acid compared to the target antigen.
In another embodiment, the variant antigen comprises at least one variant amino acid residue in an epitope to which the antibody binds.
In yet another embodiment, the variant antigen comprises at least one variant amino acid residue in a surface that contacts the antibody heavy chain.
In another embodiment, the variant antigen is encoded by a transgene in the animal.
In one particular embodiment, the transgene comprises a ubiquitin promoter for expression of the variant antigen.
In yet another embodiment, the target antigen is a carbohydrate antigen or hapten.
In one embodiment, the carbohydrate antigen is selected from a glycoprotein, glycolipid, polysaccharide and glycoconjugate antigen.
In another embodiment, the carbohydrate antigen is a cancer cell antigen.
In one particular embodiment, the target antigen is a cancer neo-antigen.
In another embodiment, the carbohydrate antigen is a bacterial or viral carbohydrate antigen.
In one embodiment of the method described herein, the animal is a mouse or rat.
In one aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a target antigen, the method comprising:
In another aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a conformationally-flexible target antigen, the method comprising:
In yet another aspect, there is provided a method for providing an antibody having improved specificity and/or affinity for a target antigen, the method comprising:
In yet another aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a conformationally-flexible target antigen, the method comprising:
In yet another aspect, there is provided a method for generating an antibody having improved specificity and/or affinity for a target antigen, the method comprising:
In yet another aspect, there is provided a method for providing an antibody having improved specificity and/or affinity for a conformationally-flexible target antigen, the method comprising:
In one embodiment, the target antigen is a conformationally-flexible antigen. In another embodiment, the conformationally-flexible antigen is selected from the group consisting of: HIV envelope protein, circumsporozoite protein (CSP), merozoite surface protein 2 (MSP2) and GAD65.
In another aspect, there is provided a B-cell isolated according to the method described herein.
In a further aspect, there is provided an isolated antibody expressed by the isolated B-cell as described herein.
In yet another aspect, there is provided a hybridoma that is derived from the isolated B-cell described herein.
In another aspect, there is provided an isolated monoclonal antibody obtained from the hybridoma as described herein.
In another aspect, there is provided a composition comprising the isolated antibody as described herein.
In one embodiment, the composition is a therapeutic composition comprising a pharmaceutically acceptable carrier.
In another aspect, there is provided a transgenic animal comprising B-cells encoding an antibody for a target antigen, and wherein the animal has been genetically modified to express a variant antigen that is structurally related to the target antigen.
In one embodiment, the transgenic animal is a transgenic mouse.
In one specific embodiment, the B-cells in the transgenic mouse have been genetically modified to encode the antibody for the target antigen.
Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein.
Data representative of 2 independent experiments per timepoint with 2-3 mice per group.
Key to the Sequence Listing
SEQ ID NO: 1 Amino acid sequence for wild-type HEL protein.
SEQ ID NO: 2 Amino acid sequence for a mutant flexible HEL protein.
SEQ ID NO: 3 Amino acid sequence for a mutant rigid HEL protein.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in immunology, immunohistochemistry, protein chemistry, biochemistry and chemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edn, Cold Spring Harbour Laboratory Press (2001), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The description and definitions of immunoglobulins, antibodies and fragments thereof herein may be further understood by the discussion in Kabat, 1987 and/or 1991, Bork et al., 1994 and/or Chothia and Lesk, 1987 and/or 1989 or Al-Lazikani et al., 1997.
Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the disclosure, as described herein.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
As used herein, the singular forms of “a”, “and” and “the” include plural forms of these words, unless the context clearly dictates otherwise.
The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.
As used herein, the term “target” shall be understood to refer to an antigen against which it is desired to produce an antibody. A target antigen in one embodiment is a foreign antigen. As used herein, the term “antigen” shall be understood to mean any composition of matter against which an immunoglobulin response (e.g., an antibody response) could potentially be raised. Exemplary antigens include proteins, peptides, polypeptides, carbohydrates, phosphate groups, phosphor-peptides or polypeptides, glyscosylated peptides or peptides, etc.
Functionally important vaccine targets are often highly disordered or surrounded by flexible loops. In one embodiment, the target antigen may be a conformationally-flexible antigen. Suitable examples of conformationally-flexible antigens include, but are not limited to, HIV envelope protein, circumsporozoite protein (CSP), merozoite surface protein 2(MSP2) and GAD65. In one example, the Root Mean Square Deviation of the target antigen is at least 0.5, 1, 2, 3, 4, 5 Angstroms. There are techniques known in the art to generate a conformationally-flexible antigen. For example, an antigen may be made conformationally-flexible by mutation or by denaturation such as by varying temperature or pH or by the addition of adjuvants.
As used herein, the term “affinity” shall be understood to refer to the strength of interaction between the binding sites of the antibodies disclosed herein with the antigen. In one example described herein, strength of interaction may be determined by measuring the strength of binding reaction through affinity constants which are well known in the art. In one example, strength of binding between the immunoglobulin homodimers described herein and the antigen may be determined by measuring the equilibrium binding constant (KD). The method described herein provides that affinity may be measured using any suitable techniques, including any suitable optical analytical techniques for measuring biomolecular interactions known in the art. In one example, optical analytical techniques may include: Bio-Layer Interferometry. In another example, the optical analytical technique may include: dual polarisation interferometry, static light scattering, dynamic light scattering, surface plasmon resonance, fluorescence polarisation/anisotropy, fluorescence correlation spectroscopy or nuclear magnetic resonance.
The term “basic affinity” as used herein refers to the affinity of an antibody for a target antigen prior to performing the method as described herein. As a result of performing the method described herein, the antibody will have an improved affinity for the target antigen. As understood in the art, KD and affinity are inversely related. The KD value relates to the concentration of antibody (for example, the amount of antibody needed for a particular experiment) and so the lower the KD value (lower concentration), the higher the affinity of the antibody. Thus, an improvement or increase in affinity relates to a lower numerical KD figure. For example, a decrease in KD from 1×10−6 M to 1×10−7 M relates to an improvement or increase in affinity.
Accordingly, the basic affinity of the antibody prior to performing the method described herein is lower than a desired range for an intended purpose. For example, desirable antibody affinities for therapeutic antibodies may be represented by a KD in the range of low nanomolar (nM) to picomolar (pM), and to the femtomolar (fM) range. Accordingly, if a desirable antibody affinity is in the low nM range, then a basic affinity will be less than this, and may be, for example, in the μM to high nM range. Thus, in one embodiment, the basic affinity of the antibody to the target antigen is low affinity relative to a desired affinity. In one embodiment, low affinity relative to a desired affinity may be at least 10-fold, at least 100-fold, at least 1000 fold, or at least 10,000-fold lower affinity than the desired affinity. In one embodiment, the basic affinity for the target antigen is a KD of about 10−6 M to about 10−8 M. Thus, as used herein the term “improved affinity” refers to the affinity of the antibody for the target antigen after performing the method as described herein. In certain embodiments, the improved affinity will be at least 10-fold, at least 100-fold, at least 1000-fold or at least 10,000-fold or more improvement over the basic affinity for the target antigen.
As used herein, the term “variant antigen that is structurally related to the target antigen” refers to an antigen that has a closely related structure to the target antigen, such that prior to performing the method as described herein, the antibody on the B-cell cross-reacts with both the target antigen and the variant antigen. In one embodiment, the variant antigen is a self-antigen. For example, in one embodiment, the antibody has a basic specificity (i.e. the specificity of the antibody prior to performing the method as described herein) that is represented by a less than 10-fold difference in binding affinity for the target antigen compared to the binding affinity for the variant antigen. In another embodiment, the basic specificity may be a binding affinity that is about 10−6 to about 10−7 for both the target antigen and the variant antigen.
For example, for a peptide or polypeptide antigen, the variant antigen that is structurally related to the target antigen may comprise one or more amino acid residue variants compared to the target antigen that result in a change in, for example, the primary, secondary or tertiary structure of the molecule. In certain embodiments, the variant residue may be in a linear epitope, a surface that contacts the antibody, or a conformational epitope, or in any other region of a peptide or polypeptide that affects the structure of the molecule. For carbohydrate antigens, there are techniques known in the art to structurally modify carbohydrate antigens. In the context of transgenic animals, modification of carbohydrate antigens may be achieved through the modification or deletion of, for example, enzymes in carbohydrate biosynthesis pathways.
Antibody specificity is the degree to which an antibody differentiates between two antigens. A simple approach measures the relative binding affinities of an antibody to the target antigen and one or more variant antigens. Discrimination depends on the range of variants to which the antibody binds, on the binding affinity, and on the stringency of the conditions under which the assay is conducted. Such binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry using transfected cells expressing the antigen.
As used herein, the term “basic specificity” refers to the specificity of the antibody for binding the target antigen and variant antigen prior to performing the method as described herein. Thus, the term “basic specificity” refers to an antibody that is cross-reactive with both the target antigen and variant antigen.
As used herein, the term “improved specificity” refers to the specificity of the antibody after performing the method described herein. Accordingly, the isolated B-cells expressing antibody with improved specificity will typically exhibit increased affinity for the target antigen and a decreased affinity to the variant antigen as compared to the basic specificity of the antibody. In one embodiment, improved specificity for the target antigen is an increase in the ratio of the affinity for the target antigen to the affinity for the variant antigen by at least 10-fold, or at least 100-fold, or at least 1000-fold, or at least 10,000-fold.
All mice used in the experiments were bred at Australian BioResources and held at the Garvan Institute of Medical Research in specific pathogen-free environments. The Garvan Animal Ethics Committee approved all mice protocols and procedures. C57BL/6 (non-transgenic) mice were purchased from the Australian BioResources (Moss Vale, New South Wales). HyHEL10-transgenic (SWHEL) mice have been described previously (Phan et al., 2003 J. Exp. Med. 197, 845-860 (2003)). These mice carry a single copy VH10 anti-HEL heavy chain variable region coding exon targeted to the endogenous Ighb allele plus multiple copies VH10-κ anti-HEL light chain transgene. SWHEL mice on a CD45.1 congenic (Ptprca/a) C57BL/6 background were also homozygous Rag1−/−, which prevented endogenous Ig variable region gene rearrangements so that all B-cells expressed the HyHEL10 B-cell receptor (BCR).
HEL3X is an R21Q, R73E, and D101R triply mutated HEL protein. HEL3X binds to HyHEL10 with relatively low affinity of Ka=1.19×1107 M−1. Transgenic mice on the C57BL/6 background expressed HEL3X as an integral cell surface protein, by addition at the C-terminus of the transmembrane segment and cytoplasmic tail of the H2Kb class I major histocompatibility protein. The transgene was controlled by the human ubiquitin C (UBC) promoter, resulting in membrane bound HEL3X expression on the surface of all nucleated and anucleate cells. HEL3X membrane expression was confirmed using flow cytometric binding to HyHEL9 on RBC and WBC.
HELR21Q,R73E,C76S,C94S (HEL2X-flex) is mutant version of HEL, designed with four mutations compared to mature wild-type HEL. WT HEL amino acid residues R21 and R73 both have positively charged side chains and are contained within the binding epitope of HyHEL10. The R21Q and R73E found in HEL2X-flex both cause unfavourable charge reversals in the side chains of these residues and thus directly decrease HyHEL10 binding affinity. In contrast, mutations C76S and C94S remove a cysteine disulphide bond from the HEL protein, causing structural flexibility in the HEL protein in the region containing the HyHEL10 binding epitope.
HELR21Q,R73E (HEL2X-rigid) is another variant that has the same contact residue mutations as HEL2X-flex but has the disulfide bond, giving it a more rigid conformational structure.
Recipient mice of 8-12 weeks of age were lethally irradiated (2×425 cGy) using an XRAD 320 Biological Irradiator (Precision X-Ray, North Branford, Conn., UA). Femoral, humeral, and tibial bone marrow cells were aspirated into B-cell medium (BCM) comprising RPMI (Gibco, Carlsbad, Calif., USA) with 10% heat-inactivated Fetal Calf Serum (FCS) (Gibco), 2 mM L-glutamine, 100 U/mL penicillin RPMI media (Gibco). Fifteen hours after irradiation recipient mice were transplanted with an intravenous injection of 5-10×106 bone marrow cells. For mHEL3X transgenic (CD45.2+) recipients, injected bone marrow cells were 80% of SWHEL.Rag1−/− (CD45.1+) origin and 20% of mHEL3X transgenic (CD45.2+) origin. As previously observed, CD45.2+ pre-B-cells lacking pre-rearranged Igh and Igk genes proliferated more than their SWHEL (CD45.1+) Ig transgenic counterparts, so that only ˜5% of B220+ in chimeric recipients were CD45.1+ anti-HEL B-cells. Initially (
Five-to-seven-micron sections were cut using a Leica CM1900 cryostat, fixed in acetone and blocked with 30% normal horse serum. To stain HyHEL10 B-cells, sections were incubated with 200 ng/mL HEL (Sigma, St Louis, Mo., USA), polyclonal rabbit anti-HEL sera, and anti-rabbit-IgG-FITC (Rockland Immunochemicals, Pottstown, Pa., USA). T-cells were stained with anti-CD3-biotin (eBiosciences, San Diego, Calif., USA) and streptavidin-AlexaFlour 555 (Invitrogen, Carlsbad, Calif., USA). Follicular B-cells were stained with anti-IgD-AlexaFlour 647 (Biolegend, San Diego, Calif., USA). For GC analysis, GCs were stained with anti-CD16/CD32-PE (BD Pharmingen, San Diego, Calif., USA) and CD3-biotin stains were followed by streptavidin-BV421 (BD Pharmingen). Stained tissue sections were imaged using a Zeiss Leica DM5500 microscope. AxioVision software was used for image capture and Adobe Photoshop used to compile the images. Images shown are 200× magnification.
B-cell responses in vitro were determined by culturing fresh splenocytes overnight at 37° C. in BCM following red-blood-cell lysis. B-cells were stimulated with one of the following additives: HELWT (200 ng/mL), LPS (2.5 μg/mL, Sigma), recombinant mouse IL-4 (10 ng/mL, R&D Systems, Minneapolis, Minn.) or anti-IgM mAb (5 μg/mL, Southern Biotech, Birmingham, Ala., USA). Cells were then surface stained to detect the upregulation of CD86 by flow cytometry.
Alternatively, fresh splenocytes were resuspended at 2×107 cells/mL in PBS containing 5% FCS and CFSE labeled with a final concentration of 11 μM for 5 minutes at room temperature. Cells were washed in PBS containing 5% FCS and then resuspended in BCM. B-cells were stimulated with anti-CD40 mAb (5 μg/mL, BD Pharminigen), LPS, anti-IgM mAb, IL-4, HELWT plus LPS, anti-CD40 mAb plus IL-4, HELWT plus IL-4 or anti-IgM mAb plus IL-4. Cells were cultured for 3 days at 37° C. proliferation was assessed as loss of CFSE staining via flow cytometry.
Purified HELWT (SEQ ID NO: 1) was purchased from Sigma-Aldrich. Recombinant HEL3X and DEL proteins were made as secreted proteins in Pichia pastoris yeast (Invitrogen) and purified from culture supernatants by ion exchange chromatography as previously described (Phan et al. 2003; Paus et al. 2006; Chan et al. 2012 Immunity 37, 893-904 (2012).
In order to generate the HELR21Q,R73E,C76S,94S (HEL2X-flex, SEQ ID NO: 2) and HELR21Q,R73E (HEL2X-rigid, SEQ ID NO: 3) variants, DNA encoding the HEL2X-flex amino acid sequence or the HEL2X-rigid amino acid sequence was cloned into the pCEP4 expression vector. Transient expression was then carried out in the Expi293™ transient expression system (Thermo Fisher Scientific) following the manufacturer's instructions.
Affinity chromatography medium was prepared by covalent coupling of anti-HEL VHH domain D2L19 (De Genst et al. 2006) to CNBr-Activated Sepharose 4B (GE Healthcare).
Soluble HEL2X-flex or HEL2X-rigid was then purified from the culture supernatant using the D2L19-coupled sepharose followed by elution with 0.1 M Glycine pH 2.7. Purified protein was then dialysed into PBS using snakeskin 3.5 kDa MWCO dialysis tubing (Pierce) and used for conjugation to SRBC as described in Example 6 for DEL and other related antigens.
For the purposes of crystallography, DEL lacking a poly-histidine affinity tag was purified from duck eggs. Proteins were stored in PBS at 1-2.5 mg/mL at −80° C. Prior to use samples were thawed and stored at 4° C. for a maximum of 8 months.
HEL proteins were desalted into conjugation buffer (distilled water with 0.35 M DMannitol [Sigma] and 0.01 M Sodium Chloride [Sigma]). For this process PD-10 columns (Amersham, Piscataway, N.J., USA) were equilibrated with 30 mL Conjugation buffer. One hundred micrograms of protein was loaded onto each column and pushed through the column using 2.5 mL Conjugation buffer. For elution of the protein, 3.5 mL conjugation buffer was added and the HEL protein collected as fractions in the following volumes; 250 μL, 1000 μL, 250 μL, 250 μL, 250 μL. Protein concentrations of each 4 fraction were determined by spectrophotometry.
For conjugation, SRBC were washed in 30 mL of PBS per 6-8×109 cells. Washing was performed three times by centrifugation at 2,300 rpm (1,111 g) for 5 min at 4° C. in PBS and then once in conjugation buffer. SRBC were then resuspended in a final volume of 1 mL conjugation buffer in a 50 mL Falcon tube containing 10 μg/mL of protein for conjugation, unless otherwise stated, which had first been buffer exchanged by gel filtration on PD10 columns into the conjugation buffer. The solution was mixed on a platform rocker on ice for 10 minutes. One hundred microliters of 100 mg/mL N-(3-Dimethylaminopropyl)-N-ethylcarbodimide hydrochloride (Sigma) was then added and the solution was mixed for a further 30 minutes on ice. SRBCs were then washed four times in 30 mL PBS. Confirmation of successful conjugation was performed by flow cytometric analysis of SRBC using AlexaFluor 647-conjugated HyHEL9 antibody. 2×108 conjugated or unconjugated SRBC were injected into the lateral tail vein of each chimeric mouse.
On the day of harvest organs were collected into BCM, cell suspensions passed through a 70 μm cell strainer (Falcon, Corning, N.Y., USA) and centrifuged 1 500 rpm (440 g) for 5 min at 4° C. Fc receptors were blocked with unlabeled anti-CD16/32 (ebioscience) before staining. To detect HEL3X-binding cells, cells were stained with 2 μg/mL (0.14 μM) HEL3X, followed by AlexaFluor 647-conjugated HyHEL9. Since this concentration of HEL3X approximates the Ka of the unmutated HyHEL10 receptor on the B-cells, it occupied approximately half of the binding sites.
For DEL-N terminal biotinylation, DEL was conjugated to 5 M excess of NHS biotin (Sigma) at pH 6.5 overnight on ice. For panels where DEL-biotin staining was concurrently used with HyHEL9, HyHEL9 stains were followed with HEL4X at 2 μg/mL to block any unbound HyHEL9 binding sites without binding to the HyHEL10 BCR. DEL-biotin staining then followed at 2 μg/mL.
Anti-IgG1-FITC (BD Pharmingen) stains were followed by 5% mouse serum before staining for other surface molecules. Cells were filtered using 35 μm filter round-bottom FACS tubes (BD Pharmingen) immediately before data acquisition on a LSR II analyzer (BD Pharmingen).
Forward- and side-scatter threshold gates were applied to remove red blood cells and debris and approximately 5-7×106 events were collected per sample. Cytometer files were analyzed with FlowJo software (FlowJo LLC, Ashland, Oreg., USA).
BrdU staining was performed as described previously (16). Briefly mice were given drinking water shielded from light containing 0.8 mg/mL BrdU (Sigma). Spleens and bone marrow were prepared as for flow cytometry. Following surface staining cells were fixed and permeabilized, DNA denatured and then stained with anti-BrdU FITC using BrdU staining kit (BD Phaminigen) as per the manufacturer's directions.
Mutant and wild-type HyHEL10 heavy (IgH) and wild-type HyHEL10 kappa-light (IgK) chain FAb sequences were synthesized and cloned into pCEP4 expression vector via KpnI and BamHI restriction sites. The heavy chain was C-terminally his-tagged for purification purposes. Fab arms were transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific, Boston, Mass., USA) according to the manufacturer's recommendations. Lipid-DNA complexes were prepared using a 1H:2L chain ratio, as previously described (28). Fab was purified from cell culture supernatant using HisTrap FF crude columns (GE Healthcare, Little Chalfont, UK) according to the manufacturer's instructions. After dialysis against PBS Fabs were concentrated using spin filters (EMD Millipore, Billerica, Mass., USA), inspected on SDS-PAGE and their concentrations determined by spectrometry (absorbance at 280 nm).
Purified HyHEL10 FAbs were buffer exchanged into PBS using equilibrated ZebaSpin columns (Thermo Fisher Scientific). FAb samples were requantified and incubated with EZ-Link NHS-PEG4-Biotinylation reagent (Thermo Fisher Scientific) at a 5:1 biotin-toprotein ratio. Free biotin was removed from the samples by repeating the buffer exchange step in a second zebaspin column equilibrated with PBS.
Affinity of interactions between biotinylated FAbs and purified lysozyme proteins (DEL, HEL3X, HEL2X-flex and HEL2X-rigid) by Biolayer Interferometry (BLItz, ForteBio, Menlo Park, Calif., USA). Streptavidin biosensors were rehydrated in PBS containing 0.1% w/v BSA for 1 hr at RT. Biotinylated FAb was loaded onto the sensors “on-line” using an advanced kinetics protocol, and global fits were obtained for the binding kinetics by running associations and dissociations of Lysozyme proteins at a suitable range of molar concentrations. The global dissociation constant (KD) for each 1:1 FAb-lysozyme interaction was determined using the BlitzPro 1.2.1.3 software.
Cell suspensions were prepared and germinal center B-cells identified as for flow cytometry. Single-cell sorting into 96-well plates (Thermo Fisher Scientific) was performed on a FACSAria or FACSAriaIII (BD Pharminigen). B-cells from each mouse were analyzed individually to ensure over-representation of one particular clone did not affect mutation analysis. The VDJH exon of the HyHEL10 heavy chain gene was amplified from genomic DNA by PCR, sequenced, and analyzed.
ELISA detection of serum concentrations of IgG1 antibodies binding to HEL3X/DEL were measured. High-binding plates (Corning, Corning, N.Y., USA) were coated with HEL3X or DEL and bound serum antibody quantified using the same IgH chain isotype-specific secondary antibodies used for flow cytometry. Antibody levels were quantified against HyHEL10 standards.
The complex comprising DEL purified from duck eggs (isoform DEL-I) and the Fab arm of HyHEL10I29F,S52T,Y53F (HH10*3) was prepared by gel filtration chromatography in which a 2:1 ratio of DEL:HH10*3 was applied to an S200 26/60 column (GE Healthcare) plumbed with 25 mM Tris (pH 8.5), 150 mM NaCl. Crystals of HH10*3-DEL and other HyHEL10 complexes were grown by hanging drop vapor diffusion whereby 2 L of protein complex (at ˜6.5 mg/mL) was combined with an equal volume of well solution comprising 100 mM sodium citrate (pH 4.75 and 17% v/v) PEG3350 (Hampton Research, Aliso Viejo, Calif., USA). Crystals grew over several weeks. Crystals were briefly swum (10 sec) in well solution doped with glycerol to ˜25% (v/v) prior to being flash frozen in N2 (1 sec) for data collection.
Diffraction data were collected at the Australian Synchrotron on beamline MX2 at 100 K. Diffraction data were indexed and integrated using iMOSFLM, the space group determined with POINTLESS, and scaling performed with AIMLESS. Structures were solved via molecular replacement using PHASER and employing PDB entries 3D9A (Fab) and 5V8G (DEL-I) as search models. Rigid-body and restrained B-factor refinement were performed with REFMAC5, part of the CCP4 suite of crystallography software. Models were inspected and compared with electron density maps, and where necessary modified, using COOT. Validation was performed using the MOLPROBITY server.
GraphPad Prism 6 (GraphPad Software, San Diego, USA) was used for data analysis. When the data were normally distributed, an unpaired Student's t-test was performed for analysis. When data was not normally distributed Welsh's correction was applied. For all tests, P<0.05 was considered as being statistically significant. Unless otherwise stated error bars represent arithmetic mean. Flow cytometric plots of multiple samples are presented as mean and standard error or mean. For all figures, data points indicate individual mice. * represents P<0.05, ** represents P<0.01, *** represents P<0.001, **** represents P<0.0001.
Bone marrow chimeric mice were engineered (
It was firstly tested if self-reactive SWHEL B-cells could respond to a foreign antigen that perfectly mimicked self. Sheep red blood cells (SRBCs) were covalently coupled with self-antigen at surface densities either the same as on the endogenous mouse red blood cells (MRBCs) or 30 fold higher (
Next the response of self-reactive SWHEL B-cells to DEL, which differs from self-antigen at four residues that contact the HyHEL10 H-chain (
indicates data missing or illegible when filed
To determine if rapid selection for mutant GC B-cells with decreased affinity for self was followed by affinity maturation towards foreign, antibody mutations were analyzed 4, 7, and 11 days after SWHEL B-cells were challenged with DEL-SRBC (
The I29F mutation became paired with S52T or Y53F mutations in CDR2, starting as a small subset of self-reactive cells on day 7 but becoming the most prevalent as pairs or a trio by day 11. S52T or Y53F were rarely found individually but combined with the I29F foundation mutation increased foreign-self discrimination, retaining 1×106 M−1 affinity for self but progressively increasing foreign affinity to 6×109 M−1. Strong epistatic (non-additive) effects were observed. For example, the I29F S52T YF3F trio increased the apparent AAG for binding foreign antigen by −3.3 kcal/mol, compared with −1.6 kcal/mol expected for additive effects of the individual mutations (Table 1). This trio of mutations became even more prevalent when self-reactive SWHEL B-cells were recruited at the outset of the GC reaction and analyzed 15 days later (
A different, less optimal evolutionary trajectory prevailed when SWHEL B-cells were not self-reactive, dominated by acquisition of a CDR2 mutation, Y58F, either alone, paired, or in trio with S52T and Y53F (
To understand how these three mutations conferred a 5000-fold differential binding to foreign over self, we used X-ray crystallography to analyze the structure of HyHEL10I29F, S52T, Y53F complexed with DEL (
Anergic B-cells in the mHEL3×tg mice were next identified within a polyclonal repertoire that possessed micromolar affinity for the same self antigen and tested if they too could resolve antigenic mimicry. HEL3X-binding B-cells comprised 2.7% of IgD+IgMl® anergic B-cells and 0.5% of all spleen B-cells (
The findings here provide evidence for autoantibody redemption in human antibodies by showing mutation away from self precedes mutation towards foreign to create unique epistatic trajectories. Self-reactivity, rather than being a barrier to immunization, directed cells down an alternative trajectory, which produced a higher final affinity against the foreign immunogen.
The affinity maturation pathways of an antibody that cross-reacts with a conformationally flexible protein antigen as well as a self-antigen were investigated. Specifically, the role of epitope flexibility in both non self-reactive and self-reactive systems were explored concurrently.
To further investigate the role of self-reactivity constraints on broadly neutralizing antibody production bone marrow chimeric mice in which the majority of mature B-cells were polyclonal and CD45.2+ were engineered in accordance with Example 2. However, 0.1% of mature follicular B-cells expressed the HyHEL10, antibody as described previously in Example 16. In one group of chimeric mice, mHEL3X, a defined low affinity self protein was displayed on all cells as an integral membrane protein (Chan, et al. 2012). As described above in Example 16, when the mice were immunized with a similar affinity protein, structurally similar to the self-antigen (duck egg lysozyme, DEL) self-reactive HyHEL10 B cells are able to acquire mutations that specifically lowered their affinity to the self-antigen while maintaining foreign binding. Thus to further this phenomenon the structure of HEL3X was solved and compared directly to DEL. This revealed the backbone of the self and foreign antigens to be remarkably similar with the main differences relating largely to surface residues, particularly around location 73.
As described above in Example 16, mice were immunized with sheep red blood cells (SRBC) conjugated to the foreign antigen identical (HEL3X) or slightly differing from self (DEL), both with a near identical affinity to HyHEL10 (1/KD=1.2×107 M−1 vs 2.5×107 M−1) to investigate the importance of similarity between the self and foreign antigen. Although the overall GC and memory response was comparable with these two different antigens when the self-antigen and the foreign antigen were identical HyHEL10 B cells were significantly less frequent in the GC and memory compartment Analysis with the serum IgG1 compartment revealed that mice with self-HEL3X immunized with foreign DEL were actually able to generate 26 fold higher IgG1 antibody titers than those without self-mHEL3X, as was shown in Example 16. In contrast, although the mice immunized with foreign HEL3X were able to form 70 fold higher HEL3X IgG1 antibody titers than those mice immunized with unconjugated SRBC, it remained 10 fold lower than that achieved in the non self-reactive mice.
As seen previously in Example 16, when mice with self-mHEL3X were immunized with foreign DEL, this rapidly led for the acquisition of a mutational trajectory which resulted in the formation of clones that had dramatically decreased affinity for the self-antigen while concurrently increasing foreign binding affinity. When the control non-tg mice were immunized with foreign HEL3X, where the HyHEL10 cells responded in the absence of self-mHEL3X, high frequencies of HyHEL10 GC cells accumulated with Y53D (84% of cells), Y58F (10%) or both (5%. Y53D increases HEL3X affinity 100-fold (Phan, et al. 2006, Chan, et al. 2012). By contrast Y53D was virtually absent among HyHEL10 GC cells in HEL3tg mice where they were exposed to foreign and self-HEL3X. In the presence of self-mHEL3X, 61% of HyHEL10 GC cells elicited by of HEL3X-SRBC carried S52R, including 37% where S52R was paired with Y53F. Bio-layer interferometry revealed that the S52R mutation, alone or in combination with the Y53F mutation, completely abolished measurable binding to the highest concentration of HEL3X tested, representing at least 100-fold lower affinity. Thus when foreign erythrocytes carried a with identical amino acid sequence to a protein on self-erythrocytes, despite reactivation of anergic B cells into the germinal center subsequent affinity maturation of HyHEL10 B cells in GC was completely suppressed. Instead the cells were selected for loss of affinity. This is similar to the what has been seen previously when HEL was the self and foreign antigen whereby HyHEL10 GC B cells inserted a glycosylation to decrease, but not entirely remove, self and foreign binding (Sabouri, et al. 2014), however in this scenario, where the B cell affinity to the self and foreign antigen began at a much lower, physiological affinity, these mutations completely abolished binding to both antigens.
HEL is characterized to have four disulphide bonds all of which contribute to the structural stability of the molecule (Inaka, et al. 1991, Buck, et al. 1995, Yokota, et al. 2000). To investigate the role of epitope flexibility in neutralizing antibody responses a variant of HELR21Q,R73E,C76S,C94S (HEL2X-flex) lacking one of these disulphide bonds (
To determine the effect of epitope flexibility, the responses of HEL2X-flex to DEL, with both antigens being extremely similar in regards to HyHEL10 affinity (1/KD=1.6×107 M−1 for HEL2X-flex vs 1/KD=2.5×107 M−1 for DEL) and having comparable contact residue changes from the self-antigen (
Although HEL2X-flex and DEL have near identical affinity for HyHEL10 direct comparison between them is somewhat confounded by the structural differences between the two proteins. To precisely determine the effect of epitope flexibility on neutralizing antibody production, a variant of HEL HELR21Q,R73E (HEL2X-rigid) that had the same contact residue mutations as HEL2X-flex but maintained the disulfide bond, giving it a more rigid conformational structure (
Given the dramatic differences in the mutational hierarchy achieved by HyHEL10 neutralizing the rigid or flexible variant of the epitope, the crystals of several HyHEL10 complexes were explored to further understand this variation. Crystals of the highest affinity neutralizing clone enriched in the self-reactive HyHEL10 GC B cells, HyHEL10L4F, Y33H, S56N, Y58F, in combination with HEL2X-flex were generated to further elucidate how the antibody was able to neutralize the flexible epitope of HEL2X-flex while avoiding binding to the self-antigen. This was compared to the structure of the HyHEL10L4F, Y33H, S56N, Y58F antibody alone. A complex of the HyHEL10 antibody complexed with HEL2X-rigid, which has similar foreign binding affinity as the HyHEL10L4F, Y33H, 56N, Y58F HEL2X-flex complex was also generated as a control. Despite these two antibodies having similar foreign binding affinities, the flexible nature of the disulfide bond meant that the antibody antigen complex behaved in a drastically different manner in the two situations (
The findings here extend the evidence that autoantibody redemption could be utilized as a potential strategy to generate bNAs for challenging antigens, including those utilizing epitope flexibility. GC B cells are able to concurrently overcome the challenges imposed by molecular mimicry and epitope flexibility to generate high affinity neutralizing antibodies. However a high mutational load including non-canonical framework region mutations are required to overcome these challenges concurrently. This suggests that potentially the limitations currently preventing bNA production for important pathogen neutralization may in fact be probabilistic, rather than biophysical or thermodynamic. This supports previous evidence suggesting that a high frequency of germline precursors may be a limiting factor in HIV bNA production (Abbott, et al. 2018) and may help to explain the highly mutated and atypical nature of HIV bNAs and explain why they are only generated in a limited number of patients (Klein, et al. 2013, West, et al. 2014).
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2018901070 | Mar 2018 | AU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/AU2019/050283 | 3/29/2019 | WO | 00 |
