Universal or normalized antibody frameworks for improved functionality and manufacturability

Abstract

To provide methods of designing and manufacturing universal or normalized sequence templates for rabbit monoclonal antibodies for diagnostic applications.SOLUTION: Provided is a method of making a universal or normalized rabbit antibody comprising: selecting a wild-type rabbit antibody; comparing the wild-type antibody sequence to a normalized antibody sequence comprising a set of amino acid residues in the variable framework and / or joining regions that are most frequently-occurring at the same position in rabbit antibodies known to be thermally stable and / or having a long term stability of at least 12 months, and identifying it; and mutating said one or more amino acid residues of the wild type antibody identified in the previous step to the corresponding amino acid residue of the normalized sequence to obtain a universal or normalized rabbit antibody, the binding affinity of the antibody to its target remaining within an acceptable range when compared to the binding affinity of the unmodified wild- type antibody.SELECTED DRAWING: Figure 6A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 682,795, filed June 8, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to sequence listing submitted as an ASCII text file (.TXT) A Sequence Listing (entitled "P34814WO_Sequence_ST25") in the form of an ASCII compliant text file (22,104 bytes in size, created on April 26, 2019) has been submitted contemporaneously with the present application, and the contents of the Sequence Listing are incorporated herein by reference in their entirety.

[0003] The present invention provides methods for designing universal or normalized sequence templates for producing rabbit monoclonal antibodies for diagnostic applications by modifying the variable regions. The present invention further provides methods for optimizing desirable antibody properties such as thermal stability, long-term stability, expression, deamination / oxidation and / or aggregation that may adversely affect diagnostic assays, without adversely affecting binding specificity to the target. The present invention further provides universal or normalized antibody templates or frameworks, and rabbit monoclonal antibodies obtained therefrom. [Background technology]

[0004] There is a need to develop methods to improve reproducible binding ratios, as well as methods to standardize antibody framework regions for stability and developability for use in diagnostic assays such as immunohistochemistry.

[0005] In principle, antibody engineering methods can be used to standardize antibody templates such as the framework and constant regions while still maintaining antibody specificity and affinity.

[0006] The complementarity determining regions ("CDRs") of an antibody are usually not modified, since they are the main determinants of the specificity and affinity of the antibody to the target antigen. However, in some cases, the CDR sequences themselves may be a source of antibody instability.

[0007] In fact, most of the accumulated knowledge in antibody engineering concerns human and mouse antibodies, although rabbit monoclonal antibodies are used in several diagnostic applications both as primary antigen detection reagents and as reagents for secondary signal generation or amplification.

[0008] Primary antibodies are used to select specific tumor markers with exquisite specificity, and secondary antibodies are used to generate detection signals. Each antibody is unique in terms of specificity, affinity, production level, stability, and suitability for binding. This uniqueness comes from the amino acid sequence, posing a challenge to standardization of diagnostic assay development and production. The inventors have utilized protein engineering to solve this problem, focusing their efforts on standardizing antibody framework regions for stability in particular, and antibody developability more generally.

[0009] There are many advantages to using rabbit monoclonal antibodies. By using rabbit monoclonal antibodies, the problem of certain proteins being recognized as self-antigens in humans and mice is avoided. In addition, rabbit monoclonal antibodies have improved immune responses to small epitopes, recognize a more diverse range of epitopes, and have better responses to mouse antigens. Thus, rabbit monoclonal antibodies give a better response to antigens than monoclonal antibodies from other hosts such as mice.

[0010] Moreover, the use of rabbit monoclonal antibodies provides more accurate immunohistochemistry results, which may be due to the unique immune system of rabbits. Rabbits can generate a broader range of high affinity antibodies than mice. Therefore, it is more likely to find a rabbit antibody that can function in a range of applications than to find an equally suitable mouse antibody. In addition, many smaller peptides that elicit poor responses in mice produce favorable responses in rabbits. For these reasons, rabbit monoclonal antibodies are becoming more preferred in research and clinical applications.

[0011] US 2004 / 0086979, incorporated herein by reference, discloses a method for expressing a rabbit antibody, the method comprising: (a) identifying surface-exposed amino acids in a framework region of a parent rabbit antibody that differ from amino acids at corresponding positions in the non-rabbit antibody by comparing the amino acid sequence of the framework region of the parent rabbit antibody with the amino acid sequence of the framework region of a non-rabbit antibody; and (b) substituting the identified amino acids with amino acids at corresponding positions in the non-rabbit antibody to express the rabbit antibody.

[0012] US2005 / 030331, incorporated herein by reference, discloses a method for humanizing a rabbit monoclonal antibody, comprising: (a) comparing the amino acid sequences of the heavy and light chain variable domains of a parent rabbit antibody with the amino acid sequences of the heavy and light chain variable domains of an analogous human antibody; and (b) altering amino acids in the framework regions of the heavy and light chain variable domains of the rabbit antibody such that the altered framework regions are more similar in sequence to the equivalent framework regions of the analogous human antibody, the altered amino acids being either not involved in complementarity determining region (CDR) contacts, interchain contacts, or buried residues having substantially different side chains.

[0013] U.S. Patent No. 7,462,697, incorporated herein by reference, identifies tolerant positions for mutations in a parent rabbit antibody by comparing the amino acid sequence of the rabbit antibody in a) to the amino acid sequences of multiple related antibodies obtained from the same rabbit as the parent rabbit antibody, wherein the parent and related antibodies i) bind to the same antigen, ii) each comprise heavy chain variable domains having at least 90% overall amino acid sequence identity to each other, iii. each comprise light chain variable domains having at least 90% overall amino acid sequence identity to each other, iv. have H3 CDRs that are identical in length and sequence except for 0, 1, or 2 amino acid substitutions to each other, and v. have L3 CDRs that are identical in length and sequence except for 0, 1, or 2 amino acid substitutions to each other. The present invention discloses a method for humanizing a rabbit antibody comprising: (a) determining whether the human antibody has a CDR; (b) aligning the amino acid sequence of the parent rabbit antibody with the amino acid sequence of a human antibody, wherein the human antibody is one of the 10 human antibodies that are most homologous to the parent rabbit antibody; and (c) replacing the amino acids present at the mutation-tolerant positions with the corresponding amino acids of the human antibody to generate the humanized antibody.

[0014] Therefore, there is a need to provide a standardized, universal, or normalized (used interchangeably) rabbit monoclonal antibody framework or template that optimizes desirable antibody characteristics such as thermal stability. Improved thermal stability and reduced aggregation translate into extended shelf life, which are important for standardization of diagnostic assays. Summary of the Invention

[0015] The present invention provides a method for designing and producing a normalized rabbit antibody, comprising selecting a wild-type rabbit antibody and mutating the wild-type rabbit antibody to obtain a normalized rabbit antibody to optimize desired properties.

[0016] In one aspect of the disclosure, the method includes: selecting a wild-type rabbit antibody; comparing the wild-type antibody sequence with a normalized antibody that comprises a set of amino acid residues in the variable framework and / or junction regions that occur most frequently at the same positions in rabbit antibodies known to be thermostable and / or have long-term stability for at least 12 months; identifying one or more amino acid residues in the wild-type antibody that differ from the normalized sequence; and mutating the one or more amino acid residues of the wild-type antibody identified in step (b) to the corresponding amino acid residues of the normalized sequence to obtain a universal or normalized rabbit antibody, wherein the binding affinity of the antibody to its target remains within an acceptable range when compared to the binding affinity of the unmodified wild-type antibody. In some embodiments, the universal or normalized rabbit antibody comprises an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10 in the VH chain. In some embodiments, the universal or normalized rabbit antibody comprises an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17 in the Vk chain. In some embodiments, the universal or normalized rabbit antibody comprises an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10 and an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17. In some embodiments, the universal or normalized rabbit antibody exhibits improved thermal stability compared to a wild-type rabbit antibody. In some embodiments, the universal or normalized rabbit antibody exhibits improved long-term stability compared to a wild-type rabbit antibody. In some embodiments, the universal or normalized rabbit antibody exhibits improved deamination compared to a wild-type rabbit antibody. In some embodiments, the universal or normalized rabbit antibody exhibits improved expression compared to a wild-type rabbit antibody. In some embodiments, the universal or normalized rabbit antibody is a primary rabbit antibody. In some embodiments, the mutation is in the J1 region of the kappa chain. In some embodiments, the mutation is in the FR4 region of the kappa chain.In some embodiments, the mutations are in the J2, J4, and / or J6 regions of the heavy chain. In some embodiments, the binding affinity of the antibody to its target remains within an acceptable range when compared to the binding affinity of the unmodified wild-type antibody. In some embodiments, the mutations are in the complementarity determining regions.

[0017] In one aspect, the invention features universal or normalized rabbit antibodies obtained by the methods of the disclosure.

[0018] In one embodiment of the disclosure is a universal or normalized rabbit antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, and 10 within the VH region.

[0019] In one embodiment of the disclosure is a universal or normalized rabbit antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 13, 14, 15, 16, and 17 within the Vk region.

[0020] In one embodiment of the disclosure is a universal or normalized rabbit antibody comprising an amino acid sequence within the VH region selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, and 10, and an amino acid sequence within the Vk region selected from the group consisting of SEQ ID NOs: 2, 12, 13, 14, 15, 16, and 17.

[0021] In some embodiments of the disclosed methods, the mutated universal or normalized antibody has improved properties for diagnostic use. In some embodiments, the improved properties are selected from the group consisting of thermal stability, long-term stability, reduced aggregation and reduced oxidation / deamidation. In some embodiments of the disclosed methods, the mutation is not in a complementarity determining region.

[0022] In one embodiment of the disclosure, a method of improving one or more properties in an antibody for diagnostic use comprises selecting a wild-type rabbit antibody that is deficient in one or more characteristics selected from the group consisting of recombinant expression, thermal stability, long-term stability, aggregation, and oxidation / deamidation, comparing the wild-type antibody sequence to a normalized antibody sequence comprising a set of amino acid residues in the variable framework and / or junction regions that occur most frequently at the same position in a set of rabbit antibodies known to have the characteristics within an acceptable range, and identifying one or more amino acid residues in the wild-type antibody that differ from the normalized sequence, and mutating the identified one or more amino acid residues of the wild-type antibody to the corresponding amino acid residues of the normalized sequence, wherein the one or more characteristics are improved compared to the unmodified wild-type antibody. In one aspect of the disclosure, a method of producing a recombinant rabbit monoclonal antibody includes selecting a wild-type rabbit antibody, comparing the wild-type antibody sequence to a normalized antibody sequence comprising a set of amino acid residues in the junction region that occur most frequently at the same position in a set of rabbit antibodies known to have characteristics within an acceptable range selected from the group consisting of variable framework / thermal stability, long-term stability, reduced aggregation, and reduced oxidation / deamidation, preparing a standard expression vector encoding a complementarity determining region containing a change to the wild-type sequence at one or more of the identified residues, expressing the vector in a suitable host cell, and purifying the antibody from the step. In some embodiments, the set of amino acid residues in the normalized sequence occur most frequently at the same position in at least 50 rabbit antibodies with the relevant characteristic. In some embodiments, the set of amino acid residues in the normalized sequence occur most frequently at the same position in at least 100 rabbit antibodies with the relevant characteristic. In some embodiments, the set of amino acid residues in the normalized sequence occur most frequently at the same position in at least 150 rabbit antibodies with the relevant characteristic. In some embodiments, the set of amino acid residues of the normalized sequences occur most frequently at the same positions in at least 200 rabbit antibodies with related characteristics.In some embodiments, the set of amino acid residues in the normalized sequences occur most frequently at the same positions in at least 250 rabbit antibodies with related features, hi some embodiments, the set of amino acid residues in the normalized sequences occur most frequently at the same positions in at least 300 rabbit antibodies with related features.

[0023] The inventors have surprisingly found that the antibodies obtained according to the invention exhibit excellent properties without substantially affecting binding affinity and specificity for the target.

[0024] The present disclosure further provides a normalized monoclonal rabbit antibody obtained by selecting a wild-type rabbit antibody and mutating the wild-type rabbit antibody.

[0025] Further objects, features, and advantages of the present invention will become apparent from the detailed description that follows. [Brief description of the drawings]

[0026] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the U.S. Patent and Trademark Office upon request and payment of the necessary fee.

[0027] [Figure 1A]FIG. 1 shows (A) an overview of the protein engineering strategy employed by the present invention, and (B) a schematic diagram of how the script program is used to generate mutants based on a universal template. These mutants for the light chain (pink) and heavy chain (purple) were gene synthesized and then cloned into either the pTT5 or pDV3a vectors, followed by transient transfection of HEK293 cells. Cultures were grown in shake flasks for 6 days, and antibody supernatants were collected and purified as required. The antibody supernatants were serially diluted to determine the antibody concentration and used to determine binding affinity. The thermostability of the antibody supernatants was tested by ELISA. The purified antibodies were used to measure the melting and aggregation temperatures. Immunohistochemistry procedures were performed on control tissues. [Figure 1B] FIG. 1 shows (A) an overview of the protein engineering strategy employed by the present invention, and (B) a schematic diagram of how the script program is used to generate mutants based on a universal template. These mutants for the light chain (pink) and heavy chain (purple) were gene synthesized and then cloned into either the pTT5 or pDV3a vectors, followed by transient transfection of HEK293 cells. Cultures were grown in shake flasks for 6 days, and antibody supernatants were collected and purified as required. The antibody supernatants were serially diluted to determine the antibody concentration and used to determine binding affinity. The thermostability of the antibody supernatants was tested by ELISA. The purified antibodies were used to measure the melting and aggregation temperatures. Immunohistochemistry procedures were performed on control tissues.

[0028] [Figure 2A] FIG. 2 shows (A) the VH domains of rabbit (yellow) and mouse (green) antibodies, and (B) a close-up of the Tyr corners of the VH domains. [Figure 2B] FIG. 2 shows (A) the VH domains of rabbit (yellow) and mouse (green) antibodies, and (B) a close-up of the Tyr corners of the VH domains.

[0029] [Figure 3-1]FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Figure 3-2] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Figure 3-3] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Diagram 3-4] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Figure 3-5] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Diagram 3-6] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Diagram 3-7] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Diagram 3-8] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Diagram 3-9] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence. [Figure 3-10] FIG. 3 shows the distribution of amino acids in the rabbit antibody sequence.

[0030] [Figure 4]Figure 4 shows the universal or normalized antibody templates of the invention. Rows: (NO) numbering system (mainly IMGT) and approximate locations of FRs and CDRs, (IK) important Vk locations, (TK) universal or normalized Vk template, (IH) important VH chain locations, (TH) universal or normalized VH template. IK and IH rows: (c) predicted CDR contacts, (d) buried and predicted CDR contacts, (2) buried polar region 2, (b) buried, (0) no amino acid residues. (-) no preference, (i) putative interchain contacts, (t) buried tyrosine corners, (v) turns, (x) surface residues that can be changed with very low risk of altering affinity, (u / c / b) vaguely defined CDRs, where these residues can be conservatively altered with a high risk of altering affinity, (s) residue VH84 at the first position after the signal peptide (putative CDR contact) or in the DE loop, which is absent in some rabbit VH chains.

[0031] [Figure 5A] FIG. 5 shows binding curves for (A) VH and Vk series mutants and (B) VH mutants paired with wild-type Vk, (C) Vk mutants paired with wild-type VH, and (D) synthetic lethality determinants. [Figure 5B] FIG. 5 shows binding curves for (A) VH and Vk series mutants and (B) VH mutants paired with wild-type Vk, (C) Vk mutants paired with wild-type VH, and (D) synthetic lethality determinants. [Figure 5C] FIG. 5 shows binding curves for (A) VH and Vk series mutants and (B) VH mutants paired with wild-type Vk, (C) Vk mutants paired with wild-type VH, and (D) synthetic lethality determinants. [Figure 5D] FIG. 5 shows binding curves for (A) VH and Vk series mutants and (B) VH mutants paired with wild-type Vk, (C) Vk mutants paired with wild-type VH, and (D) synthetic lethality determinants.

[0032] [Figure 6A] FIG. 6 shows (A) thermal stability curves for wild-type ("WT") Vk with VH mutants, (B) thermal stability curves for WT VH and Vk mutants, and (C) thermal stability curves for xCD2 WT versus the most mutated VH and Vk. [Figure 6B] FIG. 6 shows (A) thermal stability curves for wild-type ("WT") Vk with VH mutants, (B) thermal stability curves for WT VH and Vk mutants, and (C) thermal stability curves for xCD2 WT versus the most mutated VH and Vk. [Figure 6C] FIG. 6 shows (A) thermal stability curves for wild-type ("WT") Vk with VH mutants, (B) thermal stability curves for WT VH and Vk mutants, and (C) thermal stability curves for xCD2 WT versus the most mutated VH and Vk.

[0033] [Figure 7A] FIG. 7 shows (A) the effect of conversion to universal or normalized templates on expression levels, melting points, and aggregation points ("Tagg266" and "Tagg473"), as well as similar staining patterns of WT xCD2 antibody and xCD2 antibody converted to universal or normalized templates of the invention. [Figure 7B] FIG. 7 shows (A) the effect of conversion to universal or normalized templates on expression levels, melting points, and aggregation points ("Tagg266" and "Tagg473"), as well as similar staining patterns of WT xCD2 antibody and xCD2 antibody converted to universal or normalized templates of the invention.

[0034] Abbreviation "FR" refers to "framework region."

[0035] "CDR" refers to "complementarity determining region."

[0036] "V" refers to the variable region.

[0037] "Vk" refers to the variable kappa chain (light chain).

[0038] "Vλ" refers to the variable lambda chain (light chain).

[0039] "VH" refers to the variable heavy chain.

[0040] "J" refers to the "binding region." DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0042] Antibody: A polypeptide that contains at least a light or heavy immunoglobulin variable region and specifically binds an epitope of an antigen. Antibodies include monoclonal antibodies, polyclonal antibodies, or antibody fragments, and others known in the art. In some instances, the antibody is linked or conjugated to another molecule, such as a nanoparticle (e.g., gold nanoparticle) or an enzyme (e.g., alkaline phosphatase).

[0043] Antibodies are composed of heavy and light chains, each of which has a variable region called the variable heavy (VH) region and the variable light (VL) region. Collectively, the VH and VL regions are responsible for binding the antigen recognized by the antibody. This includes intact immunoglobulins and their variants and portions well known in the art, such as Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are linked by a linker, whereas in dsFv, the chains are mutated to introduce disulfide bonds to stabilize the association of the chains. The term also includes recombinant forms such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (such as bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology, 3rd Ed., W.H. Freeman & Co., New York, 1997.

[0044] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes from a mouse or rabbit, or from a cell transfected with the light and heavy chain genes of a single antibody, e.g., HEK293 cells. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells and immune spleen cells. These fused cells and their progeny are called "hybridomas." Monoclonal antibodies include humanized monoclonal antibodies.

[0045] A primary (1°) antibody is an antibody that is immunospecific for one or more components of a biological sample. Primary antibodies are frequently used in diagnostic applications that require the detection and analysis of a biomarker in a sample, such as immunohistochemistry, immunocytochemistry, flow cytometry, and the like. Secondary (2°) antibodies are antibodies that are immunospecific for another antibody (or a component of another antibody) and are frequently used in diagnostic applications that rely on the indirect detection of a biomarker of interest. For example, in immunohistochemistry and immunocytochemistry applications, a primary antibody is used to mediate the deposition of a detectable moiety on a histological or cytological sample in proximity to the biomarker to which the primary antibody is bound. In some cases, the detectable moiety is directly attached to the primary antibody and is therefore deposited on the sample when the biomarker-specific reagent binds to its target (generally referred to as direct labeling). In other embodiments, deposition of the detectable moiety is effected by binding a secondary antibody to a primary antibody bound to the sample, followed by the use of a set of detection reagents that bind to or otherwise react with the secondary antibody in a manner that results in deposition of the detectable moiety (generally referred to as indirect labeling methods). Non-limiting examples of commercially available reagents or kits for use in indirect histochemical or cytochemical detection methods include the VENT ANA ultraView Detection System (secondary antibodies conjugated to enzymes, including HRP and AP), the VENT ANA iVIEW Detection System (biotinylated anti-species secondary antibodies and streptavidin-conjugated enzymes), the VENTANA OptiView Detection System (OptiView) (anti-species secondary antibodies conjugated to haptens and anti-hapten tertiary antibodies conjugated to enzyme multimers), the VENTANA Amplification Kit (unconjugated secondary antibodies that can be used with any of the previous VENTANA Detection Systems to amplify the number of enzymes at the site of primary antibody binding), and the VENTANA OptiView Amplification System (anti-species secondary antibodies conjugated to haptens, anti-hapten tertiary antibodies conjugated to enzyme multimers, and tyramide conjugated to the same hapten. In use, the secondary antibodies are contacted with the sample resulting in binding to the primary antibodies.The sample is then incubated with an anti-hapten antibody to allow the enzyme to associate with the secondary antibody. The sample is then incubated with tyramide to allow deposition of additional hapten molecules. The sample is then incubated again with the anti-hapten antibody to allow deposition of additional enzyme molecules. The sample is then incubated with a detectable moiety to allow pigmentation), VENT ANA DISCOVERY, DISCOVERY OmniMap, DISCOVERY UltraMap anti-hapten antibody, secondary antibody, chromogen, fluorophore, and dye kits, each of which is available from Ventana Medical Systems, Inc. (Tucson, Arizona), PowerVision and PowerVision+IHC Detection Systems (secondary antibodies that polymerize directly with HRP or AP into compact polymers with high enzyme-to-antibody ratios), and DAKO EnVision™+System (enzyme-labeled polymers that bind to secondary antibodies).

[0046] Regardless of the type of antibody or detection system used, each antibody is unique in terms of specificity, affinity, production level, stability, and suitability for binding, making assay development and manufacturing standardization a challenge. The inventors have used protein engineering to solve this problem, focusing on a) cloning hybridomas to convert them into recombinant antibodies, b) modifying antibody sequences to obtain higher and more reproducible binding ratios, and c) standardizing antibody framework regions for stability and, more generally, antibody developability.

[0047] The inventors have surprisingly discovered that the methods of the invention can be used to produce normalized antibodies for improved reproducibility and development.

[0048] FIG. 1A shows an overview of an exemplary protein engineering strategy. Hybridomas can lose antibody production over time, and therefore conversion to a recombinant version of the antibody may be necessary for certain clones. With recombinant antibodies, protein engineering can also introduce site-specific conjugation for reliable labeling, and optimize antibody composition for developability, stability, and other desired properties required for downstream applications. Current methods for conjugation rely on the natural properties available in antibodies, thus resulting in a random distribution of bioconjugates. Introducing site-specific conjugation sites can greatly improve manufacturability and product composition. With engineered recombinant antibodies under control, antibody clones can finally be introduced into targeted integrated systems based on CHO cells. This can allow large-scale expression, production, and manufacturing that rivals the amount of antibodies produced by hybridomas.

[0049] Figure 1B shows that cDNA from an existing antibody vector or hybridoma clone can be used as the starting template from which the light (pink) and heavy (purple) chains are amplified using specifically designed primers and polymerase chain reaction. After cloning the antibody chains into either the pTT5 or pDV3a vector, HEK293 cells were transiently transfected and cultures were grown in shake flasks for 6 days. Antibody supernatants were harvested and purified, and purified antibodies were characterized by ELISA assays to determine antigen binding and by immunohistochemistry to confirm functionality.

[0050] The inventors have generated a "universal or normalized stable scaffold template" and presented its application towards stabilization of rabbit monoclonal antibodies. Specifically, the inventors have discovered that a) there is no need to extend the use of hybridomas and there is no risk of loss of specific antibody expression due to hybridoma instability, and b) the variable regions of the antibodies can be redesigned to introduce desirable formulation properties such as stability without losing specificity and affinity.

[0051] In principle, engineering can be used to standardize antibody scaffolds while maintaining antibody specificity and affinity, but in practice, most of the accumulated antibody engineering knowledge concerns human and mouse antibodies. We have examined the structure and sequence of rabbit antibodies, found many similarities with human and mouse antibodies, and created a slightly modified IMGT numbering system for rabbit variable heavy (VH) and variable kappa light (Vk) chains to generate structural alignments. The most frequently occurring amino acids (Aa) within framework (FR) sequences can lead to higher stability. Therefore, for positions that are not critical for binding, frequently occurring Aa or Aa present in rabbit antibodies with no known stability issues can be used to construct a universal or normalized antibody scaffold that can act as a template for converting unstable antibodies.

[0052] We searched for amino acid sequences that occur frequently in rabbit antibodies from germline, with the view that these sequences would provide higher stability and other desirable properties.For sequence positions that do not have strong bias, we used amino acids that occur frequently.In addition, we used amino acids that exist in antibodies that do not have known stability problems.The loosely defined CDRs can generally be left as is.

[0053] We first compared the sequences and structures of the rabbit Vk and VH regions with their human and mouse counterparts and confirmed that individual rabbit antibody amino acid residues have similar structural roles as their corresponding human and mouse counterparts.

[0054] We first compared the sequences and structures of rabbit Vk and VH regions with their human and mouse counterparts.

[0055] The antibodies according to the invention are produced from one or more universal or normalized antibody templates covering the frameworks of the kappa and heavy variable regions (Vk, Vlambda, and VH, respectively). The framework regions are found between the hypervariable regions of the variable chains of antibodies. The framework regions form a beta-sheet (beta-barrel) structure that acts as a scaffold to hold the hypervariable regions in position to contact the antigen or target.

[0056] These universal or normalized antibody templates of the present invention were generated based on the analysis of the variable region structures and sequences of existing rabbit monoclonal antibodies.

[0057] In one embodiment, the disclosure provides a method by which one of skill in the art can vary as many residues as possible from a candidate antibody to the corresponding residues in a universal or normalized template to improve desired properties without appreciable loss of binding affinity. In another embodiment, the method according to the invention is applicable to CDR sequences that increase stability without loss of affinity.

[0058] In one embodiment, binding affinity is measured using the dissociation constant (K D ) is determined by evaluating the K D can be assessed using surface-based (heterogeneous) methods, including surface plasmon resonance (SPR), biolayer interferometry (BLI), and enzyme-linked immunosorbent assay (ELISA) Schuck 1997, Gauglitz 2008, Friguet et al. 1985.

[0059] Generally, the universal or normalized K of an antibody D is 10 -6 ~10 -12 (M), but in either case, there can be an improvement compared to the wild-type antibody.

[0060] We collected approximately 300 amino acid sequences from antibodies against various antigens and peptides from antibodies independently isolated by Spring Bioscience. We also collected nine VH / Vk sequences of published rabbit antibody structures, 4HBC, 4HT1, 4J01, 4J04, 4MA3, 404Y, 4ZTP, 5C0N, 5DRN, which were downloaded from the RCSB Protein Databank. The distribution of amino acids in these sequences is shown in Figure 3, in which the intensity of green coloring is proportional to the occurrence of the amino acid at the indicated position.

[0061] We further collected 39 VH and 65 Vk1 functional germline sequences (not analyzed in Figure 3).

[0062] The VH and Vk sequences were then aligned primarily using the IMGT numbering system (see Lefranc et al., 2003, "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains") and published structural alignments for mouse and human antibodies (see Honegger and Pluckthun, 2001, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modelling and analysis tool").

[0063] All framework amino acid positions and position-dependent structural features generally applicable to all human, mouse and rabbit antibodies were annotated and confirmed as previously described (Chothia et al. 1998; Narciso et al. 2011), except for a) the frequent presence of an additional disulfide bond formed between VH FR2 C40 and C55, b) an internal CDR3 disulfide bond in several Vk1 sequences, c) an interdomain disulfide bond between a Cys residue in Vk FR3 (kabbat C80) or in FR4 (last position) and a Cys residue in CK, and d) a truncated DE loop in many rabbit VH FR3 regions that can be left without residue VH85 or both residues VH84 and VH85.

[0064] Using these numbering systems together, we then calculated the frequency of occurrence for each of the 20 standard amino acid residues at each of the VH and Vk structural alignment positions. The proposed universal or normalized Vk and VH templates are shown in Figure 4.

[0065] For kappa chain framework 4 ("FR4"), the exemplary sequence FGGGTEVVVK (SEQ ID NO: 18) from the J1 junction region was selected.

[0066] For the heavy chain, the exemplary sequence WGPGTLVTVSS (SEQ ID NO: 19) from junction regions J2, J4, and J6 was selected.

[0067] These Vk and VH FR4 sequences are found in most cloned antibodies and have no known stability problems.

[0068] The numbering system adopted is also shown in Figure 4, which applies to both the VH and Vk chains. Dark blue coloring indicates 100% conservation in the germline. For FR4, the sequences correspond to the rabbit germline KJ1 and HJ2 / 4 / 6, respectively. Dark green coloring indicates 89% occurrence in the antibodies provided by Spring Bioscience. Light green indicates residues commonly found in antibodies with no known stability issues. Finally, "-" indicates amino acid positions that should be conserved as in the original wild type sequence.

[0069] In one embodiment, the recombinant universal or normalized antibodies of the invention are produced by any host species suitable for the production of diagnostic antibodies. In one embodiment, the host cell is a mammalian cell. In a specific embodiment, the host cell is a HEK293F cell. In another specific embodiment, the cell is a CHO cell. In a further embodiment, the host cell is a yeast, bacterial (e.g., E. coli) or filamentous fungal cell. In a further embodiment, the host cell is an insect cell. Purification of the recombinant universal or normalized antibodies can be achieved using any known method, including chromatography and affinity chromatography.

[0070] In another embodiment, the antibodies of the invention are rabbit, mouse, or human antibodies.

[0071] In another embodiment, the antibody of the invention is a rabbit antibody.

[0072] In an embodiment, the antibodies of the invention are primary rabbit antibodies.

[0073] In an embodiment, the antibody of the invention is a secondary rabbit antibody.

[0074] In embodiments, the antibodies of the invention can be humanized.

[0075] In another embodiment, the antibody is an antibody fragment or a single chain antibody, including Fab and single chain Fv (scFv).

[0076] In another embodiment, the antibody is a chimeric antibody having constant regions derived from different antibodies and / or species.

[0077] In embodiments, antibodies of the invention are of any isotype, including IgA, IgM, IgG (all subtypes), IgA and IgE.

[0078] In embodiments, the antibodies of the invention exhibit improved thermal stability, long term stability, recombinant expression / potency, deamination / oxidation, and / or aggregation compared to the wild-type unmodified antibody.

[0079] Stability assays known in the art can be used to measure thermal stability / denaturation. In one embodiment, immunohistochemistry can be used. In another embodiment, ELISA can be used. In another embodiment, 2D electrophoresis is used. In another embodiment, differential scanning calorimetry and / or circular dichroism spectroscopy can be used. In further embodiments, nano differential scanning fluorimetry can be used. See, e.g., Vermeer and Norde, 2000; Svilenov et al. 2018; Strutz 2016; Amin et al. 2014. In one embodiment, an increase in melting point correlates with improved thermal stability.

[0080] Protein aggregation can be measured using a variety of known techniques, including mass spectrometry, size exclusion chromatography (SEC), dynamic light scattering (DLS), photo-obscuration (LO), dynamic imaging particle analysis (DIPA) techniques such as microflow imaging (MFI), and Coulter counter (CC). Engelsman et al., Strategies for the Assessment of Protein Aggregates in Pharmaceutical Biotech Product Development; Pharm. Res. 2011; 28(4):920-33.

[0081] In embodiments, the antibodies of the invention exhibit improved thermostability.

[0082] In another embodiment, the antibodies of the invention exhibit improved long term stability, including at least 6 months, preferably at least 1 year, more preferably at least 2 years, and most preferably at least 3 years at temperatures between -20 and -70°C. In one embodiment, the antibodies are lyophilized.

[0083] In another embodiment, the antibody of the present invention exhibits improved expression, for example as determined by increased titer from recombinant expression compared to the corresponding unmodified wild-type antibody. In one embodiment, the titer is at least 0.5 g / L, more preferably at least 0.75 g / L. In another embodiment, increased expression can be measured by taking OD280 readings. In yet other embodiments, surface plasmon resonance (SPR) can also be used to measure concentration, and protein gel electrophoresis with standards can be used to determine antibody concentration.

[0084] In another embodiment, the antibody of the present invention exhibits improved deamination / oxidation (i.e., reduced deamidation / oxidation). Deamidation is a spontaneous reaction that occurs when the side chains of asparagine and glutamine are modified, which can result in cyclic intermediate compounds. Oxidation occurs primarily at cysteine ​​and methionine, and these sulfur-containing residues result in the formation of intrachain or interchain disulfide bonds. Methods for assessing oxidation are described in detail in Yan.2009. Tryptophan is also easily oxidized. The loss of tryptophan can be elucidated by the identification of Trp decomposition compounds, including oxyindolylalanine (Oia), 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid (PIC), N-formylkynurenine (NFK), dioxyindolylalanine (DiOia), kynurenine (Kyn), and 5-hydroxytryptophan (5-OH-Trp).

[0085] In another embodiment, the antibodies of the invention exhibit improved aggregation (ie, reduced aggregation).

[0086] In an embodiment, an antibody of the invention comprises an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10, which sequence includes the universal or normalized or normalized Vk template (SEQ ID NO: 1) and mutants thereof.

[0087] In an embodiment, an antibody of the invention comprises an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17, which describe the universal or normalized VH template (SEQ ID NO: 2) and mutants thereof.

[0088] In an embodiment, the antibody of the invention comprises an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10 and an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17.

[0089] In an embodiment, the antibody of the invention comprises an amino acid sequence according to SEQ ID NO:1.

[0090] In an embodiment, the antibody of the invention comprises an amino acid sequence according to SEQ ID NO:2.

[0091] In an embodiment, the antibody of the invention comprises an amino acid sequence according to SEQ ID NO:1 and SEQ ID NO:2.

[0092] The above-mentioned amino acid sequences disclosed herein can be further modified by substitution of one or more residues. Such substitutions can be of a conservative nature, for example, when an amino acid is replaced by an amino acid of similar structure and characteristics, such as when a hydrophobic amino acid is replaced by another hydrophobic amino acid.

[0093] More conservative would be the replacement of an amino acid of the same or similar size and chemical properties, such as when leucine is replaced by isoleucine. Studies of sequence variation in families of naturally occurring homologous proteins have shown that certain amino acid substitutions are often more tolerated than others, and these often correlate with similarities in size, charge, polarity, and hydrophobicity between the original amino acid and its replacement, which is the basis for defining "conservative substitutions."

[0094] Conservative substitutions can be exchanges within one of the following five groups: group 1 - small aliphatic, non-polar or slightly polar residues (Ala, Ser, Thr, Pro, Gly); group 2 - polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); group 3 - polar, positively charged residues (His, Arg, Lys); group 4 - large, aliphatic, non-polar residues (Met, Leu, Ile, Val, Cys); and group 5 - large, aromatic residues (Phe, Tyr, Trp).

[0095] Less conservative substitutions include the replacement of one amino acid with another amino acid of similar characteristics, but which differs somewhat in size, such as the replacement of an alanine with an isoleucine residue.

[0096] Very non-conservative substitutions may involve replacing an acidic amino acid with one that is polar or even basic in nature, however such "very important" substitutions cannot be dismissed as potentially ineffective because chemical effects are not entirely predictable and very important substitutions may well produce fortuitous effects not predictable from simple chemical principles.

[0097] In one embodiment, the invention includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% percent identity to any of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, or 10, or SEQ ID NOs: 2, 12, 13, 14, 15, 16, 17, 18, or 19. Percent identity can be determined, for example, by comparing sequence information using the GAP computer program, version 6.0, described by Devereux et al. 1984 and available from the University of Wisconsin Genetics Computer Group (UWGCG). The GAP program utilizes the alignment method of Neddleman and Wunsch 1970 as modified by Smith and Waterman 1981. In another embodiment, the invention comprises an amino acid sequence having a percent identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% to any of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, or 10, or sequences 2, 12, 13, 14, 15, 16, 17, 18, or 19, and exhibits improved properties such as thermal stability, long-term stability, and expression.

[0098] In a specific embodiment, the universal or normalized antibody comprises a wild-type Vk chain and a VH chain having at least one mutation.

[0099] In a specific embodiment, the universal or normalized or normalized antibody comprises a wild-type Vk chain and a VH chain with a Y52W mutation.

[0100] In another specific embodiment, the universal or normalized or normalized antibody comprises a wild type Vk chain and a VH chain with all of the following mutations: V4L, _83S, S96T, A54G, N101T, and Y52W, where _ indicates the amino acid positions that should be preserved as in the original wild type sequence. In yet another specific embodiment, the universal or normalized or normalized l antibody comprises a wild type Vk chain and a VH chain with all of the following mutations: V4L, R48K, _83S, S96T, A54G, and N101T.

[0101] In a specific embodiment, the universal or normalized antibody comprises a Vk chain with only one mutation and a wild-type VH chain. In another embodiment, the universal or normalized or normalized antibody comprises a wild-type Vk chain or Vlambda chain and a mutated VH chain. In another specific embodiment, the universal or normalized antibody comprises a wild-type Vk chain or Vlambda chain and a VH chain with one or more of the following mutations: V4L, R48K, _83S, S96T, A54G, N101T, or Y52W.

[0102] In another embodiment, the universal or normalized antibody comprises a Vk chain or Vlambda chain with at least one mutation and a VH chain with at least one mutation. In a specific embodiment, the Vk chain mutation is A2Q, S20T, S46P, A40S, Q52L, or P69S. In another specific embodiment, the Vk chain comprises at least two of the above mutations, with the proviso that the mutation is not S20T or Q52L. In another specific embodiment, the Vk chain comprises all of the following mutations: A2Q, S20T, S46P, A40S, and P69S. In another specific embodiment, the Vk chain comprises all of the following mutations: A2Q, S46P, A40S, Q52L, and P69S.

[0103] In another embodiment, the universal or normalized antibody comprises a Vk chain having one or more of the following mutations: A2Q, S20T, A40S, S46P, Q52L or P69S, with the proviso that the mutation is not S20T or Q52L, and the VH chain has one or more of the following mutations: V4L, R48K, _83S, S96T, A54G, N101T, and Y52W.

[0104] In another embodiment, the universal or normalized antibody has a Vk or Vlambda chain with at least one mutation and one or more of the following mutations: V4L, R48K, _83S, S96T, A54G, N101T, and Y52W.

[0105] In another embodiment, the universal or normalized antibody is used in diagnostic assay.In one embodiment, the assay is immunohistochemistry (IHC).In another embodiment, the assay is enzyme-linked immunosorbent assay (ELISA).In another embodiment, the diagnostic assay is immunocytochemistry (ICC).In a further embodiment, the diagnostic assay is flow cytometry or FACS.In yet a further embodiment, the diagnostic assay is radioimmunoassay (RIA).

[0106] The methods of the invention are not limited to use with antibodies known or found to be problematic with stability or aggregation or other properties addressed above, hi another embodiment, the methods of the invention can be used to standardize a panel of antibodies for an immunoassay. The following examples serve to illustrate certain embodiments of the present disclosure and are not intended to limit the disclosure, in fact other rabbit antibodies can be modified using the same methods. EXAMPLES

[0107] Example 1 - Mutagenesis for conversion to a universal or normalized template

[0108] A rabbit monoclonal antibody produced by Spring Bioscience was selected against the 9-mer peptide. M , T of 73.4C agg 266 73.8C T agg Expression yields of 473 and 0.51 mg / ml.

[0109] DNA sequences encoding the Vk and VH of selected rabbit monoclonal antibodies were obtained from Spring Bioscience, and expression vectors were constructed.

[0110] The expression vector was based on the pDV2 / pRK-Fc system ((Shang et al. 2015; Tesar and Hotzel 2013)) modified to express rabbit IgG constant regions instead of the original human sequences.

[0111] Concentrations were determined by comparing the absorbance values ​​of the sample antibody measured at several dilutions with the absorbance values ​​of a standard curve.

[0112] A series of VH and Vk chains were synthesized by making both individual and multiple sequence changes from the original wild-type antibody to a universal or normalized template (see FIG. 5A showing the VH and Vk series).

[0113] In the CDR3 and FR4 regions, the original IMGT residue C104 was maintained as in the wild type antibody. The wild type FR4 sequence was perfectly matched to that of the universal or normalized template. FR4 is identical to the universal or normalized template. (N0, IM, TH) Same as Figure 5. VH variants: (hwt) WT HC. Vk variants: (kwt) wild type light chain.

[0114] The VH and Vk series were expressed in HEK293 cells, such that each mutated heavy chain was paired with a wild-type kappa chain and vice versa.

[0115] ELISA was used to measure antibody concentrations in rabbits and to compare antibody binding among all mutants as described below.

[0116] Example 2 - ELISA Assay

[0117] Antibody concentrations and EC50s were measured via ELISA.

[0118] Concentrations were determined by comparing the absorbance values ​​of the sample antibody measured at several dilutions with the absorbance values ​​of a standard curve.

[0119] Using gene synthesis, different VH and Vk single and combinatorial mutants were generated and then cloned into pTT5 or pDV3a vectors, where CDR3 and FR4 were unchanged and FR4 was identical to the universal or normalized template.

[0120] Table 1 describes the wild-type and mutant sequences that were assayed. TIFF2025041680000002.tif148170

[0121] Notably, all the universal or normalized VH template-altered clones (Figure 6, "h.all") have the same affinity as the wild-type antibody.

[0122] In contrast, as shown in Figure 6C, Vk single mutations did not affect binding, but the combination of all Vk mutations ("k.all") abolished binding. It is hypothesized that at least two of the Vk mutations were synthetic lethal and were identified after testing as A40S and Q52L. As shown in Figure 6D, k.all without Q52L (-Q52L) and k.all without A40S (-A40S) did not restore binding to Vk with all mutations (k.all), whereas k.all without both A40S and Q52L (-A40S-Q52L) restored binding to levels similar to that of wild-type Vk (kwt).

[0123] A 3 μg / mL solution of goat anti-rabbit IgG (obtained from Jackson ImmunoResearch Laboratories, Inc. of West Grove, Pennsylvania ("Jackson Imm"); 411-005-003) in phosphate buffered saline ("PBS") was added to the wells of a round-bottom microtiter plate (Greiner 650061) in a volume of 100 μl / well. The plate was then sealed and stored overnight at 4° C.

[0124] The wells were emptied and refilled with 150 μl of 3% bovine serum albumin ("BSA") in PBS and incubated with shaking for 1 hour at room temperature. The wells were then emptied and 100 μl / well of serial dilutions of standard Chromopure Rabbit IgG whole molecule (Jackson Imm; 011.000.003) and sample culture supernatants were added.

[0125] Standards and unknowns were allowed to bind for 2 hours at room temperature with shaking. Wells were then emptied and washed at least four times with PBS + 0.5% Tween 20 ("PBST"). Horseradish peroxidase ("HRP")-conjugated goat anti-rabbit IG (Jackson Imm 111-035-046) was diluted 1 / 2000 in PBS and added in a volume of 100 μl / well.

[0126] The HRP conjugate was incubated for 45 minutes at room temperature with shaking, then the wells were emptied and washed at least four times with PBST.

[0127] TMB (Thermo Fisher catalogue no. 002023) was added at 100 μl / well and the plate was incubated at room temperature without shaking until the blue colour was stable (approximately 30 min).

[0128] Signal was read at 650 nm using a microtiter plate reader set and the plate was shaken for 3-5 seconds before reading.

[0129] Antibody binding curves were generated according to the ELISA protocol described above, except that microtiter plates were first prepared by plating 100 μl / well of a 0.5 μg / ml solution of the specific antigen in PBS.

[0130] Example 3 - Thermal Stability Analysis

[0131] To examine the stability of the mutant and wild-type sequences described above, thermostability was selected as a desirable property for optimization.

[0132] Thermostability can be measured quickly and accurately, and lack of thermostability can lead to additional problems associated with antibodies, such as aggregation or degradation.

[0133] Cell culture supernatants were diluted to 10ng / ml and 20μ aliquots were dispensed into 8-tube PCR strips. Each strip contained up to 8 different cell culture supernatants. Several strips were prepared equally, spun in a PCR strip centrifuge, and placed on ice.

[0134] The thermocycler was programmed for several successive 10 minute temperature holds, and the strips were placed in the thermocycler successively, each time removing the previous strip and replacing it with a new one, which was then centrifuged after incubation and placed back on ice.

[0135] Control strips were left on ice and treated strips were stored at 4° C. overnight.

[0136] The next day, ELISA was performed using microtiter plates coated with the specific antigens.

[0137] It was observed that several single mutations in VH improved thermal stability. In particular, the point mutation Y52W, as shown in Figure 6A, resulted in improved thermal stability. However, the stability benefit was additive, and maximum stability was achieved even in the absence of Y52W, provided that all other mutations were included simultaneously.

[0138] Figure 6B shows the thermal stability curves of WT VH and Vk mutants. The results indicate that the Vk mutants provided little to no improvement. Figure 6C shows xCD2 WT versus the most mutated VH and Vk sequences. The results indicate that the greatest stability was achieved by combining fully mutated VH and Vk with the most tolerated mutations.

[0139] The improvement in stability was unexpected since modifications of residues remote from the binding site can have significant effects on binding affinity.

[0140] Conversion to a universal or normalized template also allows for the determination of expression levels and T m Figure 7A shows that antibody preparations resulted in higher yields of the universal or normalized template antibody (17.3 mg) than the wild-type antibody (4.5 mg), i.e., a three-fold higher yield was obtained for the universal or normalized template clone, and a higher T for the universal or normalized template antibody (75.5°C) than the wild-type antibody (73.1°C). m The T of the universal or normalized template antibody (77.8°C) was higher than that of the wild-type antibody (73.4°C). agg 266 is higher, with a T of the universal or normalized template antibody (78.4°C) than the wild-type antibody (73.8°C). agg Figure 7B shows that the staining intensity of the universal or normalized template antibody is comparable to that of the wild-type antibody.

[0141] Advantages of the present invention can include that recombinant antibodies provide a reliable product source and starting point for efforts in protein engineering to improve bioconjugation, stability, and manufacturability. References The following references are incorporated herein by reference in their entireties: LeFranc MP, et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Dev.Comp.Immunol.2003;2(1):55-77. Honegger and Pluckthun, Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool. J. Mol. Biol. 2001;309(3):657-70. Chothiaら,1998,“Structural determinants in the sequences of immunoglobulin variable domain,”J Mol Biol.278(2):457-479。 Narciso JEら,Analysis of the antibody structure based on high-resolution crystallographic studies.N.Biotechnol.2011;28(5):435-47。 Schuck,P.Reliable determination of binding affinity and kinetics using surface plasmon resonance biosensors.Current Opinion in Biotechnology 8,498-502(1997)。 Gauglitz,G.Direct optical detection in bioanalysis:an update.Analytical and Bioanalytical Chemistry 398,2363-2372,doi:10.l007 / s00216-010-3904-4(2010)。 Friguet,B.,Chaffotte,A.F.,Djavadi-Ohaniance,F.&Goldberg,M.E.Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay.Journal of Immunological Methods.1985;77,305-319。 Vermeer and Norde,The thermal stability of immunoglobulin:unfolding and aggregation of A multi-domain protein.Biophys.J.2000;78(1):394-404。 Svilenovら,Isothermal chemical denaturation as A complementary tool to overcome limitations of thermal differential scanning fluorimetry in predicting physical stability of protein formulations;Biopharmaceutics.2018;125:106-113。 Strutzら.Exploring protein stability by NanoDSF;Biopysical J.2016;110(3):393A)。 Aminら,Protein aggregation,particle formation characterization&rheology.Curr.Op.in Colloid and Interface Science.2014;19(5):438-449)。 Devereuxら,Nucl.Acids Res.1984;12:387。 Neddleman and Wunsch,J.Mol.Biol.1970;48:443。 Smith and Waterman,Adv.Appl.Math,1981;2.482。 Yonglei Shang,Devin Tesar,Isidro Hotzel.Modular protein expression by RNA trans-splicing enables flexible expression of antibody formats in mammalian cells from A dual-host phage display vector.Protein Engineering,Design and Selection,Volume 28,Issue 10,1 October 2015,Pages 437-444。 Tesar D,Hotzel I.A dual host vector for Fab phage display and expression of native IgG in mammalian cells.Protein Eng Des Sel.2013 Oct;26(lO):655-62.doi:10.1093 / protein / gzt050.Epub 2013 Sep 24。 Yan,Analysis of oxidative Modification of Proteins.Curr.Protoc.Protein Sci.2009;56:14.4.1-14.4.28

Claims

1. 1. A method for producing a universal or normalized rabbit antibody, comprising: (a) selecting a wild-type rabbit antibody; (b) comparing said wild-type antibody sequence to a normalized antibody sequence that comprises in the variable framework and / or junction regions a set of amino acid residues that occur most frequently at the same positions in rabbit antibodies known to be thermostable and / or known to have long-term stability of at least 12 months, and identifying one or more amino acid residues in said wild-type antibody that differ from said normalized sequence; (c) mutating the one or more amino acid residues of the wild-type antibody identified in step (b) to a corresponding amino acid residue of the normalized sequence to obtain a universal or normalized rabbit antibody; wherein the binding affinity of said antibody to its target remains within an acceptable range when compared to the binding affinity of said unmodified wild-type antibody.

2. The method of claim 1, wherein the universal or normalized rabbit antibody comprises an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10 in the VH chain.

3. The method of claim 1, wherein the universal or normalized rabbit antibody comprises an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17 in the Vk chain.

4. 2. The method of claim 1, wherein the universal or normalized rabbit antibody is: (a) an amino acid sequence according to SEQ ID NO: 1, 4, 5, 6, 7, 8, 9, or 10, and (b) comprising an amino acid sequence according to SEQ ID NO: 2, 12, 13, 14, 15, 16, or 17.

5. The method of any one of claims 1 to 4, wherein the universal or normalized rabbit antibody exhibits improved thermal stability compared to the wild-type rabbit antibody.

6. The method of any one of claims 1 to 5, wherein the universal or normalized rabbit antibody exhibits improved long-term stability compared to the wild-type rabbit antibody.

7. The method of any one of claims 1 to 6, wherein the universal or normalized rabbit antibody exhibits improved deamination compared to the wild-type rabbit antibody.

8. The method of any one of claims 1 to 7, wherein the universal or normalized rabbit antibody exhibits improved expression compared to the wild-type rabbit antibody.

9. The method of any one of claims 1 to 8, wherein the universal or normalized rabbit antibody is a primary rabbit antibody.

10. The method of claim 1 , wherein the mutation is in the J1 region of the kappa chain.

11. The method of claim 1 , wherein the mutation is in the FR4 region of the kappa chain.

12. 2. The method of claim 1, wherein the mutation is in the J2, J4, and / or J6 region of the heavy chain.

13. 2. The method of claim 1, wherein the binding affinity of the antibody to its target remains within an acceptable range when compared to the binding affinity of the unmodified wild-type antibody.

14. The method of claim 1 , wherein the mutation is in a complementarity determining region.

15. A universal or normalized rabbit antibody obtained by the method according to any one of claims 1 to 14.

16. A universal or normalized rabbit antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, and 10 of the VH region.

17. A universal or normalized rabbit antibody comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 12, 13, 14, 15, 16, and 17 of the Vk region.

18. A universal or normalized rabbit antibody comprising an amino acid sequence of the VH region selected from the group consisting of SEQ ID NOs: 1, 4, 5, 6, 7, 8, 9, and 10, and an amino acid sequence of the Vk region selected from the group consisting of SEQ ID NOs: 2, 12, 13, 14, 15, 16, and 17.

19. The method of claim 1 , wherein the mutated universal or normalized antibody has improved properties for diagnostic use.

20. 20. The method of claim 19, wherein the improved property is selected from the group consisting of thermal stability, long term stability, reduced aggregation and reduced oxidation / deamidation.

21. The method of claim 1 , wherein the mutation is not in the complementarity determining region.

22. 1. A method for improving one or more properties in an antibody for diagnostic use, comprising the steps of: (a) selecting a wild-type rabbit antibody that is deficient in one or more properties selected from the group consisting of recombinant expression, thermostability, long-term stability, aggregation, and oxidation / deamidation; (b) comparing said wild-type antibody sequence to a normalized antibody sequence that comprises in the variable framework and / or junction regions a set of amino acid residues that occur most frequently at the same positions in a set of rabbit antibodies known to have the characteristics of (a) within an acceptable range, and identifying one or more amino acid residues in said wild-type antibody that differ from said normalized sequence; (c) mutating the one or more amino acid residues of the wild-type antibody identified in step (b) to a corresponding amino acid residue of the normalized sequence, wherein the one or more characteristics in step (a) are improved compared to the unmodified wild-type antibody.

23. A method for producing a recombinant rabbit monoclonal antibody, comprising the steps of: (a) selecting a wild-type rabbit antibody; (b) comparing said wild-type antibody sequence to a normalized antibody sequence that comprises a set of amino acid residues in the variable framework and / or interface regions that occur most frequently at the same positions in a set of rabbit antibodies known to have characteristics within an acceptable range selected from the group consisting of thermal stability, long-term stability, reduced aggregation, and reduced oxidation / deamidation, and identifying one or more amino acid residues in said wild-type antibody that differ from said normalized sequence; (c) preparing a standard expression vector encoding a complementarity determining region containing an alteration to the wild-type sequence of one or more of the residues identified in step (b); (d) expressing the vector in a suitable host cell; (e) purifying the antibody from step (d).

24. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 50 rabbit antibodies having said relevant characteristics.

25. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 100 rabbit antibodies having said relevant characteristics.

26. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 150 rabbit antibodies having said relevant characteristics.

27. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 200 rabbit antibodies having said relevant characteristics.

28. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 250 rabbit antibodies having said relevant characteristics.

29. 24. The method of any one of claims 1-14 or 19-23, wherein said set of amino acid residues of said normalized sequences occur most frequently at the same positions in at least 300 rabbit antibodies having said relevant characteristics.

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