Rabbit antibodies against human immunoglobulin G

Abstract

Disclosed are anti-human IgG antibodies and antigen-binding portions thereof obtained from rabbits and methods of using such antibodies and portions thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Patent Application No. 63 / 033,073, filed June 1, 2020, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on May 24, 2021, is named 022548_WO063_SL.txt and is 84,819 bytes in size. [Background technology]

[0003] Therapeutic monoclonal antibodies (mAbs) have become one of the fastest-growing drug classes in recent years, being approved for the treatment of a variety of indications, from cancer to autoimmune diseases. Preclinical pharmacokinetic characterization of these therapeutic mAbs is often performed in non-human primates to demonstrate efficacy and safety before initiating clinical trials. Cynomolgus monkeys are the preferred non-human primate for such preclinical studies because they often exhibit sufficient levels of cross-reactivity with the therapeutic antibody's target (Non-Patent Document 1). However, cynomolgus monkey immunoglobulins also exhibit high sequence homology with human immunoglobulins. Due to the high level of IgG protein sequence homology, the lack of high-quality reagent antibodies capable of distinguishing human therapeutic molecules in serum from cynomolgus monkey immunoglobulins presents a significant challenge for the bioanalytical measurement of human therapeutic antibodies in non-human primate serum samples (Non-Patent Document 2). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Iwasaki et al., Drug Metab Pharmacokinet. (2019) 34:55-63 [Non-patent document 2] Stubenrauch et al., J Pharm Biomed Anal. (2009) 49:1003-8 Summary of the Invention [Problem to be solved by the invention]

[0005] Currently, evaluation of the pharmacokinetics (PK) and pharmacodynamics (PD) of therapeutic antibodies in preclinical studies utilizes drug-specific anti-idiotypic antibodies, which require a lot of effort and time to develop. Each drug candidate requires its own anti-idiotypic antibody. There are few options for universal reagents that can detect all human IgG-based therapeutic antibodies in preclinical studies. Therefore, to accurately measure the levels of human IgG-derived therapeutic mAbs in non-human primates during preclinical studies, it is necessary to develop monoclonal antibodies that are universally specific for human IgG but do not bind to monkey IgG.

[0006] The present disclosure provides an isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human IgG, wherein the antibody or portion comprises heavy chain complementarity-determining regions (CDRs) 1-3 and light chain CDRs 1-3, each comprising SEQ ID NOs: 27-32, 33-38, 39-44, 45-50, 51-56, or 57-62. The antibody can be a rabbit antibody or modified from such a molecule (including, for example, chimeric antibodies with an Fc domain from a non-rabbit species, such as mouse, rat, or human). [Means for solving the problem]

[0007] In some embodiments, the antibody or portion comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) comprising SEQ ID NOs: 13 and 19, SEQ ID NOs: 14 and 20, SEQ ID NOs: 15 and 21, SEQ ID NOs: 16 and 22, SEQ ID NOs: 17 and 23, or SEQ ID NOs: 18 and 24, respectively. The antibody can be a rabbit antibody or is modified from such a molecule.

[0008] In some embodiments, the antibody comprises the heavy chain constant region amino acid sequence of SEQ ID NO: 25 and / or the light chain constant region amino acid sequence of SEQ ID NO: 26. In further embodiments, the antibody comprises heavy and light chains having the amino acid sequences of SEQ ID NOs: 63 and 69, SEQ ID NOs: 64 and 70, SEQ ID NOs: 65 and 71, SEQ ID NOs: 66 and 72, SEQ ID NOs: 67 and 73, or SEQ ID NOs: 68 and 74, respectively, with or without leader sequences.

[0009] In certain embodiments, the antibody or antigen-binding portion comprises a detectable label.

[0010] The present disclosure also provides compositions or kits comprising the monoclonal antibodies or antigen-binding portions in aqueous buffer solution.

[0011] In other aspects, the present disclosure provides isolated nucleic acid molecules encoding the heavy chain, light chain, or both of the present monoclonal antibodies or antigen-binding portions. In some embodiments, the nucleic acid molecules comprise SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and / or 12. In further embodiments, the nucleic acid molecules comprise SEQ ID NOs: 1 and 7, 2 and 8, 3 and 9, 4 and 10, 5 and 11, or 6 and 12. Also provided herein are expression constructs comprising nucleic acid molecules and host cells (e.g., mammalian cells) comprising nucleotide sequences encoding the heavy and light chains of the present monoclonal antibodies or antigen-binding portions. The present disclosure also provides methods for making an antibody or antigen-binding portion thereof, comprising culturing host cells under conditions allowing expression of the heavy and light chains of the antibody or portion, and isolating the antibody or portion from the cultured cells or cell culture supernatant.

[0012] In another aspect, the present disclosure provides a method for detecting human IgG or a fragment thereof in a sample, comprising contacting the sample with one or more monoclonal antibodies or antigen-binding portions described herein. The sample (e.g., a tissue sample such as a blood, serum, or plasma sample, or a biopsy sample) can be obtained, for example, from an animal administered an antibody or fragment thereof (e.g., a Fab or F(ab')2 fragment) comprising a human IgG constant region (e.g., a human IgG1, IgG2, IgG3, or IgG4 constant region). The animal can be, for example, a non-human primate such as a cynomolgus monkey or a rhesus monkey.

[0013] Other features, objects, and advantages of the present invention will become apparent in the following detailed description. It should be understood, however, that the detailed description, while indicating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description. [Brief explanation of the drawings]

[0014] [Figure 1] 1 is a panel of Biacore sensogram graphs showing binding of six rabbit recombinant antibody clones to whole human IgG1 (hIgG1), whole human IgG4 (hIgG4), human Fab (hFab), and cynomolgus monkey IgG (cynoIgG). [Figure 2A] Figure 2A shows the alignment of the heavy chain (Figure 2A) and light chain (Figure 2B) amino acid sequences of six rabbit recombinant antibody clones using the Clustal Omega and Kabat numbering systems, respectively. CDRs (see also Zhang and Ho, MABS (2017) 9(3):419-29) are underlined and in bold. The leader sequence, start of the variable domain, and start of the constant region are marked as indicated. [Figure 2B] Figure 2A shows the alignment of the heavy chain (Figure 2A) and light chain (Figure 2B) amino acid sequences of six rabbit recombinant antibody clones using the Clustal Omega and Kabat numbering systems, respectively. CDRs (see also Zhang and Ho, MABS (2017) 9(3):419-29) are underlined and in bold. The leader sequence, start of the variable domain, and start of the constant region are marked as indicated. [Figure 2C] FIG. 1 shows a phylogenetic tree of the VH and VL sequences of six rabbit antibody clones. [Figure 3A] Figure 3A shows epitope determination of six rabbit anti-hIgG clones by Biacore competition assay (Figure 3A) and Biacore kinetic assay sensorgram (Figure 3B). Antibodies MCA5748G (Bio-Rad), 19B1, 11F9, and mouse anti-hIgG mAb (Southern Biotech catalog no. 9042-01) were first biotinylated and captured on a Biacore SA chip at 500-600 RU. Eight 1:2 serial dilutions (80-0 nM) of hIgG1 antibody (isatuximab; "isa") were mixed with each of the competing antibodies (240 nM) prior to use. If the premixed competing antibody binds to hIgG1 at the same site on the chip surface as the antibody captured, hIgG1 will not be able to bind to the captured antibody. [Figure 3B]Figure 3A shows epitope determination of six rabbit anti-hIgG clones by Biacore competition assay (Figure 3A) and Biacore kinetic assay sensorgram (Figure 3B). Antibodies MCA5748G (Bio-Rad), 19B1, 11F9, and mouse anti-hIgG mAb (Southern Biotech catalog no. 9042-01) were first biotinylated and captured on a Biacore SA chip at 500-600 RU. Eight 1:2 serial dilutions (80-0 nM) of hIgG1 antibody (isatuximab; "isa") were mixed with each of the competing antibodies (240 nM) prior to use. If the premixed competing antibody binds to hIgG1 at the same site on the chip surface as the antibody captured, hIgG1 will not be able to bind to the captured antibody. [Figure 4A] Figure 4A shows an overlay graph of hIgG4 in an assay matrix containing 0%, 4%, 10%, and 25% cynomolgus monkey serum using clone 16F5 as the capture reagent; Figure 4B shows a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum; and Figure 4C shows a quality control graph in the same assay matrix containing 4% cynomolgus monkey serum. Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4), which served as the assay standard, was diluted 1:4 over the range of 1200 to 0.30 ng / mL. Alexa Fluor 647-conjugated goat anti-human IgG was used for detection. [Figure 4B]Figure 4A shows an overlay graph of hIgG4 in an assay matrix containing 0%, 4%, 10%, and 25% cynomolgus monkey serum using clone 16F5 as the capture reagent; Figure 4B shows a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum; and Figure 4C shows a quality control graph in the same assay matrix containing 4% cynomolgus monkey serum. Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4), which served as the assay standard, was diluted 1:4 over the range of 1200 to 0.30 ng / mL. Alexa Fluor 647-conjugated goat anti-human IgG was used for detection. [Figure 4C] Figure 4A shows an overlay graph of hIgG4 in an assay matrix containing 0%, 4%, 10%, and 25% cynomolgus monkey serum using clone 16F5 as the capture reagent; Figure 4B shows a standard curve graph of hIgG4 in an assay matrix containing 4% cynomolgus monkey serum; and Figure 4C shows a quality control graph in the same assay matrix containing 4% cynomolgus monkey serum. Clone 16F5 was biotinylated and used as the capture reagent. Human IgG4 antibody (hIgG4), which served as the assay standard, was diluted 1:4 over the range of 1200 to 0.30 ng / mL. Alexa Fluor 647-conjugated goat anti-human IgG was used for detection. [Figure 5A] Figure 5 shows a comparison of different clones used as capture reagents for the detection of hIgG4 and hFab. Figure 5A shows the standard curves of all six rabbit anti-hIgG mAb clones and the Gyrolab™ and MCA5748G capture agents for the detection of hIgG4. Figure 5B shows the same set of capture reagents used for the detection of hIgG Fab. It should be noted that hFab cannot be detected by either the Gyrolab™ or the MCA5748G capture agent. [Figure 5B]Figure 5 shows a comparison of different clones used as capture reagents for the detection of hIgG4 and hFab. Figure 5A shows the standard curves of all six rabbit anti-hIgG mAb clones and the Gyrolab™ and MCA5748G capture agents for the detection of hIgG4. Figure 5B shows the same set of capture reagents used for the detection of hIgG Fab. It should be noted that hFab cannot be detected by either the Gyrolab™ or the MCA5748G capture agent. [Figure 6A] Figure 1 shows a comparison of four antibodies used in the precipitation and acid dissociation (PandA) assay. The dynamic range and sensitivity of two rabbit anti-human IgG clones, 2C5 and 11G5, were compared to a commercially available mouse anti-Fc mAb, clone JDC-10 (Southern Biotech, catalog no. 9040-01). pAb: rabbit polyclonal anti-drug antibody (drug: monoclonal antibody with human IgG4 constant region). ECL: electrochemiluminescence. [Figure 6B] Figure 1 shows the performance of clones 2C5 and 11G5 in human, rat, monkey and mouse serum matrices in the PandA assay, respectively. Pos ctrl: positive control. [Figure 6C] Figure 1 shows the performance of clones 2C5 and 11G5 in human, rat, monkey and mouse serum matrices in the PandA assay, respectively. Pos ctrl: positive control. DETAILED DESCRIPTION OF THE INVENTION

[0015] The present disclosure provides rabbit monoclonal antibodies that bind with high affinity to human immunoglobulin G and its Fab fragments, but do not detectably bind to IgG from non-human primates such as monkeys (e.g., cynomolgus or rhesus monkeys). These rabbit antibodies are particularly useful as reagents for detecting human IgG or its fragments in preclinical pharmaceutical studies of human IgG-based therapeutic antibodies in cynomolgus monkeys or other non-human primates. For example, these rabbit antibodies can be used to study therapeutic antibodies that are fully human IgG antibodies, humanized IgG antibodies, chimeric antibodies with human IgG constant regions, and Fab fragments thereof. The rabbit antibodies can also be used in preclinical immunohistochemistry studies and in the development of manufacturing processes for human IgG-based therapeutic antibodies (e.g., whole antibodies, including monospecific, bispecific, and trispecific antibodies, and Fab fragments thereof).

[0016] The rabbit monoclonal antibodies bind to three unique epitopes on human IgG, which are distinct from the epitope bound by the commercially available mouse anti-hIgG antibody MCA5748G. Rabbit monoclonal antibodies offer several advantages over traditional mouse monoclonal antibodies, including higher binding affinity and specificity and more diverse epitope recognition. The rabbit immune system is evolutionarily distinct from that of rodents and uses different mechanisms to generate, diversify, and optimize the affinity of the antibodies it produces. In addition, the rabbit immune system can recognize smaller epitopes that are not immunogenic in mice, while maintaining the ability to generate a strong immune response. Therefore, the rabbit antibodies described herein are advantageous over mouse antibodies.

[0017] Rabbit anti-hIgG antibody The present disclosure provides antibodies that specifically (i.e., with high affinity) bind to human IgG and antigen-binding portions thereof (e.g., Fab and F(ab')2) and do not detectably bind to immunoglobulins (e.g., IgG) of other species (e.g., mouse, rat, rabbit, non-human primate, or dog) commonly used in preclinical studies.

[0018] As used herein, the term "affinity" refers to a measure of the attractive force between an antigen and an antibody. The intrinsic attractive effect of an antibody for an antigen is determined by the binding affinity equilibrium constant (K D ) is generally expressed as a high binding affinity, i.e., K D An antibody is said to specifically bind an antigen when the K is ≦100 nM (e.g., ≦10 nM or ≦1 nM). D The binding affinity constant can be measured by surface plasmon resonance (Biacore®) using, for example, a Biacore® T200 from Biacore. The binding affinity of a particular antibody-antigen interaction can also be demonstrated by a standard concentration-response curve using, for example, a Gyrolab™ xPlore from Gyros Protein Technologies. In some embodiments, the antibody exhibits a K D It binds to human IgG and Fab fragments derived therefrom at 2 nM or less, but shows no detectable binding to cynomolgus monkey IgG.

[0019] The antibodies exemplified herein bind to three different epitopes on hIgG and its Fab fragment. As used herein, the term "epitope" refers to the portion (determinant) of an antigen that specifically binds to a related molecule, such as an antibody or bispecific binding molecule. Epitope determinants generally consist of chemically active surface groupings of molecules, such as amino acids or carbohydrate or sugar side chains, and generally have specific three-dimensional structural and charge characteristics. Epitopes can be "linear" or "conformational." In a linear epitope, all of the points of interaction between a protein (e.g., an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction occur between amino acid residues on the protein that are separated from each other in the primary amino acid sequence. Once a desired epitope on an antigen has been determined, antibodies to that epitope can be generated using techniques well known to those skilled in the art. For example, antibodies to linear epitopes can be generated by, for example, immunizing animals with peptides containing the amino acid residues of the linear epitope. Antibodies to conformational epitopes can be generated by, for example, immunizing animals with a minidomain containing the relevant amino acid residues of the conformational epitope. Antibodies to specific epitopes can also be generated by, for example, immunizing animals with a target molecule of interest (e.g., IgG or Fab) or a relevant portion thereof and screening for binding to the epitope.

[0020] Methods known to those skilled in the art, including but not limited to competitive assays, epitope binning, and alanine scanning, can be used to determine whether an antibody binds to the same epitope or competes for binding with an anti-hIgG antibody of the present disclosure. In some embodiments, an anti-hIgG antibody of the present disclosure is bound to hIgG under saturating conditions, and then the ability of a test antibody to bind to hIgG is measured. If the test antibody can bind to hIgG simultaneously with a reference anti-IgG antibody, the test antibody binds to a different epitope than the reference anti-IgG antibody. However, if the test antibody cannot simultaneously bind to hIgG, the test antibody binds to the same epitope as, an overlapping epitope, or an adjacent epitope bound by an anti-IgG antibody of the present disclosure. This experiment can be performed, for example, using ELISA, RIA, BIACORE™, SPR, biolayer interferometry, or flow cytometry. To test whether an anti-hIgG antibody cross-competes with another anti-IgG antibody, the above-described competitive method can be used in two directions: to determine whether a known antibody blocks the test antibody, and vice versa. Competition experiments can be carried out, for example, using a Biacore® T200 instrument.

[0021] The antigen-binding portion of the anti-hIgG antibody disclosed herein can be used instead of the complete antibody. The term "antigen-binding portion" refers to one or more portions or fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., human IgG or a fragment thereof). Examples of antigen-binding portions include, but are not limited to, (i) a Fab fragment: a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment: a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment consisting of the VH domain; and (vi) an isolated complementarity-determining region (CDR) capable of specifically binding to an antigen. Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be connected by a synthetic linker, which allows recombinant methods to generate a single protein chain in which the VL and VH domains pair to form a monovalent molecule (known as a single-chain Fv (scFv)). Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain but use a linker that is too short to allow pairing between the two domains on the same chain; instead, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies or recombinant DNA technology.

[0022] The antibodies bind to one, more, or all of the human IgG subtypes hIgG1, hIgG2, hIgG3, and hIgG4. In certain embodiments, the antibodies bind all of the above subtypes. It is understood that the antibodies described herein can bind to humanized and / or chimeric antibodies containing sequences derived from human IgG.

[0023] In some embodiments, the present disclosure provides an anti-hIgG monoclonal antibody, or antigen-binding portion thereof, wherein the heavy chain CDRs 1-3 and the light chain CDRs 1-3 comprise SEQ ID NOs: 27-32, 33-38, 39-44, 45-50, 51-56, or 57-62, respectively. The antibody framework can be derived from an antibody of a rabbit or another species (e.g., a mouse, a human, or a rat).

[0024] In some embodiments, the disclosure provides an anti-hIgG monoclonal antibody, or antigen-binding portion thereof, whose heavy chain variable domain (VH) and light chain variable domain (VL) comprise SEQ ID NOs: 13 and 19, 14 and 20, 15 and 21, 16 and 22, 17 and 23, or 18 and 24, respectively. The constant region of the antibody can be derived from a rabbit or another species (e.g., mouse, human, or rat) antibody.

[0025] In some embodiments, the present disclosure provides: a) a heavy chain (HC) comprising the amino acid sequences of SEQ ID NOs: 13 and 25 and a light chain (LC) comprising the amino acid sequences of SEQ ID NOs: 19 and 26; b) an HC comprising the amino acid sequences of SEQ ID NOs: 14 and 25 and an LC comprising the amino acid sequences of SEQ ID NOs: 20 and 26; c) HC comprising the amino acid sequences of SEQ ID NOs: 15 and 25 and LC comprising the amino acid sequences of SEQ ID NOs: 21 and 26; d) HC comprising the amino acid sequences of SEQ ID NOs: 16 and 25 and LC comprising the amino acid sequences of SEQ ID NOs: 22 and 26; e) an HC comprising the amino acid sequences of SEQ ID NOs: 17 and 25 and an LC comprising the amino acid sequences of SEQ ID NOs: 23 and 26; or f) HC comprising the amino acid sequences of SEQ ID NOs: 18 and 25 and LC comprising the amino acid sequences of SEQ ID NOs: 24 and 26 The present invention provides an anti-hIgG monoclonal antibody comprising:

[0026] In some embodiments, the anti-hIgG antibody or antigen-binding portion has a VH amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13, 14, 15, 16, 17, or 18.

[0027] In some embodiments, the anti-hIgG antibody or antigen-binding portion has a VL amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19, 20, 21, 22, 23, or 24.

[0028] In some embodiments, the anti-hIgG antibody or antigen-binding portion has VH and VL amino acid sequences that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 13 and 19, 14 and 20, 15 and 21, 16 and 22, 17 and 23, or 18 and 24, respectively.

[0029] In some embodiments, the anti-hIgG antibody has an HC and LC comprising SEQ ID NOs: 63 and 69, 64 and 70, 65 and 71, 66 and 72, 67 and 73, or 68 and 74, respectively, with or without the leader sequence.

[0030] In some embodiments, an anti-hIgG antibody or antigen-binding portion of the disclosure comprises the HCDR1-3 and LCDR1-3, VH and VL or HC and LC amino acid sequences of antibody 2C5, 9E6, 11F9, 11G5, 16F5, or 19B1.

[0031] Amino acid numbering and CDR assignments can follow the definitions in Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD (1987 and 1991). See also Zhang, supra.

[0032] The anti-hIgG antibodies or antigen-binding portions of the present disclosure can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or portion thereof is derivatized so that IgG binding is not adversely affected by the derivatization or labeling. For example, an antibody or antibody portion of the present disclosure can be operably linked (by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or a detectable label or tag. Examples include radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 90 Y, 99 Tc, 111 In, 125 I, 131 Examples of suitable labels include, but are not limited to, fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores, phycoerythrin, or Alexa Fluor® dyes), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and magnetic agents such as gadolinium chelates. In some embodiments, labels are attached by spacer arms of various lengths to reduce potential steric hindrance.

[0033] The anti-hIgG antibodies and antigen-binding portions of the present disclosure are useful for detecting and / or measuring levels of human IgG or Fab in samples derived from animals (e.g., non-human primates such as cynomolgus or rhesus monkeys). In some embodiments, the antibodies and antigen-binding portions can be used to detect and / or measure levels of human IgG or Fab in samples derived from humans. Suitable detection and measurement methods include immunological methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and immunohistology. In some embodiments, the antibodies and antigen-binding portions can be used to detect and / or measure levels of human IgG or Fab in samples derived from humans for preclinical or clinical immunohistochemistry (IHC) studies.

[0034] The rabbit antibodies described herein can bind to different epitopes, so they can be used alone or in pairs to detect human IgG in any host animal to meet the needs of therapeutic monoclonal antibody development in preclinical trials. For example, antibodies 16F5, 11F9, and 11G5 / 19B1 / 2C5 / 9E6 bind to three different epitopes that are different from the epitope of MCA5748G. Therefore, a pair of antibodies selected from antibodies binding to two different epitopes can be used together to, for example, increase assay sensitivity and specificity. For example, 16F5 can be used together with 11F9; 16F5 or 11F9 can be used together with 11G5, 19B1, 2C5, or 9E6; MCA5748G can be used with any one of 16F5, 11F9, 11G5, 19B1, 2C5, and 9E6. The pair of antibodies is labeled differently.

[0035] Preparation of anti-hIgG antibody The present anti-hIgG antibodies can be produced by well-known hybridoma technology in which rabbit B cells producing the antibody of interest are fused with immortalized cells to form antibody-producing hybridoma cell lines.

[0036] Alternatively, the present hIgG antibody or antigen-binding portion thereof is produced by recombinant technology using a host cell containing a nucleotide sequence encoding the heavy and light chains of the antibody or portion. Thus, the present disclosure also provides nucleic acid molecules and sequences encoding the anti-IgG antibody or antigen-binding portion thereof described herein. The nucleotide sequences encoding the heavy and light chain amino acid sequences are introduced into the host cell on two different vectors or on the same vector. They are expressed under the transcriptional control of one promoter or two separate promoters.

[0037] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to a nucleotide sequence encoding (i) SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; or (ii) SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26.

[0038] In the context of amino acid and nucleic acid sequences, the term "percent sequence identity" refers to the residues that are identical in two sequences when aligned for maximum correspondence.For example, the length of sequence identity comparison is at least about 9 nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, generally at least about 28 nucleotides, more usually at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides.There are many different algorithms known to those skilled in the art that can be used to measure the identity of amino acid and nucleotide sequences.For example, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs of Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wisconsin. For example, FASTA, including the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (see, e.g., Pearson, Methods Enzymol. (1990) 183:63-98; Pearson, Methods Mol. Biol. (2000) 132:185-219; Pearson, Methods Enzymol. (1996) 266:227-58; and Pearson, J. Mol. Biol. (1998) 276:71-84; incorporated herein by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with default parameters (word size 6 and NOPAM factor in the scoring matrix) or using Gap with default parameters provided in GCG Version 6.1, incorporated herein by reference.

[0039] In certain embodiments, the disclosure provides a nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NOs: 1 and 7, 2 and 8, 3 and 9, 4 and 10, 5 and 11, or 6 and 12.

[0040] In any of the above embodiments, the nucleic acid molecule is isolated. A nucleic acid molecule referred to herein as "isolated" or "purified" is (1) separated from the nucleic acid of the genomic DNA or cellular RNA of its original source; and / or (2) is a non-naturally occurring nucleic acid.

[0041] In a further aspect, the present disclosure provides vectors suitable for expressing one or both chains of an antibody or antigen-binding portion thereof described herein. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0042] The present disclosure provides vectors comprising nucleic acid molecules encoding the heavy chain, light chain, or both the heavy and light chains of an anti-hIgG antibody, or antigen-binding portion thereof, described herein. The vectors can further comprise expression control sequences.

[0043] As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary to affect the expression and processing of ligated coding sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosomal binding site, and a transcription termination sequence; in eukaryotes, such control sequences generally include a promoter and a transcription termination sequence. The term "control sequence" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0044] In some embodiments, the nucleic acid molecules described herein comprise a nucleotide sequence encoding the VH domain of an anti-IgG antibody or antigen-binding portion described herein joined in-frame to a nucleotide sequence encoding a heavy chain constant region from any source. Similarly, the nucleic acid molecules described herein can comprise a nucleotide sequence encoding the VL domain of an anti-IgG antibody or antigen-binding portion described herein joined in-frame to a nucleotide sequence encoding a light chain constant region from any source.

[0045] In a further embodiment of the present disclosure, nucleic acid molecules encoding VH and / or VL are "converted" into full-length antibody genes. In some embodiments, nucleic acid molecules encoding the VH or VL domains are converted into full-length antibody genes by inserting them into expression vectors already encoding heavy chain constant (CH) or light chain constant (CL) regions, respectively, such that the VH segment is operably linked to the CH segment within the vector and / or the VL segment is operably linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding the VH and / or VL domains are converted into full-length antibody genes by joining, e.g., ligating, the nucleic acid molecules encoding the VH and / or VL domains to nucleic acid molecules encoding the CH and / or CL regions using standard molecular biology techniques. The nucleic acid molecules encoding the full-length heavy and / or light chains are then expressed from the transfected cells, and anti-IgG antibodies are isolated.

[0046] In some embodiments, the framework regions are mutated so that the resulting framework regions have the amino acid sequence of the corresponding germline gene. Mutations can be made in the framework regions or constant regions, for example, to increase the half-life of an anti-IgG antibody. See, for example, PCT Publication WO 00 / 09560. Mutations in the framework regions or constant regions can also be made to modify the immunogenicity of the antibody and / or to provide sites for covalent or non-covalent binding to another molecule. According to the present disclosure, the antibody can have mutations in any one or more of the CDRs or framework regions of the variable domain or in the constant region.

[0047] The present disclosure also provides methods for producing the antibody compositions and antibodies and antigen-binding portions thereof described herein. In some embodiments, the present disclosure relates to a method for producing the anti-IgG antibodies or antigen-binding portions described herein, comprising the steps of: providing a recombinant host cell containing a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof and a nucleotide sequence encoding the light chain or antigen-binding portion thereof of the anti-IgG antibodies or antigen-binding portions described herein; culturing the host cell under conditions suitable for expression of the antibody or antigen-binding portion; and isolating the resulting antibody or antigen-binding portion. The antibody or antigen-binding portion produced by such expression in such a recombinant host cell is referred to herein as a "recombinant" antibody or antigen-binding portion. The present disclosure also provides progeny cells of such host cells, and the antibodies or antigen-binding portions produced thereby.

[0048] As used herein, the term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. By definition, a recombinant host cell does not exist in nature. The present disclosure provides host cells that can include, for example, a vector described herein. The present disclosure also provides host cells that include, for example, a nucleotide sequence encoding the heavy chain or antigen-binding portion thereof, a nucleotide sequence encoding the light chain or antigen-binding portion thereof, or both, of an anti-IgG antibody or antigen-binding portion thereof described herein. It should be understood that "recombinant host cell" and "host cell" refer not only to the particular subject cell but also to the progeny of that cell. Because some modifications may exist in subsequent generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still intended to be included within the scope of the term "host cell" herein.

[0049] Nucleic acid molecules encoding anti-IgG antibodies and their antigen-binding portions, as well as vectors containing these nucleic acid molecules, can be used to transfect suitable mammalian, plant, bacterial, or yeast host cells. Transformation can be performed by any known method for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known to those skilled in the art, and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells using viral vectors.

[0050] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation patterns from each other. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present disclosure, regardless of the glycosylation state of the antibody, and more generally, regardless of post-translational modifications.

[0051] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In the case of conflicts, the present specification, including definitions, will control. Generally, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, bacteriology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used by those of ordinary skill in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished by those skilled in the art or as described herein. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise," or variations such as "has," "having," "comprises," or "comprising," will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many documents are cited in this specification, this citation is not an admission that any of these documents form part of the common general knowledge of those skilled in the art.

[0052] In order that the present disclosure may be better understood, the following examples are set forth. These examples are for illustrative purposes only and should not be construed as limiting the scope of the disclosure in any way. [Example]

[0053] The following example describes an experiment in which rabbits were immunized with human IgG Fab, splenocytes were isolated, and then used to select human IgG-specific B cells. Cynomolgus monkey IgG was used as a counterscreening agent to obtain six human IgG- and Fab-specific mAb clones. These clones demonstrated superior binding affinity and targeted epitopes distinct from the only commercially available anti-hIgG mAb clone, MCA5748G, of murine origin. These rabbit anti-hIgG mAb clones were evaluated using the Gyrolab™ assay and found to be suitable for use as capture reagents in comprehensive pharmacokinetic assays in the presence of cynomolgus monkey serum. The materials and methods for the experiments described herein are as follows.

[0054] Chemicals and Reagents The therapeutic antibodies used in these experiments were humanized therapeutic monoclonal antibody IgG1 (hIgG1), humanized development candidate monoclonal antibody IgG4 (hIgG4), and in-house research reagent human Fab (hFab). Cynomolgus monkey IgG (cynoIgG) was purified by protein A affinity purification from cynomolgus monkey serum purchased from Innovative Research (Novi, MI 48377). Mouse anti-human IgG monoclonal antibody MCA5748G (Stubenrauch et al., J Pharm Biomed Anal. (2009) 49:1003-8) was purchased from BioRad Laboratories (Hercules, CA). Cell culture medium and phosphate-buffered saline (PBS) were purchased from ThermoFisher Scientific (Waltham, MA). All other chemicals were of analytical quality.

[0055] ELISA to determine binding specificity Enzyme-linked immunosorbent assays (ELISAs) were used to assess the specificity of antibody clones for binding to human immunoglobulin (IgG), using cynoIgG as a control. ELISAs were performed at room temperature on microtiter plates from ThermoFisher Scientific (Waltham, MA) that were first coated with hIgG1, hIgG4, or cynoIgG in PBS for 1 hour. After washing three times with phosphate-buffered saline-polysorbate 20 (Tween 20), the plates were blocked with PBS / 3% bovine serum albumin for 1 hour. The plates were then washed again and incubated with anti-human IgG antibody clones for 1 hour. After another washing step, bound antibody was detected with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody from Southern Biotech (Birmingham, AL) according to the manufacturer's instructions.

[0056] Biacore assays to determine binding specificity and kinetics The specificity of the rabbit anti-human IgG monoclonal antibody was evaluated in a second assay system described elsewhere (Chu et al., Sci Rep. (2015) 4:7360). These experiments were performed on a Biacore® T200 instrument (Biacore, Uppsala, Sweden) using Biacore streptavidin or CM5 sensor chips. Coating of the streptavidin chip with antibody was achieved by injecting amine-coupled biotinylated target antibodies using the EZ-Link™ Amine-PEG11-Biotin reagent from ThermoFisher Scientific (Waltham, MA) according to the manufacturer's instructions. In the case of the CM5 chip, the target antigen or antibody was coupled to the chip surface by standard amine coupling using the Biacore amine coupling kit. Unless otherwise stated, all binding and kinetic assays were performed in HBS-EP+ buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% vol / vol Surfactant P20) at 25° C. Dissociation constants (KD) were calculated using a 1:1 Langmuir fitting model using Biacore's BIAevaluation software V4.1.

[0057] Rabbit immunization and B cell cloning Two rabbits were immunized with a total of five antigen injections using the hIgG Fab described in Wu et al., Nat Cancer (2020) 1:86-98. Complete Freund's adjuvant (CFA) was used for the primary injection, and incomplete Freund's adjuvant (IFA) was used for the four booster immunizations. Both CFA and IFA were from ThermoFisher Scientific (Waltham, MA). Serum titers were monitored by ELISA using the antigen protein. Rabbits with higher ELISA titers were selected for splenectomy.

[0058] For B cell isolation, fresh splenocytes were isolated from the spleen. Approximately 1.2 × 10 8Splenocytes were cultured overnight in a specialized B cell medium customized by Yurogen (Worcester, MA) before sorting. Splenocytes were processed using Yurogen's SMab™ platform to enrich for antigen-recognizing B cells. Antigen-selected B cells were seeded at 1 cell / well in 96-well plates and cultured for 10-14 days.

[0059] B cell clones that recognized the antigen were identified and confirmed using a direct ELISA coated with hIgG4, and purified cynoIgG was used for counterscreening in ELISA. Antigen-specific B cell clones were ranked and selected according to the positive / negative ELISA signal ratio, and the heavy and light chains of the IgG coding sequences were amplified by RT-PCR. The heavy and light chain PCR products were combined and used to directly transfect HEK293F cells. Transiently expressed recombinant rabbit IgG clones were then further confirmed for specific binding to hIgG1, hIgG4, and hFab by ELISA and Biacore binding assays. Once specific binding to hIgG1, hIgG4, and hFab was confirmed, PCR products from selected positive B cells were cloned into a mammalian expression vector to expand antibody production in HEK293F cells. Recombinant rabbit mAb clones produced by HEK293F transfection were purified using protein A chromatography for further evaluation.

[0060] Gyrolab™ Assay Gyrolab™ xPlore, Bioaffy 1000nL CD, Rexxip A, and Rexxip F buffers from Gyros Protein Technologies (Uppsala, Sweden) were used for all experiments (Fraley et al., Bioanalysis (2013) 5:1765-74). Biotinylated capture antibodies were diluted to 0.1-0.2 μg / μL in Rexxip A buffer and run on a streptavidin bead column within the Bioaffy CD microstructure. Standard curve and quality control (QC) samples were prepared by spiking the indicated ranges of hIgG4 or hFab into Rexxip A buffer containing various amounts of cynomolgus monkey serum. Standard curve samples, QC samples, mock samples, and assay reagents were added to a PCR plate and loaded into the Gyrolab™ instrument. One duplicate of the standard curve, QC sample, or mock sample was loaded onto two CD microstructures by the Gyros instrument and then run through the bead column. Alexa fluor 647-labeled goat anti-human IgG (Fc) antibody purchased from Southern Biotech (Birmingham, AL) was used as the detection reagent at 2 μg / mL in Rexxip F buffer. A wash solution of PBS containing 0.01% v / v Tween-20 was run through the column before each run to pre-wet the streptavidin beads, and any unbound reagent was washed off after each step of the assay. Sample concentrations were determined by data collection at a 1% photomultiplier level. Results were analyzed using the Gyrolab™ Evaluator Program with a five-parameter fit and 1 / Y2 weighting as instructed by the manufacturer. [Example]

[0061] Isolation of a human IgG-specific rabbit antibody clone This example describes experiments in which six rabbit antibody clones that recognize both human IgG and Fab, but not cynomolgus monkey IgG, were developed using rabbit B cell cloning techniques.

[0062] Hybridoma screening and display methodologies have been used to develop rabbit monoclonal antibodies, but both have several drawbacks: hybridoma technology has low cell fusion efficiency, and display methods eliminate the natural cognate pairs of heavy and light chains (Zhang et al., Front Immunol. (2017) 8:494). To overcome these problems, single B cell-based antibody gene cloning technology (or single B cell cloning) has recently been developed (Seeber et al., PLoS ONE (2014) 9:e86184; Rashidian et al., "Single B Cell Cloning and Production of Rabbit Monoclonal Antibodies," in Zielonka and Krah (eds.) Genotype Phenotype Coupling. Methods in Molecular Biology, Vol. 2070, Humana, New York, NY, 2020).

[0063] Briefly, single B cell cloning consists of the following steps: (i) isolating specific single B cells from peripheral blood or lymphoid tissues by antigen-based FACS sorting; (ii) growing and expanding the single B cells for two weeks; (iii) performing RT-PCR using antibody-specific primers to amplify and sequence the antibody genes; (iv) cloning the antibody genes into an expression vector and producing recombinant monoclonal antibodies in mammalian cell lines (e.g., HEK293, CHO cells); and (v) purifying the recombinant monoclonal antibodies and evaluating them by ELISA and other in vitro assays.

[0064] Rabbits were immunized with human IgG1 Fab, and the resulting splenocytes were selected with biotinylated hIgG1, a humanized whole IgG1 molecule. A total of 530 primary B cells were seeded at single cell in a 96-well plate and grown for 2 weeks. B cell culture media containing monoclonal rabbit IgG antibodies were screened by direct ELISA using hIgG4, hIgG1, and cynoIgG. Based on the ELISA hIgG1 / cynoIgG and hIgG4 / cynoIgG signal values, 17 clones with both hIgG1 and hIgG4 OD450 > 0.9 and CynoIgG OD450 < 0.2 were selected (Table 1), and their antibody coding sequences were amplified by PCR.

[0065] [Table 1]

[0066] Of these 17 clones, 11 were able to obtain both heavy and light chain PCR products. The heavy and light chain PCR products from each clone were combined in a 1:1 ratio and used to directly transfect HEK293F cells. The cell culture medium from the transfected HEK293F cells was further confirmed by the same ELISA screening assay. Table 2 shows the binding of six rabbit recombinant antibody clones that specifically bind to whole human IgG (hIgG) and Fab but not to cynomolgus monkey IgG (cynoIgG), as indicated by the signal ratios of hIgG1 or hIgG4 to cynoIgG binding determined by ELISA assay. These six clones exhibited excellent hIgG4 / cynoIgG and hIgG1 / cynoIgG ELISA signal ratios ranging from 4.94 to 14.99.

[0067] [Table 2]

[0068] These six clones were further analyzed for direct binding to hIgG1, hIgG4, hFab, and cynoIgG by Biacore, as shown in Figure 1. All six clones showed varying levels of binding to hIgG1, hIgG4, and hFab, but not to cynoIgG. These data indicate that these clones are specific for human IgG and Fab, but not for cynoIgG. [Example]

[0069] Rabbit anti-hIgG antibody sequence To characterize the six clones, the heavy and light chain PCR products were subjected to DNA sequencing. The deduced amino acid sequences of the heavy and light chain variable regions were aligned using the EMBL-EBI web-based Clustal Omega (Sievers et al., Mol Syst Biol. (2011) 7:539). As shown in Figures 2A and 2B, all six clones possessed unique amino acid sequences for both the heavy and light chains. A phylogenetic tree based on Clustal Omega was constructed to visualize the relative distances (Figure 2C). In particular, clone 16F5 was highly divergent from the other five clones in both the heavy and light chains, while clone 11F9 was the next most divergent clone in the heavy chain but not in the light chain.

[0070] Table 3 shows the nucleotide sequences encoding antibodies 2C5, 9E6, 11F9, 11G5, 16F5 and 19B1 (SEQ ID NO:).

[0071] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0072] Table 4 shows the deduced amino acid sequences of the variable domains of antibodies 2C5, 9E6, 11F9, 11G5, 16F5 and 19B1. Complementarity determining regions (CDRs) are bolded and underlined.

[0073] [Table 4]

[0074] Table 5 shows the heavy and light chain constant region amino acid sequences (CH and CL, respectively) of six antibodies: 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1.

[0075] [Table 5]

[0076] Table 6 shows the amino acid sequences of the heavy chain CDRs (HCDRs) and light chain CDRs (LCDRs) of antibodies 2C5, 9E6, 11F9, 11G5, 16F5, and 19B1, where the CDRs are defined according to the Kabat numbering system. The sequence numbers are shown in parentheses.

[0077] [Table 6]

[0078] Table 7 shows the sequence number information for antibodies 2C5, 9E6, 11F9, 11G5, 16F5 and 19B1. All sequences in the table are amino acid sequences except when indicated by "nt" (nucleotides).

[0079] [Table 7] [Example]

[0080] Binding affinity and epitope of rabbit anti-hIgG antibodies The amplified PCR products containing the heavy and light chain antibody coding sequences were cloned into expression vectors and used for transient transfection and antibody purification. Mouse anti-rabbit mAb was directly immobilized onto a CM5 chip using amine coupling (Chu et al., Sci Rep. (2015) 4:7360). Purified rabbit mAb clones were then injected, followed by 1:2 serial dilutions of hFab, hIgG1, hIgG4, and cynoIgG ranging from 80 to 0 nM.

[0081] As illustrated in Table 8, all clones exhibited subnanomolar or stronger binding affinities (KD) for hIgG1, hIgG4, and hFab, but not for cynoIgG. However, under the same conditions, the control commercially available clone MCA5748G (Bio-Rad, Hercules, CA) was able to bind to hIgG1 and hIgG4, but not to hFab and cynoIgG.

[0082] [Table 8]

[0083] To determine whether the six rabbit anti-hIgG clones possessed epitopes distinct from those of the only commercially available clone, MCA5748G, we biotinylated the MCA5748G, 19B1, and 11F9 clones, as well as a mouse anti-hIgG mAb (assay control). These biotinylated antibodies were then injected into different flow cells of a streptavidin chip to reach a range of 500–600 RU, followed by a 1:2 serial dilution of hIgG1 antibodies ranging from 80 to 0 nM in the presence of a competitor rabbit mAb clone at 240 nM. As shown in Figures 3A and 3B, when MCA5748G was used as the capture antibody on the chip surface, competition was observed only with clone MAC5748G itself, indicating that this commercially available mouse anti-hIgG clone, MAC5748G, possesses an epitope distinct from all six of the rabbit anti-hIgG clones disclosed herein.

[0084] When rabbit anti-hIgG clone 19B1 was used as the capture antibody on the chip surface, competition was observed with clones 11G5, 19B1, 2C5, and 9E6. Thus, the epitopes of these four clones were identical but distinct from those of clones 11F9 and 16F5. Furthermore, when clone 11F9 was captured on the chip surface, competition was observed only with clone 11F9 itself, but not with clone 16F5 or any of the other four clones sharing the same epitope. Thus, clone 16F5 also possessed an epitope distinct from that of clone 11F9. These results indicated that clones 16F5 and 11F9 each possessed a unique epitope, while clones 11G5, 19B1, 2C5, and 9E6 shared the same epitope. Paratopes within the CDRs determine the antibody-binding epitopes, with the heavy chain playing a predominant role, and the epitope classification of the six rabbit anti-hIgG clones correlates well with the phylogenetic tree relationships (Fig. 2C).

[0085] Additional Biacore experiments demonstrated that all six rabbit anti-hIgG clones were capable of binding to each of the 10 different human IgGs and two Fabs tested. The 10 antibodies included those with human IgG1 and IgG4. Because these rabbit anti-hIgG mAb clones were generated using Fab as the immunogen, all of them should bind within the Fab region of IgG. Because different human IgG subtypes (IgG1, IgG2, IgG3, and IgG4) are classified by the Fc sequence, which is not part of the Fab region, all six of the rabbit anti-hIgG antibody clones should be able to recognize all subtypes of human IgG. This will be a major advantage if these rabbit anti-hIgG mAbs are used to detect therapeutic human IgG molecules of different subtypes (e.g., IgG1, IgG2, and IgG4) during preclinical trials and / or development.

[0086] In summary, amino acid sequence alignment demonstrated that all six clones obtained herein were unique and diverse. They bound to three distinct epitope groups (16F5; 11F9; and 11G5 / 19B1 / 2C5 / 9E6), demonstrated superior binding affinity, and targeted different epitopes than the only commercially available anti-hIgG antibody clone of murine origin, MCA5748G. Clones with different binding epitopes can be used to develop pairwise human IgG detection assays, such as "sandwich" ELISAs and Gyrolab™ assays. [Example]

[0087] Specific detection of human IgG by rabbit antibodies in the Gyrolab assay To test whether rabbit anti-hIgG mAb could detect human IgG molecules in the presence of monkey serum, biotinylated clone 16F6 was first used as a capture reagent. Gyrolab™ xPlore, Bioaffy 1000nL CD, Rexxip A, and Rexxip F buffers from Gyros Protein Technologies (Uppsala, Sweden) were used for all experiments. The biotinylated capture antibody was diluted to 0.1–0.2 μg / μL in Rexxip A buffer and run on a streptavidin bead column within the Bioaffy CD microstructure. Standard curves and quality control (QC) samples were prepared by spiking hIgG4 or hFab at the indicated ranges into Gyrolab™ Rexxip A buffer mixed with 0, 4%, 10%, and 25% cynomolgus monkey serum. Human IgG4 antibody hIgG4 was used to prepare a standard curve of 1:4 dilutions ranging from 1200 to 0.3 ng / mL in the assay matrix.

[0088] As shown in Figure 4A, no matrix effect was observed at cynomolgus monkey serum concentrations between 0 and 4% over the range of 1200 and 0.3 ng / mL. When the assay matrix contained 10% and 25% cynomolgus monkey serum, some slight background signal was observed in the lower half of the standard curve. Therefore, an assay matrix containing 4% cynomolgus monkey serum was further tested with three QCs (750, 40, and 0.6 ng / mL hIgG4) using the same standard curve. As shown in Figure 4B and Figure 4C, a typical standard curve was obtained, with two of the three QCs meeting both <20% bias and <20% CV. However, the higher QC missed the bias cutoff by only 0.9% (20.9%), and its CV was typical (4.56%).

[0089] The suitability of other rabbit anti-hIgG clones for use as capture reagents in the Gyrolab™ assay was further tested. All six rabbit anti-hIgG clones were biotinylated, as was the commercially available mouse anti-hIgG mAb MCA5748G clone, and used at similar concentrations in the Gyrolab™ assay. The Gyrolab™ comprehensive PK assay capture reagent was used as a control. As shown in Figures 5A and 5B, when the whole antibody molecule hIgG4 was used as the standard (same range, 1200–0.3 ng / mL), all capture reagents generated concentration-dependent curves, with the highest background observed for the commercially available clone MCA5748G. However, when the Fab antibody molecule hFab was used as the standard (same range, 1200–0.3 ng / mL), all six rabbit anti-hIgG capture reagents similarly generated concentration-dependent curves. In contrast, the commercially available mouse anti-hIgG clone MAC5748G and the control Gyrolab™ capture reagent failed to generate concentration-dependent curves. [Example]

[0090] Dynamic range of rabbit mAb clones in the Gyrolab assay One advantage of the Gyrolab™ assay is that it exhibits a significantly greater dynamic range compared to surface-based assay platforms such as ELISA and MSD (Fraley et al., Bioanalysis (2015) 5:1765-74). Again using the human whole antibody hIgG4, a linear 6-point assay standard was prepared with 1:5 dilutions ranging from 5000 to 0.32 ng / mL, as shown in Table 9.

[0091] [Table 9]

[0092] A similar Gyrolab™ assay procedure was performed, and data were then extracted and compared. At the upper limit (5000 ng / mL), Gyrolab™ capture and rabbit mAb clones 9E6, 2C5, and 19B1 all had acceptable bias (<20) and signal-to-noise ratios greater than 100. The three rabbit anti-hIgG clones showed signal-to-noise ratios superior to Gyrolab™ capture. At the high endpoint of 5000 ng / mL, clone 11G5 had an acceptable average bias, but its signal-to-noise ratio was low (63.3%), which was attributed to a relatively high blank signal level (7.4). For the same reason, the commercially available mAb clone MCA5748 also had a very poor signal-to-noise ratio (24.0) at the upper limit.

[0093] These data suggested that clones 9E6, 2C5, and 19B1 exhibited a similar dynamic range with a better signal-to-noise ratio when compared to the Gyrolab™ capture reagent. The dynamic range of detection with clones 9E6, 2C5, and 19B1 was significantly better than that of the commercially available mouse anti-hIgG clone MCA5748G. Clones 16F5 and 11F9 performed well with an average bias of <20% from 0 to 1000 ng / mL, but did not perform at the 5000 ng / mL data point. This may be due to the fact that these two clones possess different binding epitopes than the other four clones.

[0094] Thus, when this experiment was performed using clone 16F5 as the capture reagent in the initial Gyrolab™ assay, a concentration-dependent assay curve was generated in an assay matrix containing up to 25% cynomolgus monkey serum. The assay performed well in an assay matrix containing 4% cynomolgus monkey serum over a range of 0.30 to 1200 ng / mL. In an expanded Gyrolab™ evaluation assay, all six rabbit mAb clones demonstrated the ability to serve as capture reagents for the detection of both total human IgG and Fab molecules. However, two control capture reagents, the Gyros capture reagent and the commercially available clone MCA5748G, were only able to detect total human IgG and not Fab molecules.

[0095] Six rabbit anti-human IgG mAb clones were developed that could bind to both whole human IgG and Fab molecules without binding to cynomolgus monkey IgG, as summarized in Table 10. These six clones belonged to three different epitope groups and were distinct from the commercially available clone MCA5748G. Each of these clones was tested for the detection of human IgG and Fab in the Gyrolab™ assay, and three of them exhibited a greater dynamic range and signal-to-noise ratio.

[0096] [Table 10] [Example]

[0097] Use of a rabbit anti-human IgG monoclonal antibody clone as a comprehensive positive control for the PandA assay. Rabbit polyclonal anti-drug antibodies (pAbs) are commonly used as performance controls for anti-drug antibody (ADA) detection. However, the generation of pAbs is time-consuming and requires repeated use of experimental animals. Furthermore, pAbs often exhibit low sensitivity as controls. This example describes a study comparing two recombinantly produced rabbit anti-human IgG mAbs, 2C5 and 11G5, with a commercially available mouse anti-Fc mAb (JDC-10; Southern Biotech catalog number 9040-01) as a positive control in the PandA assay.

[0098] 2C5 and 11G5 were diluted with JDC-10 in monkey plasma pool at 5 μg / mL, 1 μg / mL, 0.75 μg / mL, 0.5 μg / mL and 0 μg / mL, respectively.

[0099] Plasma samples containing diluted antibody were first diluted 1:5 in assay buffer (300 mM acetic acid, 2% BSA) containing excess drug (human monoclonal antibody of the IgG4 isotype; 10–50 μg / mL) and incubated in a polypropylene plate at 450 rpm for 1 hour at 37°C to allow complex formation between the drug and the added antibody in the sample. Then, 3% PEG in borate (pH 8.0) was added to each sample and incubated overnight at 2–8°C. The final concentration of PEG buffer in each sample was 1.5%.

[0100] The next day, the plates were centrifuged at 4000 rpm for 20 minutes to precipitate the complexes into a pellet. The pellet was resuspended in 1.5% PEG in borate (pH 8.0) and centrifuged a second time at 4000 rpm for 20 minutes. The wash cycle was repeated three times. After the final centrifugation, each sample was suspended in 100 μL of 300 mM acetic acid and further diluted 1:10 (20 μL sample + 180 μL acetic acid) for a final sample dilution of 1:50. The diluted samples were added in duplicate to wells of an MSD High Bind plate at 25 μL per well and incubated for 1 hour at 24°C with shaking at 450 rpm.

[0101] After incubation, the plates were washed with 1x plate wash buffer and blocked with 3% milk in PBS at 24°C for 1 hour with shaking. The plates were then washed, and 100 ng / mL sulfo-TAG-Drug was added to the samples and incubated at 24°C for 1 hour with shaking. After the final incubation, the plates were washed with 0.05% Tween in PBS. Read buffer T 2x was then added, and the plates were read on a Sector PR2400. The electrochemiluminescence (ECL) signal in each sample was proportional to the anti-drug antibody.

[0102] As shown in Figure 6A and Table 11, the two rabbit anti-hIgG clones 2C5 and 11G5 demonstrated greater dynamic range and sensitivity compared to the commercially available anti-Fc mAb clone JDC-10.

[0103] [Table 11]

[0104] Clones 2C5 and 11G5 also served as performance controls and were tested in human, rat, monkey, and mouse plasma and serum matrices. The data show that both 2C5 (Figure 6B) and 11G5 (Figure 6C) can distinguish between human, monkey, mouse, and rat serum, with 2C5 being more sensitive than 11G5 (Table 12). In human matrices, background was increased for both clones, as expected.

[0105] [Table 12]

[0106] The above results demonstrated that recombinantly produced rabbit anti-human IgG mAbs serve as excellent global positive controls for preclinical assays, especially for PandA formats (e.g., monkey, rat, and mouse matrices). These antibodies performed well in both plasma and serum matrices. The dynamic range and assay sensitivity of the rabbit IgG mAbs were improved.

Claims

1. 1. An isolated monoclonal antibody or antigen-binding portion thereof that specifically binds to human IgG, the antibody or portion thereof SEQ ID NOs: 27 to 32, SEQ ID NOs: 33 to 38, SEQ ID NOs: 39 to 44, SEQ ID NOs: 45 to 50, SEQ ID NOs: 51 to 56 or SEQ ID NOs: 57-62 and a light chain CDR1-3, each of which comprises:

2. the antibody or portion thereof SEQ ID NOs: 13 and 19, SEQ ID NOs: 14 and 20, SEQ ID NOs: 15 and 21, SEQ ID NOs: 16 and 22, SEQ ID NOs: 17 and 23, or SEQ ID NOs: 18 and 24 2. The monoclonal antibody or antigen-binding portion of claim 1, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), each comprising:

3. The monoclonal antibody according to claim 1 or 2, wherein the antibody is a rabbit IgG antibody.

4. The monoclonal antibody according to any one of claims 1 to 3, comprising the heavy chain constant region amino acid sequence of SEQ ID NO: 25 and / or the light chain constant region amino acid sequence of SEQ ID NO:

26.

5. with or without a leader sequence, SEQ ID NOs: 63 and 69, SEQ ID NOs: 64 and 70, SEQ ID NOs: 65 and 71, SEQ ID NOs: 66 and 72, SEQ ID NOs: 67 and 73, or SEQ ID NOs: 68 and 74 2. The monoclonal antibody of claim 1, comprising a heavy chain and a light chain each having the amino acid sequence:

6. The monoclonal antibody or antigen-binding portion of any one of claims 1 to 5, further comprising a detectable label.

7. A composition or kit comprising the monoclonal antibody or antigen-binding portion of any one of claims 1 to 6 in an aqueous buffer solution.

8. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen-binding portion of any one of claims 1 to 6.

9. 9. The nucleic acid molecule of claim 8, comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and / or 12.

10. SEQ ID NOs: 1 and 7, SEQ ID NOs: 2 and 8, SEQ ID NOs: 3 and 9, SEQ ID NOs: 4 and 10, SEQ ID NOs: 5 and 11, or SEQ ID NOs: 6 and 12 The nucleic acid molecule of claim 9, comprising:

11. An expression construct comprising the nucleic acid molecule of any one of claims 8 to 10.

12. A host cell comprising nucleotide sequences encoding the heavy and light chains of the monoclonal antibody or antigen-binding portion of any one of claims 1 to 6.

13. The host cell of claim 12 , wherein the host cell is a mammalian cell.

14. 14. A method for producing an antibody or antigen-binding portion thereof, comprising culturing the host cell of claim 13 under conditions that allow expression of the heavy and light chains of the antibody or portion, and isolating the antibody or portion from the cultured cell or cell culture supernatant.

15. A method for detecting human IgG or fragments thereof in a sample, comprising contacting the sample with one or more monoclonal antibodies or antigen-binding portions thereof according to any one of claims 1 to 6.

16. 16. The method of claim 15, wherein the sample is obtained from an animal administered an antibody or fragment thereof comprising a human IgG constant region.

17. 17. The method of claim 16, wherein the human IgG constant region is a human IgG1, IgG2, IgG3, or IgG4 constant region.

18. 17. The method of claim 16, wherein the animal is administered a Fab or F(ab')2 fragment of an antibody comprising a human IgG1, IgG2, IgG3, or IgG4 constant region.

19. The method according to any one of claims 15 to 18, wherein the sample is a tissue sample.

20. The method of any one of claims 16 to 19, wherein the animal is a non-human primate.

21. The method of claim 19, wherein the tissue sample is a blood, serum or plasma sample.

22. The method of claim 20, wherein the non-human primate is a cynomolgus monkey or a rhesus monkey.

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