Homologous dimerization peptide and antibody containing same

Abstract

Homologous dimerization (HD) peptides with improved self-binding are described. The HD peptides may comprise an amino acid sequence having a reversed configuration compared to the corresponding naturally occurring HD peptide, or may comprise the amino acid sequence of a naturally occurring HD peptide with one or more hydrophilic substitutions. Additionally, dimers of HD peptides are described. Antibodies comprising said HD peptides or HD dimers, as well as methods of making and using said antibodies, are also disclosed.

Description

[Technical field]

[0001] The present invention relates to homologous dimerization peptides, antibodies comprising said homologous dimerization peptides, and methods and uses thereof. [Background technology]

[0002] Antibodies have been hailed as "magic bullets" for fighting disease and have emerged as major therapeutic tools for treating chronic diseases such as cancer and autoimmune disorders. Notable success stories include Herceptin® in the treatment of breast cancer and Rituxan® in the treatment of non-Hodgkin's lymphoma. The key advantage of antibodies in treating disease lies in their ability to target disease-causing cells or molecules while sparing healthy tissues and the body's normal products.

[0003] Currently, there are 16 FDA-approved monoclonal antibodies with annual sales of $62 billion. This is expected to grow to 70 approved products generating $120 billion in annual sales by 2025. Furthermore, the newest and most successful cancer therapy of the last decade, monoclonal antibodies (mAbs) directed against suppressor molecules on the surface of cancer cells such as PD-L1, have proven to be able to release the brakes on cancer patients' immune systems, resulting in high therapeutic activity in cancers that are not well treated by traditional chemotherapy. This goal alone is expected to generate $35 billion in annual sales by 2025.

[0004] However, antibodies that exhibit the desired specificity in laboratory studies often fail in preclinical and clinical evaluation due to inefficient targeting, low biological activity, poor therapeutic efficacy, and / or unacceptable side effects, due in part to the fact that antibodies represent only one arm of immune defense, with T cells providing the other strategy in immune defense.

[0005] Antibodies are an ideal platform for targeting and delivery devices. Antibodies have been used as delivery devices (ADCs) for several biologically active molecules, such as toxins, drugs and cytokines. In some cases, fragments of antibodies, such as antigen-binding fragments (Fabs) or single-chain variable fragments (scFvs), are preferred for better tissue penetration. Despite the fact that there are currently several approved ADCs, their development is very costly and requires long approval times.

[0006] The preferred therapeutic mAb format is human (or humanized) IgG1, and mAb therapeutic activity is conferred by its binding function (agonist, antagonist) or effector function [complement-mediated cytotoxicity (C'MC), antibody-dependent cell-mediated toxicity (ADCC) or induction of apoptosis]. mAbs are already adapted for long-term survival in blood, possess sites that facilitate vascular and tissue penetration, and are functionally associated with several defense mechanisms of innate immunity.

[0007] It is known that the main mechanism by which therapeutic antibodies are effective against their target cells is by inducing cell death, i.e., antibody-induced apoptosis. Such induced apoptosis is typically triggered by crosslinking receptors that are part of the cell's apoptotic signaling pathway. For example, crosslinking of B cell antigen receptors by antibodies induces apoptosis in B cell tumors (Ghetie M., et al., 1997). Crosslinking of cell receptors also increases the binding avidity of the antibody to its target antigen, and thus likely increases all cell surface-dependent therapeutic mechanisms such as complement-mediated killing and complement-dependent opsonization and phagocytosis, antibody-dependent cellular cytotoxicity (ADCC), and, when using antibodies that target cell receptors, inhibiting cell proliferation or enhancing changes in metabolic pathways within the cell through increased binding to cell receptors and blocking the cell receptor.

[0008] The therapeutic properties of an antibody can be enhanced in terms of affinity for its target antigen by using Fab libraries aimed at "evolving" native antibodies. This can occasionally result in an increase in affinity of as much as 100-fold to 1000-fold. However, this does not overcome the fundamental nature of monoclonal antibody binding. With respect to protein epitopes, a monoclonal antibody typically binds a single epitope with one arm, and when the mAb dissociates from its target, the next target is typically too far away for the mAb to rebind. Such an obstacle can be overcome by using targeting systems where the mAb can span the distance between epitopes, which typically requires a very high antigen density clustered on the membrane. One example is cell surface immunoglobulins on B cells. Unfortunately, there are few therapeutic targets of this nature.

[0009] Antigen binding can be enhanced by simultaneously engaging epitopes on adjacent target antigens, increasing the chances that a mAb will crosslink its target. For many targets, this crosslinking is a powerful means of triggering apoptosis. The likelihood of crosslinking can be increased by increasing the valency of antibodies, such as pentameric IgM antibodies. This can also be done by recombinant means to create multimeric immunoglobulin molecules from IgG (Xiao-Yun Liu, Laurentiu M. Pop, Lydia Tsai, Iliodora V. Pop and Ellen S. Vitetta, Int. J. Cancer 129, 497-506 (2011)). However, for most therapeutic targets, crosslinking cannot be achieved even by increasing the valency and size of the immunoglobulin molecules.

[0010] Valency and avidity are increased in a rare class of self-binding or homophilic antibodies, variously known as "autophilic antibodies" or "autobodies", identified in Nature (Kang, CY, Cheng, HL, Rudikoff, S. and Kohler, HJ Exp. Med. 165:1332, (1987); Xiyun, AN, Evans, SV, Kaminki, MJ, Fillies, SFD, Reisfeld, RA, Noughton, AN and Chapman, PBJ Immunol. 157:1582-1588 (1996)). This arises as a result of secondary interactions (to antigen binding) and can incorporate multiple IgGs and span any distance on the cell surface between targets. They can form dimers and / or polymers by non-covalent interactions with self. One example of an autophilic antibody is TEPC-15 (T15), which targets the normally cryptic determinants of phosphorylcholine on apoptotic cells and atherosclerotic lesions (Binder, J., et al., 2003; Kang, CY, et al., 1988). Dimerization or multimerization can only be induced after the modified antibody has attached to its cell surface target, i.e., after "differential oligomerization." In solution, an autophilic antibody can be in equilibrium between its monomeric and dimeric forms (Kaveri S., et al., 1990). Unfortunately, Nature has produced only a few of these types of antibodies, and they are directed against a limited number of targets.

[0011] A peptide in the heavy chain region of the TEPC-15 (T15) antibody was identified as being self-binding and conferring greater therapeutic activity to the antibody (Kang, CYBrunck, TK, Kieber-Emmons, T., Blalock, JE and Kohler, H., Science, 240:1034-1036, 1988). Such peptides are known as "autophilic peptides" or "homologous dimerization (HD) peptides." The elucidation of this and other peptide sequences with similar ability to induce antibody self-association has provided an opportunity to confer the same property of self-association to other antibodies targeting different antigens. More recently, the nature of self-association has been investigated and preferences for synthetic forms of peptides forming secondary and tertiary features have been deduced (Bost KL, Blalock JE. Viral Immunol. 2(4), 229-238 (1989); Kohler, H, Immunotherapy (2013) 5(3), 235-246).

[0012] In an effort to enhance the antigen detection and / or therapeutic efficacy of known antibodies, hybrid molecules containing two different covalent binding domains have been proposed. For example, US Patent Application Publication No. 2003 / 0103984 (Kohler) and US Patent Application Publication No. 2004 / 0185039 (Kohler) disclose fusion proteins containing an antibody domain and a peptide domain, where the peptide domain can have autophilic activity. WO2009 / 002939 discloses an immunoglobulin component having binding affinity for the CD-20 antigen fused to an autophilic peptide. WO2009 / 108803 discloses a method and kit for detecting an analyte in a sample using an antibody conjugated to an autophilic peptide.

[0013] However, there remains a need to improve the sensitivity and effectiveness of antibodies for the detection, prevention and / or treatment of disease. Summary of the Invention

[0014] The present invention relates to homologous dimerization (HD) peptides with improved biological activity of self-association or homologous dimerization. The present invention also relates to fusion proteins (e.g., chemically conjugated or recombinant antibodies) comprising said HD peptides. The fusion protein comprising an immunoglobulin component or an antibody and an HD peptide may be recombinant, or the HD peptide may be conjugated thereto in a manner that does not interfere with antigen binding and allows for favorable conformational changes in the HD peptide sequence, thereby conferring dimerization activity. The HD peptides disclosed herein may be used to enhance the potency of therapeutic antibodies or to increase binding sensitivity and / or avidity for other applications such as diagnostics.

[0015] In one aspect, a homologous dimerization (HD) peptide is provided that comprises an amino acid sequence in a reversed configuration compared to a corresponding naturally occurring HD peptide or a conservative variant thereof, hi some embodiments, the corresponding naturally occurring HD peptide may be a T15 HD peptide or an R24 HD peptide.

[0016] In some embodiments, the HD peptide can comprise an amino acid sequence having at least 90% sequence identity to RSVIFRGKVSASYETTYDNAKNRSA (SEQ ID NO: 4) or AYNISSGGSSIYAY (SEQ ID NO: 6), ie, the reverse amino acid sequence of the T15 and R24HD peptides.

[0017] In a further aspect, there is provided a homologous dimerization (HD) peptide, or a conservative variant thereof, comprising one or more substitutions that confer improved self-binding properties. The one or more substitutions may be hydrophilic substitutions. Thus, the HD peptides described herein may comprise one or more amino acid substitutions, where the one or more substitutions increase the hydropathy of the HD peptide.

[0018] In some embodiments, a homologous dimerization (HD) peptide may comprise the amino acid sequence ASRNKANDYTTEYSASVKGRFIVSR (SEQ ID NO:1) with one or more substitutions at nucleotide positions 4, 7, and 18. The one or more amino acid substitutions at position 4 may be a K or a conserved amino acid substitution of K, the one or more amino acid substitutions at position 7 may be a R or a conserved amino acid substitution of R, and the one or more amino acid substitutions at position 18 may be a H or a conserved amino acid substitution of H.

[0019] In some embodiments, the one or more substitutions may be N4K, N7R and / or K18H.

[0020] In some embodiments, the HD peptide may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO:8, 9, or 10.

[0021] In yet another aspect, a homologous dimerization (HD) peptide dimer is provided that comprises the above-mentioned HD peptide. The HD peptides of the dimer may be linked by a linker. In some embodiments, the linker may be gly-gly.

[0022] Further provided is a homologous dimerization (HD) peptide dimer comprising a first HD peptide and a second HD peptide, wherein the first and second HD peptides are derived from a naturally occurring HD peptide, or the first and second HD peptides are derived from the reverse sequence of a naturally occurring HD peptide.

[0023] In some embodiments, the HD peptide dimer can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5 or 7, i.e., a dimer of T15 or R24HD peptide linked by a gly-gly linker.

[0024] In another embodiment, the HD peptide dimer can comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 11, 12, 13, 14, or 15. In one embodiment, the HD peptide dimer comprises an amino acid sequence having 80-100% sequence identity to SEQ ID NO: 5, 7, 11, 12, 13, 14, 15, 17, or 19.

[0025] The present invention further provides antibodies or antigen-binding fragments comprising an HD peptide or HD peptide dimer as described herein fused thereto. Antibodies having an HD peptide or HD peptide dimer as described herein have been found to confer improved binding and therapeutic properties.

[0026] In some embodiments, the antibody or antigen-binding fragment is a humanized IgG. For example, the antibody or antigen-binding fragment may be a humanized IgG1, humanized IgG4, or humanized IgG3.

[0027] The HD peptide or HD peptide dimer may be located at any suitable site in the antibody. In some embodiments, the HD peptide or HD peptide dimer may be fused to the nucleotide affinity site of the antibody or antigen-binding fragment. For example, the HD peptide or HD peptide dimer may be fused via a lysine, cysteine, or carbohydrate.

[0028] In some embodiments, the HD peptide or HD peptide dimer is positioned immediately following the CDR3 of the heavy or light chain of the antibody. In alternative embodiments, the HD peptide or HD peptide dimer is positioned immediately following the C-terminus of the heavy or light chain constant region of the antibody. In alternative embodiments, the HD peptide or HD peptide dimer is positioned immediately following the C-terminus of the heavy chain variable region of the antibody. In alternative embodiments, the HD peptide or HD peptide dimer is positioned directly at the C-terminus of the Fc region of the antibody.

[0029] The antibodies described herein can be formed by conjugating HD peptides or HD peptide dimers thereto. Alternatively, the antibodies can be produced by recombinant methods. The present invention also relates to methods for producing recombinant antibodies and peptides capable of self-associating forming lattices and cross-linking their target antigens. Such cross-linking can then result in enhanced cell signaling, enhanced receptor blockade, internalization of antigen / antibody complexes, and even induction of apoptosis.

[0030] In some embodiments, the attachment position of the HD peptide or HD peptide dimer to the antibody may be preceded by a spacer, such as gly-gly.

[0031] The antibody or antigen-binding fragment (Fab) may be a single chain antibody (scFv), a bispecific antibody (BsAb) or an antibody-like peptide.

[0032] In some embodiments, the antibody is a humanized monoclonal antibody. The antibody may be a Her-2 neu antibody, such as Herceptin. The antibody may be a CD-20 antibody, such as Rituxin. The antibody may be a vascular endothelial growth factor antibody, such as Avastin. The antibody may be a checkpoint inhibitor antibody, such as PD-L1.

[0033] Additionally, compositions comprising one or more of the antibodies or antigen-binding fragments described herein and a pharma- ceutically acceptable carrier are provided.

[0034] Further provided is an expression vector comprising a first nucleic acid sequence encoding an HD peptide or HD peptide dimer described herein, in some embodiments, the expression vector further comprises a second nucleic acid sequence encoding an antibody or antigen-binding fragment, such that when the first and second nucleic acid sequences are expressed, the HD peptide or HD peptide dimer and the antibody or antigen-binding fragment are expressed as a fusion protein.

[0035] Methods for producing a homodimerizing (HD) antibody are provided, comprising expressing an expression vector described herein in a host cell. For example, the host cell may be an animal cell, a yeast cell, or a plant cell.

[0036] An isolated host cell transformed with an expression vector described herein is provided.

[0037] Methods are provided for enhancing antibody binding and / or efficacy comprising conjugating an HD peptide or HD peptide dimer described herein to an antibody or recombinantly expressing an antibody with the HD peptide or HD peptide dimer.

[0038] Provided is a method of treating a patient suffering from a disease or condition, the method comprising administering an antibody or antigen-binding fragment described herein, the disease or condition may be selected from the list consisting of cancer, an autoimmune disorder, an inflammatory disorder, a neurodegenerative disease, a cardiovascular disease, and graft or transplant rejection.

[0039] Provided is a method for detecting an analyte in a sample, the method comprising contacting the analyte with an antibody or antigen-binding fragment to the analyte, where the antibody or antigen-binding fragment is fused to an HD peptide or HD peptide dimer as described herein, and detecting a complex formed by the analyte and the antibody fused to the HD peptide or HD peptide dimer.

[0040] Kits for detecting an analyte in a sample are provided that include an antibody or antigen-binding fragment to the analyte, the antibody or antigen-binding fragment fused to an HD peptide or HD peptide dimer described herein, and instructions for use in detecting the analyte.

[0041] Phage display libraries are provided that contain antibodies or antigen-binding fragments linked to the HD peptides or HD peptide dimers described herein.

[0042] The antibodies described herein may be used in therapy, e.g., in the prevention and / or treatment of disease. The antibodies described herein may also be used in diagnosis, e.g., in in vitro diagnostic assays.

[0043] This summary of the invention does not necessarily describe all features of the invention.

[0044] These and other features of the present invention will become more apparent from the following description taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0045] [Figure 1A] FIG. 2 shows the amino acid sequence (aa 50-70) of the homophilic domain in the T15 antibody, showing (from left to right) CDR2 and framework 3. [Figure 1B] FIG. 13 shows the carbon background trace of the T15 sequence aa50-70 within the fold of the MPC 603 Fab structure. [Figure 1C] FIG. 1 shows a ball representation of the T15 aa50-70 MPC603 structure. [Figure 1D] FIG. 1 shows an alignment of T15 peptides showing hydropathic interactions.

[0046] [Diagram 2] FIG. 1 shows the hydropathy profile of the T15 HD peptide (T15) and its reverse sequence (rs-T15) using the Kyte-Doolittle algorithm with a window of 7 residues.

[0047] [Diagram 3] FIG. 3 shows the hydropathic alignment of the T15 peptide profiled in FIG. 2 and its reverse sequence (rs-T15).

[0048] [Figure 4]Figure 2 shows amino acid hydropathy analysis of T15 peptides in three germline sequences derived from heavy chain CDR2 / framework 3 of Mopc antibodies with no, little or high HD activity. Top peptide: T15 peptide with amino acids contributing to self-binding shown in greyscale. Hydropathy scores of individual amino acids shown above or below. Middle peptide: low HD binding, amino acid substitutions from T15 shown in greyscale. Bottom peptide: no HD binding (Mpc 167), amino acid substitutions from T15 shown in greyscale.

[0049] [Diagram 5] Figure 1 shows the temperature-dependent self-association of G11, S107 and G9 antibodies at 4°C and 37°C under non-physiological conditions (see Bryan, JA and Kohler, H. Physical and Biological Properties of Homophilic Therapeutic Antibodies, Cancer Immunology Immunotherapy, 60:507, 2010). The time required for the meniscus to reach equilibrium after horizontal displacement from above and below was measured. Error bars represent the percent variation of two runs.

[0050] [Figure 6] Figure 1 shows human lymphoma cells where binding of Rituxin and HD-Rituxin was detected by flow cytometry: Rituxin identifies two primary populations of cancer cells, one with low antigen density (second arrow) and a second with higher density (third arrow).

[0051] [Figure 7]Figure 1 shows complement-dependent cytotoxicity of Rituxin and HD-Rituxin. Rituxin can mediate C'MC against three different cell lines (Raji, Ramon and JOK1) with different antigen densities (light bars). C'MC with HD-Rituxin is significantly enhanced, demonstrating higher levels of killing at lower antibody concentrations (dark bars). Antibody-dependent cell-mediated cytotoxicity (ADCC) is also enhanced with HD antibodies (similar results were obtained with HD-Herceptin, data not shown).

[0052] [Figure 8] FIG. 1 shows the efficacy of Herceptin and HD-Herceptin in a nude mouse model of low antigen expressing breast cancer (MCF-7) 7 days after tumor injection.

[0053] [Figure 9] 1 shows the HD peptide sequence from antibody R24, with the amino acid interactions within the hairpin loop that result in self-binding also shown.

[0054] [Figure 10] FIG. 1 shows the hydropathy profile of the R24 HD peptide (R24) using the Kyte-Doolittle algorithm.

[0055] [Figure 11] 1 shows detection of PSA antibodies using electrochemiluminescence (e.g., for diagnostics). The graph shows the signal generated with anti-PSA antibodies modified by HD technology and with unmodified antibodies.

[0056] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The following description is of a preferred embodiment.

[0058] The present disclosure relates to homologous dimerization (HD) peptides. More specifically, the present disclosure relates to artificial or synthetic homologous dimerization (HD) peptides. The HD peptides of the present disclosure have improved biological activity of self-association or homologous dimerization.

[0059] In one aspect, the HD peptide may be an artificial or synthetic HD peptide derived from a naturally occurring HD peptide or a conservative variant thereof. For example, the HD peptide may comprise an amino acid sequence in a reversed arrangement compared to the corresponding naturally occurring HD peptide or a conservative variant thereof. In one embodiment, the artificial or synthetic HD peptide may comprise an amino acid sequence in a reversed arrangement to that of the T15 HD peptide or the R24 HD peptide.

[0060] In a further aspect, there is provided a homologous dimerization (HD) peptide, or a conservative variant thereof, comprising one or more substitutions that confer improved self-binding properties. The one or more substitutions may be hydrophilic substitutions. The homologous dimerization (HD) peptide comprises one or more amino acid substitutions, wherein the one or more substitutions increase the hydropathy of the HD peptide.

[0061] In some embodiments, a homologous dimerization (HD) peptide can comprise the amino acid sequence ASRNKANDYTTEYSASVKGRFIVSR (SEQ ID NO: 1) with one or more substitutions at nucleotide positions 4, 7, and 18. The one or more amino acid substitutions at position 4 can be a substitution of K or a substitution of a conserved amino acid for K, the one or more amino acid substitutions at position 7 can be a substitution of R or a substitution of a conserved amino acid for R, and the one or more amino acid substitutions at position 18 can be a substitution of H or a substitution of a conserved amino acid for H. An HD peptide can comprise an amino acid sequence having 80% to 100% sequence identity to SEQ ID NO: 8, 9, or 10.

[0062] Also provided are artificial or synthetic HD peptide dimers, which may include dimers of naturally occurring HD peptides, dimers of reverse sequences of naturally occurring HD peptides, or conservative variants thereof. The homologous dimerization (HD) peptide dimer may include a first HD peptide and a second HD peptide, where the first and second HD peptides are derived from a naturally occurring HD peptide, or the first and second HD peptides are derived from the reverse sequence of a naturally occurring HD peptide. The first and second HD peptides of the dimer may be linked by a linker. The HD peptide in the dimer may further include one or more substitutions as described herein. The homologous dimerization (HD) peptide dimer may include an amino acid sequence having 80-100% sequence identity to SEQ ID NO: 5, 7, 11, 12, 13, 14, 15, 17, or 19.

[0063] Thus, the artificial or synthetic HD dimer may comprise a first HD peptide and a second HD peptide, where the first, second or first and second HD peptides are derived from a naturally occurring HD peptide or a conservative variant thereof. Furthermore, the artificial or synthetic HD may be an HD dimer and may comprise a first HD peptide and a second HD peptide, where the first, second or first and second HD peptides are derived from the reverse sequence of a naturally occurring HD peptide or a conservative variant thereof. For example, the first and second HD peptides may be derived from a T15 HD peptide, an R24 HD peptide, a reverse T15 HD peptide, a reverse R24 HD peptide or a conservative variant thereof. For example, the first and second HD peptides of the dimer may comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 5, 7, 11, 12 or 13.

[0064] The HD peptides within a dimer may be linked by a linker, hi some embodiments, the linker may be gly-gly.

[0065] The disclosure further provides fusion proteins (e.g., chemically conjugated or recombinant antibodies) comprising the HD peptides and / or HD dimers described herein. The fusion proteins may include an immunoglobulin component or an antibody, and the HD peptide or HD peptide dimer may be recombinant, or the HD peptide or HD peptide dimer may be conjugated thereto in a manner that does not interfere with antigen binding but allows for favorable conformational changes in the HD peptide sequence, thereby conferring dimerization activity. The HD peptides and HD peptide dimers disclosed herein may be used to enhance the potency of therapeutic antibodies or to increase binding sensitivity and / or avidity for other uses, such as diagnostics.

[0066] The term "homodimerization", also called "autophilic", refers to an entity that self-associates. For example, a "homodimerizing antibody" or an "autophilic antibody" is an antibody that binds to itself. The term "homodimerization (HD) peptide" or "autophilic peptide" is a peptide that allows, for example, an antibody or other immunoglobulin to self-associate or bind to itself.

[0067] The term "naturally occurring HD peptide" or "native HD peptide" refers to an HD peptide found in nature. For example, "T15" refers to an anti-phosphorylcholine antibody, and T15 HD peptide refers to the HD peptide found in the T15 antibody having the amino acid sequence SRNKADYTTEYSASVKGRFIVSR (SEQ ID NO: 1). "R24" refers to an antibody that recognizes disialoganglioside GD3 (J. Biol. Chem 374:5597-55604, 1999), and R24 HD peptide refers to the HD peptide found in the R24 antibody having the amino acid sequence VAYISSGGSSINYA (SEQ ID NO: 3).

[0068] The term "reverse configuration" or "reverse sequence" with respect to an HD peptide sequence means that the amino acid sequence of a naturally occurring HD peptide is in reverse order, i.e., the N-terminus becomes the C-terminus and the C-terminus becomes the N-terminus. A reverse sequence can be obtained by reading a peptide or protein sequence in the reverse direction. A sequence read in the reverse direction (retrosequence) is a new peptide (retropeptide) or protein sequence (retroprotein).

[0069] The term "antibody" generally refers to a heavy or light chain immunoglobulin molecule or any functional combination or fragment thereof that comprises an antigen-binding site, e.g., an antigen-binding fragment (Fab). Antibodies are preferably specific for cellular receptors, membrane structures such as proteins, glycoproteins, polysaccharides or carbohydrates, and normal or tumor cells. Antibodies can be full-length immunoglobulin molecules or variable domain fragments of antibodies. The term "antibody" encompasses nanobodies, bispecific antibodies and diabodies, Fv and Fab, F(ab)2, camelids and other antigen-binding scaffolds.

[0070] The term "chimeric" refers to a combination of components derived from different genetic sources or species. For example, a chimeric antibody according to the present disclosure may contain immunoglobulin portions derived from one antibody and an HD peptide as described herein. A chimeric antibody may also contain immunoglobulin portions and HD peptides derived from more than one source or species.

[0071] A "conservative variant" with respect to an amino acid sequence refers to a variant of the amino acid sequence having one or more conservative substitutions.

[0072] As used herein, the term "conserved substitution" or "conservative substitution" and grammatical variations thereof refer to the presence of an amino acid residue in the sequence of an HD peptide that is different from the described substitution or described residue (i.e., a non-polar residue substituting a non-polar residue, an aromatic residue substituting an aromatic residue, a polar uncharged residue substituting a polar uncharged residue, a charged residue substituting a charged residue), but is of the same class of amino acid. Additionally, conservative substitutions can include residues having the same sign and generally similar magnitude of interface hydropathy value as the residue substituting the wild-type residue.

[0073] As used herein, the term "non-polar residue" refers to glycine (G, Gly), alanine (A, Ala), valine (V, Val), leucine (L, Leu), isoleucine (I, Ile), and proline (P, Pro), the term "aromatic residue" refers to phenylalanine (F, Phe), tyrosine (Y, Tyr), and tryptophan (W, Trp), and the term "polar, uncharged residue" refers to serine (R, S). The term "charged residue" refers to the negatively charged amino acids aspartic acid (D, Asp) and glutamic acid (E, Glu), and the positively charged amino acids lysine (K, Lys), arginine (R, Arg) and histidine (H, His). Other classifications of amino acids may be as follows: Amino acids with hydrophobic side chains (aliphatic): alanine (A, Ala), isoleucine (I, Ile), leucine (L, Leu), methionine (M, Met) and valine (V, Val); Amino acids with hydrophobic side chains (aromatic): phenylalanine (F, Phe), tryptophan (W, Trp), tyrosine (Y, Tyr); Amino acids with polar neutral side chains: asparagine (N, Asn), cysteine ​​(C, Cys), glutamine (Q, Gln), serine (S, Ser) and threonine (T, Thr); Amino acids with charged side chains (acidic): aspartic acid (D, Asp), glutamic acid (E, Glu); Amino acids with charged side chains (basic): arginine (R, Arg); histidine (H, His); lysine (K, Lys), glycine (G, Gly) and proline (P, Pro).

[0074] Conservative amino acid substitutions are likely to have a similar effect on the activity of the resulting HA protein variant or modified HA protein as the original substitution or modification. Further information on conservative substitutions can be found, for example, in Ben Bassat et al. (J. Bacteriol, 169:751-757, 1987), O'Regan et al. (Gene, 77:237-251, 1989), Sahin-Toth et al. (Protein ScL, 3:240-247, 1994), Hochuli et al. (Bio / Technology, 6:1321-1325, 1988) and in widely used genetics and molecular biology textbooks.

[0075] Blosum matrices are commonly used to determine the relatedness of polypeptide sequences. Blosum matrices were created using a large database of trusted alignments (BLOCKS database), in which pairwise sequence alignments related by less than some threshold percent identity were counted (Henikoff et al., Proc. Natl. Acad. Sci. USA, 89:10915-10919, 1992). For highly conserved target frequency in the BLOSUM90 matrix, a threshold of 90% identity was used. For the BLOSUM65 matrix, a threshold of 65% identity was used. A score of 0 or greater in the Blosum matrix is ​​considered a "conservative substitution" at the selected percent identity. The following table shows exemplary conservative amino acid substitutions: Table 1.

[0076] [Table 1]

[0077] Without wishing to be bound by theory, it is believed that increasing hydropathy (by using the hydropathy index for individual amino acids) may increase the level of binding, while decreasing hydropathy may decrease self-binding.

[0078] In some embodiments, the HD peptide sequence may be modified to enhance the cross-linking ability of the HD antibodies described herein. In one embodiment, such functionally enhanced peptides are determined by generating a series of synthetic peptides with substitutions at each amino acid position in the template sequence and then testing this library of peptides for self-affinity binding or binding to the original peptide sequence. Those peptides with better binding than the original sequence are then conjugated to immunoglobulins and the resulting conjugates are tested for potency, specificity, and undesirable ability to induce aggregation. In one particular embodiment, the T15 peptide sequence is altered and the modified sequence is selected to enhance function. In another embodiment of the invention, the self-binding ability of the peptide can be increased by increasing the complementarity of the sequence as described in U.S. Pat. No. 4,863,857 (issued to Blalock et al.). The self-binding ability and / or tolerance of the peptide can also be enhanced by humanizing the self-binding peptide sequence derived from a non-human animal. Humanizing the peptide sequence includes optimizing the sequence for expression or functionality in humans. Examples and methods for humanizing peptides and proteins are described elsewhere (Roque-Navarro et al., 2003; Caldas et al., 2003; Leger et al., 1997; Isaacs and Waldmann, 1994; Miles et al. 1989; Veeraraghavan et al., 2004; Dean et al., 2004; Hakenberg et al., 2003; Gonzales et al., 2004; and H. Schellekens, 2002).

[0079] The term "expression construct" refers to a recombinant nucleic acid sequence that includes a nucleic acid sequence that encodes a peptide or protein to be expressed. The nucleic acid encoding the peptide or protein to be expressed is operably linked to one or more regulatory nucleic acid sequences that facilitate expression of the peptide or protein to be expressed. Nucleic acid sequences are operably linked when they are in a functional relationship. Regulatory nucleic acid sequences are illustratively promoters, enhancers, DNA and / or RNA polymerase binding sites, ribosome binding sites, polyadenylation signals, transcription initiation sites, transcription termination sites, or internal ribosome entry sites (IRES). The expression construct can be incorporated into a vector, such as an expression vector and / or a cloning vector. The term "vector" refers to a recombinant nucleic acid vehicle for transferring a nucleic acid. Exemplary vectors are plasmids, cosmids, viruses, and bacteriophages. Specific vectors are known in the art, and the skilled artisan will recognize the appropriate vector for a particular purpose.

[0080] Homologous dimerization (HD) peptides

[0081] This application describes homologous dimerization (HD) peptides with improved self-binding, exemplified by the T15 and R24 HD peptides (see Example 1). Analysis of the self-binding and hydropathic properties of the T15 and other HD peptides has yielded motifs useful for predicting amino acid substitutions that may result in improved self-binding.

[0082] Exemplary homologous dimerization peptides described herein are summarized in Table 2 below.

[0083] [Table 2]

[0084] A substantially identical amino acid sequence of an immunoglobulin component has an amino acid sequence that is at least 70%, 80%, 85%, 90%, more preferably 95%, 96%, 97%, 98%, 99% or more percent identical to an amino acid sequence disclosed herein in certain embodiments of the invention, where the substantially identical protein retains substantially similar or better function compared to the reference protein to which it is substantially identical. As will be appreciated by those of skill in the art, the degeneracy of the genetic code is such that more than one nucleic acid may encode a particular immunoglobulin component, and these alternative sequences are considered to be within the scope of the present invention.

[0085] The amino acid sequence substantially identical to the 25-mer of SEQ ID NO: 4 and 8-10 has at least 20 consecutive amino acids, more preferably at least 22 consecutive amino acids, with an amino acid sequence that is at least 70%, 80%, 85%, 90%, more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 20 or more consecutive amino acids of the specified self-affinity amino acid sequence. The amino acid sequence substantially identical to the 14-mer of SEQ ID NO: 3 and 6 has at least 10 consecutive amino acids, more preferably at least 8 consecutive amino acids, with an amino acid sequence that is at least 70%, 80%, 85%, 90%, more preferably 95%, 96%, 97%, 98%, 99% or 100% identical to 10 or more consecutive amino acids of the specified self-affinity amino acid sequence. The same applies to each amino acid sequence in the HD dimer, for example, SEQ ID NO: 5, 7, 11, 12 and 13. The linker does not necessarily have to be gly-gly, but may be any other suitable linker.

[0086] An amino acid sequence having at least 70%, 80%, 85%, 90%, more preferably 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity or similarity to the sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19, or any amount of identity or similarity therebetween.

[0087] Substantially identical amino acid sequences may contain one or more conservative substitutions and may be referred to as conservative variants. Peptides that are substantially identical to the HD peptides described herein retain substantially similar or better autophilic function compared to a substantially identical reference autophilic peptide.

[0088] Homodimerizing antibodies (also called autoaffinity, autobinding antibodies or HD antibodies)

[0089] The present disclosure describes the generation of antibody-peptide fusion proteins that enhance the biological and immunological activity of antibodies without altering the antibody specificity for the corresponding antigen. Specifically, the present disclosure provides for the generation of antibody fusion proteins that contain a full or partial autophilic 24mer peptide from T15 or a 17mer peptide from R24. The HD peptides can be provided in the inverted configuration, as dimers, or with hydrophilic substitutions.

[0090] CDRs from any antibody may be used in the peptide / antibody conjugates described herein. Preferred antibodies are those that bind to immunomodulatory or checkpoint inhibitors. For example, the CDR sequences may encode antibodies specific for PD-1 / PD-1L or CTLA-4 and express activity for T cell activation. Any limitations on peptide length are practical limitations associated with peptide synthesis, not limitations associated with the practice of the methods of the present disclosure. Other preferred CDR sequences are derived from FDA approved antibodies such as Rituxin, Herceptin and / or Avastin.

[0091] Recombinant monoclonal antibodies are made with genes of CDRs (antigen-binding sequences) of non-human species (typically mouse) inserted into a human antibody framework for the primary purpose of reducing immunogenicity. These recombinant antibodies have the ability to interact with effector cells and human complement, but therapeutic activity, if any, is usually conferred by the original CDR sequences due to the nature of the antigen / antibody interaction.

[0092] HD peptides bound to antibodies allow the formation of a cell surface lattice incorporating both antibodies bound to their targets and antibodies bound solely by self-binding interactions with antibodies bound to other targets. This not only allows for lattice formation and cross-linking of cell surface antigens, but also allows for a higher than typical 1:1 ratio of antibodies bound to antigen epitopes. Based on flow cytometry analysis, this ratio can be as high as 50-100 fold, at least in some antigen-antibody systems. In fact, this large increase in binding of HD-modified antibodies is a hallmark of HD antibodies and is used as an initial assay for the evaluation of recombinant and chemically conjugated antibodies.

[0093] The discovery of homologous dimerization sequences (HDs) that allow antibodies to self-associate, form lattices, and cross-link target antigens provides an opportunity to confer therapeutic activity to recombinant monoclonal antibodies that is independent of the antigen-binding CDR sequences and is therefore applicable to many antigen / antibody systems. Such peptide sequences have previously been chemically conjugated to antibodies via nucleotide binding sites and inserted at the C-terminus of the Fc region of the fusion protein. The former methodology allows HD peptides to be inserted into antibodies in a site-specific manner, but is not a scalable manufacturing process. In contrast, the latter approach is scalable, but does not allow optimal activity. For optimal HD activity in antibodies, the peptide needs to interact with HD peptides on neighboring antibodies at multiple amino acid positions. This self-association can be reduced by the tendency of the peptide to form a hairpin structure near the c-terminus of the HD peptide. Unexpectedly, it was found that HD peptide sequences can be inserted into recombinant antibodies or by site-specific conjugation, by careful selection of the peptide position, for example by reversing the order of amino acids in the HD peptide, without loss of antigen binding or reduction of effector function. The problem of loss of HD activity upon insertion into the C-terminus of the Fc region in previous recombinant antibody approaches was overcome by reversing the usual sequence order (N- to C-terminus), which does not allow the C-terminus of the HD peptide to form its preferred hairpin structure. Similar results can also be achieved by dimerizing the HD peptide in either the forward or reverse orientation and linking the two HD peptides with a linker such as gly-gly.

[0094] Incorporation of HD technology into other antibody formats other than the prototypical human or humanized IgG1 antibody formats will greatly enhance binding and therefore improve their therapeutic or diagnostic activity, including but not limited to nanobodies, bispecifics and diabodies, Fv and Fab, F(ab)2, camelid and other antigen-binding scaffolds (reviewed in Hoglan Yu, Abhiskek Saxena, Sachdev S. Sidhu, Donghui Wu, Frontiers of Immunology 8:Article 38, 2017).

[0095] The group of recombinant antibody forms modified in the Fc region to increase or decrease serum half-life, antibody-dependent cellular cytotoxicity, complement binding or complement-mediated killing also represents antibody forms whose increased antigen binding by HD technology may enhance therapeutic or diagnostic activity (reviewed in Abhishek Saxena & Donghui Wu, Frontiers in Immunology, 7:Article 580, 2016). The recombinant antibody forms of the present invention are not limited by these reference forms.

[0096] The antibodies according to the invention can only spontaneously bind to themselves after first binding to their target antigen. The homodimerized antibodies of the invention preferably non-covalently bind to other such conjugated antibodies when bound to their target antigen, usually a cell surface transmembrane receptor.

[0097] The homodimerized antibodies of the present invention typically comprise an antibody conjugated with one or more peptides having an HD peptide sequence. The homodimerized antibodies of the present invention can comprise virtually any immunoglobulin. In some embodiments, the antibody binds to a target involved in a disease or disorder, and target binding has a therapeutic effect on the disease or disorder. The target antigen can comprise a cell surface antigen, including a transmembrane receptor. In certain embodiments, the Ig component of the antibody can comprise a monoclonal antibody.

[0098] The present invention provides antibodies with self-binding properties that mimic those of rare, naturally occurring autoaffinity antibodies, thereby offering a simple and attractive alternative to covalent dimerization and other engineering approaches aimed at enhancing the therapeutic potential of antibodies.

[0099] Expression system

[0100] The present invention provides an isolated host cell transformed with an expression vector encoding an immunoglobulin heavy chain having an antigen-binding domain and an HD peptide. In a particular embodiment, the isolated host cell is also transformed with an expression vector encoding an immunoglobulin light chain having an antigen-binding domain, and the antigen-binding domain of the immunoglobulin heavy chain and the antigen-binding domain of the immunoglobulin light chain together form an antigen-binding site. The isolated host cell for producing the recombinant autophilic antibody of the present invention may be in vitro. Expression systems for HD antibody expression illustratively include eukaryotic cells such as mammalian cells, plant cells, insect cells, yeast cells and amphibian cells, as well as prokaryotic expression systems such as bacteria. Those skilled in the art can select a particular expression system to use for producing a recombinant HD antibody.

[0101] Position of HD peptides

[0102] As seen in US Patent Publication No. 20030103984, it was previously thought to be practical only to insert HD peptide sequences at the c-terminus of the Fc region. This was due to concerns that inserting HD peptides into other regions of the mAb may reduce or interfere with antigen binding or potential therapeutic activities such as ADCC or complement fixation. Indeed, published data showed that the 24-mer HD sequence derived from the T15 antibody expressed a three-dimensional conformation when synthesized and assayed, and encoding it into an antibody would not only negate the 3D structure of the peptide but could also impair antigen binding of nearby CDRs. Self-association activity in Fc-terminal constructs (Kohler, H, Rector, K & Amick J., Hybridoma 2012(6):395-402) was described compared to nucleotide affinity site chemical conjugates, but activity was reduced (data not shown).

[0103] In a preferred embodiment of the invention, the HD peptide is located at one of three positions within an antibody, for example, IgG.

[0104] The first is immediately after the CDR3 of the heavy or light chain. In this form, the Ig molecule can be expressed as a single chain Fv with the HD peptide encoded in any of the optimized configurations described herein at the most c-terminal part preceded by a gly-gly or similar spacer. Despite being close to the antigen-binding part of the antibody, this position can confer maximum self-association without reducing antigen binding. The retention of antigen binding at this position was completely unexpected. Because we, the inventors of the original technology, thought this was completely impossible, we actually previously linked the sequence to the c-terminus of the Fc region of a whole IgG, which is the furthest part where we could separate antigen binding from self-association.

[0105] The second site is immediately c-terminal to the heavy or light chain constant region preceded by a gly-gly spacer. This may be designated Fab. In this form, the Fab may also be part of a naive or immunized phage expression library assayed for binding to the target antigen. The use of HD peptides allows for enhanced binding and identification of even lower affinity antibodies.

[0106] The third site is where the HD sequence is cloned into the c-terminus of the Fc region preceded by a gly-gly or other spacer. In the embodiment where the HD peptide is a dimer of HD peptides, in the normal or inverted configuration, the separation of the two HD peptides by a gly-gly or alternative spacer is particularly relevant for c-terminal construction of antibodies to overcome steric inhibition. In initial tests, this form of HD peptide has a higher interaction avidity.

[0107] Methods for generating homodimerizing (autophilic) antibodies

[0108] HD peptide modified antibodies made by various site-specific chemical conjugation methods can also be used to make fully active self-binding antibodies. Numerous methods for attaching HD peptides to antibody molecules will be known to those skilled in the art. One method is the use of chemical cross-linking, such as the affinity cross-linking method described in US Patent Publication No. 20040185039. Such methods were initially adopted because they allow peptides to be linked to the nucleotide binding site of antibodies located at the end of the heavy chain of the Fab. This affinity site allows for the insertion of peptides without reducing the affinity of binding and preserves the self-association activity of the peptides. Even though conjugation via carbohydrates resulted in more peptides bound to the antibody, such site-specific conjugates near the antigen-binding region were more active when compared to conjugation of HD-peptides to carbohydrates (J. Immunol Methods: (2005) 304: 100-106, Photo-activated affinity-site cross-linking of antibodies using tryptophan containing peptides, Mike Russ, Dingyuan Lou, Heinz Kohler). However, due to elements of the methodology, namely the photoactivation step, the manufacturing process for conjugation for this method is not scalable and is only useful for generating small amounts of conjugates.

[0109] A second method of site-specific conjugation, the smart tag technology, has been developed for use with antibody-drug conjugates (ADCs) (Wu, P., et al., Site-specific chemical modification of recombinant proteins produced in mammalian cells by using for genetically encoded aldehyde tag, Proc Natl Acad Sci USA, 2009.106(9):p.3000-5). The use of such technology or similar site-specific technologies with HD peptides can overcome the scalability problem.

[0110] Alternatively, recombinant methods may be used. For example, a fusion gene may be prepared comprising a nucleic acid sequence encoding an antibody and a nucleic acid sequence encoding a peptide, the nucleic acid sequence encoding the peptide being located inside the nucleic acid sequence encoding the antibody at a site whereby when the fusion is expressed, the fusion protein formed thereby comprises the antibody and the peptide, and the peptide is attached to the antibody at a site that does not interfere with antigen binding of the antibody, and the fusion gene is expressed to form the fusion protein. In particular, a fusion protein may be formed by providing a gene encoding an antibody, the gene being mutated to contain a restriction site, the restriction site being located immediately C-terminal to the CDR3 of the antibody, or immediately C-terminal to the heavy chain variable region of the antibody, or on the C-terminus of the antibody. Humanized antibodies, generated by CDR exchange of mouse or other human or non-human sources with human IgG frameworks encoding HD peptides, may be expressed by the fusion gene.

[0111] Methods for making fusion proteins are described, for example, in the following U.S. patents: U.S. Patent No. 5,563,046 to Mascarenhas et al.; U.S. Patent No. 5,645,835 to Fell, Jr.; U.S. Patent No. 5,668,225 to Murphy; U.S. Patent No. 5,698,679 to Nemazee; U.S. Patent No. 5,763,733 to Whitlow et al.; U.S. Patent No. 5,811,265 to Quertermous et al.; No. 5,908,626 to Hang et al.; No. 5,969,109 to Bona et al.; No. 6,008,319 to Epstein et al.; No. 6,117,656 to Seed; No. 6,121,424 to Whitlow et al.; No. 6,132,992 to Ledbetter et al.; No. 6,207,804 to Huston et al.; and No. 6,224,870 to Segal. Methods for making Ig fusion proteins are described, for example, in Antibody Engineering, 2nd Edition. ed.: Carl A. K. Borrebaeck, Oxford University Press 1995, and Molecular Cloning: A Laboratory Manual, Second Ed., Cold Spring Harbor Press, 1989.

[0112] In certain embodiments, a DNA sequence encoding an HD peptide described herein, or a substantially identical HD peptide, is inserted in frame with a DNA sequence encoding an immunoglobulin heavy chain and / or an immunoglobulin light chain, and the fusion protein (or HD antibody) expressed from the DNA sequence comprises an immunoglobulin heavy chain and / or an immunoglobulin light chain bearing the HD peptide.

[0113] Nucleic acids encoding immunoglobulin heavy or light chains are well known, and any of a variety of nucleic acids encoding immunoglobulin heavy or light chains can be used to produce the recombinant chimeric HD antibodies of the invention. Particular nucleic acids encoding human constant heavy and / or light chains, particularly human gamma constant heavy and kappa constant light chains, are described herein. Nucleic acids encoding human gamma constant heavy and / or kappa constant light chains can be obtained from commercial sources, such as vector pAc-k-CH3 available from Progen Biotechnik GmbH. Nucleic acids encoding the proteins and / or peptides described herein, including human gamma constant heavy and / or kappa constant light chains, can be generated using recombinant techniques, such as cloning or synthesis. Particular immunoglobulin constant heavy and / or kappa constant light chains are described, for example, in U.S. Patent Nos. 5,736,137 and 6,194,551.

[0114] IgG fusion proteins

[0115] Ig fusion proteins have the advantage of combining an antibody combined with a molecule that contributes specificity and / or antibody effector function to its unique properties. The ability to generate this family of proteins was first demonstrated when c-myc was substituted for the Fc of an antibody molecule (Neuberger MS, Williams GT and Fox R O. Nature 125:604, 1984), but there are now many examples. Ab fusion proteins can be achieved in several different ways. In one approach, non-Ig sequences are substituted for the variable region, and the molecule substituting the V region provides the specificity of targeting by the antibody that contributes to properties such as effector function and improved pharmacokinetics. Examples include IL-2 and CD4. Alternatively, the non-Ig sequence can be substituted or attached to the constant region. The resulting molecule retains the binding specificity of the original antibody but gains characteristics from the attached protein. Depending on the position of the substitution, different antibody-associated effector functions and biological properties are retained.

[0116] Vectors for the construction of IgG fusion proteins

[0117] A series of vectors have been generated that allow the fusion of proteins at different positions within the antibody molecule, thereby facilitating the construction of fusion proteins with different properties. Using these vectors, it is possible to generate a family of fusion proteins with molecules of different molecular weights, valencies, and thus different subsets of the functional properties of the antibody molecule.

[0118] As a specific example of a method to facilitate construction of fusion genes, site-directed mutagenesis was used to generate unique restriction enzyme sites in the human IgG3 heavy chain gene. In this particular example, restriction sites were generated at the 3' end of the CH1 exon, immediately after the hinge at the 5' end of the CH2 exon, and at the 3' end of the CH3 exon. The restriction sites thus generated were SnaBI at the end of CH1 by replacing TtgGTg with TacGTa, Pvu II at the beginning of CH2 by replacing CAcCTG with CAgCTG, and SspI at the end of CH3 replacing AATgag with AATatt. These manipulations provided unique blunt-end cloning sites at these positions. In all cases, the restriction sites were positioned such that after cleavage, Ig would contribute the first base of the codon. A human IgG3 with an extended hinge region of 62 amino acids was selected for use as an immunoglobulin, and if present, this hinge should provide spacing and flexibility, thereby facilitating simultaneous binding of antigen and receptor. An EcoR I site was also introduced 3' of the IgG3 gene to provide a 3' cloning site and polyA addition signal. These restriction sites were originally designed for use with growth factors, but can be used to place any new sequence into the antibody at a given location. Also, by using these cloning cassettes, the variable regions can be easily altered. Similar techniques can be used to generate appropriate restriction sites for other antibody genes.

[0119] Generation of fusion genes

[0120] As a first step in the generation of a fusion protein, a blunt-end restriction site must be introduced at the desired position at the 5' end of the gene to be fused. In order to maintain the correct reading frame, the site must be positioned so that it contributes two bases to the codon after cleavage. If the goal is to create a fusion protein with the entire molecule, the restriction site is usually introduced at the location of any post-translational processing, such as after the leader sequence. Alternatively, if the goal is to use only a portion of the protein, the blunt-end site can be introduced anywhere within the gene, but care must always be taken to maintain the correct reading frame. Additionally, if there is carboxyl-terminal post-translational processing of the fusion protein, it is often desirable to introduce a stop codon at this processing site.

[0121] The main concern when generating fusion proteins is to maintain the biological activity of all components. The generation of fusion proteins with antibodies is facilitated by the domain structure of antibodies, with all cloning sites located immediately after the intact domains. This arrangement should ensure correct folding of the immunoglobulin. The folding of the linked protein depends on its structure and where it is fused. Whenever structural information is available, it is desirable to generate fusions at positions that maintain the structural integrity of the linked protein.

[0122] To generate sufficient amounts of protein for functional analysis, it is desirable to have the protein secreted into the medium, and although previously reported examples have demonstrated that assembled fusion proteins are both assembled and secreted, this remains a concern when designing additional fusion proteins.

[0123] A method for designing a fusion gene containing a biologically active peptide as part of a heavy or light chain gene can use established antibody engineering protocols (Antibody Engineering, 2nd Edition. ed.: Carl A. K. Borrebaeck, Oxford University Press 1995. Chapter 9, pages 267-293). The peptide can be fused to either the N-terminal or C-terminal residue of the H or L chain. Expression of such fusion genes is typically carried out in mammalian cell lines, although other expression systems, such as bacterial or yeast expression systems, may also be used.

[0124] Pharmaceutical Compositions

[0125] The present invention also relates to a composition comprising a homodimerized antibody of the present invention and a pharma- ceutically acceptable carrier.

[0126] The antibodies of the present invention are useful in pharmaceutical compositions for systemic administration to humans and animals, such as unit dosage forms, sterile solutions or suspensions, sterile parenteral solutions or suspensions, oral solutions or suspensions, oil-in-water or water-in-oil emulsions, containing an appropriate amount of active ingredient. Topical application can be in the form of ointments, creams, lotions, jellies, sprays, washes, etc. The compositions are useful in pharmaceutical compositions (% by weight) of active ingredient with a carrier or vehicle in the composition of about 1-20%, preferably about 5-15%.

[0127] The parenteral solutions or suspensions described above may be administered transdermally, or more concentrated sustained release forms may be administered if desired. The fusion proteins of the present invention may be administered intravenously, intramuscularly, intraperitoneally, or topically. Thus, incorporation of the active compound into a sustained release matrix may be practiced for transdermal administration. Acceptable pharmaceutical carriers for the purposes of the present invention are those known in the art that do not adversely affect the drug, the host, or the materials comprising the drug delivery device. The carrier may also contain preservatives, stabilizers, wetting agents, emulsifiers, and the like, along with the penetration enhancers of the present invention. The effective dosage for a mammal may vary due to factors such as the age, weight activity level, or condition of the subject to be treated. Typically, the effective dosage of the compounds according to the present invention is about 10 to 500 mg when administered in solution at least once a day. Administration may be repeated at appropriate intervals.

[0128] Conjugated autoaffinity antibodies can non-covalently bind to other autoaffinity antibodies when bound to their target antigen. However, premature formation of antibody dimers or multimers can lead to difficulties in manufacturing, such as during purification and concentration, as well as drawbacks in administration that can result in side effects. Therefore, compositions containing the lipophilic antibody-peptide conjugates of the present invention are formulated in solution and prior to administration to reduce this dimerization potential and maximize monomeric properties. For example, it has been found that solution dimerization can be reduced or mitigated by using hypertonic compositions. In some embodiments, salt concentrations of 0.5M or higher, low levels of SDS or various other detergents such as anionic ones (see U.S. Pat. No. 5,151,266, incorporated herein by reference), or modification of the antibody to reduce its isoelectric point, for example by using succinic anhydride (see U.S. Pat. No. 5,322,678, incorporated herein by reference), can be used to formulate the composition.

[0129] Immunoassays

[0130] According to the present invention, there is provided an immunoassay comprising contacting an analyte in a biological or environmental sample with an antibody conjugated to an autoaffinity peptide, and then detecting a complex formed by the analyte and the antibody conjugated to the autoaffinity peptide.

[0131] In certain embodiments, the assays of the present invention are characterized by detecting antigens expressed at low levels, such as on the cell surface, using antibodies that contain an autoaffinity peptide, either naturally or by conjugation of the autoaffinity peptide to the antibody.

[0132] The use of highly specific antibodies is common in many diagnostic applications. The binding of the antibody may be detected directly by several means, or a secondary antibody is required for signal enhancement and detection. Previously, signal detection was directly related to the binding amount of the antibody bound to the target either monovalently or bivalently. In the present invention, the enhanced signal strength is due to the multivalent binding interaction of the self-affinity peptide-conjugated antibody bound to the target analyte.

[0133] Whether directly or indirectly detected, the autoaffinity peptide-conjugated antibody greatly enhances signal detection due to lattice formation and more antibodies surrounding the target.Therefore, naturally occurring autoaffinity antibodies and non-naturally occurring autoaffinity peptide-conjugated antibodies can be directly labeled with a detectable label that results in enhanced signals in immunoassays.Similarly, indirect labeling, such as labeling of secondary antibodies, results in increased signals in immunoassays due to increased numbers of secondary antibodies binding to primary autoaffinity peptide-conjugated antibodies.

[0134] In an embodiment of the assay of the present invention, naturally occurring autoaffinity antibodies or antibodies conjugated to homologous dimerization (HD) peptides are used to enhance signal detection of antigens immobilized on a substrate such as plastic in an assay such as an ELISA.

[0135] In an embodiment of the assay of the present invention, naturally occurring autoaffinity antibodies or antibodies conjugated to homologous dimerization (HD) peptides are used to enhance the "off-rate" or increase the avidity of the antigen coated or bound to a polymer chip and detected by surface plasmon resonance.

[0136] In an embodiment of the assay of the present invention, naturally occurring autophilic antibodies or antibodies conjugated to homologous dimerization (HD) peptides are used to enhance the binding and localization detection of target-bound antibodies by fluorescence or other signal detection methods in vivo, e.g., in xenograft tumor animal models.

[0137] Assays according to embodiments of the present invention may include virtually any immunoglobulin conjugated to one or more homologous dimerization (HD) peptides to enhance detection of an analyte.

[0138] The term "analyte" refers to any molecule or compound that is specifically recognized by an antibody conjugated to an autoaffinity peptide, illustratively including proteins, peptides, haptens, carbohydrates, lipids, gangliosides, and combinations thereof. In certain embodiments, the analyte may be a mammalian analyte, illustratively including proteins, peptides, haptens, carbohydrates, lipids, gangliosides, or combinations thereof, produced, for example, by normal or abnormal cells of a mammal.

[0139] In further particular embodiments, the analyte may be a non-mammalian analyte such as a protein, peptide, hapten, carbohydrate, lipid, ganglioside or combinations thereof produced by a microorganism, such as a bacterium or a virus. Thus, particular assays of the invention for the detection of microorganisms and / or microbial products are provided. For example, the assays for the detection of microorganisms and / or microbial products are used to assay samples obtained from humans or non-human animals to detect infection. In further embodiments, the assays for the detection of microorganisms and / or microbial products are used to assay samples obtained from an environment or subject to be tested for contamination with microorganisms and / or microbial products.

[0140] In an embodiment of the assay of the invention, the target analytes are B cell receptor, CD20, Her2, ganglioside GM2, glycolic acid ganglioside GM3, GD3 ganglioside, caspase, oxidized low density lipoprotein, phosphocholine, EGFR, CD32B, HLADR1, CD19, EpCAM, PSA and bacterial antigens, e.g., Staphylococcus aureus antigens.

[0141] In some embodiments of the present invention, diagnostic and prognostic immunoassays are provided. The term "diagnostic immunoassay" refers to an immunoassay that allows for the determination of the presence or amount of an analyte that is indicative of a disease or pathological condition in an animal or human subject. The term "prognostic immunoassay" refers to an immunoassay that allows for the determination of the presence or amount of an analyte that is indicative of the progression of a disease or pathological condition in an animal or human subject.

[0142] kit

[0143] In an embodiment of the invention, a kit is provided for use in performing an assay using an antibody-homologous dimerization (HD) peptide conjugate. In a particular embodiment, the kit comprises an antibody-homologous dimerization (HD) peptide conjugate and instructions for use in detecting an analyte in a sample.

[0144] Treatment

[0145] Methods of enhancing apoptosis, complement fixation, effector cell-mediated killing of a target, or preventing the onset or progression of a disease state using the homologous dimerization (HD) antibody of the present invention or a composition comprising the homologous dimerization (HD) antibody are also contemplated. In one embodiment, the autoaffinity conjugate of the present invention or a composition containing the autoaffinity conjugate of the present invention is administered to a subject. Upon administration, the antibody binds to the target cell and enhances apoptosis, complement fixation, effector cell-mediated killing of the target, or prevents the target antigen or cell from stimulating or further enhancing the development of a disease state. In a further embodiment, a second anti-autoaffinity peptide antibody can be administered, allowing time for the autoaffinity conjugate to bind to the target cell and enhance apoptosis, complement fixation, effector cell-mediated killing of the target, or prevents the target antigen or cell from further enhancing a disease state, and for the autoaffinity conjugate to be cleared from normal tissue.

[0146] In some embodiments, a patient suffering from a debilitating or potentially life-threatening disease or condition is administered at least one of the subject homologous dimerization (HD) antibodies in an amount effective to alleviate the symptoms of the disease or condition. The disease or condition contemplated for treatment with the antibodies of the invention may be a malignancy, neoplasm, cancer, an autoimmune disorder, Alzheimer's disease or other neurodegenerative condition, or graft or transplant rejection.

[0147] In some embodiments, a method for enhancing apoptosis of a target cell in a patient includes administering a first homodimerization (HD) antibody-peptide conjugate and a second antibody that recognizes the peptide domain of the conjugate. In this embodiment, the antibody-peptide conjugate recognizes the extracellular region of a transmembrane receptor of the target cell. Due to its homodimerization property, the antibody-peptide conjugate may bind to the target more avidly than a corresponding antibody lacking the self-binding peptide domain. Furthermore, whenever an autophilic antibody binds to two or more receptors, an apoptotic signal may be triggered in the cell since the receptors are in closer proximity to the self-binding property of the antibody. If the peptide domain of the conjugate presents an exposed epitope, a second antibody specific for the autophilic peptide may be administered and bind to the modified antibody, enhancing the cross-linking process and even causing temporary clearance of the target antigen. If the target antigen is a receptor, clearance from the cell surface, endocytosis and degradation then require the synthesis of new receptor protein, which means that the biological function of the receptor is more effectively inhibited over a long period of time than using either a simple blocking antibody or a small molecule inhibitor. Alternatively, the second antibody can carry a radiolabel or other potential therapeutic agent, so that when administered, it can attack the target cells. The key to using this second antibody is the specificity of the antibody. The homologous dimerization (HD) peptide occurs naturally, but only on a small number of mouse immunoglobulins. Therefore, an antibody specific for this peptide would have the necessary selectivity to be used in vivo.

[0148] disease

[0149] The disease or condition contemplated for treatment with the antibodies of the invention may be a malignancy, a neoplasm, a cancer, atherosclerosis, an autoimmune disorder, Alzheimer's disease or other neurodegenerative conditions, graft or transplant rejection, or any other disease or condition that responds to antibody therapy.

[0150] dose

[0151] The homologous dimerization (HD) antibodies described herein may be administered in one or more dosages that are substantially the same as or less than that practicable for the unmodified antibody. [Table 3]

[0152] The following examples are presented to illustrate certain aspects of the invention and are not intended to limit the scope of the invention.

[0153] The invention is further illustrated in the following examples.

[0154] example

[0155] Example 1 - HD peptides with optimized self-association potential

[0156] The 24-mer peptide sequence derived from T15 was analyzed and compared with other anti-phosphorylcholine antibodies of the same germline configuration that have limited or no HD activity using individual amino acid hydropathy scores and global peptide hydropathy plots (Kyte-Doolittle), a method of protein analysis commonly used for non-immunoglobulin proteins.

[0157] As shown in Figure 1, the T15 peptide has a preference to form hairpin structures at the C-terminus, which may impair its ability to self-bind (Figure 1B). In the original HD antibody (T15), the HD sequence was inserted at the end of CDR3 and at the first part of CH1, a position that does not allow secondary conformations. We hypothesized that in a recombinant form of the antibody with the HD peptide inserted at the c-terminus of the antibody, the HD peptide would be free to form hairpin structures, reducing self-binding and its ability to enhance the therapeutic activity of the antibody through lattice formation. Similarly, the T15 peptide chemically conjugated to the antibody, in its normal N- to C-terminal configuration, would also have the ability to form secondary conformations with reduced self-binding. Furthermore, the small HD peptide sequence may have steric restrictions on self-binding in the context of the large antibody protein.

[0158] The first comparison was with the known T15 peptide configuration in either forward or reversed order (see Figures 2 and 3). Inspection of the original T15 sequence (left panel of Figure 2) shows that self-binding correlates with the sequence hydropathy plot of the peptide. The first half of the peptide was derived from the CDR2 of the antibody and the second half from the framework between the CDRs. In that case, the first half would contain the amino acids that confer self-binding and are altered from the germline sequence. Importantly, the more hydropathic residues in the second half serve to bind to the less hydropathic residues in the first half to initiate the self-binding process. Surprisingly, when we examined the 24mer peptide in the reversed sequence (right panel of Figure 2), it was also able to adopt the required self-binding configuration despite the presence of a hairpin structure at the c-terminus. This then allows tethering of the peptide via the former's c-terminus (now the n-terminus) to an antibody with the more significant first half of the peptide, which determines self-binding and makes it more feasible to further tether from the antibody and search for the corresponding self-binding sequence in other peptide / antibody conjugates.

[0159] Identification of the hydropathy contribution of individual amino acids to self-binding to construct an optimizable motif was performed by comparing the amino acid differences in three germline sequences extracted from the heavy chain CDR2 / framework 3 of Mopc antibodies with no, little or high HD activity. The individual amino acid hydropathy scores of the amino acids (available at http: / / gcat.davidson.edu / DGPB / kd / aminoacidscores.htm) were plotted against the known positions of the amino acids involved in self-binding (see FIG. 4). As shown, amino acid changes in the T15 peptide that allow self-binding (high HD activity) are associated with unique changes in the hydropathy score of the substituted amino acid, i.e., a more hydrophilic substitution that allows self-binding into a typically more hydrophobic framework region. This allows prediction that substitution of other amino acids into the previous CDR2 region of the T15 peptide should preserve the hydropathy properties of the individual amino acids. It was also found that further hydrophilic substitutions in the preceding CDR2 region of the T15 peptide could increase the potential binding to the more hydrophobic preceding framework portion of the peptide, and vice versa. Substitution of more hydrophobic amino acids into the framework 3 portion would allow more self-binding to the more hydrophilic portion of the T15 peptide. This hydrophobic-hydrophilic interaction serves to initiate the self-binding process, which then leads to the specific amino acids involved in self-binding and form other non-covalent interactions. The entire process confers the relative affinity of the self-binding process. Some exemplary single amino acid conservative substitutions are shown in Table 2 (e.g., T15-var1, T15-var2, and T15-var3).

[0160] Without wishing to be bound by theory, it is believed that by increasing the hydropathy of the HD peptide (e.g., by amino acid substitution), the binding properties of the HD peptide may be modulated, e.g., the binding properties of the HD peptide may be increased. Furthermore, it is believed that the affinity for self-binding may be optimized to avoid the formation of antibody dimers before the antibody binds to its target. Thus, the affinity of the HD peptide may be increased compared to a naturally occurring HD peptide, but still be lower than the binding affinity of an antibody to its target. For example, the binding affinity of an HD peptide may be increased by 10 -4 , 10 -5 , 10 -6 , 10 -7 or 10 -8 It could be.

[0161] The potential increase in self-binding can be easily measured by synthetic peptide synthesis of single amino acid mutants, labeling of hydrogen atoms with tritium, and then measuring binding to immobilized T15 peptide. Positive changes in single amino acid substitution peptides can be incorporated as well as double or triple amino acid changes. It should be noted that the end result is a self-binding peptide with higher self-binding affinity, but not so much that it causes antibody aggregation.

[0162] T15 is not the only source of naturally occurring HD antibodies and HD peptides. A well-studied example is that of R24 against disialoganglioside GD3. Similar to T15, the increase in potency in the original murine antibody was associated with the presence of a self-binding peptide that allows the formation of a lattice at the cell surface, which increases the therapeutic effect. As shown in Figure 9, the R24 sequence, which is responsible for lattice formation, self-binds in an antiparallel manner. Similar to T15, the R24 peptide is shorter than T15, but shows a corresponding hydropathy plot where one end of the peptide is more hydropathic than the other end (see Figure 10). The plot is essentially the inverse of T15, where the hydropathy changes from low to high instead of from high to low. Thus, the same principles for promoting self-binding via sequence inversion, forming dimers, and creating single conservative amino acid substitutions with altered hydropathic properties serve to increase self-binding.

[0163] Example 2 - Identification of antibodies with autoaffinity / homologous dimerization (HD) activity

[0164] The following method uses the specific biophysical properties of antibodies incorporating HD peptides to test for HD activity in antibody preparations or phage libraries of scFv or Fab. This method can also be used as a quality assurance (QA) release assay for HD antibodies.

[0165] antibody viscosity

[0166] Microdilution tubes (USA Scientific, Ocala, Fla., containing 1 mg / ml HPC G9 or HPC G11 in PBS) were mounted on standard microscope slides. They were equilibrated at 4° C., 20° C., and 37° C., sealed, then positioned vertically, then horizontally, and then photographed at each temperature.

[0167] Viscosity equilibrium measurement over time

[0168] Microdilution tubes containing 1 mg / ml antibody in PBS were equilibrated at either 4° C. or 37° C. The tubes were placed vertically for 3 seconds, then placed horizontally and photographed. The time required for the meniscus to stop moving was measured using a stopwatch.

[0169] result

[0170] Temperature-dependent equilibrium of homodimers: HPC G9 / HPC G11 are isoforms of IgG anti-phosphocholine antibodies, and HPC G11 shares idiotype and sequence with CDR2 / FR3 of TEPC-15. Therefore, HPC G11 is homophilic, whereas HPC G9 is not homophilic. Since temperature is known to affect the physical properties of proteins, including antibodies, the behavior of HPC G9 and HPC G11 was compared at 4°C, 20°C, and 37°C using size exclusion chromatography. The amount of antibody eluted in the excluded volume was compared to the amount in the encapsulated volume.

[0171] Ratio of excluded / included HPC G11 at different temperatures. There was no significant change in the ratio of excluded protein with HPC G9 at 4°C, 20°C and 37°C (data not shown), but the ratio of HPC G11 increased with temperature. These findings support the idea that the degree of dimerization of homophilic G11 in solution is higher at physiological temperatures than at non-physiological temperatures.

[0172] Viscosity difference

[0173] The viscosity of HPC G9 and HPC G11 was also compared at different temperatures. A volume of 500 ml of each antibody was placed in a tube in a rack and equilibrated in a horizontal position for 30 minutes at 4°C, 20°C, and 37°C. The tube was then moved to an upright vertical position for 1 second, then returned to a horizontal position, and photographed. The position of the meniscus was measured, and the ratio of these measurements was calculated for HPC G9 or HPC G11. At 4°C, the ratio was 1.45, at 20°C, the ratio was 0.92, and at 37°C, the ratio was 0.54. The tilt of the tube after equilibration was reversed (i.e., the bottom of the tube was lifted vertically for 1 second, then returned to a horizontal position, and photographed). The ratio of HPC G9 to HPC G11 was 1.5 at 4°C, 1.41 at 20°C, and 2.06 at 37°C.

[0174] To exclude the possibility that the viscosity of G11 is intrinsic, S107, a mouse monoclonal IgA antibody known to be hemophilic, was tested. For comparison, another mouse antibody, G9, was included in the viscosity analysis. The tubes were first placed inverted for 3 seconds and then returned to a horizontal position. The time required for the meniscus to stop moving was measured for each antibody. The recorded number of seconds for the meniscus of these antibodies to stop moving at 4°C and 37°C is shown in Figure 5. The longer the time required for the movement to stop, the more viscous the solution. As shown in Figure 5, the time difference recorded at both temperatures is smaller for G11 and S107 than for G9. The ratios of time at 4°C divided by time at 37°C for G11, S107, and 1F7 are identical, but the ratio for G9 is more than double. G11 and S107 are facultative homophilic polymers, while 1F7 IgM is a covalent pentamer. It is interesting to note that the homophilic polymer and the covalent IgM polymer have similar but different viscosities than the monomeric G9 bivalent antibody. This inherent viscosity of homophilic antibodies is independent of Ig class and may be part of their inherent dimerization ability responsible for their superior targeting.

[0175] Example 3 - Generation and characterization of a recombinant therapeutic HD antibody (rituximab) with a reverse configuration

[0176] The following example illustrates the generation of a reverse-sequence HD antibody and the changes in the therapeutic properties of the recombinant HD form of the antibody compared to the native antibody. To test the potential of the HD peptide, molecular biology techniques were used to generate a chimeric version of the rituximab antibody (chRituximab), and a chimeric HD antibody identical to chRituximab, except that the HD peptide (rs-T15) was added to the C-terminus of each variable region of the heavy chain.

[0177] Materials and Methods

[0178] cell line

[0179] JOK-1 cells were provided by Affimed Inc. JOK-1 cells were grown in RPMI-1640 containing Glutamax (Gibco) supplemented with 10% FBS-Premium-HI (Aleken Biologicals) and 1% penicillin / streptomycin (Gibco). Raji and Ramos cells were obtained from American Type Culture Collection (ATCC) numbers HB-9645, CCL-86, CRL-1596, and TIB-152, respectively. Raji and Ramos cells were maintained in RPMI-1640 medium (ATCC) containing HEPES supplemented with 10% FBS-Premium-HI (Aleken Biologicals) and 1% penicillin / streptomycin (Gibco). Rituximab cells were maintained in HEPES-containing RPMI-1640 medium (ATCC) supplemented with 10% FBS-low-IgG (Gibco), 1% penicillin / streptomycin (Gibco) and 0.5% Glutamax (Gibco). CHO-S cells were purchased from Invitrogen and grown in CD CHO medium supplemented with 1% HT supplement (Gibco), 2% Glutamax (Gibco) and 100 U / ml penicillin / streptomycin (Gibco). After introduction of vector DNA, CHO-S cells were grown as above with the addition of 1.2 mg / ml 418 (Invivogen) for selection. All cells were maintained at 37°C and 5% CO2.

[0180] Construction of chimeric antibody genes

[0181] The heavy and light chain variable regions were synthesized from the published rituximab sequence. Rituximab heavy chain:

number

number

[0182] The Rituximab heavy chain variable region was amplified from a cDNA pool by PCR using primers modVHRituximabfwd and modVHRituximabrev. All oligos were purchased from Operon. The Rituximab light chain variable region was amplified from a cDNA pool by PCR using primers modVLRituximabfwd and modVLRituximabrev. The heavy and light chain PCR products were cloned into the XhoI-NheI and SacI-HindIII sites of vector pAc-k-CH3 (Progen Biotechnik GmbH) to form pAc-k-RituximabH and RituximabK, respectively. Clones were verified by sequencing in both directions. All restriction enzymes were purchased from Takara or New England Biolabs. Taq polymerase (Promega) was used for all PCRs. All enzymatic reactions were performed using the manufacturer's protocols.

[0183] Construction of antibody expression vectors

[0184] A DNA sequence encoding the reverse sequence of the T15 peptide was constructed using Oligos LongT15fwd, LongT15rev and PrimerB in a nested PCR. The resulting PCR product was cloned into the SalI-NotI sites of MCSB of pIRES (Clontech) to form pHD. The complete heavy and light chains of pAc-k-RituximabH and pAc-k-RituximabK were PCR amplified using primers modVHXfwd and modVHXrev, or VKXfwd and VKXrev, respectively. The light chain was cloned into the NheI-XhoI sites of MCSA of vector pHD and the heavy chain was cloned into the SalI-NotI sites of the resulting vector to form pchRituximab-HD. Clones were verified by sequencing in both directions. To generate pchRituximab (anti-CD20 without the T15 peptide), pchRituximab-HD and pIRES were digested with NotI and ClaI. The approximately 6 Kb DNA fragments from pchRituximab-HD and pIRES were gel purified from 1% agarose gels using a Qiaquick kit (Qiagen) and ligated together to form rituximab. Clones were verified by sequencing in both directions. All vector constructs were introduced into E. coli (XL-10 cells from Stratagene) using the heat shock protocol provided. Plasmids were purified from 3 ml overnight bacterial cultures using a Qiagen mini-prep kit. Vectors pchRituximab and pchRituximab-HD were electroporated into CHO-S cells using a 4 mm gap cuvette in an Eppendorf Multiporator set at 580V and 40 μs. They were allowed to recover for 2 days before initiating selection.

[0185] Recombinant antibody purification

[0186] Cell culture supernatants were harvested every 3–5 days depending on cell density. Cell suspensions were centrifuged at low speed (480–740 × g) for 7–10 min and the supernatants were kept at −20 °C before further processing. After rapid thawing at 37 °C, the supernatants were passed through a 0.2 μm filter (Corning) by vacuum filtration to remove cell debris, and then the filtered supernatant was passed through a HiTrap Protein G HP column (GE Healthcare). Bound antibodies were eluted with 0.1 M glycine buffer pH 2.7, collected in 1 mL fractions, and the pH was neutralized with 50 μL of 1 M Tris pH 9. The elution profile was determined by reading the UV absorbance at 280 (data not shown). Fractions with significant protein content were then pooled and concentrated using an Amicon Ultra centrifugal filter device 50,000 MW cutoff (Millipore) according to the manufacturer's instructions.

[0187] cell surface binding

[0188] 3 × 10 of Raji, Ramos, or JOK-1 cells 5 1000 / well were seeded in a 24-well plate and incubated overnight at 37°C and 5% CO2. The cells were then harvested and washed twice with PBS. The cells were resuspended in 1 mL of PBS and incubated with either chRituximab or HD chRituximab at increasing concentrations (1 μg, 5 μg, 10 μg, 20 μg / mL) and incubated at 4°C for 30 min. Excess antibody was removed by washing the cells twice with PBS, and the cells were then resuspended in 1 mL solution of FITC-conjugated goat anti-human (Sigma, 1:1000) and incubated at 4°C for 30 min. After washing twice, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Specific mean fluorescence intensity was determined by using the formula: specific MFI = MFI (primary Ab + goat anti-human FITC) - MFI (goat anti-human FITC).

[0189] Apoptosis assay

[0190] 2 × 10 of Raji, Ramos, or JOK-1 cells 5 100 μL / well were seeded into a 24-well plate and incubated overnight at 37° C. and 5% CO2. Cells were then treated with increasing concentrations of Ab for 20 h at 37° C. Cells were harvested, washed once with PBS, and resuspended in 100 μL of 1× annexin binding buffer containing 3 μL of Annexin V Alexa Fluor 488 conjugate (Invitrogen) and a final concentration of 4 μg / mL of propidium iodide (Sigma) to detect apoptosis and cell death, respectively. After 20 min of incubation at 37° C., cells were diluted with 150 μL of 1× annexin binding buffer and analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). The percentage of apoptotic cells was determined by gating on the healthy population in untreated control samples and using the formula: percent apoptotic cells=(1-(live treated target cells / live untreated target cells))*100.

[0191] CDC Assay

[0192] 2×10 5 Cells were seeded in 24-well plates and incubated overnight at 37°C and 5% CO2. Cells were then treated with increasing concentrations of Abs in the presence of 5% rabbit HLA-ABC complement enriched serum (Sigma) for 2 h at 37°C. Cells were harvested, washed once with PBS, and resuspended in 200 μL of PBS containing 50 nM calcein-AM (Biochemica) and 4 μg / mL propidium iodide (Sigma). After 20 min of incubation at 37°C, cell viability was analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Percent death was determined by the formula: Percent dead cells = (1-(live treated target cells / live untreated target cells))*100.

[0193] PBMC separation

[0194] Peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats (Kentucky Blood Center, Lexington KY) of healthy donors by Ficoll-Hypaque density gradient centrifugation. PBMCs were diluted to 6×106 cells / mL in hRPMI (10% FBS, low IgG) culture medium and maintained for up to 3 days. PBMC viability and daily cell population dynamics were analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience) prior to the experiment.

[0195] ADCC assay

[0196] Target cells (Raji, Ramos, or JOK-1) were harvested from T75 flasks and resuspended in 1 mL of medium containing 400 nM calcein-AM (Biochemica) and 8 μL of TFL2 dye (OncoImmunin), used according to the manufacturer's instructions. Target cells were labeled for 45 min at 37°C, washed twice with medium, and then diluted to 6 × 10 5 Effector cells (PBMCs) were then harvested from the T75 flask and resuspended to a density of 1.2 × 10 cells / mL. 7 Target cells and effector cells were mixed at an E:T ratio of 20:1, and then 250 μL of the cell mixture was dispensed into individual 5 mL round-bottom tubes and incubated with increasing concentrations of Ab for 2 hours at 37° C. After incubation, target cell viability was analyzed by flow cytometry (BD FACSCalibur Instrument, BD Bioscience). Percent death was determined by the formula: Percent dead cells=(1-(live treated target cells / live untreated target cells))*100.

[0197] result

[0198] Fluorescence-activated cell sorting (FACS)

[0199] To verify that recombinant chRituximab and chRituximab-HD antibody are functional, their ability to bind to cells from the human B cell line JOK-1 was tested using fluorescence-activated cell sorting (FACS). In Figure 6, the lower panel shows the mean fluorescence intensity (MFI) of staining with chRituximab-HD antibody, while the upper panel represents staining using chRituximab, a non-HD antibody. Binding of chRituximab-HD antibody was approximately 4-fold higher than that of chRituximab.

[0200] Induction of apoptosis is dependent on receptor cross-linking

[0201] One of the proposed mechanisms of HD antibodies is receptor cross-linking induction of apoptosis. The induction of apoptosis of chRituximab and HD antibodies was compared in three cell lines, Raji, Ramos and JOK-1. The addition of chRituximab induces apoptosis in about 30% of cells in some cell lines, especially at the highest dose. HD antibodies induce significantly more apoptosis than the unmodified chimera. Similarly, HD antibodies are more potent inducers of apoptosis in Ramos cells and other B lymphoma cells. Table 4 shows the apoptotic effects of two versions of Rituximab over a range of concentrations. It is interesting to note that the enhancing effect is much more pronounced at lower concentrations of Abs. For example, after treatment of Raji cells with 5 μg / mL of either antibody, the percentage of apoptotic cells is 2.5-fold higher after HD treatment, but only slightly less than 2-fold higher after treatment with 20 μg / mL.

[0202] [Table 4]

[0203] Comparison of complement-dependent cytotoxicity (CDC)

[0204] The CDC activity of chRituximab and chRituximab-HD was compared (see Figures 7A-C). CDC is induced after binding of complement components to the Fc region of the antibody and is potent in IgG1 isotypes, which is the isotype of the HD construct. The enhancing effect was observed in all cell lines. As seen in Figure 7A, for example, at 5 μg / mL, there was virtually no CDC activity in Raji cells carrying the chimera, but 35% of the cells were killed by the HD mAb. This correlates with the highest improvement in efficacy in apoptosis. It is interesting to note that the potency of the HD antibody reaches a plateau at 5 μg / ml in Ramos cells (Figure 7B). chRituximab appears to reach a plateau at 10 μg / ml, but does not reach the potency of the HD Ab at any of the levels tested, suggesting that even higher doses do not reach the killing capacity of the HD Ab at 5 μg / ml.

[0205] ADCC Comparison

[0206] The chimeric antibodies were tested for their ability to induce antibody-dependent cellular cytotoxicity (ADCC). The HD antibody induces significantly more ADCC than chRituximab in Raji and Ramos cells at 1 μg / ml and 3 μg / ml.

[0207] Inhibition of lymphoma growth in vitro

[0208] To estimate the in vivo killing potential of these anti-CD20 antibodies against tumor cells, the anti-proliferative effects of chRituximab and chRituximab-HD were tested in Raji and Ramos cell lines. The assay measures the level of fluorescent dye that binds to nucleic acids (see Methods and Materials). The HD antibody inhibited proliferation to a greater extent in both cell lines at all concentrations tested.

[0209] Example 4 - In vivo characterization of HD anti-Her-2 antibody (trastuzumab) with reverse configuration

[0210] The HD form of Herceptin (Trastuzumab) was produced as described in Example 3. CDR sequences were extracted from the heavy and light chain variable regions. Herceptin light chain (1 and 2)

number

number

[0211] To evaluate the HD antibody for efficacy in a relevant human tumor model, treatment was initiated in a nude mouse model of low antigen expressing breast cancer (MCF-7) 7 days after tumor injection.

[0212] As shown in Figure 8, Herceptin showed no therapeutic effect, with tumor measurements identical to controls (top line). In contrast, HD-Herceptin dramatically inhibited tumor growth (bottom line). Herceptin's lack of activity in this model was expected due to its failure to recognize low antigen expressing breast cancer. Histological examination of HD-Herceptin treated tumors revealed few viable cancer cells, but abundant inflammatory cells.

[0213] When extrapolated to human doses, the maximum effective dose of HD-Herceptin was found to be 10 μg, while Herceptin was ineffective up to 100 μg / dose (based on the assumption of a 75 kg human subject).

[0214] Example 5 - Characterization of HD anti-Vegf (Avastin) antibodies with reverse configuration

[0215] The biosimilar bevacizumab antibody was converted into its recombinant HD form as described in Example 3 using CDRs extracted from the variable heavy and variable light chain sequences. Bevacizumab light chain

number

number

[0216] VEGF is produced by healthy and neoplastic cells. Its activity is mediated by two receptor tyrosine kinases. VEGF signaling is often the rate-limiting step in physiological and pathological angiogenesis. Bevacizumab has been studied as an antiangiogenic cancer therapeutic as a single agent and in combination with chemotherapy in patients with stage III and IV colon cancer.

[0217] Summary of in vivo tumor model evaluation of HD bevacizumab: a) inhibiting tumor growth (in renal cell carcinoma and prostate cancer); b) controlling tumors (in colon cancer); c) prevention of tumor recurrence; d) reducing / preventing metastasis (both renal and colonic); e) reducing the proliferation of A498 human renal carcinoma cells.

[0218] In vivo evaluations have shown that other types of cancer can be treated with HD-anti-Vegf, not only inhibiting proliferation but also regressing the cancer.

[0219] Example 6 - Characterization of HD anti-PD-L1 antibodies with T15 dimer configuration

[0220] The CDRs from the following variable heavy and light chain sequences of atezolizumab (anti-PD-L1 mAb) were used to construct a humanized IgG1 (as described in Example 3) incorporating a reversed configuration of T15 dimers (see Table 2) on the Fc terminus of the antibody. Heavy Chain:

number

number

[0221] HD-atezolizumab was tested against the parental antibody against melanoma cells bearing the PD-L1 antigen. Human PD-L1 expression was assessed by flow cytometry. Briefly, melanoma cell suspensions were prepared and washed with fluorescence-activated cell sorter buffer consisting of phosphate-buffered saline (PBS; pH 7.2) containing 2% fetal bovine serum. Cells were incubated with anti-PD-L1 antibody (2 μg / mL) or mouse IgG1 isotype control (2 μg / mL) for 60 min at 4°C, washed three times, and incubated with FITC-labeled secondary antibody (BD PharMingen, San Jose, CA) for 30 min at 4°C. Cells were then washed three more times with PBS, fixed with 2% formalin, and assessed for fluorescence (FACScalibur flow cytometer; BD Bioscience, San Diego, CA).

[0222] Results were expressed as mean fluorescence index (MFI) or fold increase in binding (background subtracted) - see Table 5 below.

[0223] [Table 5]

[0224] The results demonstrate a greater than 100-fold increase in binding to cell surface PD-L1 by the HD form of the antibody, which necessarily results in an increased ability to block tumor cell-mediated T cell suppression.

[0225] Example 7 - HD Technology for in vitro diagnostic applications

[0226] A monoclonal antibody against PSA was recombinantly HD-modified, then labeled for electrochemiluminescence detection and utilized in an antigen detection assay (Anal. Sci. 2009, May 25(5):587-97). The unmodified antibody was compared to an HD-modified antibody in which the T15 peptide was linked to the end of the antibody light chain.

[0227] The HD-modified antibody provided a much higher signal to noise ratio than the unmodified antibody, making the assay more sensitive, especially at lower antigen concentrations (see FIG. 11).

[0228] The results demonstrate that HD modified antibodies can be used in any configuration (direct or indirect detection) with any detection method, including more standard ELISA. Due to higher signal generation, HD modified antibodies can be used to increase the signal and shorten the time to readout of the assay.

[0229] The use of the word "a" or "an" when used herein in conjunction with the term "comprising" can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more." Any element expressed in the singular also includes its plural. Any element expressed in the plural also includes its singular. The term "plurality," as used herein, means more than one, e.g., two or more, three or more, four or more, etc.

[0230] As used herein, the terms "comprising," "having," "including," "containing," and grammatical variations thereof are inclusive or open ended and do not exclude additional unrecited elements and / or method steps. The term "consisting essentially of," when used herein in connection with a composition, use, or method, indicates that additional elements, method steps, or both additional elements and method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used herein in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. As used herein, the term "about," when used to describe a recited value, means within 10% of the recited value.

[0231] All citations are incorporated herein by reference.

[0232] The present invention has been described with respect to one or more embodiments. However, it will be apparent to those skilled in the art that many variations and modifications can be made without departing from the scope of the invention as defined in the claims. The claims should not be limited by the preferred embodiments set forth in the examples, but should be accorded the broadest interpretation consistent with the description as a whole.

Claims

1. An HD peptide comprising an amino acid sequence in reverse configuration compared to a corresponding naturally occurring homodimerization (HD) peptide or a conservative variant having at least 90% sequence identity therewith.

2. The HD peptide according to claim 1, wherein the corresponding naturally occurring HD peptide is a T15 HD peptide or an R24 HD peptide, and optionally, the HD peptide comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 4, SEQ ID NO: 6 or SEQ ID NO:

18.

3. The HD peptide according to claim 1, having one or more amino acid substitutions, wherein the one or more substitutions increase the hydropathy of the HD peptide.

4. A homodimerization (HD) peptide comprising an amino acid sequence of a naturally occurring HD peptide and comprising an amino acid sequence containing one or more amino acid substitutions, wherein the one or more substitutions increase the hydropathy of the homodimerization (HD) peptide.

5. The HD peptide according to claim 4, comprising an amino acid sequence of SEQ ID NO: 1 and having one or more amino acid substitutions at positions 4, 7 and 18, and optionally, the one or more amino acid substitutions at position 4 are substitutions to K or to a conserved amino acid of K, the one or more amino acid substitutions at position 7 are substitutions to R or to a conserved amino acid of R, the one or more amino acid substitutions at position 18 are substitutions to H or to a conserved amino acid of H, and optionally, comprising an amino acid sequence having 80% to 100% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO:

10.

6. A homodimerization (HD) peptide dimer comprising a first HD peptide and a second HD peptide, wherein the first and / or second HD peptide comprises the HD peptide according to claim 1.

7. The HD peptide dimer according to claim 6, wherein the first and second HD peptides of the dimer are linked by a linker such as gly-gly.

8. The homodimerization (HD) peptide dimer according to claim 6, comprising an amino acid sequence having 80% to 100% sequence identity with SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO:

19.

9. An antibody or antigen-binding fragment comprising the HD peptide according to any one of claims 1 to 5 or the HD peptide dimer according to any one of claims 6 to 8, which is optionally a humanized IgG such as humanized IgG1, humanized IgG4 or humanized IgG3.

10. The antibody or antigen-binding fragment according to claim 9, wherein the HD peptide or HD peptide dimer is fused to the nucleotide affinity site of the antibody or antigen-binding fragment, and optionally, the HD peptide or HD peptide dimer is fused via lysine, cysteine or a carbohydrate.

11. The HD peptide or HD peptide dimer is positioned immediately after the CDR3 of the heavy chain or light chain of the antibody, positioned immediately after the C-terminus of the constant region of the heavy chain or light chain of the antibody, positioned immediately after the variable region of the heavy chain of the antibody, or positioned immediately after the C-terminus of the Fc region of the antibody, The antibody or antigen-binding fragment according to claim 9.

12. The antibody or antigen-binding fragment according to claim 9, wherein the HD peptide or HD peptide dimer is conjugated to the antibody, and the antibody may be a chimeric recombinant antibody.

13. The antibody or antigen-binding fragment according to claim 9, wherein a spacer such as gly-gly precedes the HD peptide or HD peptide dimer.

14. The antibody is a single-chain antibody (scFv), bispecific antibody (BsAb) or antibody-like peptide, and optionally, the antibody is a humanized monoclonal antibody, and optionally, the antibody is a Her-2neu antibody (such as Herceptin), a CD-20 antibody (such as Rituximab), a vascular endothelial growth factor antibody (such as Avastin), or a checkpoint inhibitor antibody (such as PD-L1).

15. A composition comprising one or more antibodies or antigen-binding fragments according to claim 9 and a pharmaceutically acceptable carrier.

16. A first nucleic acid sequence encoding an HD peptide according to any one of claims 1 to 5 or an HD peptide dimer according to any one of claims 6 to 8, optionally further comprising a second nucleic acid sequence encoding an antibody or antigen-binding fragment, whereby when the first and second nucleic acid sequences are expressed, the HD peptide and the antibody or antigen-binding fragment are expressed as a fusion protein, an expression vector.

17. A method for generating a homodimerization (HD) antibody, comprising the step of expressing the expression vector according to claim 16 in a host cell.

18. An isolated host cell transformed with the expression vector according to claim 16.

19. A method for enhancing the binding and / or efficacy of an antibody, comprising: conjugating an HD peptide according to any one of claims 1 to 5 or an HD dimer according to any one of claims 6 to 8 to the antibody; or recombinantly expressing a chimeric antibody comprising the HD peptide A method comprising.

20. A pharmaceutical composition comprising the antibody or antigen-binding fragment according to claim 9 for treating a patient suffering from a disease or condition, optionally wherein the disease or condition is selected from the list consisting of cancer, autoimmune disorder, inflammatory disorder, neurodegenerative disease, cardiovascular disease, graft or transplant rejection, a pharmaceutical composition.

21. A method for detecting an analyte in a sample, comprising: contacting the analyte with an antibody or antigen-binding fragment against the analyte, wherein the antibody or antigen-binding fragment is fused to an HD peptide according to any one of claims 1 to 5 or an HD peptide dimer according to any one of claims 6 to 8; detecting a complex formed by the analyte and the antibody fused to the HD peptide A method comprising.

22. A kit for detecting an analyte in a sample, comprising: an antibody or antigen-binding fragment against the analyte, which is an antibody or antigen-binding fragment fused to an HD peptide according to any one of claims 1 to 5 or an HD peptide dimer according to any one of claims 6 to 8; instructions for use in detecting the analyte A kit comprising.

23. A phage display library comprising an HD peptide according to any one of claims 1 to 5 or an antibody or antigen-binding fragment linked to an HD peptide dimer according to any one of claims 6 to 8.

24. A therapeutic or diagnostic composition comprising the antibody or antigen-binding fragment according to claim 9.

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