AU2017100957A4 - IGM antibody, composition and kit comprising it for treatment or diagnosis of a disease - Google Patents
IGM antibody, composition and kit comprising it for treatment or diagnosis of a disease Download PDFInfo
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- AU2017100957A4 AU2017100957A4 AU2017100957A AU2017100957A AU2017100957A4 AU 2017100957 A4 AU2017100957 A4 AU 2017100957A4 AU 2017100957 A AU2017100957 A AU 2017100957A AU 2017100957 A AU2017100957 A AU 2017100957A AU 2017100957 A4 AU2017100957 A4 AU 2017100957A4
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- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C07K16/1036—Retroviridae, e.g. leukemia viruses
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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Abstract
The present invention provides an IgM antibody for use in a method for treatment or 5 diagnosis of a disease in a subject. The subject especially but not exclusively is a human and the disease includes cancer, an infectious disease, an autoimmune disease or an allergic disease. In another aspect, the present invention refers to a pharmaceutical composition for use in a method for treatment of a disease and a kit for diagnosis of a disease in a subject such as in a human comprising the IgM antibody. 10 The inventor unexpectedly found that the IgM antibodies are highly advantageous over IgG antibodies of at least similar specificity which cannot access the target antigen and, thus, fail to sufficiently treat or diagnose the disease. 9270519_1 (GHMatters) P106323.AU Antibody combining site Antigen Fc Light chain 4- Heavy chain Fig. 1A Fig. 1B 1gM ~ilsomerize IgG naked antigen blocked antigen blocked antigen blocked antigen Fig. 1C
Description
IGM antibody, composition and kit comprising it for treatment or diagnosis of a 2017100957 12 Μ 2017 disease
The present invention provides an IgM antibody for use in a method for treatment or diagnosis of a disease in a subject. The subject, especially but not exclusively, is a human 5 and the disease includes cancer, an infectious disease, an autoimmune disease or an allergic disease. In another aspect, the present invention refers to a pharmaceutical composition for use in a method for treatment of a disease and a kit for diagnosis of a disease in a subject such as in a human comprising the IgM antibody.
BACKGROUND OF THE INVENTION 10 Antibodies are serum proteins produced by the body in response to an infection caused by a bacterium or a virus, or to an insult from a chemical compound foreign to the body. The natural role of antibodies is to bind to the infectious or foreign agent so as to inactivate it or to remove it from the body. The basic structure of an antibody is a bisymmetrical molecule also named Ig basic unit, each half comprising a short polypeptide (LC) joined to a longer 15 polypeptide (HC). Both polypeptides have a “variable” region at the N-terminal end and a “constant” region at the other end, both regions made up of domains. The variable region has a single domain but the constant region has 1 (in LC) or 3-4 (HC) domains. The variable regions of LC and HC form the antibody combining site (also called antigen binding site or paratope) that governs the binding specificity of the protein. 20 There are 5 classes of antibodies distinguished by the HC constant region. IgM is the primordial member, being the first to evolve in nature and the first to appear in the body during an infection, while IgG comes second both in evolution and during a response. Other classes (IgA and IgE) follow accordingly. IgG is in fact derived from IgM due to a cellular “switch” mechanism in the antibody producer cell. Importantly in this process, while 25 the HC constant region is substituted, the same variable region in both LC and HC is retained by the IgG molecule. However, important amino-acid changes are often found in the IgG variable region due to somatic mutations that occur during the switch process and these endow the antibody with higher affinities for the antigen.
Thus, while IgM antibodies usually have low intrinsic affinities for the antigen (<107 M"1 per 30 antibody combining site), IgG antibodies usually have much higher affinities (109 to 1014 M"1). Teleologically, IgM serves to provide an immediate first-line defense against an infection while IgG comes in weeks later to deal more effectively and more specifically with 1
9270519_1 (GHMatters) P106323.AU the infection. Despite the low affinity of the individual combining sites, which handicaps their binding to small or soluble antigens, IgM antibodies can nevertheless bind very well to bacteria and other large particulates (e.g. eukaryotic cells) that have multiple copies of the antigen. IgG is a monomer and can because of the high intrinsic affinities bind stably 5 through just a single combining site. The small size of IgG is advantageous and assumed to allow the molecule to traverse into inter-vascular spaces and tissues that are inaccessible by IgM. 2017100957 12 Μ 2017
IgG antibodies are extremely specific and precise as they are able to discriminate between closely related antigens with differences as small as the stereo-isomeric groups of a 10 monosaccharide. They are mono-specific. The structural basis of this exquisite specificity, as traditionally explained, is that the antibody combining site fits the antigen stereo-chemically like a lock and key (Eisen, H.N. and Chakraborty, A.K., Evolving concepts of specificity in immune reactions. Proc Natl Acad Sci U S A., 107(52): p. 22373-80). The combining site is pre-configured before antigen contact, determined only 15 by the amino-acid sequences of the variable regions of both LC and HC and not by the antigen. The combining site is thus a fairly rigid structure so that antigen is only bound when the combining site is able to dock with the antigen in the first place, and secondly, when there is sufficient physical complementariness between the two surfaces to allow bonding forces to operate at close range to hold the antigen in place. 20 This “lock-and-key” model has also been used to explain how IgM antibodies bind to their target i.e. via an inflexible pre-configured combining site. Recently, a small subset of IgM antibodies were found to be poly-specific (or poly-reactive) i.e. capable of binding to more than one type of antigen (Notkins, A.L., Polyreactivity of antibody molecules. Trends Immunol., 2004, 25(4): p. 174-9). This was explained by the “conformational flexibility” 25 hypothesis in that the combining sites of these antibodies were able to constantly isomerize between several types of conformations, each conformation again pre-configured before antigen contact, and which fits the antigen like a lock-and-key (James, L.C. and Tawfik, D.S., Conformational diversity and protein evolution-a 60-year-old hypothesis revisited. Trends Biochem Sci., 2003, 28(7): p. 361-8). In addition, 30 there is yet another group of IgM antibodies that is found at low levels in humans - the “natural antibodies” (Coutinho, A. et al., Natural autoantibodies. Curr. Opin. Immunol., 1995, 7: p. 812-818). These antibodies have low binding activities to a wide variety of antigens particularly the self- or auto-antigens i.e. quite non-specific, but little else is known. 2
9270519_1 (GHMatters) P106323.AU
Their exquisite specificity makes antibodies valuable both as high-precision diagnostic probes - whether to detect cancers in whole body scans or to identify cells and sub-cellular structures by histopathology for various diseases - and as high-precision therapeutic missiles, whether to annihilate microorganisms or cancerous and autoimmune cells. Thus, 5 a number of “designer” antibodies have been provided, which term “designer antibody” is used to distinguish them from the animal or human sera that contain the naturally-elicited polyclonal antibodies. 2017100957 12 Μ 2017
IgG are predestinated for the above-referenced applications as: (a) the individual combining sites have higher affinities for the antigen i.e. bind much better; (b) the antibody 10 is five-fold smaller which allows it to navigate through tissues better and stay longer in the circulation. In contrast, there are problems with IgM antibodies known to the skilled person mainly precluding their application, namely: (a) the individual combining sites have weak affinities for the antigen; (b) the antibody is generally less specific for the antigen i.e. some can cross-react with other antigens of a similar structure; (c) the large size of the molecule 15 hinders its passage through inter-vascular spaces; (d) its size, making it more difficult to produce them especially in large scale and to re-engineer them; (e) it is also more difficult to isolate and purify them due to its chemical characteristics besides being extremely fragile; (f) they are more quickly cleared by the body, and (g) they bear a higher anaphylactic potential. 20 Accordingly, of the 51 designer antibodies approved by the FDA in 2014 for therapeutic use (IMGT Repertoire [IG and TR] www.imgt.org/mAb-DB/index), and the 53 new designer antibodies released in 2016 (Reichert, J.M., Antibodies to watch in 2016. MAbs., 2015. 8(2): p. 197-204), all of them are IgG antibodies (or fragments of these) (See also Adler, M.J. and Dimitrov, D.S., Therapeutic antibodies against cancer. Hematol Oncol Clin North 25 Am., 2012, 26(3): p. 447-81, vii). Of the few IgM antibodies that have been investigated, one human and another mouse but both specific for a common antigen (lipid A) in the cell wall (endotoxin) of Gram-negative bacteria, were developed for the treatment of bacterial sepsis in the late ‘80s; however, both failed to gain FDA approval due to a questionable efficacy (among other concerns) (Marks, L., The birth pangs of monoclonal antibody 30 therapeutics: the failure and legacy of Centoxin. MAbs., 2012, 4(3): p. 403-12). The rationale for using these antibodies is not because they have an advantage over IgG antibodies in overcoming the occluded antigen - a property not known to these inventors nor is the antigen occluded - but rather because they happen to be around in the first place and then found to bind satisfactorily to the antigen. The reason why IgM antibodies can 3
9270519 1 (GHMallers) P106323.AU bind very well to this particular antigen is because it is present in multiple copies in the soluble material released from the bacteria to the circulation that is easily accessible, i.e. in situations where IgG can also bind. Furthermore, the endotoxin bound by IgM is efficiently cleared by the body. For reasons unclear, IgM hybridomas are produced abundantly and 5 more frequently than expected from the cell-fusion process especially for certain antigens including ones that do not have repeating structures. This is true both for mouse hybridomas created from mouse cells using the traditional methodology described by Kohler and Milstein in 1975, and also, more recently, for human hybridomas created from human cells grown in “humanized” mice. 2017100957 12 Μ 2017 10 In diagnostic pathology too, IgG antibodies are mainly used as the reagent probe for the simple reason that these have higher affinities than IgM as well as the perception that, being smaller, they can access the antigen better. Again, if IgM antibodies were used for certain antigens, this was for the same reasons and in situations as mentioned above.
However, not all designer IgG antibodies in the market or used by researchers perform 15 satisfactorily according to expectation. This particularly relates to certain types of antigens. Indeed, it is quite common to encounter an IgG antibody which satisfies all the criteria of a good antibody in terms of specificity and binding strength when assessed against the purified or recombinant or synthetic antigen, but fails to bind to the native antigen found in living cells or in formalin-fixed tissues. 20 It seems in these situations, that the access to the native antigen is somehow blocked. In some cases, a solution commonly employed by tissue pathologists is to first retrieve the antigen by heating the tissue in a microwave or by enzyme digestion. For example, localization of osteopontin in paraffin-embedded bone specimens to specific organelles in osteoblasts, osteocytes, or the bone matrix by immunochemical staining with specific IgG 25 antibodies has presented great difficulties and conflicting results (Devoll, R.E., et al., Improved immunohistochemical staining of osteopontin (OPN) in paraffin-embedded archival bone specimens following antigen retrieval: anti-human OPN antibody recognizes multiple molecular forms. Calcif Tissue Int., 1997, 60(4): p. 380-6). Another example is the antibody-detection of the nuclear enzyme, telomerase, in tumor cells (Leung, D.T., et al., 30 Nuclear telomerase is less accessible to antibody probing than known nuclear antigens: retrieval with new immunostaining buffer. Histochem Cell Biol., 2005, 123(1): p. 105-12). A solution to these problems so far is to use antigen retrieval in various forms (Gown, A.M., Unmasking the mysteries of antigen or epitope retrieval and formalin fixation. Am J Clin Pathol., 2004, 121(2): p. 172-4, Denda, T, et al., Optimal antigen retrieval for ethanol-fixed 4
9270519_1 (GHMatters) P106323.AU cytologic smears. Cancer Cytopathol., 120(3): p. 167-76), but this is time-consuming and expensive. 2017100957 12 Μ 2017
In addition, despite intense investigations over the last several decades, no vaccine has been found that can effectively prevent HIV infection. The major obstacle is the fact the 5 HIV virus is extremely elusive and smart in finding ways to escape the immune forces. A case in point is this: IgG antibodies to the viral surface protein, gp120, a glycoprotein, have long been found to neutralize the virus effectively and are protective. However, it was recently realized that these same antibodies were unable to bind to this viral protein in some patient isolates. It was revealed in these cases that the virus managed to shield the 10 antigen with glycans (carbohydrates) (van Gils, M.J., et al., Longer V1V2 region with increased number of potential N-linked glycosylation sites in the HIV-1 envelope glycoprotein protects against HIV-specific neutralizing antibodies. J Virol., 2011, 85(14): p. 6986-95, Burton, D.R. and Hangartner, L. Broadly neutralizing antibodies to HIV and their role in vaccine design. Annu. Rev. Immunol. 2016. 34: p. 635-59). 15 There, thus, remains a strong need for the means and methods for therapy and diagnosis in situations, in which IgG antibodies fail to provide satisfactory results such as the treatment of HIV where new treatment options are urgently required.
SUMMARY OF THE INVENTION
The present invention provides in a first aspect an IgM antibody for use in a method for 20 treatment or diagnosis of a disease in a subject, which disease cannot be treated or diagnosed with an IgG antibody with at least similar specificity. In a second aspect, the present invention provides an IgM antibody for use in a method for treatment or diagnosis of a disease in a subject, wherein the antibody binds to an antigen hidden for an IgG antibody with at least similar specificity. The subject is preferably a human and the disease 25 in particular includes cancer, an infectious disease, an autoimmune disease or an allergic disease, in particular cancer like colorectal cancer or a human immunodeficiency virus (HIV) infection.
The IgM antibody can be selected from a Fab, Fab’ or F(ab’)2 fragment and conjugated to at least one of nano-microspheres, human serum albumin or a toxic agent and/or can be a 30 structurally modified IgM in form of a hybrid IgG-like antibody.
In particular, the IgM antibody is a human, humanized or chimeric antibody and used in a method for treatment of a disease and wherein the disease is a HIV infection. The IgM 5
9270519J (GHMallers) P106323.AU antibody can also be a murine antibody used in a method for diagnosis of a disease, which disease is cancer, which IgM antibody in particular recognizes telomerase. 2017100957 12 Μ 2017
In another aspect, the present invention refers to a pharmaceutical composition for use in a method for treatment of a disease. The pharmaceutical composition comprises an IgM 5 antibody as described above and at least one pharmaceutically tolerable excipient.
In a further aspect, the present invention refers to a kit for diagnosis of a disease in a subject, in particular a human comprising an IgM antibody as described above.
The inventors unexpectedly found that the IgM antibodies provided herein are highly advantageous as IgG antibodies of similar specificity failed, in particular because the 10 antigen might not be accessible for the IgG antibody, i.e. hidden for the IgG antibody. This finding is surprising considering the widely held prejudices in the respective technical field against the use of IgM antibodies. IgM which are even about 5-times bigger than IgG unexpectedly proved to be able to access the target antigen in the situations as claimed in the present invention. 15 The inventors assume that this might be based on a particular binding mechanism of the IgM antibodies which seems to be different from the one used by IgG antibodies. Specifically, this relates to the docking of the antigen by the antibody at close-range where the antibody combining sites are within striking distance of the antigen, i.e. in situations where the antibody has already managed to navigate through any distant obstacles to 20 arrive at the antigen’s proximity. Thus, while IgG is able to bind to an antigen when the latter is presented in an open space (unobstructed), it is unable to do so when the antigen is, for example, obstructed by neighboring structures due to the rigidity of the antibody combining site. The antibody site fits the antigen like a lock-and-key. In contrast, the inventors postulate that the antibody combining site of IgM appears to be flexible and 25 isomerizes between states in which the variable regions of the HC and LC constantly associate with and dissociate from each other, so that the opportunity avails for the dissociated chains to individually maneuver around the obstacle to reach the antigen and ultimately form a stable association with it. Thus, IgM antibodies are particularly advantageous in the treatment and diagnosis of diseases which cannot be treated or 30 diagnosed with IgG antibodies with at least similar specificity and with an antigen not accessible for them, respectively. Besides, the simultaneous engagement of several sites together as in IgM antibodies can significantly increase the overall affinity (avidity) for the target antigen. 6
9270519 1 (GHMatiers) P106323.AU
BRIEF DESCRIPTION OF THE DRAWINGS 2017100957 12 Μ 2017
Fig. 1A is a schematic representation of the basic structure of an Ig basic functional unit.
Fig. 1B is a schematic representation of the basic structure of an IgM antibody.
Fig. 1C is a schematic representation of the different docking mechanisms assumed by the 5 inventors, namely to explain why IgG is able to bind to antigen when the latter is presented in an open space (unobstructed), but is unable to do so when the antigen is obstructed by neighboring structures due to the rigidity of the antibody combining site. The antibody site fits the antigen like a lock-and-key. In contrast, the inventors postulate that the antibody combining site of IgM is flexible and isomerizes between states in which the variable 10 regions of the HC and LC constantly associate with and dissociate from each other.
Fig. 2 is a diagram referring to the results of a direct ELISA assay showing the binding of the three IgM (Mab1, Mab23, Mab50) and the single IgG (Mab12) mAbs (at a fixed dilution) to GTP-HSA or GMP-BSA compared to the carrier protein alone (HSA or BSA respectively). Abbreviations: ELISA = enzyme-linked immunosorbent assay; mAb = monoclonal antibody; 15 GTP = guanosine triphosphate; GMP = guanosine monophosphate; HSA = human serum albumin; BSA= bovine serum albumin; (see later); MSA= mouse serum albumin.
Fig. 3 is a diagram and shows results obtained with two human purified anti-GTP antibodies (in serial dilutions) sharing the same specificity as Mab12 by inhibition ELISA (positive inhibition). Control using two human sera which had no anti-GTP activity did not 20 inhibit the binding of Mab12 to the coated antigen (GTP-HSA).
Fig. 4 shows diagrams obtained with an inhibition ELISA providing a comparison between the fine specificities of Mab12, Mab23 and Mab50 by inhibition ELISA using various analogues (or inhibitors, in serial dilutions) to inhibit the binding of these antibodies to the coated antigen (GTP-BSA). HSA is the control negative, other analogues used are 25 indicated in the graph. Oligo-G = polymer of GMP (linear structure); Oligo-GC = mixed polymer of alternating GMP and CMP (linear but can form circles).
Fig. 5A to 5C refer to the results of the cell-immune-fluorescence seen under the fluorescence microscope showing the staining pattern on HEp2 cells of various mouse mAbs and human anti-GTP antibodies (Hu 1 - Hu 5) (Fig. 5A), and control antibodies 30 known to bind to specific cellular structures (Fig. 5B). Also shown are the results of inhibition of Mab12 staining by Mab23 and Mab50 (Fig. 5C).
Fig. 6A to 6B refer to the results of the cell-immune-fluorescence seen under the 7
9270519_1 (GHMatters) P106323.AU fluorescence microscope showing the staining pattern on skeletal muscle of various mAbs and human IgG anti-GTP antibodies (Fig. 6A), and control antibodies known to bind to specific cellular structures (Fig. 6B). 2017100957 12 Μ 2017
Fig. 7A to 7B refer to the results of the cell-immune-fluorescence seen under the 5 fluorescence microscope with the smooth muscle, which has both a tubulin and a keratin network, both containing an abundance of GTP residues. The figures show the staining pattern on smooth muscle of various mAbs and human IgG anti-GTP antibodies (Fig. 7A), including the binding by Mab12 following prior digestion of the muscle by proteinase K, and control antibodies known to bind to specific cellular structures (Fig. 7B). 10 Fig. 8A to 8D show diagrams with the titration results of the supernatant obtained from various antibodies following absorption with various types of antigen-bead preparations, showing the residual anti-GTP ELISA activity. Antibodies: Mab12 (IgG), purified human anti-GTP (IgG), Mab23 (IgM) and Mab50 (IgM). Antigen preparations: magnetic beads coupled with GTP-HSA, magnetic beads coupled with GTP-MSA, magnetic beads coupled 15 with HSA or glycine, Sepharose beads coupled with GTP-HSA, Sepharose beads coupled with GTP-MSA, and Sepharose beads coupled with glycine. MB = magnetic beads; Sep = Sepharose beads; Glycine-MB, glycine-Sep and HSA-MB are control-negative absorbents.
Fig. 9Ato 9G show the different types and preparations of GTP or GMP antigens used. Of the synthetic antigens, there are two types: one in which the GTP (Fig. 9A) or GMP (Fig. 20 9B) hapten exists as free (uncoupled) individual molecules in solution, and the other, where the hapten is insolubilized by conjugating it at various densities to a carrier protein but still used as a liquid suspension. The protein carriers are small soluble proteins e.g. bovine serum albumin (BSA) (Fig. 9C). These hapten-carrier compounds are used by themselves suspended in solution, or are covalently coupled to large inorganic 25 microspheres: magnetic particles (1 pm) (Fig. 9E) and Sepharose beads (45-165 pm) (Fig. 9D). These conjugates are used for binding studies in solution. In the case of the free hapten, different versions were also made: G-G-G polymers (20 GMP residue long) (Fig. 9G), and G-C-G-C co-polymers (also 20 residue long) (Fig. 9F) which contain mixtures of cytosine (C) and G.
30 DESCRIPTION OF THE INVENTION
The following description and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and for referring to 8
9270519_1 (GHMatters) P106323.AU preferred embodiments thereof. The technical terms used in the present application have the meaning as commonly understood by a respective skilled person unless specifically defined otherwise. 2017100957 12 Μ 2017
As used herein, “comprising” means including the following elements but not excluding 5 others. “Essentially consisting of means that the material consists of the respective element along with usually and unavoidable impurities such as side products and components usually resulting from the respective preparation or method for obtaining the material such as traces of further components. “Consisting of means that the material is solely consist of, i.e. is formed by the respective element. 10 The present invention provides in a first aspect an IgM antibody for use in a method for treatment or diagnosis of a disease in a subject, which disease cannot be treated or diagnosed with an IgG antibody with at least similar specificity. In another aspect, an IgM antibody for use in a method for treatment or diagnosis of a disease in a subject is provided, wherein the antibody binds to an antigen hidden for an IgG antibody with at least 15 similar specificity.
The expression “cannot be treated/diagnosed” means that no sufficient therapeutic effect is achievable with the IgG antibody or that the disease cannot be diagnosed with sufficient sensitivity and specificity of the diagnostic test.
The treatment of the disease includes the administration of the antibody to the subject in 20 effective amounts such as in form of a pharmaceutical composition.
The antibody can be administered by various routes of administration, typically parenteral. This is intended to include intravenous, intramuscular, subcutaneous or rectal administration.
The antibody preferably binds to and in particular neutralizes a target antigen, i.e. it 25 modulates and in particular inhibits at least one effect and more preferably any effect it has biologically. The target antigen which is bound by the IgM antibody can be, for example, a HIV antigen like the viral protein gp120 or a region within gp120 like the V1/V2 region, EGFR (Epidermal growth factor receptor), KRAS (Ki-ras2 Kirsten rat sarcoma viral oncogene homolog), NRAS (Neuroblastoma RAS viral oncogene homolog) or BRAF 30 (v-Raf murine sarcoma viral oncogene homolog B), CD23 (IgE receptor), or the IL-6 receptor.
Diagnosis of a disease includes the detection of a target antigen in a sample like cells in form of a tissue such as a tissue after fixation, in particular chemical fixation, like 9
9270519J (GHMallers) P106323.AU formalin-fixed tissues or of bone specimens such as osteoblasts, osteocytes or bone matrix such as in form of paraffin-embedded bone specimens of a subject. Diagnosis includes diagnostic pathology to identify cells or cellular structures in tissue sections and/or whole-body imaging including detecting a target antigen in a sample like tissue, living cells, 5 fixed cells like methanol-fixed cells, or in fixed tissues like formalin-fixed tissues, or in paraffin-embedded bone specimens (e.g. osteoblasts, osteocytes, bone matrix). For example, diagnosis may include the determination of osteopontin, for example, in bone specimens such as for diagnosis of bone diseases like osteoporosis or the determination of target antigens expressed on granulocytes (NCA, CD15, CD66, and CD67) such as the 10 NCA-90 granulocytic antigen for whole body imaging to diagnose osteomyelitis or the determination of nuclear antigens like PCNA (Proliferating cell nuclear antigen), telomerase, ER and Ki-67 in tissues or the determination of phosphorylcholine. Preferably diagnosis of the disease includes steps of: 2017100957 12M2017 - obtaining a sample from the subject; which subject is preferably a human; 15 - determining the amount or presence of the target antigen in the sample; - optionally comparing the amount with a reference value; and - diagnosing the disease based on the amount or presence of the target antigen in the sample.
The skilled person is aware of the terms “IgM” and “IgG” representing known classes of 20 antibodies, wherein IgG consists of four polypeptides - two heavy chains and two light chains joined to form a "Y" shaped molecule (Fig. 1 A), i.e. one Ig basic functional unit. IgM are pentameric with five Ig units, i.e. they consist of five IgG-related molecules linked by means of a joining peptide and disulfide chains (Fig. 1B). Both differ in size and molecular weight, charge, amino acid composition and carbohydrate content. The pentameric nature 25 of IgM antibodies poses problems due to the sterical hindrance as mentioned above.
In particular, the present invention provides a solution to the problem of using IgG antibodies as a diagnostic or therapeutic probe in the scenarios described above where it seems that IgG antibodies were structurally blocked from docking with the target antigen even though this is well within their reach. The inventor surprisingly and unexpectedly 30 found that the above-mentioned problems can be solved with the IgM antibodies of the present invention. The inventors in particular unexpectedly found a special property of these antibodies not known previously: IgM can bind to certain antigens that appear blocked and inaccessible to IgG antibodies. The mechanism involved is not completely 10
9270519 1 (GHMatiers) P106323.AU known. It is important to note, first and foremost, that this phenomenon does not relate to the IgM pentamer as a whole which is known to be associated with disadvantages compared to IgG in terms of tissue penetration. Instead, this concerns only the individual antibody combining sites of the two classes of antibodies. The inventors assume without 5 being bound by theory that the IgM combining site is not a rigid pre-configured receptor like the IgG combining site but may exist instead, in a state of constant flux in which the variable regions of light chain (LC) and heavy chain (HC) constantly associate with and constantly dissociate from each other. That is, the inventors posit that IgM docks with antigen differently from the lock-and-key approach of IgG. The dissociated HC variable 10 region of IgM is assumed to have the ability to probe for an antigen on its own without LC, which, because of its size and flexibility, allows it to navigate through close-range obstacles that surround certain types of antigens - structures that obstruct the passage of an intact and rigid IgG combining site as schematically presented in Fig. 1C. The LC variable region may then follow the same docking pathway and complete the tether around the antigen. 15 This is similar to the binding mechanism of single-chain or single-domain antibodies (sometimes called camelid antibodies) found in some animals, e.g. sharks and camels (Muyldermans, S., "Nanobodies: Natural Single-Domain Antibodies". Annual Review of Biochemistry, 2013, 82: p. 775-797). 2017100957 12 Μ 2017
The inventors speculate that the low inter-chain affinity between the variable regions of HC 20 and LC in IgM antibodies might make the IgM antibody combining site flexible and different from that of IgG, which in most cases might also reflect the low affinity of the antibody combining site for the antigen. The inventors presume, at the least, the first CH domain has a role in this. The importance of the CH region in influencing the affinity and specificity of an antibody has in fact been proposed by some investigators previously (Torres M. and 25 Casadevall, A., The immunoglobulin constant region contributes to the affinity and specificity. Trends in Immunol., 2007, 29: p. 91-97).
The term “antibody” as used in this application generally includes the intact antibody or any fragment thereof with retained ability to bind to a target antigen like a Fab, Fab’ or F(ab’)2 fragment. Such fragments can be produced by proteolytically cleaving an intact antibody or 30 by synthetic or recombinant techniques. The term antibody generally also covers variants thereof like murine, human, humanized, chimeric or engineered antibodies including fragments thereof. ’’Intact” as used herein means the full, i.e. complete antibody in contrast to fragments thereof.
IgG antibody as used herein means in particular an IgG antibody produced during the 11
9270519_1 (GHMatters) P106323.AU normal course of an infection in a patient, in particular a human. The IgM antibody can in embodiments of the present invention be a “natural" IgM antibody as usually present in the circulation of normal humans and other mammalian species even in the absence of prior immunization, or an “immune” IgM obtained by intentional immunization of a host with a 5 particular target antigen. 2017100957 12 Μ 2017
The IgM antibody is preferably a human, humanized or chimeric antibody when used in a method for treatment of a disease.
In particular embodiments of the present invention, the IgM antibody for use in a method for treatment of a disease is selected from a human, humanized or chimeric intact antibody, 10 Fab, Fab’ or F(ab’)2 fragment or tetramers of Fab and so on. Antibody fragments containing only the combining site are Fv or scFv, wherein antibodies containing the combining site together with the first constant domain of LC and HC are Fab, Fab’ or Fab-dimer. These fragments generally retain the binding specificity of the intact antibody. The smallest active unit defined as IgM antibody herein is the Fab or Fab’ fragment. 15 In embodiments of the present invention, the IgM antibody for use in the method for treatment of a disease is an intact antibody which is humanized or chimeric, also referenced as designer antibody, in order to exploit the useful effector (secondary) functions of IgM such as an efficient opsonization and complement-fixation. In other embodiments of the present invention, fragments are used as IgM antibody, in particular 20 based on Fab fragments like one of Fab, Fab’ or F(ab’)2 or Fab tetramers of the antibody to enable better navigation and easier handling. Since the intrinsic affinity of the IgM Fab is low, the Fab-dimer F(ab’)2 may be preferred to allow multivalent binding. The Fab-dimer, which includes CH2, can be generated by digesting the whole molecule with trypsin, pepsin or 2-mercaptoethylamine-HCI, or it can be constructed using standard protein engineering 25 methods. Larger polymers of Fab, e.g. tetramers, can be similarly constructed.
In the method for diagnosis of a disease, the IgM antibody can be a murine antibody without the need for humanization or the need to generate fragments, i.e. the IgM antibody is preferably a murine intact antibody.
The IgM antibody and the IgG antibody can independently of each other comprise kappa 30 or lambda light chains. “Specificity” refers to the feature or ability of an antibody not to cross-react with antigens other than a target antigen, i.e. it means the ability of an antibody to recognize and differentiate a target antigen by "specifically binding" to the target antigen. More specifically, 12
9270519_1 (GHMatters) P106323.AU it refers to the degree to which an antibody can differentiate and discriminate between the target antigen and non-target homologous or heterologous antigens. Antibodies are generally said to be "specifically binding" if they do not significantly cross-react with non-target antigens. Thus, the IgG antibody has “at least similar specificity” compared to 5 the IgM antibody if it binds to the same target antigen with at least a similar degree of specificity and with at least a similar degree of “specific binding” respectively. 2017100957 12 Μ 2017
Antibody specificity can, for example, be derived from the binding affinity of the antibody to a target antigen and a non-target antigen, because the binding affinity to the target antigen will be higher than the binding affinity to non-target antigens. In particular, antibody 10 specificity might be determined, for example, by evaluating the cross-reactivity of the antibody to non-target antigens. For example, cross-reactivity might be expressed as the ratio of the concentration of the target antigen and of the concentration of a non-target antigen to achieve the same response under the same conditions such as measured with a direct ELISA. For example, a cross-reactivity of 1:200 ratio conveys that a 200 fold 15 increase in concentration of the non-target antigen is necessary to achieve the same response as the target antigen under the same conditions. For example, an antibody is said to specifically bind a target antigen if it exhibits a cross-reactivity ratio of less than 1:1, i.e. of less than 1.0, or of less than 1:20 such as less than 1:100, 1:200, 1:500, 1:1000, 1:5000, 1:10,000 to a non-target antigen. A non-target antigen can be, for example, a 20 homologous or heterologous antigen with at most 80% sequence identity which can be determined using a suitable computer algorithm, as known in the art.
Based thereon, an IgG antibody can be considered for having “at least a similar specificity” compared to the IgM antibody if the cross reactivity ratio as determined with an identical target antigen and an identical non-target antigen, namely the ratio of the concentration of 25 the target antigen and of the concentration of the non-target antigen to achieve the same response for the IgG antibody amounts to at most 110% of the ratio as determined for the IgM antibody for the same target and non-target antigen or is even lower.
Alternatively, the specificity of IgM and IgG can be derived and, hence, whether the IgG has at least a similar specificity can be determined by means of an inhibition type assay 30 like an inhibition ELISA, namely by comparing the binding of both, IgG antibody and IgM antibody, to the target antigen in the presence of a non-target antigen or, alternatively, in the presence of the other antibody in predetermined dilutions. For example, non-target antigen is co-incubated with either the IgG or IgM antibody in the presence of immobilized target antigen, i.e. immobilized on a solid support such as coated on the surface of a 13
9270519 1 (GHMatlers) P106323.AU micro-plate. Alternatively, the IgM antibody is co-incubated with the IgG antibody in the presence of immobilized target antigen. 2017100957 12 Μ 2017
Generally, the skilled person is aware that antibodies bind to the antigen at an epitope region of the antigen. “Epitope" is, thus, the region formed by amino acid residues in the 5 target antigen which are important for mediation the binding with the antibody, in particular which are involved in the direct binding interaction such as bound by the antibody. Alternatively, the epitope can be a particular sugar or a carbohydrate group. An “antigen hidden for an IgG antibody” means that the antigen, in particular the epitope, is located within a region unexposed and not accessible, respectively, for the IgG antibody. In 10 particular the epitope is located such that it is prevented from an interaction with the IgG antibody, i.e. not or poorly accessible for the IgG antibody with at least similar specificity compared to the IgM antibody. This includes embodiments, in which the antigen is in or on the surface of a cell circulating in the blood or within a tissue, in particular the epitope, is at least partially obstructed, shielded, covered or concealed, conjugated to protein carriers, or 15 modified like mutated. Mutated means a change in the epitope amino acid residues compared to the natural epitope sequence in particular with a sequence identity of at most 90%. Then the IgG antibodies are ineffective because these cannot bind to the, for example, blocked or modified (mutated) antigen, anymore.
The IgM antibody has a suitable affinity for the target antigen. The IgM antibody can, for 20 example, bind to the target antigen with a KD of between 1 x 10"6 and 1 x 10'9 mol/liter or even lower. The affinity can be determined by surface plasmon resonance or the Kinexa method, as known to those of skill in the art.
As used herein, the “antibody” can be polyclonal or monoclonal and can be isolated, recombinant and/or synthetic. In embodiments of the present invention, the IgM antibody is 25 a monoclonal antibody. Methods for preparing antibodies are generally known to one skilled in the art and include immunization of host species, hybridoma technology, phage-display technology and others. A preferred method for preparing a polyclonal antibody comprises a step of immunizing a host species such as an animal like a goat, rat or mouse, in particular a mouse, comprising 30 injecting an immunogen suitable to induce the formation of the antibody into the host species, optionally repeating the injection and optionally utilizing adjuvants to increase the immunological response like Freund’s adjuvant, mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, or 14
9270519_1 (GHMatters) P106323.AU oil emulsions. The method further comprises steps of isolating the polyclonal antibodies contained in the sera of the immunized host species and purifying the antibody. 2017100957 12 Μ 2017
Monoclonal antibodies can be prepared using standard hybridoma technology. In particular, monoclonal antibodies can be obtained by any technique that provides for the production 5 of antibody molecules by continuous cell lines in culture. The method for producing a monoclonal antibody in particular comprises a step of immunizing a host species, in particular a mouse, with an immunogen, obtaining an immunocyte (B lymphocyte) preferably a spleen cell from the animal; and fusing the immunocyte and a myeloma, so as to prepare a hybridoma. The method further comprises steps of culturing the hybridoma 10 and recovering the antibody from the culture and purifying the antibody.
The antibody may be a recombinant antibody, which preparation further includes recombinant DNA methods and involves molecular cloning and expression of gene segments in cells, viruses or yeasts. The method for preparing the antibody can then include the step of providing a host cell, transgenic animal or transgenic plant capable of 15 expressing said antibody in recoverable amounts.
Chimeric or humanized antibodies can be produced through standard techniques. The antibodies can be re-engineered to become more “human” by replacing the entire HC constant with a human equivalent (“chimeric” antibody) or further modified by replacing the “framework” residues in the variable regions with human equivalents (“humanized” 20 antibody). Full human antibodies can be generated from isolated human B cells by immortalization with the Epstein-Barr virus, using the phage-display technology, or more recently, by making hybridomas from mice (“humanized”) in which the antibody genes have been replaced with human equivalents.
Once produced, the antibodies can be assayed for recognition of the target antigen by 25 standard immunoassay methods including ELISA techniques, immunofluorescence, immunohistochemistry, and Western blotting.
The epitope can be confirmed by means of methods known to the skilled person including, but not limited to, chemical structural analysis, mutagenesis studies, protection assays or X-ray crystallography. 30 The IgM antibody can in embodiments of the present invention be bi- or poly-specific (poly-reactive), i.e. have binding specificities for two or more different target antigens. Bispecific or poly-specific antibodies can be monoclonal, preferably human or humanized antibodies. Methods for making bispecific antibodies are known in the art. In particular, the 15
9270519_1 (GHMatters) P106323.AU recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities. The constantly changing association and dissociation between the variable regions of the HC and LC is assumed to allow these antibodies not only to adopt different 5 conformations, but also to successfully dock with different antigens using the single-polypeptide approach. 2017100957 12 Μ 2017
The IgM antibody is in embodiments of the present invention conjugated to at least one carrier, which carrier is in particular selected from nano-microspheres, human serum albumin or fragments thereof, bovine serum albumin or mouse serum albumin. In a 10 particular embodiment of the present invention, the IgM antibody is present in form of a Fab, Fab’ or F(ab’)2 fragment and conjugated to nano-microspheres or human serum albumin or fragments thereof.
In an embodiment of the present invention, the Fab, Fab’ or F(ab’)2 fragment is conjugated to nano-microspheres. In such embodiment, the Fab, Fab’ or F(ab’)2 fragments are 15 mounted onto nano-microspheres to form a coat using covalent coupling such as through the carboxyl group in Fab or by other standard procedures such as histidine - nickel affinity.
Nano-microspheres preferably have an average diameter of less than 1000 nm and can have a substantially spherical form. "Diameter" preferably refers to the Feret (or Feret's) diameter at the thickest point of such particle. The Feret diameter is a measure of an object 20 size along a specified direction and can be defined as the distance between the two parallel planes restricting the object perpendicular to that direction. The Feret diameter can be determined, for example, with microscopic methods. I.e. if the Feret diameters measured for the different directions of a particle differ, the “diameter” referred to in the present application always refers to the highest value measured. “Average diameter” refers 25 to the average of “diameter” preferably measured with at least 10 particles.
In an alternative embodiment, the carrier is human serum albumin or a fragment thereof. The advantage of using this protein is the fact that it is not foreign and hence will not elicit anaphylactic reactions. In addition, it is relatively small (55 kDa) but has at least a dozen sites available for coupling to the carboxyl group of the ligand. 30 In other embodiments of the present invention, the IgM antibody is a structurally modified IgM in particular in the form of a hybrid IgG-like antibody. In a hybrid IgG-like antibody one or more of the constant regions of IgM may be derived from IgG. In a preferred hybrid IgG-like antibody, the IgM Fab fragment is fused to the CH2-CH3 domains of a truncated 16
9270519_1 (GHMatters) P106323.AU ‘heavy chain’ of IgG. The hybrid IgG-like antibody can additionally or alternatively be conjugated to nano-microspheres or human serum albumin or fragments thereof. This antibody will have the “mouth” of an IgM but the “tail” of an IgG and the associated effector functions. 2017100957 12 Μ 2017 5 A dimer or trimer of this hybrid IgG-like antibody - which is still smaller than an intact IgM -can be constructed for more effective binding. Polymerization can be achieved in several ways e.g. by di-sulfide bonding, or the hybrid IgG-like antibody can be coated onto nano-microspheres, human serum albumin or fragments thereof, bovine serum albumin or mouse serum albumin to form a polymer of antibody combining sites. 10 In additional or alternative embodiments, the IgM antibody can be conjugated, in particular covalently coupled, to a toxic agent. A toxic agent is a compound with toxic effects on specific cells and can be a compound able to kill specific cells.
The subject is preferably a human. The disease can be selected from, for example, cancer such as colorectal cancer, an infectious disease such as HIV infection, an autoimmune 15 disease or an allergic disease such as asthma. In particular embodiments of the present invention, the IgM antibody is for use in a method for treatment of a disease, which disease is a HIV infection. Preferably, the IgM antibody recognizes gp120 of HIV, further preferably specifically binds to gp120 of HIV, in particular recognizes and preferably specifically binds to the V1/V2 region of gp120 of HIV. HIV as used herein includes the respective subtypes 20 and groups within the subtypes of HIV, in particular it is HIV1.
The envelope coding sequence including the sequence of gp120 is known to the skilled person (e.g. Gen Bank Accession No. AF038399.1).
In other embodiments of the present invention, the disease is cancer and the IgM antibody recognizes, in particular specifically binds at, EGFR, KRAS, NRAS or BRAF found in tumor 25 cells, in particular the disease is colorectal cancer and the IgM antibody in particular recognizes EGFR.
For example, patients treated with the IgG anti-EGFR antibody often develop resistance to it within months, a reason being the fact that EGFR becomes mutated (Arena, S. et al., Emergence of Multiple EGFR Extracellular Mutations during Cetuximab Treatment in 30 Colorectal Cancer. Clin Cancer Res., 2015, 21 (9): p. 2157-66).
In further embodiments of the present invention, the disease is asthma and the IgM antibody recognizes, in particular specifically binds to, the IgE receptor (CD23) found in mast cells. 17
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In further embodiments of the present invention, the disease is an autoimmune disease, and the IgM antibody recognizes, in particular specifically binds to, the IL-6 receptor. 2017100957 12 Μ 2017
In further embodiments of the present invention, the IgM antibody recognizes guanosine triphosphate. 5 In a particular embodiment of the present invention, the IgM antibodies are natural IgM antibodies and the method for treatment of the disease includes administering natural IgM antibodies to a subject, in particular in a late stage of the disease. The natural IgM antibodies are derived from an external source like from other subjects with the same disease who have recovered. The subject is preferably a human. 10 This treatment might be highly advantageous considering that IgM is produced during the natural course after an infection as a first-line defense and quickly replaced by IgG after about a month or the like. Thus, only IgG but no IgM antibodies are present in the patient in a late stage of the disease. At this convalescence stage, most patients recover but, at least for some types of infection and in some patients, the disease still persists unabated. The 15 inventors assume that one probable reason for this is the ineffectiveness of IgG, because the target antigen becomes blocked or mutated.
In another aspect, the present invention refers to a pharmaceutical composition for use in a method for treatment of a disease in a subject, preferably a human. The pharmaceutical composition comprises an IgM antibody as described above and at least one 20 pharmaceutically tolerable excipient. A pharmaceutically tolerable excipient is an excipient which can be taken by the subject and does not have a negative impact on the therapeutic effect of the antibody, and can include any suitable excipient, such as, but not limited to, diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative, adjuvant or the like. 25 The skilled person is able to select suitable pharmaceutically tolerable excipients depending on the form of the pharmaceutical composition and is aware of methods for manufacturing pharmaceutical compositions as well as able to select a suitable method for preparing the pharmaceutical composition depending on the kind of excipients and the form of the pharmaceutical composition. 30 The antibody will be formulated for therapeutic usage by standard methods, e.g., by addition of the at least one pharmaceutically tolerable excipient such as diluent, binder, stabilizer, buffer, salt, lipophilic solvent, preservative and/or adjuvant. 18
9270519_1 (GHMatters) P106323.AU
Besides treatment, the IgM antibody is used in a method for diagnosis of a disease like in diagnostic pathology in situations where IgG antibodies cannot function due to their inability to bind to the blocked or altered target antigen. An area where this is highly advantageous is the detection of the various nuclear antigens that are often hidden in cells 5 in fixed tissues e.g. PCNA or telomerase. The diagnosis can be carried out with a sample like a tissue or other cytological sample from the subject, in particular a human. 2017100957 12 Μ 2017
In a particular embodiment of the present invention, the IgM antibody is used in a method for diagnosis of a disease, which disease is cancer and wherein the IgM antibody recognizes PCNA, telomerase, ER or Ki-67, in particular telomerase such as in 10 immunopathology.
In another embodiment of the present invention, the IgM antibody is used in a method for diagnosis of a disease, which disease is osteomyelitis and wherein the IgM antibody recognizes the NCA-90 granulocytic antigen such as for whole body imaging.
The IgM antibody can be used in a method of laboratory diagnosis of a disease selected 15 from cancer, an infectious disease, an autoimmune disease or an allergic disease. The diagnosis is based on the examination of a tissue or other cytological sample from the subject. In particular, the conventional diagnosis requires the use of an IgG antibody as probe and an antigen-retrieval reagent to unmask the target antigen, i.e. for antigen retrieval. To “unmask” the antigen means making the antigen accessible for the IgG 20 antibody by means of antigen retrieval methods, in particular by means of the antigen-retrieval reagent, wherein the skilled person is aware of antigen-retrieval reagents and respective systems or kits.
In another aspect, the present invention refers to a kit for diagnosis of a disease in a subject, preferably a human, comprising an IgM antibody as described above. 25 The kit can be used to detect a target antigen in a biological sample like a tissue, in particular a fixed tissue after chemical fixation, in standard immunoassay methods including ELISA techniques, radioimmunoassays, immunofluorescence, immunohistochemistry, and Western blotting.
Although the following examples refer to specific IgM antibodies, the skilled person will 30 understand that the observations are general and relate to all types of specificities. This is because it is plausible that the mechanism is common to all IgMs rather than each type of IgM having its own modus operandi. This applies for poly-specific IgM antibodies, too. 19
9270519_1 (GHMatters) P106323.AU EXAMPLES EXAMPLE 1 2017100957 12 Μ 2017
Experimental data regarding the failure of IgG antibodies in therapy and diagnosis
EXAMPLE 1A 5 Binding of IgG to phosphorylcholine (PC) A mouse IgG monoclonal antibody (named Mab2) has been provided that was highly specific for PC, another antigen or hapten (small chemical group). The inventors found that in ELISA immunoassays, it could only bind to this hapten when presented in the original form used to generate it (a natural antigen from the parasite, Trichinella spiralis), or to the 10 un-conjugated (free) PC, but it could not bind to PC conjugated to unrelated protein carriers such as BSA. This antibody, which contains several somatic mutations in the combining site, is thus carrier-specific but importantly, this indirectly demonstrates that the antibody combining site is rigid and inflexible, locked in a predetermined configuration. A Fv fragment containing only the variable regions of HC and LC constructed from the 15 antibody showed no carrier specificity, but the Fab fragment retained this, suggesting that the first constant domain of HC i.e. the antibody class, determines the antibody carrier specificity (Tam, F.C. et al., Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues. J Immunol Methods., 2007, 321(1-2): p. 152-63). 20 Similar carrier-specific IgG antibodies which recognize both PC and the adjoining carrier have been described, but no such anti-PC antibody of the IgM class is known.
From the above explanations, if follows that an IgM antibody of the present invention will plausibly overcome the above disadvantages of IgG antibodies under the same conditions.
EXAMPLE1B 25 Binding of IgG to telomerase and other nuclear antigens A mouse IgG monoclonal antibody (Mab 476) was produced from mice that were hyperimmunized against a recombinant segment (TERT) of the human nuclear enzyme, telomerase. This mAb had extensive somatic mutations and bound extremely well to TERT in an ELISA, but of pertinence, it could not bind at all to the native antigen located in the 30 nucleus of HeLa cells or in human liver carcinoma cells. Binding was however restored when the methanol-fixed cells or the formalin-fixed tissue sections were first treated with a 20
9270519 1 (GHMatters) P106323.AU chemical agent to retrieve the antigen. These results demonstrate that Mab 476 is somehow blocked from binding to the target antigen embedded in the cell or in tissues, whereas if the antigen is presented openly in solution, it could bind extremely well (Leung, D.T. et al., Nuclear telomerase is less accessible to antibody probing than known nuclear 5 antigens: retrieval with new immunostaining buffer. Histochem Cell Biol., 2005, 123(1): p. 105-12). 2017100957 12 Μ 2017
Similar experiences were reported of other IgG antibodies with other specificities. Thus, a study carried out recently found 9 out of 10 nuclear antigens (e.g. PCNA, ER and Ki-67) in formalin-fixed tissues could not be stained by IgG antibodies but binding was restored after 10 enzyme digestion or heat treatment used to retrieve the antigen. Very few antigens could be stained without antigen retrieval (Denda, T. et al., Optimal antigen retrieval for ethanol-fixed cytologic smears. Cancer Cytopathol., 2012, 120(3): p. 167-76, van Gils, M.J. et al., Longer V1V2 region with increased number of potential N-linked glycosylation sites in the HIV-1 envelope glycoprotein protects against HIV-specific neutralizing antibodies. J 15 Virol., 2011,85(14): p. 6986-95).
The skilled person will appreciate and understand that IgM will overcome the disadvantages of IgG under the above described conditions. Likewise, the skilled person will understand that IgM for HIV gp120 antigen are able to bind to the gp120 viral protein even if the antigen is hidden for the IgG antibody, namely in case the virus managed to 20 shield the antigen with glycans (carbohydrates). Still further, it is plausible for a skilled person that the IgM of the present invention is also able to localize osteopontin even in paraffin-embedded bone specimens (e.g. osteoblasts, osteocytes, bone matrix) by immunochemical staining, i.e. where specific IgG antibodies failed and led to conflicting results obviating the need for antigen retrieval. 25 EXAMPLE 2
Experimental data for IgM antibodies of the present invention
Reagents and methodologies applied: Antigens
The target antigen is a very small chemical compound (or hapten) called guanosine triphosphate (GTP), which has a molecular weight of 523 Da. The main reactive 30 component is guanosine (G), and mono-phosphates of guanosine (GMP) are antigenically as reactive as GTP. In nature, GMP is found commonly in many different forms in living cells e.g. double-stranded (ds) DNA or the separated constituents called single stranded (ss) DNA found in the cell nucleus, and RNA including mRNAand tRNA found in ribosomes, 21
9270519 1 (GHMallers) P106323.AU while GTP, which plays the important role of an on-off switch for many cellular activities, is found in the cell membrane and cytoskeleton including tubulin and keratin. 2017100957 12 Μ 2017
For the experiment, various types of GTP or GMP antigens were obtained or prepared both from natural sources (native antigens) and by chemical synthesis (synthetic antigens) (see 5 Fig. 9A to 9G). Of the synthetic antigens, there are two types: one in which the GTP or GMP hapten exists as free (uncoupled) individual molecules in solution, and the other, where the hapten is insolubilized by conjugating it at various densities to a carrier protein but still used as a liquid suspension. The protein carriers are small soluble proteins e.g. bovine serum albumin (BSA), human serum albumin (HSA), and mouse serum albumin 10 (MSA). These hapten-carrier compounds are used by themselves suspended in solution, or are covalently coupled to large inorganic microspheres: magnetic particles (1 pm diameter) and Sepharose beads (45-165 pm diameter). These conjugates are used for binding studies in solution. In the case of the free hapten, different versions were also made: G-G-G polymers (20 GMP residue long), and G-C-G-C co-polymers (also 20 15 residue long) which contain mixtures of cytosine (C) and G.
Of the natural antigens, the first type comprises several types of small, soluble compounds suspended in solution e.g. double-stranded DNA, single-stranded DNA, and tRNA. The second type comprises bigger entities: (a) single cell suspensions of a human cell line (HEp2) prepared as a cytological smear, and (b) formalin-fixed tissue sections of rat 20 smooth muscle or rat skeletal muscle.
Antibodies
Three IgM (Mab1, Mab23, Mab50) and one IgG (Mab12) monoclonal antibody (mAb) have been produced using the hybridoma technology from BALB/c mice that were hyperimmunized against GTP-BSA (Niu, H. et al., Cells that produce deleterious 25 autoreactive antibodies are vulnerable to suicide. J Immunol., 2008,181(3): p. 2246-57). In addition, GTP-affinity chromatography has been used for purifying the IgG antibodies individually from 9 autoimmune patients whose sera showed high levels of anti-GTP activity (Ma, C.H. et al., Antibodies to guanosine triphosphate misidentified as anti-double-stranded DNA antibodies in a patient with antinuclear antibody-negative lupus, 30 due to buckling of insolubilized assay DNA. Arthritis Rheum., 2004, 50(5): p. 1533-8). Mouse hyperimmune sera containing high levels of IgG anti-GTP antibodies were obtained from the GTP-BSA immunized mice.
Assay Methods 22
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Direct ELISA 2017100957 12 Μ 2017
This was used to quantitatively determine the binding (or lack of binding) of an unknown antibody to the target antigen, GTP. This antigen, in a suitable soluble form e.g. GTP-BSA or GTP-HSA, was coated on 96-well micro-plates. Binding of the unknown antibody was 5 detected by first incubating the antibody (in serial dilutions) in the antigen-coated well, the plate washed, and a developer then added to reveal the presence of any bound antibody via enzyme-substrate reaction.
Inhibition ELISA
This is a variation of the above assay and was used to detect (a) how related an unknown 10 antigen is to GTP, or (b) whether an unknown antibody has anti-GTP activity, by the ability of the antigen or antibody (in serial dilutions) to block the binding of a known anti-GTP antibody (at a selected concentration) to the coated GTP-BSA. This can detect all types of antigens and antibodies including antigens that are too small to coat on the micro-plates such as free GTP. Here, the unknown antigen or antibody was co-incubated with the 15 anti-GTP antibody in the coated well, and the assay was performed as before.
Cell immune-fluorescence (IF)
This has been used to detect antibodies that can bind to GTP structures in HEp2 cells derived from a human epithelioid cell line. These cells were spread out singly to form a smear on a microscope slide. Examples of cellular structures which contain GTP are the 20 tubulin and keratin networks. Binding was revealed by first incubating the unknown antibody with the cells, and then using a second antibody which has a fluorescein tag to develop the assay; this antibody which binds to the first (unknown) antibody was revealed by the fluorescence seen under a fluorescence microscope.
Tissue immunopathology 25 This method was used to detect antibodies that can bind to GTP structures in formalin-fixed tissues derived from rat skeletal or rat smooth muscle. The tissues were prepared as micro-mm-thin sections on microscope slides. GTP was found along the tropomyosin fibres in skeletal muscle, and in both tubulin and tropomyosin in smooth muscle. The assay was performed and scored basically as for cell immune-fluorescence. 30 Magnetic/Sepharose bead absorption
Here, binding of an unknown antibody to different types and preparations of GTP antigens (e.g. GTP conjugated to magnetic or Sepharose beads at low or high densities (see Fig. 23
9270519J (GHMatters) P106323.AU 9Α to 9G) was revealed by first incubating the antibody (at a selected concentration) with the particular antigen, the antigen then separated by magnetic force or centrifugation, and the supernatant containing any unbound (unreacted) antibody was then titrated in the direct ELISA. 2017100957 12 Μ 2017 5 Results and Discussion
All three IgM (Mab1, Mab23, Mab50) and the single IgG (Mab12) mAbs were found to be specific for GTP. In particular, guanosine (G) was the main component recognized since GMP and deoxy-GTP (see below) were also bound by these antibodies. Thus, all antibodies bound better to GTP-BSA, GMP-HSA or GMP-MSA than the respective carrier 10 protein (BSA, HSA or MSA) (e.g. Fig. 2). However, some background binding to the carrier was seen with the IgM mAbs, particularly Mab1 and Mab50, but none of these are poly-reactive because they did not bind to dsDNA (see later) or thyroglobulin (data not shown) that known poly-reactive antibodies bind to.
Human IgG antibodies specific for GTP were affinity-purified individually from the serum of 15 patients with autoimmune disease; these showed the same specificity as Mab12 by inhibition ELISA (Fig. 3).
The fine specificity of Mab23 (IgM), Mab50 (IgM) and Mab12 (IgG) were examined more closely by inhibition ELISA using various GTP analogues. As shown (Fig. 4), Mab12 showed very high affinity for GTP, while Mab50 had low affinity, and Mab23 was 20 intermediate. This is shown by the overall analysis and specifically, by the fact that Mab12 bound extremely well to free GTP which was not the case with the IgM mAbs. In contrast, if GTP (or GMP) is presented in multiple copies on a protein carrier such as BSA or MSA, the IgM mAbs could now bind because of their multivalence. This is also the reason why the IgM mAbs were able to bind to oligo-G or oligo-GC polymers. Significantly, with both these 25 polymers, Mab12 could not bind. This the first indication that access to the target antigenic site is somehow blocked in these complex structures for Mab12 but not the IgMs. Obstruction could come from neighboring residues e.g. G-G-G or C-G-C. Again, for the same reason, ssDNA which contains multiple copies of GMP arranged in a random mixed polymer of G, C, A and T residues, was bound only by the IgM mAbs but not Mab12. 30 dsDNA was not bound by any of the mAbs because here, G is hidden inside the two strands of DNA that are bonded together.
Smears of single cells made from HEp2 cells are routinely used to detect anti-nuclear antibodies from patients suspected of having an autoimmune disease. These 24
9270519J (GHMallers) P106323.AU auto-antibodies, which are diagnostic because healthy people do not have them, include antibodies specific for dsDNA in the nucleus, and cytoskeletal structures in the cytoplasm. As shown (Fig. 5Ato 5C), both IgM and IgG antibodies could indeed stain the cytoplasm of the cell like natural auto-antibodies to tubulin or cytokeratin; in addition, the IgG antibodies 5 also stained the nucleolus (possibly, pre-RNA). The IgM antibodies used were: Mab1, Mab23, and Mab50, while the IgG antibodies include Mab12, 9 lots of human anti-GTP antibodies purified from individual autoimmune patients, and 1 lot of mouse anti-GTP hyperimmune serum. Inhibition analysis showed that Mab12, Mab23 and Mab50 target a common antigen in the cell (Fig. 5C); this is probably tubulin, which is known to contain an 10 abundance of GTP residues. 2017100957 12 Μ 2017
Another natural source of GTP used in the study is skeletal muscle prepared as a formalin-fixed tissue section. This is routinely used to detect auto-antibodies to skeletal muscle, which is diagnostic of an autoimmune disease called Myasthenia gravis. No anti-GTP antibody has been examined on this tissue previously. Thus, for the first time (Fig. 15 6A and 6B), we showed that Mab1, Mab23 and Mab50 stained the tissue just like normal anti-skeletal muscle auto-antibodies derived from patients. The target antigen is tropomyosin (muscle fibre), which forms a striation-like network in the tissue. The more remarkable discovery is this: none of the IgG antibodies - Mab12, 9 preparations of human anti-GTP antibodies purified from 9 different patients, and a mouse anti-GTP hyperimmune 20 serum - all of which showed excellent binding on HEp2 cells previously, were reactive with this tissue. The possibility that the IgM reactivity was non-specific is ruled unlikely by the fact the staining could be blocked by GTP-BSA added to the reaction mixture. This suggests that the GTP antigen in the tissue was somehow blocked for binding by the IgG antibodies, but IgM antibodies somehow managed not only to overcome this obstacle, but 25 also find two or more antigenic sites close enough together for multivalent binding. This is possible because GTP is found every 6 angstrom apart along the tropomyosin strand, and two strands are coiled together in opposite directions.
Another type of tissue - the smooth muscle - was studied, which has both a tubulin and a keratin network, both containing an abundance of GTP residues. The results are similar to 30 those from skeletal muscle (Fig. 7A and 7B). Thus, whereas all three IgM mAbs (Mab1, Mab23 and Mab50) stained this tissue very well mimicking true anti-smooth muscle auto-antibodies, the IgG antibodies (Mab12 and 9 human anti-GTP antibodies) were not reactive. Importantly, binding by Mab12 was restored by pre-digesting the tissue with an enzyme (proteinase K), a common procedure used to retrieve (unmask) hidden antigens in 25
9270519_1 (GHMatters) P106323.AU tissues. (Similar pretreatment could not be done with the skeletal muscle because this caused high background or non-specific noise.) The antigen, both here and in the case of skeletal muscle, might be blocked by tissue-associated structures and/or non-specific obstacles resulting from the chemical process of tissue fixation. 2017100957 12M2017 5 To verify that the difference between IgM and IgG antibodies is real in their ability to access the GTP antigen in skeletal and smooth muscle, artificial antigen scaffolds have been created using magnetic microspheres of 1 pm diameter and larger Sepharose microspheres (45-165 pm). These were covalently coupled with HSA or MSA proteins which in turn had been covalently decorated with GTP at low (2.85 molecules per carrier) 10 or high (9.16) density, respectively. Controls were coated with HSA (without GTP) or glycine. The different preparations of antigen were used to examine the binding of the following antibodies: Mab23 (IgM), Mab50 (IgM), Mab12 (IgG), and a purified human anti-GTP (IgG). Following an hour of incubation between antigen and antibody, (a) the magnetic beads were sedimented by use of magnetic force, or (b) the Sepharose beads 15 were sedimented by centrifugation, and in both cases, the supernatant was removed for analysis. The supernatant contains any antibodies left in the solution that did not bind to the antigen (beads). The amount left was revealed by titration in a direct ELISA. As shown in Fig. 8A to 8D, in the case of the Sepharose beads, all four types of antibodies bound very well regardless of whether GTP-HSA or GTP-MSA was coupled, as revealed by the 20 virtual lack of antibody activity in the supernatant. In contrast, there was a difference with the magnetic particles: both the IgG antibodies (Mab12 and human anti-GTP) bound reasonably well to GTP-HSA, but not to GTP-MSA, whereas both the IgM mAbs bound well to both antigens.
However, Mab50 also bound to the control magnetic bead coated with BSAonly (probably 25 reactive with some polystyrene groups on the bead) - results for this antibody in this experiment are thus ignored. The findings are consistent with those obtained from the tissues. It appears that, in the case of the GTP-MSA coated magnetic beads, the antigen is presented in an extremely cluttered environment because of the high density of GTP molecules in MSA, as well as the fact that the carrier protein is coated densely on a small 30 curvy surface. As in the case of the skeletal and smooth muscle, in such an environment, IgG antibodies are obstructed from docking with the antigen. In contrast, the antigen is presented more openly in the case of GTP-BSA due to the much lower hapten density. This is particularly so when Sepharose beads are used, which have much bigger surfaces. In fact, at the extreme, microtiter wells coated with high densities of GTP do not present 26
9270519_1 (GHMatters) P106323.AU any problem to binding by IgG antibodies (data not shown). 2017100957 12 Μ 2017
Altogether, the findings revealed a unique hitherto-unknown property of IgM. This is the ability of these antibodies to bind to antigens that are hidden and inaccessible to IgG. This should not be confused with the well-known fact that, as whole molecules, it is IgG rather 5 than IgM that is able to permeate into tight inter-vascular spaces. Here, the inventors are concerned only about the individual antibody combining sites i.e. the ability of the antibody, via one of these sites at a given instance, to dock with the antigen at very close range. EXAMPLE 3
Preparation of IgM antibodies to HIV-1 gp120
10 EXAMPLE 3A
Expression and purification of bacterial or insect-cell derived recombinant human immunodeficiency virus type 1 (HIV-1) gp120
Bacterially derived recombinant HIV-1 gp120 (unglycosylated), employed as screening antigen, was generated as GST fusion protein in Escherichia coli BL21 using the pGEX-2T 15 expression vector (Amersham Biosciences, Piscataway, NJ) as previously described (Leung, D.T. et al., Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid. J Infect Dis., 2004, 190(2): p. 379-86). gp120 cDNA obtained from Biorbyt (Berkeley, CA) was PCR-amplified to generate DNA fragment corresponding to the full-length gp120 (residues 1-482) protein using the 20 following forward (F) primers and reverse (R) primers: F, 5’-CGT(GGATCC)ACAGAAAAATTGTGGGTCACA-3’ (containing a BamH1 site between the brackets); R, 5’-CGAT(GAATTC)CTCTTTTTTCTCTCTGCACCAC-3’ (containing an EcoR1 site between the brackets)], and subcloned into the pGEX-2T vector. The fusion protein derived from the above constructs were induced with 25 isopropyl-b-D-thiogalactopyranoside, purified by glutathione-coupled sepharose 4B column (Amersham Biosciences), and examined on SDS-PAGE by Coomassie Blue staining and Western Blotting.
Insect-cell derived recombinant HIV-1 gp120 (glycosylated), employed as both immunogen and screening antigen, was generated as V5 peptide-tagged (V5) fusion protein in Sf9 cell 30 line using the BaculoDirect Baculovirus Expression System according to manufacturer’s instruction (Invitrogen, Waltham, MA). gp120 cDNA obtained from Biorbyt (Berkeley, CA) was PCR-amplified to generate DNA fragment corresponding to the full-length gp120 27
9270519_1 (GHMatters) P106323.AU protein using the following forward (F) primers (containing a Kozak sequence between the brackets) and reverse (R) primers: F, 2017100957 12 Μ 2017 5’-(CCACCAT GGG A) ACAG AAAAATT GTGGGT CACA-3’; R, 5’-T CTTTTTT CT CT CT GCACCAC-3’, and subcloned into the entry vector 5 pCR8/GW/TOPO (Invitrogen). Purified plasmid with insert of right orientation was recombined with the BaculoDirect linear DNA by incubation with LR clonase overnight at room temperature. Sf9 cells growing at log-phase were transfected with the recombined DNA mixture by Cellfectin (Invitrogen) and selected with ganciclovir. Fusion protein was obtained from lysates of Sf9 cells where almost 100% of cells were infected at the time of 10 harvest as revealed by anti-V5 antibody staining. Cytosolic extracts were prepared by incubating the infected Sf9 cells with lysis buffer, followed by centrifugation. The fusion proteins were purified by using anti-V5 antibody-coupled sepharose 4B column (Biotool, Houston, TX), and examined on SDS-PAGE by Coomassie Blue staining and Western Blotting.
15 EXAMPLE 3B
Production of gp120-specific IgM monoclonal antibodies BALB/c mice were immunized with purified insect-cell derived gp120 fusion protein for a short period of time only (1 primer, 1 booster, over 3-4 weeks) to increase the chance of obtaining IgM clones. Spleen cells obtained from these animals were fused with NS0 20 myeloma cells as previously described (Wun, H.L. et al., Molecular mimicry: anti-DNA antibodies may arise inadvertently as a response to antibodies generated to microorganisms. Int Immunol., 2001, 13(9): p. 1099-107).
Hybridomas obtained were screened by direct-binding ELISA for reactivity against the glycosylated insect-cell protein, as well as the unglycosylated bacterial protein in parallel. 25 Both IgM and IgG clones were selected. Three types of hybridomas could be differentiated: Type-I, positive for both insect-cell and bacterial proteins; Type-ll, positive for insect-cell protein only; and Type-Ill, positive for the bacterial protein only (excluding the non-HIV spacer or carrier protein). Type I hybridomas recognize the peptide antigens (epitopes) while Type II recognize the glycosylated antigens. Type III hybridomas are of interest, 30 particularly if they are IgG antibodies - these presumably recognize the peptide antigen in the bacterial protein which is blocked by carbohydrate groups present in the insect-cell counterpart. IgM antibodies are not expected to be found here because these would behave like Type I antibodies. To separate this type of IgM antibodies from the normal 28
9270519J (GHMallers) P106323.AU
Type I IgM antibodies that recognize the naked peptide antigen found in both insect-cell and bacterially-derived fusion proteins, the carbohydrate moiety in the insect-cell antigen can be removed by enzymes and the cleansed antigen then re-examined for binding. That the IgM antibody truly recognizes a hidden antigen can be confirmed with HIV viruses 5 obtained from patients in different phases of the disease. 2017100957 12 Μ 2017
The fine specificities of the anti-gp120 IgM antibodies can be further characterized by indirect immunofluorescence assay, inhibitory/competitive ELISA, and Western Blotting, using the appropriate substrates and antigens. The neutralization activities of these antibodies can be assayed in-vitro using cloned env-pseudovirus and TZM-BL cells 10 carrying the luciferase reporter gene (Montefiori, D.C., Curr Protoc Immunol., 2005, Jan; Chapter 12:Unit 12.11).
In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, 15 i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.
It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. 20 29
9270519_1 (GHMatters) P106323.AU
Claims (5)
- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:1. A method for treatment or diagnosis of a disease selected from a cancer, an infectious disease, an autoimmune disease or an allergic disease in a subject, optionally a human subject, the method comprising (a) administering to said subject an IgM antibody, which disease cannot be treated or diagnosed with an IgG antibody with at least similar specificity or (b) administering to said subject an IgM antibody which binds to an antigen hidden for an IgG antibody with at least similar specificity, optionally wherein the IgM antibody is a murine antibody which recognizes telomerase and the method or use is diagnosis of cancer.
- 2. Use of an IgM antibody in the manufacture of a medicament for treatment or diagnosis of a disease selected from a cancer, an infectious disease, an autoimmune disease or an allergic disease in a subject, (a) which disease cannot be treated or diagnosed with an IgG antibody with at least similar specificity or (b) which IgM antibody binds to an antigen hidden for an IgG antibody with at least similar specificity, optionally wherein the IgM antibody is a murine antibody which recognizes telomerase and the method or use is diagnosis of cancer.
- 3. The method or use of claim 1 or claim 2, wherein the IgM antibody is selected from a Fab, Fab’ or F(ab’)2 fragment and conjugated to at least one of nano-microspheres, human serum albumin or a toxic agent and, or wherein the IgM antibody is a structurally modified IgM in form of a hybrid IgG-like antibody and optionally wherein the IgM antibody is a monoclonal human, humanized or chimeric antibody and wherein the infectious disease is a HIV infection, optionally, wherein the IgM antibody recognizes gp120 of HIV, optionally wherein the IgM antibody recognizes the V1/V2 region of gp120 of HIV.
- 4. The method or use of claim 1 or claim 2, wherein the disease is colorectal cancer.
- 5. The method or use of claim 1 or claim 2, for in vitro diagnosis of the disease, optionally, wherein the diagnosis is based on the examination of a tissue or other cytological sample from the subject or wherein conventional diagnosis requires the use of an IgG antibody as probe and an antigen-retrieval reagent to unmask the antigen.
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