Clinical evaluation of the M protein response in multiple myeloma

Abstract

The problem that this invention aims to solve is to provide an antibody that can distinguish between therapeutic antibodies and endogenous antibodies, and that can avoid interference caused by therapeutic antibodies during SPEP and IFE. [Solution] The present invention relates to an anti-idiotype antibody fused to human albumin.

Description

Background Art

[0001] Multiple myeloma (MM) is a blood cancer involving the clonal proliferation of malignant plasma cells. MM is the most common malignant plasma cell tumor and the second most common hematologic malignancy in the United States. The age-adjusted incidence in the United States is 5.5 patients per 100,000 people, and the annual incidence reaches approximately 6 to 7 per 100,000 people in the United Kingdom.

[0002] Plasma cells produce immunoglobulins (also called gamma globulins), which consist of heavy chains (IgG, IgA, IgM, IgD, or IgE) and light chains (kappa or lambda) that bind together. Each plasma cell produces one type of immunoglobulin (e.g., IgA kappa or IgG kappa). Normally, the body contains various different plasma cells (“polyclonal”), so the immunoglobulins in the serum also represent a wide variety of formats and specificities (polyclonal). In the case of multiple myeloma, the malignant cells are copies of only one or a distinct few plasma cells, and the immunoglobulin secreted by these cells is considered monoclonal.

[0003] This monoclonal immunoglobulin is called the M protein or paraprotein and may consist of heavy chains (mostly IgG or IgA, but also IgM, IgD, or IgE) and light chains (kappa or lambda) or a truncated form of the immunoglobulin. An increase in the M protein in the serum is used to identify B-cell malignancies such as MM.

[0004] Currently, several staging systems are used for the diagnosis and monitoring of response in multiple myeloma: a) the Durie and Salmon Staging System, and b) the International Staging System (ISS) and the International Myeloma Working Group (IMWG). The Durie and Salmon staging system includes features that assess tumor cell mass, high serum immunoglobulin (Ig) G levels, terminal organ damage, and osteolytic bone lesions. The ISS places a greater emphasis on disease burden, based on β2-microglobulin levels and serum albumin levels. The IMWG considers both molecular and cytogenetic abnormalities, and in detail, the time-dependent decline of M protein is one of the most important factors used to assess disease progression and treatment success.

[0005] Protein expressions specific to multiple myeloma include increased monoclonal (M) protein concentrations (IgG, IgA, IgD), light chain concentrations (such as kappa [κ] and lambda [λ]), elevated levels of abnormal β2-microglobulin, serum albumin, creatinine, and hemoglobin, and the presence of myeloid plasma cells (more than 5%). Measurement of protein expression produced by patients (e.g., M protein) can be achieved by numerous methods. Tests for measuring M protein include 24-hour urine collection tests, urinary protein electrophoresis (UPEP), serum protein electrophoresis (SPEP), immunofixation electrophoresis (IFE), and serum free light chain (sFLC) assays.

[0006] CD38 is an example of an antigen expressed on malignant plasma cells and other lymphocytes, and therefore represents a therapeutic target in the treatment of multiple myeloma and other immunoglobulinemias. Functions attributed to CD38 include receptor-mediated activity in adhesion and signaling events, as well as (ecto) enzymatic activity. As an ectoenzyme, CD38 utilizes NAD+ as a substrate for the formation of cyclic ADP-ribose (cADPR) and ADPR, as well as nicotinamide and nicotinic acid adenine dinucleotide phosphate (NAADP). cADPR and NAADP have been shown to function as secondary messengers for Ca2+ mobilization. By converting NAD+ to cADPR, CD38 regulates extracellular NAD+ concentration and thus regulates cell survival by regulating NAD-induced cell death (NCID). In addition to Ca2+-mediated signaling, CD38 signaling occurs via crosstalk with antigen receptor complexes on T and B cells, or with other types of receptor complexes, such as MHC molecules, and thus is involved in several cellular responses, as well as IgG switching and secretion.

[0007] A CD38-specific antibody is under development for the treatment of multiple myeloma. This CD38-specific antibody is described in International Publication No. 1999 / 62526 (Mayo Foundation); International Publication No. 200206347 (Crucell Holland); U.S. Patent Application Publication No. 2002164788 (Jonathan Ellis) (in its entirety incorporated by reference); International Publication No. 2005 / 103083 (MorphoSys AG), U.S. Patent Application No. 10 / 588,568 (in its entirety incorporated by reference), International Publication No. 2006 / 125640 (MorphoSys AG), U.S. Patent Application No. 11 / 920,830 (in its entirety incorporated by reference), and International Publication No. 2007 / 042309 (MorphoSys AG), U.S. Patent Application No. 12 / 089,806 (in its entirety incorporated by reference); International Publication No. 2006099875 (Genmab), U.S. Patent Application No. 11 / 886,932 (in its entirety incorporated by reference); International Publication No. 2011154453A1 (Genmab), U.S. Patent Application No. 13 / 702,857 (in its entirety incorporated by reference); International Publication No. 08 / 047242 (Sanofi-Aventis), U.S. Patent Application No. 12 / 441,466 (in its entirety incorporated by reference This information is incorporated by international publication brochure No. 2015066450 (Sanofi), U.S. Patent Application No. 14 / 529,719 (in its entirety incorporated by reference); international publication brochures No. 2012092616A1 and 2012092612A1 (Takeda Pharmaceutical Company Limited), U.S. Patent Application No. 13 / 341,860 and U.S. Patent Application No. 13 / 977,207 (both incorporated in their entirety by reference), and international publication brochure No. 2014178820A1 (Teva).

[0008] Treatment with anti-CD38 antibodies in MM patients can result in partial or complete clearance of the M protein produced by multiple myeloma cells. Serum protein electrophoresis (SPEP) and immunofixation electrophoresis (IFE) are both essential assays used to identify monoclonal proteins and determine their immunotypes in patients with multiple myeloma. Recent studies have demonstrated that certain therapeutic antibodies under development for the treatment of multiple myeloma are readily detectable on serum IFE and may interfere with the detection and monitoring of M protein levels (see McCudden et al., Clinical Chemistry, 56:12;1897-1904 (2010), and also Genzen et al., British Journal of Haematology (2011) 155(1) 123-125). McCudden et al. observed that incubation with siltuximab (an anti-IL-6 antibody) accompanied by an anti-drug antibody shifted the drug electrophoretic pattern, allowing the therapeutic antibody siltuximab to be distinguished from the endogenous M protein. Janssen also recently published the development of a clinical assay using a similar approach to mitigate daratumumab interference with the M protein on IFE, which shifted the complex on IFE using a mouse anti-daratumumab antibody ideally labeled with a biotin tag or Alexa-fluor tag.Axel, et al., Development of a Clinical Assay to Mitigate Daratumumab, an IgG1k Monoclonal Antibody, Interference with Serum Immunofixation and Clinical Assessement of M-protein Response in Multiple Myeloma Poster Presented at the 105th Annual Meeting of the American Association for Cancer Research (AACR), April 5-9, 2014, San Diego, California, USA; See also, Monoclonal antibodies targeting CD38 in hematological malignancies and beyond, Immunological Reviews, 270:95-112 (2016).

[0009] However, these approaches are not sufficient for all therapeutic antibodies. Novel, antibody-specific mitigation strategies are needed to avoid this potential interference with SPEP and IFE, in order to ensure documentation of effective clinical responses that meet the International Myeloma Working Group (IMWG) criteria. [Overview of the project]

[0010] The applicant discloses herein an anti-idiotype antibody against MOR202, which, when fused to human albumin, shifted the antibody in the IFE, thereby mitigating any potential interference of MOR202 with M protein-based clinical assessments.

[0011] The anti-idiotype antibody albumin fusion will be incorporated into the clinical development design of MOR202 to enhance the clinical evaluation of the M protein response.

[0012] One embodiment is an anti-idiotype antibody against MOR202. In one embodiment, the anti-idiotype antibody fuses with human albumin. In an embodiment, the anti-idiotype antibody is HCDR1 of amino acid sequence YSFSNYWIS (SEQ ID NO: 18), HCDR2 of amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 19), HCDR3 with amino acid sequence SRGAGMDY (SEQ ID NO: 20) Variable heavy chains including, LCDR1 of amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 21), LCDR2 of amino acid sequence LLIYADNNRPS (SEQ ID NO: 22), LCDR3 of amino acid sequence GSYDESSNSM (SEQ ID NO: 23) Includes variable light chains.

[0013] In one embodiment, the anti-idiotype antibody is a human antibody.

[0014] In this embodiment, the anti-idiotype antibody fusion has the following amino acid sequence [ka] It contains a heavy chain.

[0015] In this embodiment, the anti-idiotype antibody fusion has the following amino acid sequence [ka] It includes a light chain.

[0016] One embodiment is a method for evaluating a blood sample obtained from a patient undergoing treatment for multiple myeloma or other immunoglobulinemia, which includes the following: a) Obtain a blood sample from the patient, b) Incubate the blood sample with an anti-idiotype antibody. c) Perform immunofixation electrophoresis (IFE), and d) Report the results of the IFE.

[0017] In an embodiment, the patient has experienced treatment with MOR202.

[0018] In an embodiment, the sample is evaluated for total M protein level.

[0019] One aspect is a nucleic acid encoding an exemplary anti-idiotype antibody or an exemplary anti-idiotype antibody albumin fusion. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] [Figure 1] FIG. 1 shows the amino acid sequence of MOR202. [Figure 2A] FIG. 2A shows the amino acid sequence of the MOR09292 (anti-idiotype antibody against MOR202) human albumin fusion protein. [Figure 2B] FIG. 2B shows the amino acid sequence of the MOR09292 (anti-idiotype antibody against MOR202) human albumin fusion protein. [Figure 3] FIG. 3 shows a typical standard pattern for the distribution of proteins determined by serum protein electrophoresis. [Figure 4] FIG. 4 shows the serum protein electrophoresis distribution of a protein having a homogeneous spike-like peak in the focal region of the gamma globulin band common to disorders known as monoclonal gammopathies. This peak represents a single clone of plasma cells that produce a homogeneous M protein. [Figure 5] FIG. 5 shows an example of a gel after serum immunofixation electrophoresis of a healthy donor. Lane ELP = total protein stain; Lane G = anti-IgG stain; Lane A = anti-IgA stain; Lane M = anti-IgM stain; Lane K = anti-kappa stain; Lane L = anti-lambda stain. [Figure 6]Figure 6 shows serum immunofixation electrophoresis of samples from drug-free healthy donors (A and B) and drug-free MM patients (C and D). Samples were tested with or without MOR202 spiking at various concentrations (lane 1 = no MOR202 added; lane 2 = 200 μg / mL MOR202 added; lane 3 = 400 μg / mL MOR202 added; lane 4 = 800 μg / mL MOR202 added; lane 5 = 1200 μg / mL MOR202 added). Bands enclosed by dotted lines are visible only after MOR202 spiking and represent the respective molecules. Bands indicated by arrows represent endogenous M proteins. [Figure 7] Figure 7 shows serum immunofixation electrophoresis of pre-incubation of MOR202+ / -MOR0929 IgG1 and MOR09292 IgM in physiological saline. Samples prepared by pre-incubating MOR202 at a constant concentration of 1200 μg / mL (A and B) or 560 μg / mL (C) in physiological saline with its idiotype antibody MOR09292 in different formats were analyzed by IFE. A)+B): MOR202 and MOR09292 IgG1 (using anti-IgG staining (A) and anti-lambda staining (B)) (Lane 1 = MOR202; Lane 2 = MOR09292 IgG1 2400 μg / mL; Lane 3 = MOR202 + MOR09292 IgG1 600 μg / mL; Lane 4 = MOR202 + MOR09292 IgG1 1200 μg / mL; Lane 5 = MOR202 + MOR09292 IgG1 2400 μg / mL). C): MOR202 and MOR09292 IgM (using anti-IgG staining (lanes 2-4), anti-lambda staining (lanes 5-7), and anti-IgM staining (lanes 8-10)) (lanes 2 / 5 / 8 = MOR202; lanes 3 / 6 / 9 = MOR09292 IgM 560 μg / mL; lanes 4 / 7 / 10 = MOR202 + MOR09292 IgM 560 μg / mL; lane 1 = healthy donor human serum stained for total protein to assess general background signaling in serum samples). [Figure 8]Figure 8 shows serum immunofixation electrophoresis of pre-incubation of MOR202+ / -MOR09292 human albumin fusion (MOR09292-hAlb) in physiological saline and human serum. Samples were prepared by pre-incubating a constant concentration of 1200 μg / mL of MOR202 in physiological saline (lanes 2-3) or serum (lanes 4-13) with or without its idiotype antibody MOR09292-hAlb in various ratios, and then analyzed by IFE (using anti-IgG staining (lanes 2-8) or anti-lambda staining (lanes 9-13)) (lane 2=MOR202; lane 3=MOR202+MOR09292-hAlb 2400 μg / mL; lane 4=MOR202; lane 5=MOR202+MOR09292-hAlb 1200 μg / mL; lane 6=MOR202+MOR09292-hAlb 1800 μg / mL; lane 7=MOR202+MOR09292-hAlb 2400 μg / mL; Lane 8 = MOR202 + MOR09292-hAlb 3600 μg / mL; Lane 9 = MOR202; Lane 10 = MOR202 + MOR09292-hAlb 1200 μg / mL; Lane 11 = MOR202 + MOR09292-hAlb 1800 μg / mL; Lane 12 = MOR202 + MOR09292-hAlb 2400 μg / mL; Lane 13 = MOR202 + MOR09292-hAlb 3600 μg / mL; Lane 1 = Human serum from a healthy donor stained for total protein to evaluate general background signaling). [Modes for carrying out the invention]

[0021] definition The term "anti-idiotype" describes a protein or peptide that binds to the variable region of an antibody. An anti-idiotype protein can be an antibody. For example, the antibody MOR09292 binds to the variable region of MOR202.

[0022] The term "antibody" includes antibody fragments. Antibodies include monoclonal antibodies of any isotype, such as IgG, IgM, IgA, IgD, and IgE. An IgG antibody consists of two identical heavy chains and two identical light chains, which are linked by disulfide bonds. The heavy and light chains of an antibody contain a constant region and a variable region. Each variable region contains three segments called "complementarity-determining regions" ("CDRs") or "hypervariable regions," which are primarily responsible for binding to the antigen's epitope. They are called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The more highly conserved portion of the variable region outside the CDRs is called the "framework region." "Antibody fragment" means a fragment of Fv, scFv, dsFv, Fab, Fab'F(ab')2, or other fragments, which contain at least one variable heavy chain or variable light chain, each containing a CDR region and a framework region.

[0023] "CDR" is defined herein by Chothia et al., Kabat et al., or by the internal numbering convention. See Chothia C, Lesk AM. (1987) Canonical structures for the hypervariable regions of immunoglobulins. J Mol Biol., 196(4):901-17 (the entire text is incorporated by reference). See Kabat EA, Wu TT, Perry HM, Gottesman KS and Foeller C. (1991) Sequences of Proteins of Immunological Interest. 5th edit., NIH Publication no. 91-3242, US Dept. of Health and Human Services, Washington, DC (the entire text is incorporated by reference).

[0024] "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody or antibody fragment. "VL" refers to the variable region of the immunoglobulin light chain of an antibody or antibody fragment.

[0025] The "Fc region" refers to the constant region of the antibody, which in humans may be IgG1, 2, 3, 4 subclass, or other. The sequences of the human Fc region are available at IMGT, Human IGH C-REGIONs, http: / / www.imgt.org / ligmdb / (searched February 22, 2016).

[0026] As used herein, "human antibody" or "human antibody fragment" includes antibodies and antibody fragments having variable regions in which both the framework region and the CDR region are derived from human sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a sequence.

[0027] "Specific" describes a protein that recognizes an antigen and can distinguish such an antigen from one or more reference antigens. This ability can be identified by a standard ELISA assay. Typically, the determination of specificity is performed using a set of about 3 to 5 unrelated antigens, such as powdered milk, BSA, transferrin, etc., rather than a single reference antigen.

[0028] "Evaluating a blood sample" means evaluating the blood or a portion of a blood sample that is most relevant to the method. Currently, immunofixation electrophoresis is performed on the serum components of blood. However, if different blood components are to be evaluated in the future, the present invention will be directed toward a method for evaluating those blood components. Examples of blood components include plasma, serum, cells, such as red blood cells and white blood cells, and platelets. Examples of plasma include proteins, such as globulins and coagulation factors, as well as salts, sugars, fats, hormones, and vitamins.

[0029] Immunoglobulinemia is a condition characterized by significantly elevated serum immunoglobulin levels. This can be classified as polyclonal (elevated in all major immunoglobulin classes) or monoclonal (elevated in a single, homogeneous immunoglobulin).

[0030] Polyclonal immunoglobulinemia stems from chronic stimulation of the immune system. Therefore, it can be caused by chronic pyoderma; chronic viral infections, chronic bacterial infections, or chronic fungal infections; granulomatous bacterial diseases; abscesses; chronic parasitic infections; chronic rickettsial diseases, such as pancytopenia in tropical canids; chronic immune disorders, such as systemic lupus erythematosus, rheumatoid arthritis, and myositis; or certain tumor formations. Often, there is no apparent predisposition. In some animals, immunoglobulinemia can initially be monoclonal due to a dominance of a single immunoglobulin class (usually IgG).

[0031] Monoclonal immunoglobulinemia is characterized by the production of large amounts of single immunoglobulin proteins. Monoclonal immunoglobulinemia is benign (i.e., without an underlying disease) or, more commonly, associated with immunoglobulin-secreting tumors. Tumors that secrete monoclonal antibodies originate from plasma cells (myeloma). Myeloma can secrete intact proteins of any immunoglobulin class, or of immunoglobulin subunits or fragments (light chains or heavy chains). Examples of monoclonal immunoglobulinemia include: Hodgkin's disease; variants of multiple myeloma, e.g., solitary plasmacytoma of bone, extramedullary plasmacytoma, plasma cell leukemia, and non-secretory myeloma; lymphoproliferative disorders, e.g., Waldenström macroglobulinemia and lymphoma; heavy chain diseases (γ, α, μ); and amyloidosis.

[0032] The term "CD38" refers to the protein known as CD38, which has the following synonyms: ADP-ribosylcyclase 1, cADPr hydrolase 1, cyclic ADP-ribose hydrolase 1, and T10.

[0033] Human CD38 has the following amino acid sequence: [ka]

[0034] The amino acid sequence of the anti-CD38 antibody "MOR202" is shown in Figure 1. "MOR202" and "MOR03087" are used as synonyms to describe the antibody shown in Figure 1.

[0035] The DNA sequence encoding the MOR202 variable weight domain is as follows: [ka]

[0036] The DNA sequence encoding the MOR202 variable light domain is as follows: [ka]

[0037] MOR202 is disclosed in International Publication No. 2007 / 042309 and U.S. Patent Application No. 12 / 089,806 (which is incorporated in its entirety by reference). In U.S. Patent Application No. 12 / 089,806, MOR202 is an antibody comprising a variable heavy chain corresponding to SEQ ID NO: 21 and a variable light chain corresponding to SEQ ID NO: 51, and the nucleic acid encoding MOR202 is the variable heavy chain SEQ ID NO: 6 and the variable light chain SEQ ID NO: 36.

[0038] MOR202 is currently being tested in a Phase 1 / 2a trial in patients with relapsed / refractory myeloma. This study is evaluating the safety and preliminary efficacy of MOR202 as monotherapy and in combination with pomalidomide and lenalidomide, as well as dexamethasone.

[0039] The antibody MOR09292 is an anti-idiotype antibody against MOR202 and is encoded by the following nucleic acid sequence: VH: [ka] VL: [ka]

[0040] DNA encoding MOR09292-VH-CH1_HSA_6His (without leader sequence) (MOR09292-hAlb heavy chain): [ka]

[0041] DNA encoding MOR09292-VL-Lambda (without leader sequence) (MOR09292-hAlb light chain): [ka]

[0042] Human albumin has the following amino acid sequence (including the signal sequence): [ka]

[0043] The International Myeloma Working Group (IMWG) Uniform Response Criteria for Multiple Myeloma are as follows: [Table 1A] [Table 1B] [Table 1C]

[0044] Electrophoresis is a method for separating proteins based on their biochemical properties. Serum is placed on a specific medium and charged. The net charge (positive or negative), size, and shape of the proteins are commonly used to distinguish between different serum proteins.

[0045] Several subsets of serum protein electrophoresis are available. The names of these subsets are based on the methods used to separate and distinguish various serum components. In zone electrophoresis, for example, different protein subtypes are placed in distinct physical locations on a gel prepared from agar, cellulose, or other plant materials. Proteins are stained, and their concentrations are electronically calculated to provide graphic data on the absolute and relative amounts of various proteins. Further separation of protein subtypes is achieved by staining with immunoactive agents that result in immunofixation and / or immunofluorescence.

[0046] The pattern of serum protein electrophoresis results is determined by the fractionation of two major proteins: albumin and globulin. Albumin, the major protein component of serum, is produced by the liver under normal physiological conditions. Globulin constitutes a smaller fraction of the total serum protein content. The subset of these proteins and their relative amounts are, in most cases, the main focus of interpretation of serum protein electrophoresis.

[0047] The largest peak observed in serum protein electrophoresis, albumin, is located closest to the positive electrode. The next five components (globulins) are labeled alpha-1, alpha-2, beta-1, beta-2, and gamma. The peaks for these components appear near the negative electrode, with the gamma peak being closest to that electrode.

[0048] Figure 3 shows a typical standard pattern for protein distribution determined by serum protein electrophoresis.

[0049] Albumin bands represent the largest protein component in human serum. Albumin levels decrease under conditions where hepatic protein production is reduced or where the loss or breakdown of such proteins is increased. Malnutrition, severe liver disease, renal loss (e.g., in nephrotic syndrome), hormone therapy, and pregnancy can cause low albumin levels. Burns can also result in low albumin levels. Albumin levels increase, for example, in patients with relatively reduced serum water content (e.g., dehydration).

[0050] As we move to the negative portion of the gel (i.e., the anode), the next peaks are related to the alpha-1 and alpha-2 components. The alpha-1 protein fraction consists of alpha-1 antitrypsin, thyroid-binding globulin, and transcortin. Malignancies and acute inflammation (derived from acute-phase reactants) can increase the alpha-1 protein band. A decrease in the alpha-1 protein band can occur due to alpha-1 antitrypsin deficiency or decreased globulin production as a result of liver disease. Ceruloplasmin, alpha-2 macroglobulin, and haptoglobin contribute to the alpha-2 protein band. The alpha-2 component increases as an acute-phase reactant.

[0051] The beta fraction has two peaks labeled β1 and β2. Beta1 is mostly composed of transferrin, and beta2 contains beta-lipoprotein. IgA, IgM, and occasionally IgG can also be identified within the beta fraction along with complement proteins.

[0052] Most clinical interest is focused on the gamma region of the serum protein spectrum because immunoglobulins are migrated to this region. It should be noted that immunoglobulins can often be found throughout the electrophoretic spectrum. C-reactive protein (CRP) is located in the region between the beta and gamma components.

[0053] Many conditions can cause an increase in the gamma region, but some disease states cause homogeneous, spike-like peaks in the lesional area of ​​the gamma globulin band (Figure 4). This so-called "monoclonal immunoglobulinemia" constitutes a group of disorders characterized by the proliferation of homogeneous M proteins, such as single or very few clones of plasma cells that produce MM.

[0054] Immunostatic electrophoresis (IFE) is a technique that allows proteins to be immobilized after electrophoresis by forming insoluble complexes with added monoclonal or polyclonal detection antibody reagents. This process is carried out in the following four steps: 1) Separation of proteins by electrophoresis. 2) Immunofixation (immunoprecipitation) of proteins subjected to electrophoresis - Cover the appropriate electrophoresis tracks with individual antisera. The antisera diffuses into the gel and precipitates the corresponding antigen, if present. Fix the proteins in the reference track with a fixative. 3) Remove any soluble proteins that did not precipitate from the gel by blotting and washing. Trap the precipitated antigen-antibody complex within the gel matrix. 4) Visualize the precipitated protein by staining (e.g., acid violet staining).

[0055] To detect and identify suspected monoclonal components, the sample is subjected to electrophoresis simultaneously on several parallel tracks (see figure). After electrophoresis, the ELP track serves as a reference (containing total protein fixation) to provide a complete electrophoretic pattern of the serum sample's proteins. The remaining tracks allow for the characterization of monoclonal components, typically from the reaction of antiserum to the heavy chains of human IgG, IgA, and IgM, and to the free and bound kappa and lambda light chains, or from the absence of reaction. Other antisera (e.g., anti-IgD) are also possible. The immunofixed bands are then compared to the suspected bands in the reference pattern—corresponding bands should have the same electrophoretic position.

[0056] Figure 5 shows an example of a gel after serum immunofixation electrophoresis. Serum samples from healthy donors were separated 6-fold in parallel by gel electrophoresis, but each lane was stained with a different reagent. After staining, uncomplexed proteins were removed by blotting and washing. Lane ELP = total protein staining; Lane G = anti-IgG staining; Lane A = anti-IgA staining; Lane M = anti-IgM staining; Lane K = anti-kappa staining; Lane L = anti-lambda staining [Examples]

[0057] Materials and Methods IFE Immunofixation was performed using Sebia's semi-automated agarose gel electrophoresis systems Hydrasys and Hydrasys2, and Sebia's Maxikit Hydragel 9IF. The kit is designed for the detection of immunoglobulins in human serum by immunofixation electrophoresis and contains all the necessary reagents and materials, namely agarose gel, buffered strips, diluent, acid violet stain, antiserum (e.g., IgG, IgA, IgM, kappa, and lambda), fixation solution, and applicator.

[0058] To evaluate the effect of MOR202 on M protein analysis, serum samples from healthy donors and MM patients were spiked with MOR202 at various concentrations and incubated at room temperature (RT) for at least 15 minutes. Subsequently, samples spiked with or not spiked with MOR202 were analyzed using IFE, and the gels were stained with anti-IgG antiserum or anti-lambda antiserum (both staining reagents can bind to MOR202). In both staining methods, MOR202 was already detected at the lowest concentration tested, 200 μg / mL. This suggests IFE interference at or below this drug serum level (Figure 6).

[0059] To distinguish between MOR202-related assay signals and endogenous M protein spikes in IFE, a method of pre-incubating MOR202-containing samples with a MOR202-specific anti-idiotype antibody (MOR09292) was tested. The objective of this method was to demonstrate that MOR202-related assay signals could be clearly identified by electrophoresis and comparison of samples pre-incubated with and without MOR09292. To evaluate whether the electrophoretic distance was large enough to be detected, samples containing MOR202 in saline were prepared and pre-incubated with and without MOR09292. Anti-idiotype antibodies were generated and tested in IgG1 antibody format and IgM antibody format. Test samples were prepared with MOR202 at a constant concentration of 1200 μg / mL and pre-incubated for 60 minutes with and without two MOR09292 variants at various concentrations. Subsequently, the samples were analyzed, and the IFE gels were stained with anti-IgG antiserum or anti-lambda antiserum. The results showed that when test samples were pre-incubated with various forms of MOR09292, an acceptable migration distance of MOR202 drug spikes suitable for clinical sample evaluation could not be observed (Figure 7). A surprising finding was that even when the size of the drug / antibody complex was increased by approximately 3 times (MOR09292-IgG) or 7 times (MOR09292-IgM) compared to the drug antibody alone, the change in molecular weight of the complex did not result in a valid shift in the assay signal (i.e., a change in the electrophoretic pattern).

[0060] Based on these results, the MOR09292-Fab fragment was genetically fused to human albumin (MOR09292-hAlb) to generate further variants of the idiotype antibody. The new variants increased the size of the drug-antibody complex by up to 2.6 times compared to the drug antibody alone. More importantly, the incorporation of human serum albumin lowered the overall net charge of the complex. Sample preparation and testing were performed as previously described. As a result, a distinct shift in the MOR202 / MOR09292-huAlb complex can be observed when compared to the assay signal of MOR202 alone. See Figure 8.

[0061] A modified IFE assay using MOR09292-hAlb for sample pretreatment was incorporated into the clinical development of MOR202. Therefore, this assay was validated by a central laboratory responsible for M protein analysis and introduced into the clinical strategy as the "Immunofixation (IFE) Reflex Assay." To distinguish between MOR202-related signals and M protein-related signals, the IFE reflex assay was performed in addition to standard serum IFE and serum protein electrophoresis (SPE) when, for example, the following two conditions were met: a) A reduction of at least ≥40% in serum M protein levels compared to M protein concentration pretreatment, and b) At least one of the remaining M protein spikes has the same characteristics as the drug antibody MOR202 (i.e., IgG / lambda positive staining in IFE).

[0062] Case study on the use and results of IFE reflex assay within the scope of the clinical study MOR202C101 Within the scope of the first clinical study (MOR202C101) treating multiple myeloma patients with MOR202, an IFE reflex assay was performed on patient 19007 after an observation of a ≥86% reduction in serum M protein levels. For this patient, the identified M protein spike was described as IgG / lambda positive by IFE, which is the same molecular property known for MOR202. SPE was performed to detect residual concentrations of 1 or 2 g / L of latent M protein on January 12, 2016 and February 19, 2016. The IFE reflex assay was able to demonstrate that the M protein is not involved, as this assay signal is caused solely by MOR202 interference (see summary lab results in Table 1). These results demonstrate how the newly established IFE reflex assay can clearly distinguish between the M protein, and therefore disease-related assay signals, and MOR202 treatment-related assay signals. [Table 2]

[0063] Embodiment One embodiment is an anti-idiotype antibody fused to albumin. In one embodiment, the albumin is human albumin having the amino acid sequence of SEQ ID NO: 6. In one embodiment, the human albumin is a fragment of human albumin or a partial sequence of human albumin.

[0064] In one embodiment, albumin is a functional fragment of albumin. In another embodiment, human albumin is a functional fragment of human albumin. In this context, the terms albumin or human albumin “functional fragment” refer to albumin that is a fragment or variant of natural albumin or human albumin but is still functionally active in the sense that it can still fulfill the physiological role of albumin.

[0065] One embodiment is a variable heavy domain containing the following amino acid sequence [ka] and Variable light chain domain containing the following amino acid sequence [ka] This is an anti-idiotype antibody that is specific to antibodies containing [specific characteristic].

[0066] One embodiment is a variable heavy domain containing the following amino acid sequence [ka] and Variable light chain domain containing the following amino acid sequence [ka] This is an anti-idiotype antibody that is specific to an antibody having [a certain characteristic]. In one embodiment, the anti-idiotype antibody is fused to albumin. In one embodiment, the albumin is human albumin having the amino acid sequence of SEQ ID NO: 6. In one embodiment, the human albumin is a fragment or partial sequence of human albumin. In one embodiment, the human albumin is a functional fragment or partial sequence of human albumin.

[0067] In the embodiment, the anti-idiotype antibody is HCDR1 of amino acid sequence YSFSNYWIS (SEQ ID NO: 18), HCDR2 of amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 19), HCDR3 with amino acid sequence SRGAGMDY (SEQ ID NO: 20) Variable heavy chains including, LCDR1 of amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 21), LCDR2 of amino acid sequence LLIYADNNRPS (SEQ ID NO: 22), LCDR3 of amino acid sequence GSYDESSNSM (SEQ ID NO: 23) Includes variable light chains.

[0068] In one embodiment, the anti-idiotype antibody is a human antibody.

[0069] In this embodiment, the anti-idiotype antibody is a variable heavy chain with the following amino acid sequence. [ka] and Variable light chain of the following amino acid sequence [ka] Includes.

[0070] In the embodiment, the anti-idiotype antibody albumin fusion is a heavy chain amino acid sequence [ka] Includes.

[0071] In the embodiment, the anti-idiotype antibody albumin fusion is a light chain amino acid sequence [ka] Includes.

[0072] One embodiment is a method for evaluating a blood sample obtained from a patient undergoing treatment for multiple myeloma or other immunoglobulinemia, which includes the following: e) Obtain a blood sample from the patient, f) Incubate the blood sample with an anti-idiotype antibody. g) Perform immunofixation electrophoresis (IFE), and h) Report the results of the IFE.

[0073] In an embodiment of the method, the patient has the following amino acid sequence [ka] Variable weight domains including the following amino acid sequence [ka] We have experience with treatment using antibodies containing variable light chain domains.

[0074] In one embodiment of the method, the anti-idiotype antibody is fused to albumin. In one embodiment of the method, the albumin is human albumin having the amino acid sequence of SEQ ID NO: 6. In one embodiment of the method, the human albumin is a fragment of human albumin or a partial sequence of human albumin.

[0075] The exemplified anti-idiotype antibody MOR09292 is specific to MOR202. Anti-idiotype antibodies against MOR202, when fused to human albumin, shift the antibody in IFE, thus mitigating any potential interference of MOR202 to M protein-based clinical assessments. Conjugates of other anti-idiotype antibodies specific to other antibodies used in the treatment of multiple myeloma or other immunoglobulinemias are expected to yield similar results. Therefore, this means that other anti-idiotype antibody-albumin conjugates would be useful for shifting the antibody in IFE, thus mitigating any potential interference of antibodies to M protein-based clinical assessments.

[0076] In an embodiment of the method, the anti-idiotype antibody is HCDR1 of amino acid sequence YSFSNYWIS (SEQ ID NO: 18), HCDR2 of amino acid sequence WMGIIDPASSKTRYSPSFQG (SEQ ID NO: 19), HCDR3 with amino acid sequence SRGAGMDY (SEQ ID NO: 20) Variable heavy chains including, LCDR1 of amino acid sequence TGSSSNIGAGYDVH (SEQ ID NO: 21), LCDR2 of amino acid sequence LLIYADNNRPS (SEQ ID NO: 22), LCDR3 of amino acid sequence GSYDESSNSM (SEQ ID NO: 23) Includes variable light chains.

[0077] In the embodiment of this method, the anti-idiotype antibody is a human antibody.

[0078] In an embodiment of the method, the anti-idiotype antibody has the following amino acid sequence [ka] The variable heavy chain, and The following amino acid sequence [ka] Includes variable light chain.

[0079] In an embodiment of the method, the anti-idiotype antibody albumin fusion has the following heavy chain amino acid sequence [ka] Includes.

[0080] In an embodiment of the method, the anti-idiotype antibody albumin fusion has the following light chain amino acid sequence [ka] Includes.

[0081] In embodiments of the method, the sample is obtained from a patient undergoing treatment for multiple myeloma or other immunoglobulinemia. In further embodiments, the immunoglobulinemia is monoclonal immunoglobulinemia. In further embodiments, monoclonal immunoglobulinemias include: Hodgkin's disease; variants of multiple myeloma, e.g., solitary plasmacytoma of bone, extramedullary plasmacytoma, plasma cell leukemia, and nonsecretory myeloma; lymphoproliferative disorders, e.g., Waldenström macroglobulinemia and lymphoma; heavy chain disease (γ, α, μ); and amyloidosis.

[0082] In the embodiment of this method, the sample is evaluated for total M protein levels.

[0083] One embodiment is a nucleic acid encoding an exemplary anti-idiotype antibody or an anti-idiotype antibody albumin fusion. In one embodiment, the anti-idiotype antibody is MOR09292. In one embodiment, the anti-idiotype antibody is encoded by a nucleic acid sequence encoding the amino acid sequence shown in Figures 2A and 2B.

[0084] In one embodiment, the anti-idiotype antibody is encoded by the nucleic acid sequences of SEQ ID NO: 26(VH) and SEQ ID NO: 27(VL).

Claims

1. A nucleic acid encoding the heavy chain of a monoclonal anti-idiotype antibody fused to albumin, The monoclonal anti-idiotype antibody is a nucleic acid comprising a VH chain containing HCDR1 containing the amino acid sequence of SEQ ID NO: 18, HCDR2 containing the amino acid sequence of SEQ ID NO: 19, and HCDR3 containing the amino acid sequence of SEQ ID NO: 20, or a VL chain containing LCDR1 containing the amino acid sequence of SEQ ID NO: 21, LCDR2 containing the amino acid sequence of SEQ ID NO: 22, and LCDR3 containing the amino acid sequence of SEQ ID NO:

23.

2. The nucleic acid according to claim 1, wherein the monoclonal anti-idiotype antibody comprises a variable heavy chain having the amino acid sequence of SEQ ID NO:

16.

3. The nucleic acid according to claim 1, wherein the monoclonal anti-idiotype antibody comprises a variable light chain having the amino acid sequence of SEQ ID NO:

17.

4. The nucleic acid according to claim 1, wherein the monoclonal anti-idiotype antibody is IgG.

5. The nucleic acid according to claim 1, wherein the monoclonal anti-idiotype antibody is IgM.

6. The nucleic acid according to claim 1, wherein the nucleic acid encoding VH includes the sequence of sequence number 4.

7. The nucleic acid according to claim 6, wherein the nucleic acid encodes a heavy chain containing the sequence of sequence number 26.

8. The nucleic acid according to claim 1, wherein the nucleic acid encoding VL includes the sequence of sequence number 5.

9. The nucleic acid according to claim 8, wherein the nucleic acid encoding VL consists of the sequence of sequence number 5.

10. The nucleic acid according to claim 1, wherein the nucleic acid encodes a light chain containing the sequence of sequence number 27.

11. The nucleic acid according to claim 6, further comprising a nucleic acid encoding a VL containing the sequence of sequence number 5.

12. The nucleic acid according to claim 11, wherein the nucleic acid encodes a heavy chain containing the sequence of sequence number 26, and the nucleic acid encodes a light chain containing the sequence of sequence number 27.

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