Antibodies targeting B cell receptors in chronic lymphocytic leukemia and uses thereof

Abstract

The present invention provides antibodies for the treatment of chronic lymphocytic leukemia (CLL). These antibodies include R110 mutated immunoglobulin lambda variable 3-21 (IGLV3-21 R110 The antibody targets the B cell receptor (BCR) of CLL cells, which is characterized by the BCR being a marker of CLL cell proliferation and differentiation. The invention also provides nucleic acid sequences encoding the antibody, vectors comprising the same, pharmaceutical compositions, and kits with instructions for use.

Description

[Technical field]

[0001] The present invention provides antibodies for the treatment of chronic lymphocytic leukemia (CLL). These antibodies include R110 mutated immunoglobulin lambda variable 3-21 (IGLV3-21 R110 ) targets the B cell receptor (BCR) of CLL cells, which is characterized by

[0002] The invention also provides nucleic acid sequences encoding the above antibodies, vectors containing same, pharmaceutical compositions, and kits with instructions for use.

[0003] Background technology Antibody therapy has proven to be a highly effective agent for the treatment of leukemia and lymphoma resulting from malignant transformation of the B-cell lineage. The approval of monoclonal antibodies, such as rituximab, has significantly improved the response rate, long-term outcomes, and quality of life of patients with CLL.

[0004] CLL is a heterogeneous B-lymphocyte-derived malignancy that arises from the clonal expansion of a CD5-positive B-lymphocyte subpopulation and gradually accumulates in the bone marrow, lymph nodes, peripheral blood, and spleen (Rozman C, Montserrat E. Chronic lymphocytic leukemia. N Engl J Med. 1995; 333: 1052-1057). The disease is the most common type of leukemia in Western countries, typically occurring in older patients, with men at twice the risk of developing CLL compared to women (Kipps TJ, Steven-son FK, Wu CJ, Croce CM, Packham G, Wierda WG et al. Chronic lymphocytic leukemia. Nat Rev Dis Primers (2017) 3:1-12).

[0005] Clinical and biological evidence indicates that the BCR is one of the major factors in clonal selection and survival of CLL cells, as reviewed by Burger and Chiorazzi (Burger JA, Chiorazzi N. B cell receptor signaling in chronic lymphocytic leukemia. Trends Immunol 2013; 34: 592-601).

[0006] BCR is a multiprotein structure composed of non-covalently linked antigen-binding and signaling subunits. The antigen-binding subunit consists of a membrane immunoglobulin containing two identical heavy chains and two identical light chains, with one constant domain in each light chain and three constant domains in each heavy chain. Each heavy chain combines with a light chain to form an antigen-binding site. Each light and heavy chain contains a variable domain that forms the antigen-binding site. The immunoglobulin genes encoded at the Igh, Igl and Igk loci contain multiple V (variable), D (diversity) and J (joining) gene segments upstream of one or more constant exons. In developing B cells, rearrangement of immunoglobulin genes randomly assembles V, D and J gene segments to create a complete V exon at the Igh locus and V and J gene segments at the Igk or Igl locus. Through combinatorial joining of gene segments, junctional diversity, and random pairing of heavy and light chains, each individual B cell precursor generates a unique and nearly unique antigen-binding subunit, whose antigen-binding affinity can be further refined by somatic hypermutation (SHM).

[0007] The signaling portion of the BCR consists of a disulfide-linked heterodimer of Igα and Igβ (CD79a / CD79b) proteins. Igα and Igβ each contain a single immunoreceptor tyrosine-based activation motif (ITAM) at their cytoplasmic tails that initiates signal transduction after BCR aggregation upon antigen binding (Flaswinkel, H., Reth, M., 1994. Dual role of the tyrosine activation motif of the Ig-alpha protein during signal transduction via the B cell antigen receptor. EMBO J. 13, 83-89).

[0008] Antigen binding rapidly activates the Src family kinase Lyn, leading to phosphorylation of Igα / Igβ. This initiates the formation of a large signaling complex on the cytoplasmic side of the membrane composed of the BCR, various tyrosine kinases, adaptor proteins, and signaling enzymes. Proximal BCR signaling is mediated by the protein tyrosine kinase Syk (spleen tyrosine kinase), which is recruited to the phosphorylated ITAMs of Igα and Igβ, leading to signal propagation through the association of Syk with the adaptor protein SLP65 and its downstream signaling enzymes Bruton's tyrosine kinase (BTK) and phospholipase Cγ2 (PLCγ2). Signals emanating from the signaling complex activate downstream pathways, including calcium mobilization, phosphoinositide 3-kinase (PI3K), nuclear factor kappa B (NF-κB), nuclear factor of activated T cells (NF-AT), mitogen-activated protein kinase (MAPK), and rat sarcoma (RAS) signaling pathways (see also Burger JA and Chiorazzi N, 2013).

[0009] Chronic activation of mature B cells via the B cell receptor has been shown to be a key process in the formation and development of CLL (Stevenson FK, Krysov S, Davies AJ, Steele AJ, Packham G. B-cell receptor signaling in chronic lymphocytic leukemia Blood. 2011; 118: 4313-4320). This is consistent with studies using the EBV protein LMP2A as a constitutively active BCR surrogate, showing that development of the murine B1 subset is dependent on strong and long-lasting BCR stimulation (Casola S, Otipoby KI, Alimzhanov M, et al. B cell receptor signal strength determines B cell fate. Nat Immunol 2004; 5: 317-27). Furthermore, antigen-independent autonomous signaling of primary CLL B cells through the interaction of two adjacent BCRs on the cell has been identified as a key driver of CLL development, leading to increased tyrosine phosphorylation of BCR-proximal signaling molecules, regulating cyclic signaling and Ca2+ upregulation. 2+ (Duehren-von Minden M et al., Chronic lymphocytic leukemia is driven by antigen-independent cell-autonomous signalling. Nature. 2012; 489: 309-313).

[0010] It is well documented that the protein kinase Syk is constitutively phosphorylated by sustained BCR signaling, and several studies have revealed that other key molecules of the signaling pathway downstream of BCR engagement in normal B cells, such as PKC, phosphoinositide 3 kinase, and mitogen-activated protein kinase p38, are also constitutively activated in B-CLL cells, leading to deregulation of the activity or expression of several pro-survival molecules and downstream pathways (Gobessi S, Laurenti L, Longo PG, Carsetti L, Berno V, Si-ca S, et al. Inhibition of constitutive and BCR-induced Syk activation downregulates Mcl-1 and induces apoptosis in chronic lymphocytic leukemia B cells. Leukemia 2009; 23: 686-697. Ringshausen I, Schneller F, Bogner C, Hipp S, Duyster J, Peschel C, et al. Constitutively activated phosphatidylinositol-3 kinase (PI-3K) is involved in the defect of apoptosis in B-CLL: association with protein kinase C delta. Blood 2002; 100: 3741-3748. Plate JM. PI3-kinase regulates survival of chronic lymphocytic leukemia B-cells by preventing caspase 8 activation. Leuk Lymphoma 2004; 45: 1519-1529.Sainz-Perez A, Gary-Gouy H, Portier A, Davi F, Merle-Beral H, Galanaud P et al. High Mda-7 expression promotes malignant cell survival and p38 MAP kinase activation in chronic lymphocytic leukemia. Leukemia 2006; 20: 498-504). .

[0011] Constitutively activated signaling pathways, such as NF-kB or PI3K / AKT, have been shown to lead to the transcription and overexpression of key anti-apoptotic proteins, notably B-cell lymphoma 2 (Bcl-2) and several members of the inhibitor of apoptosis protein (IAP) family (Loeder S et al., A novel paradigm to trigger apoptosis in chronic lymphocytic leukemia. Cancer Res. 2009; 69: 8977-8986). In addition to Bcl-2 itself, it is well known that Mcl-1 plays an important role in apoptotic disorders in CLL cells, and BCR signaling has been reported to upregulate Mcl-1 expression via the PI3K / AKT pathway (Petlickovski A, Laurenti L, Li X, Marietti S, Chiusolo P, Sica S, Leone G, Efremov DG. Sustained signaling through the B-cell receptor induces Mcl-1 and promotes survival of chronic lymphocytic leukemia B cells. Blood. 2005; 105: 4820-4827).

[0012] Various aspects of the BCR have been recognized to distinguish the major CLL disease subtypes. For example, the level of somatic hypermutation within the variable region of the BCR immunoglobulin heavy chain (IGHV) has been used as a prognostic marker for decades. CLL patients with mutated IGHV genes (M-CLL), i.e., IGHV gene identity less than 98% with the closest germline, generally have a more indolent disease course than CLL patients with unmutated IGHV genes (U-CLL), with germline identity of 98% or more. However, exceptions to this have been observed, where the status of the mutated IGHV genes does not correlate with a specific disease course. For example, cases with IGHV3-21 genes had one of the worst clinical outcomes, despite most expressing mutated BCR. Different approaches determined by IGHV have led to the classification of approximately 30% of CLL cases into prognostically significant subsets, each with highly homogenous biological characteristics, clinical manifestations, and outcomes. This classification is based on the observation that there exists a typical BCR that carries a highly homologous heavy chain complementarity determining region 3 (H-CDR3) sequence between mutated and unmutated cases. According to this approach, CLL cases characterized by mutated IGHV3-21 are classified into the so-called subset #2 (Stamatopoulos K, Be-lessi C, Moreno C, et al. Over 20% of patients with chronic lymphocytic leukemia carry stereotyped receptors: pathogenic implications and clinical correlations. Blood. 2007; 109(1): 259-270; Agathangelidis A. et al. Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: A molecular classification with implications for targeted therapies. Blood. 2012; 119: 4467-4475).

[0013] In particular, the use of IGHV3-21 according to subset #2 involves the amino acid position 110 of the light chain (IGLV3-21 R110 ) has always been observed in association with expression of the immunoglobulin lambda variable 3-21 chain. R110 The cause of arginine 110 (R110) in BCR is a single G>C substitution at the splice site between the immunoglobulin lambda J gene and the constant gene. The presence of R110 together with germline-encoded lysine 16 (K16) of one BCR and aspartic acids (D) 50 and 52 of the tyrosine-aspartic acid-serine-aspartic acid (YDSD) motif of the adjacent BCR has been identified to enable BCR-BCR interactions and induce cell-autonomous signaling (Figures 6 and 7; Minici, C. et al., Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukemia, Nature Comm. 2017; 8:15746).

[0014] During the epigenetic, genomic, and transcriptomic characterization of a large cohort of CLL patients with a focus on the BCR light chain, IGLV3-21 R110 Approximately 60% of cases were found to carry non-stereotypic BCRs, with subset #2 being IGLV3-21 R110It was emphasized that IGLV3-21R110 represents only a minor subgroup of CLL characterized by a combination of chronic lymphocytic leukemia and IgLV3-21R110-associated leukemia (Stamatopoulos B, Smith T, Crompot E et al. The Light Chain IgLV3-21 Defines a New Poor Prognostic Subgroup in Chronic Lymphocytic Leukemia: Results of a Multicenter Study. Clin Cancer Res. 2018; 24(20): 5048-5057. Nadeu F, Royo R, Clot G et al. IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics. Blood. 2021; 137(21): 2935-2946).

[0015] Of the four alleles of the IGLV3-21 gene identified in humans, alleles IGLV3-21*01 (IMGT / LIGM-DB accession no. X71966) and IGLV3-21*04 (IMGT / LIGM-DB accession no. AC279208) encode the required K16, D50, and D52, the last two of which are most often involved in the IGLV3-21 gene expression. R110 The motif includes tyrosine at position 49 and serine at position 51 of the IGLV3-21 R110 (See FIG. 7 for the sequence.) However, IGLV3-21 was modified to replace tyrosine with phenylalanine or serine with threonine. R110 Functionally equivalent mutations of this motif have also been observed in CLL patients (see also Nadeu et al., 2021; for variants see also Figure 7). Interestingly, the alleles IGLV3-21*01 and IGLV3-21*04 are significantly underrepresented in B cells of healthy donors, whereas in patients studied by different groups all IGLV3-21 genes could be assigned to the allele IGLV3-21*01 or the allele IGLV3-21*04, indicating that these alleles are the IGLV3-21R110 These results suggest that it may be mechanistically required for the development of related CLL.

[0016] IGLV3-21 R110 A CLL subgroup, also proposed to be named subset #2L, is associated with a highly aggressive disease course. R110 The poor outcome of CLL cases does not depend on the IGHV mutation status or the nature of the heavy chain. IGHV3-21 R110 is found in mutated CLL, e.g., in subset #2 (see above), and in the context of different heavy chains, e.g., IGHV1-18, IGHV3-53 or IGHV3-64 (Nadeu et al., 2021, supra). R110 defines a group of CLLs that is not limited by traditional subset classifications based on empirically defined epigenetic stereotypes, nor by IGHV mutation status. Furthermore, the critical role of R110 as a CLL driver mutation has been confirmed by site-directed mutagenesis experiments, revealing that IGLV3-21 R110 From IGLV3-21 G110 It has been shown that reversion to BCR abrogates the autonomous signaling capacity of the BCR (see also Stamatopoulos B, Smith T, Crompot E et al. 2018).

[0017] Time to first treatment (TTFT) and overall survival (OS) with IGLV3-21 R110 Studies correlating the presence of the carrier BCR with IGLV3-21 R110 Compared with non-IGLV3-21 CLL patients, R110 Significantly shorter values ​​were shown for patients expressing IGLV3-21 R110 The need for rapid treatment for positive patients was emphasized (see also Nadeu F et al., 2021).

[0018] CLL in these patients is usually treated with chemotherapy drugs such as idelariship and ibrutinib, alone and in combination with other drugs. Idelaliship is an inhibitor of the hematopoietic cell-restricted delta isoform of PI3K, which promotes apoptosis of primary CLL cells (Hoellenriegel J, Meadows SA, Sivina M, et al. The phosphoinositide 3'-kinase delta inhibitor, CAL-101, inhibits B-cell receptor signaling and chemokine networks in chronic lymphocytic leukemia. Blood. 2011; 118: 3603-3612). Ibrutinib is a BTK inhibitor that induces apoptosis in B-cell lymphoma and CLL cells (Hermann SE, Gordon AL, Hertlein E, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011; 117: 6287-6296). Recent treatments have increasingly used monoclonal antibodies that act as CD52 antibodies, such as alemtuzumab, or obinutuzumab, rituximab, and ofatumumab, which target the cell surface B-lineage-restricted antigen CD-20. The use of these antibodies can extend remission periods by approximately 10 months. However, cutting-edge treatments for treatment are usually very stressful for patients because the therapeutic targets are essential for the survival of both normal and malignant B cells, and low blood counts, including low levels of certain white blood cells (neutropenia), are common side effects. Furthermore, high infection risk, latent infection, and off-target effects on the immune system have been reported as further side effects for chemotherapy drugs.It is generally concluded that unwanted side effects of treatments and often inadequate drug efficacy lead to a high mortality rate, since not only tumor cells but also healthy cells of the immune system are damaged.

[0019] Therefore, IGLV3-21 R110 No specific treatment has yet been found for patients with positive CLL that is free from the aforementioned side effects.

[0020] IGLV3-21 carrying BCR R110 Current research into potential antibodies targeting IGLV3-21 has focused primarily on the diagnosis of this particular subtype of CLL. As an example, Maity et al. R110 We describe immunophenotyping studies using the diagnostic anti-IGLV3-21 antibody. R110 The antibodies are disclosed to be IgG2a and Igκ antibodies (Maity PC, Bilal M, Koning MT, et al. IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling. PNAS. 2020; 117(8): 4320-4327).

[0021] A first step towards opening up treatment options is disclosed in WO 2019 / 008129, which discloses antibodies that can be used to remove CLL cells from blood samples. The antibodies of WO 2019 / 008129 are expressed as IgG antibodies in hybridoma cell lines started from a mouse host. WO 2019 / 008129 discloses only the respective variable heavy and variable light domains of such antibodies.

[0022] WO2019 / 008129 does not show that the antibodies disclosed therein can be selected between healthy and diseased tissues, nor have they been applied in vivo. Therefore, their therapeutic effect is not shown. The antibodies in WO2019 / 008129 are only disclosed by VH and VL, and therefore any effect of the remaining constant regions of the antibodies disclosed therein remains unknown.

[0023] From the above, IGLV3-21 R110 Therapeutic options that would allow improved treatment of CLL in positive patients, particularly antibodies that are specific, selective and do not cross-react with other tissues, are currently unknown but are needed.

[0024] Summary of the Invention In a first aspect, the present invention solves the above problem by providing an antibody having a heavy chain amino acid sequence of SEQ ID NO: 1 and a light chain amino acid sequence of SEQ ID NO: 2, or an antibody having a heavy chain amino acid sequence of SEQ ID NO: 11 and a light chain amino acid sequence of SEQ ID NO: 12, or an antibody comprising a variable heavy chain having a sequence selected from the list consisting of SEQ ID NO: 15 and SEQ ID NO: 20 and a variable light chain having a sequence selected from the list consisting of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, in any combination.

[0025] Such antibodies specifically, selectively, and non-cross-reactively bind IGLV3-21 to other tissues of CLL patients. R110 This new antibody binds to the BCR carrying IGLV3-21, thereby killing malignant B cells. R110 While recognizing the presence of the IGHV3-21 / IGLV3-21 R110 Combination of IGLV3-21 R110 It is also possible to treat "subset #2 CLL" characterized by BCR, which includes other CLLs, including

[0026] These antibodies were provided by IGLV3-21 R110The second aspect of the invention provides for the provision of said antibody for use in the treatment of CLL in a patient with IGLV3-21 inflammatory bowel disease by administering a therapeutically effective amount of said antibody. R110 It relates to a method of treating CLL in positive patients.

[0027] This is the IGLV3-21 R110 This is the first treatment for CLL in patients with IGLV3-21 positive, which is able to selectively kill malignant B cells without the side effects of current treatments. Furthermore, IGLV3-21 has been identified as a marker for CLL. R110 The binding to and killing of B cells characterized solely by the presence of a BCR bearing a specific IGHV allows for treatment that is independent of the requirement that such CLL belong to any of the previously identified subsets of CLL defined by a particular IGHV.

[0028] Without being bound by theory, treatment with the antibodies of the invention inhibits IGLV3-21 expression in the BCR in the absence of a costimulatory signal. R110 It is believed that IGLV3-21 induces hyperactivation of B cells through the IL-1 receptor. This ultimately results in induction of apoptosis in CLL cells bound by the antibody. Thus, treatment with the antibody of the present invention is highly selective for malignant B cells, and in particular, IGLV3-21 R110 It is much more selective for positive B cells.

[0029] In a third aspect, the present invention also relates to a DNA molecule (nucleic acid) encoding an antibody of the present invention. Accordingly, the present invention also relates to vectors and host cells comprising the nucleic acid sequence of the present invention.

[0030] In a fourth aspect, the present invention provides an IGLV3-21 expressed by B cells. R110 The fourth aspect of the invention also relates to said antibody for use in the diagnosis of CLL characterized by a BCR carrying IGLV3-21 expressed by B cells by applying said antibody to a patient tissue or blood sample. R110For methods of diagnosing CLL characterized by a BCR carrying the antibody of the present invention, these antibodies are conjugated to a detectable marker or a further antibody that specifically binds to the antibody of the present invention and is conjugated to a detectable marker is subsequently applied.

[0031] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this invention belongs. However, the following references provide those of ordinary skill in the art to which this invention belongs with general definitions of many of the terms used in this invention and may be used by reference to the extent that the definitions are consistent with the meanings commonly understood in the art. Such references include, but are not limited to, Singleton et al., Dictionary of Microbiology and Molecular Biology (2d ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); Hale & Marham, The Harper Collins Dictionary of Biology (1991); and Lackie et al., The Dictionary of Cell & Molecular Biology (3d ed. 1999); and Cellular and Molecular Immunology, Eds. Abbas, Lichtman and Pober, 2nd Edition, WB Saunders Company. Reference may be made to additional technical resources available to those of skill in the art that provide definitions of terms used herein that have meanings commonly understood in the art. For purposes of the present invention, the following terms are further defined.

[0032] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a gene" is a reference to one or more genes and includes equivalents thereof known to those skilled in the art, and so forth.

[0033] "Autonomously active" BCR is a special type of persistently active BCR. Conventional activation is based on external antigens, but autonomously active BCR results from interaction with membrane structures on the surface of the same cell. The clinical picture of CLL may show interactions between adjacent BCRs on the surface of the same cell that induce autonomous activation (e.g. M. Duehren-von Minden et al., Nature 2012).

[0034] IGLV3-21 R110 is the light chain variable region of the BCR that enables BCR-BCR interaction and induces an autonomously active BCR. R110 are characterized by a sequence identity of 80% or more to the sequence represented by SEQ ID NO: 53, in both cases having an arginine instead of a glycine at position 110 of said sequence.

[0035] The term "antibody", as used herein, is intended to denote an immunoglobulin molecule, preferably composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, which are typically linked together by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region may, for example, comprise three domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain (CL). The VH and VL regions may be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), and regions of high conservation, called framework regions (FRs). Each VH and VL typically consists of three CDRs and up to four FRs, arranged from the amino terminus to the carboxy terminus, for example in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0036] As used herein, the term "complementarity determining region (CDR; e.g., CDR1, CDR2, and CDR3)" refers to the amino acid residues of an antibody variable domain necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known mechanisms, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; "Kabat" numbering scheme), Chothia and Lesk (J Mol Biol 196: 901-917 (1987)), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; "IMGT" numbering scheme). Each complementarity determining region comprises amino acid residues defined by IMGT. Optionally, the complementarity determining region may also comprise amino acids from a CDR region defined according to Kabat and / or hypervariable loops according to the Chothia numbering system.

[0037] Depending on the amino acid sequence of the constant domain of the heavy chain, intact antibodies are assigned to different "classes". There are five major classes of intact antibodies IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that correspond to the different classes of antibodies are called "alpha", "delta", "epsilon", "gamma", and "mu", respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0038] The term "antibody" as used herein is understood to include antigen-binding fragments and variants thereof. Thus, any reference to an "antibody" in the context of the present invention is also a reference to an antigen-binding fragment and / or variant thereof, unless expressly stated otherwise, such as by specifying the complete heavy chain or complete light chain of the antibody that are combined to form the antibody.

[0039] Here, an "antigen-binding fragment" is defined as a fragment of an antibody / immunoglobulin (e.g., the variable region of an IgG) that retains the antigen-binding region. Antigen-binding fragments of the present invention include Fab, Fab', F(ab')2, and Fv fragments, bispecific antibodies, single domain antibodies (DAb), linear antibodies, single-chain antibody molecules (scFv), and multispecific, e.g., bispecific and trispecific, antibodies formed from antibody fragments (CA K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs in Molecular Biology), Oxford University Press; R. Kontermann & S. Duebel, editors (2001) Antibody Engineering (Springer Laboratory Manual), Springer Verlag). Antibodies other than "multispecific" or "multifunctional" antibodies are understood to have identical binding sites for each of them. F(ab')2 or Fab may be a C H1 Domain and C L It may be designed to minimize or completely eliminate intermolecular disulfide interactions occurring between the domains.

[0040] The "antigen binding region" of an antibody is typically found in one or more hypervariable regions of the antibody, e.g. the CDR1, CDR2 and / or CDR3 regions, however the variable "framework" regions may also play an important role in antigen binding, as they provide an anchorage for the CDRs.

[0041] "Variants" of antibodies or antigen-binding fragments contemplated in the present invention include IGLV3-21 R110 It is a molecule in which the binding activity of the antibody or antigen-binding fragment to the target antigen is maintained.

[0042] As used herein, a "humanized" antibody is defined as: (i) an antibody derived from a non-human source (e.g., a transgenic mouse carrying a heterologous immune system) and based on human germline sequences; (ii) an antibody in which amino acids in the framework regions of a non-human antibody have been partially replaced with human amino acid sequences by genetic engineering; or (iii) an antibody that is CDR-grafted, in which the CDRs of the variable domains are of non-human origin, but one or more frameworks of the variable domains are of human origin, and the constant domains (if present) are of human origin.

[0043] A "chimeric" antibody is defined herein as an antibody in which the variable domains are of non-human origin and some or all of the constant domains are of human origin.

[0044] The term "monoclonal" antibody as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the term "monoclonal" denotes the character of an antibody that is not a mixture of individual antibodies. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are typically uncontaminated by other immunoglobulins. The term "monoclonal" should not be construed as requiring production of the antibody by any particular method. The term monoclonal antibody specifically includes murine, chimeric and humanized antibodies.

[0045] As used herein, an antibody refers to an antigen of interest, such as a tumor-associated polypeptide antigen target (herein, IGLV3-21 R110An antibody that "specifically binds to," "is specific for," or "specifically recognizes" an antigen of interest, and is capable of distinguishing between such antigen and one or more reference antigens. The most common form is "specific binding." "Specifically binds," "is specific for," or "specifically recognizes" refers to the ability of an antibody to distinguish between an antigen of interest and unrelated antigens, as determined, for example, according to one of the following methods: such methods include, but are not limited to, flow cytometry, Western blot, ELISA test, RIA test, ECL test, IRMA test, immunohistological test, and peptide scan.

[0046] "Binding affinity" or "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise defined, as used herein, "binding affinity" or "affinity" refers to the inherent binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The dissociation rate constant, K D is usually expressed as the equilibrium association constant (k a ) and dissociation rate constant (k d The dissociation constant, K D "High affinity" is commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding partner (e.g., an antigen), i.e., how tightly the ligand binds to a particular protein. The affinity between a ligand and a protein is influenced by non-covalent intermolecular interactions between the two molecules. The term "high affinity" refers to the affinity of an antibody to IGLV3-21. R110 10 positive CLL BCR -9 It means that the antibody binds with an affinity (KD) equal to or less than M (monovalent affinity). The antibody may have a substantially higher affinity for the target antigen than other unrelated molecules. The affinity may be measured by a common method known in the art, for example according to Example 5.

[0047] As used herein, the term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, or combinations thereof, and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0048] An "isolated" antibody is one that has been identified and separated from components of the cells in which it is expressed. Contaminating components of the cells are substances that may interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to greater than 95% by weight, and in more preferred embodiments, greater than 99% by weight, of the antibody as measured, for example, by the Lowry method, IN-Vis spectroscopy, or SDS-Capillary Gel electrophoresis (e.g., Caliper LabChip GXII, GX90, or Biorad Bioanalyzer instruments), (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. An isolated naturally occurring antibody includes the antibody in situ, within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0049] The term immunoconjugate (also referred to as "antibody drug conjugate" or "ADC") refers to an antibody conjugated with one or more cytotoxic drugs, such as chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioisotopes (i.e., radioconjugates). Immunoconjugates have been used in the treatment of cancer for the localized delivery of cytotoxic drugs, which are agents that kill cells or inhibit cell growth or proliferation (e.g., Liu et al., Proc Natl. Acad. Sci. (1996), 93, 8618-8623). Immunoconjugates allow targeted delivery of the drug moiety to tumors and intracellular accumulation within tumors, where systemic administration of unconjugated drugs may result in unacceptable levels of toxicity to normal cells and / or tissues. Toxins used in antibody-toxin conjugates include bacterial toxins, such as diphtheria toxin, plant toxins, such as ricin, and small molecule toxins, such as geldanamycin. Toxins can produce cytotoxic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0050] The term "cancer" refers to a physiological condition or disease in which cells divide uncontrollably, leading to unregulated cell growth. A "tumor" comprises one or more cancer cells.

[0051] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a type of cytotoxicity in which secreted Ig bound to Fc gamma receptors (FcγR) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells, e.g., with cytotoxins.

[0052] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) bound to its cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al. (J. Immunol. Methods 202: 163 (1996)), may be performed. Polypeptide variants with altered Fc region amino acid sequences (polypeptides with mutated Fc regions) and increased or decreased C1q binding are described, e.g., in U.S. Pat. No. 6,194,551 and WO 1999 / 51642.

[0053] "Percent (%) sequence identity" to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical to the nucleic acid or amino acid residues in a reference polynucleotide or polypeptide sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Conservative substitutions are not considered part of the sequence identity. Alignment without gaps is preferred. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways that are understood by those skilled in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, LALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0054] Detailed Description of the Invention First Aspect of the Invention - Antibodies The antibody of the present invention is IGLV3-21 R110 The present invention is based on the discovery of novel murine antibodies that have specific affinity for BCRs bearing the IL-1 marker and can provide a therapeutic effect in a subject. The antibodies and their beneficial properties that enable the therapeutic effect are described in more detail below.

[0055] The antibodies of the present invention may be murine, humanized or chimeric antibodies and may be used in a number of contexts as more fully described herein.

[0056] According to a first aspect of the invention there is provided an antibody having a heavy chain amino acid sequence of SEQ ID NO:1 and a light chain amino acid sequence of SEQ ID NO:2, or an antibody having a heavy chain amino acid sequence of SEQ ID NO:11 and a light chain amino acid sequence of SEQ ID NO:12, or an antibody comprising a variable heavy chain having a sequence selected from the list consisting of SEQ ID NO:15 and SEQ ID NO:20 and a variable light chain having a sequence selected from the list consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, in any combination.

[0057] These antibodies may be murine, humanized or chimeric antibodies, and may be R110 - Binds specifically to BCR with high affinity.

[0058] As outlined above, these antibodies R110 -BCR. More specifically, these antibodies induce strong phosphorylation of Syk, BTK, and PI3K in the short term, inducing apoptosis and ultimately inhibiting tumor growth in vivo.

[0059] Preferred embodiments of the first aspect of the invention are characterized in further detail in Tables 1 and 2 of the Examples.

[0060] The antibody "mAb01-01" is therefore a first preferred embodiment of the first aspect of the invention and is characterized by a heavy chain corresponding to SEQ ID NO:1 and a light chain corresponding to SEQ ID NO:2.

[0061] The antibody "HC0-LC0" is therefore a second preferred embodiment of the first aspect of the invention and is characterized by a heavy chain corresponding to SEQ ID NO:11 and a light chain corresponding to SEQ ID NO:12.

[0062] The antibody "HC6-LC6" is therefore a second preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:16.

[0063] Within the scope of the third preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:13 and a light chain corresponding to SEQ ID NO:14 is preferred.

[0064] The antibody "HC6-LC7" is therefore a fourth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:17.

[0065] Within the fourth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 22 is preferred.

[0066] The antibody "HC6-LC8" is therefore a fifth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:18.

[0067] Within the scope of the fifth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 23 is preferred.

[0068] The antibody "HC6-LC9" is therefore a sixth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:19.

[0069] Within the sixth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 24 is preferred.

[0070] Thus, the antibody "HC7-LC6" is a seventh preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:16.

[0071] Within the seventh preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:14 is preferred.

[0072] Thus, the antibody "HC7-LC7" is an eighth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:17.

[0073] Within the scope of the eighth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:22 is preferred.

[0074] The antibody "HC7-LC8" is therefore a ninth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:18.

[0075] Within the ninth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:23 is preferred.

[0076] The antibody "HC7-LC9" is therefore a tenth preferred embodiment of the first aspect of the invention and is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:19.

[0077] Within the tenth preferred embodiment of the first aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:24 is preferred.

[0078] The antibodies of the first aspect of the invention are not limited to the specific peptide sequences provided. Rather, the invention encompasses variants. With reference to this disclosure and commonly available techniques and references, one skilled in the art can prepare, test and utilize functional variants of the antibodies disclosed herein, and these variants can be used to identify IGLV3-21. R110 It is understood that any antibody capable of binding to the BCR and killing a B cell is within the scope of the present invention.

[0079] Variants include, for example, antibodies having at least one altered complementarity determining region (CDR) (hypervariable) and / or framework (FR) (variable) domain / position relative to the peptide sequences disclosed herein. To further illustrate this concept, a brief description of antibody structure follows.

[0080] An antibody is composed of two peptide chains, each of which contains one (light chain) or three (heavy chain) constant domains and a variable region (VL, VH), which in each case is composed of four FR regions and three spaced apart CDRs (complementarity determining regions). An antigen-binding site is formed by one or more CDRs, while the FR regions provide the structural framework of the CDRs and play an important role in antigen binding. By changing one or more amino acid residues in the CDR or FR regions, one skilled in the art can routinely generate mutated or diversified antibody sequences.

[0081] As an example, one of skill in the art can use the antibody sequences provided herein (eg, Table 1 and / or Table 2) to design peptide variants within the scope of the invention.

[0082] Furthermore, variants may be obtained by using the antibody of the first aspect of the invention as a starting point for optimization, diversifying one or more amino acid residues in the antibody, preferably in one or more CDRs, and screening the resulting collection of antibody variants. Diversification can be performed by synthesizing a collection of DNA molecules using trinucleotide mutagenesis (TRIM) technology (Virnekiis B. et al., Nucl. Acids Res. 1994, 22: 5600.). Antibodies include, but are not limited to, molecules with modifications / mutations that result in, for example, altered half-life (e.g., modification of the Fe moiety or addition of an additional molecule, e.g., PEG), altered binding affinity, or altered ADCC or CDC activity.

[0083] Polypeptide variants may be made that preserve the overall molecular structure of the antibody peptide sequences described herein. Some rational substitutions will be recognized by those skilled in the art, depending on the properties of individual amino acids. Amino acid substitutions "conservative substitutions" are based, for example, on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved.

[0084] For example, (a) non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan, and methionine, (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, (c) positively charged (basic) amino acids include arginine, lysine, and histidine, and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Substitutions may typically be made within groups (a)-(d). Additionally, glycine and praline may be substituted for one another based on their ability to disrupt α-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine, and lysine, are conventionally found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan, and threonine are conventionally found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are conventionally found in turns. Some preferred substitutions may be made between the following groups: (i) S and T, (ii) P and G, (iii) A, V, L, and I. With the known genetic code and recombinant and synthetic DNA techniques, one of skill in the art can readily construct DNAs that encode conservative amino acid variants.

[0085] Third to tenth preferred embodiments of the antibody according to the first aspect of the invention may comprise a variable heavy chain that is at least 83.3%, 85%, 90%, 92%, 95% sequence identical to the variable heavy chain set forth in SEQ ID NO:15, or a variable heavy chain that is at least 82.5%, 85%, 90%, 92%, 95% sequence identical to the variable heavy chain set forth in SEQ ID NO:20.

[0086] Third to tenth preferred embodiments of the antibody according to the first aspect of the invention may also comprise a variable light chain that has at least 90%, 92%, 95% sequence identity to the variable light chain set forth in SEQ ID NO:16, or at least 87%, 90%, 92%, 95% sequence identity to the variable light chain set forth in SEQ ID NO:17, or at least 80.5%, 85%, 90%, 92%, 95% sequence identity to the variable light chain set forth in SEQ ID NO:18, or at least 77.7%, 80%, 85%, 90%, 92%, 95% sequence identity to the variable light chain set forth in SEQ ID NO:19.

[0087] All preferred embodiments of the antibody according to the first aspect of the invention combine a variable heavy chain sequence comprising the sequences represented by SEQ ID NO:5 (H-CDR1), SEQ ID NO:6 (H-CDR2) and SEQ ID NO:7 (H-CDR3) with a variable light chain sequence comprising the sequences represented by SEQ ID NO:8 (L-CDR1), SEQ ID NO:9 (L-CDR2) and SEQ ID NO:10 (L-CDR3).

[0088] Thus, a preferred embodiment of the first aspect of the invention is an antibody that combines a variable heavy chain sequence represented by SEQ ID NO: 15 with a variable light chain sequence comprising SEQ ID NO: 8 (L-CDR1), SEQ ID NO: 9 (L-CDR2) and SEQ ID NO: 10 (L-CDR3), or an antibody that combines a variable heavy chain sequence represented by SEQ ID NO: 20 with a variable light chain sequence comprising SEQ ID NO: 8 (L-CDR1), SEQ ID NO: 9 (L-CDR2) and SEQ ID NO: 10 (L-CDR3), or an antibody that combines a variable light chain sequence represented by SEQ ID NO: 16 with a variable heavy chain sequence comprising SEQ ID NO: 5 (H-CDR1), SEQ ID NO: 6 (H-CDR2) and SEQ ID NO: 7 (H-CDR3). or an antibody that combines the variable light chain sequence represented by SEQ ID NO: 17 with a variable heavy chain sequence comprising SEQ ID NO: 5 (H-CDR1), SEQ ID NO: 6 (H-CDR2) and SEQ ID NO: 7 (H-CDR3), or an antibody that combines the variable light chain sequence represented by SEQ ID NO: 18 with a variable heavy chain sequence comprising SEQ ID NO: 5 (H-CDR1), SEQ ID NO: 6 (H-CDR2) and SEQ ID NO: 7 (H-CDR3), or an antibody that combines the variable light chain sequence represented by SEQ ID NO: 19 with a variable heavy chain sequence comprising SEQ ID NO: 5 (H-CDR1), SEQ ID NO: 6 (H-CDR2) and SEQ ID NO: 7 (H-CDR3).

[0089] The antibody according to the first aspect of the invention is preferably an IgG of any isotype (e.g., IgG1, IgG2, IgG3, IgG4). The respective antigen-binding fragment may be, for example, a Fab, Fab', F(ab')2, or scFv.

[0090] Thus, an antibody fragment of the invention may be or may comprise an antigen-binding region that operates in one or more ways as described herein. For example, antibody HC6-LC6 (SEQ ID NO: 15 for VH chain, SEQ ID NO: 16 for VL chain) is expressed as a humanized IgG1 (SEQ ID NO: 13 for heavy chain, SEQ ID NO: 14 for light chain), antibody HC6-LC7 (SEQ ID NO: 15 for VH chain, SEQ ID NO: 17 for VL chain) is expressed as a humanized IgG1 (SEQ ID NO: 13 for heavy chain, SEQ ID NO: 22 for light chain), HC6-LC8 (SEQ ID NO: 15 for VH chain, SEQ ID NO: 18 for VL chain) is expressed as a humanized IgG1 (SEQ ID NO: 13 for heavy chain, SEQ ID NO: 23 for light chain), and antibody HC6-LC9 (SEQ ID NO: 15 for VH chain, SEQ ID NO: 19 for VL chain) is expressed as a humanized IgG1 (SEQ ID NO: 13 for heavy chain, SEQ ID NO: 24 for light chain). and antibody HC7-LC6 (SEQ ID NO: 20 for VH chain, SEQ ID NO: 16 for VL chain) was expressed as a humanized IgG1 (SEQ ID NO: 21 for heavy chain, SEQ ID NO: 14 for light chain), antibody HC7-LC7 (SEQ ID NO: 20 for VH chain, SEQ ID NO: 17 for VL chain) was expressed as a humanized IgG1 (SEQ ID NO: 21 for heavy chain, SEQ ID NO: 22 for light chain), HC7-LC8 (SEQ ID NO: 20 for VH chain, SEQ ID NO: 18 for VL chain) was expressed as a humanized IgG1 (SEQ ID NO: 21 for heavy chain, SEQ ID NO: 23 for light chain), and antibody HC7-LC9 (SEQ ID NO: 20 for VH chain, SEQ ID NO: 19 for VL chain) was expressed as a humanized IgG1 (SEQ ID NO: 21 for heavy chain, SEQ ID NO: 24 for light chain).

[0091] The most preferred antibodies of the first aspect of the present invention are the antibodies of the second, third and seventh embodiments ("HC0-LC0", "HC6-LC6" and "HC7-LC6"), and among them, the most preferred antibodies are the antibodies of the third and seventh embodiments ("HC6-LC6" and "HC7-LC6").

[0092] All of the above preferred embodiments of the first aspect of the invention are D about 10 -10It has been shown that IGLV3-21 exhibits extremely high affinity in the M range, and is a beneficial aspect of the present invention, specifically, selectively, and without cross-reactivity with other tissues of CLL patients. R110 This is particularly advantageous as it has the advantage of binding to BCRs bearing the .ALPHA.-specific .gamma.-binding domain, thereby allowing for the selective killing of malignant B cells.

[0093] With regard to the most preferred antibodies ("HC0-LC0", "HC6-LC6" and "HC7-LC6"), these are about 1.2 to 2.1 10 -10 The antibodies "HC6-LC6" and "HC7-LC6" according to the third and seventh preferred embodiments are further humanized and are particularly suitable for therapeutic applications due to their reduced immunogenicity.

[0094] All of the above embodiments of the first aspect of the invention may optionally be R110 The IGLV3-21 R110 Beyond the definition of IGLV3-21 R110 is, in a generally preferred embodiment of this first aspect of the invention, further characterized by greater than 80% sequence identity to the sequence represented by SEQ ID NO:53, said sequence having a lysine at position 16 and an aspartic acid at positions 50 and 52. In a generally more preferred embodiment of this first aspect of the invention, a tyrosine or phenylalanine at position 49 and a serine or threonine at position 51. In an even more generally preferred embodiment of this first aspect of the invention, a tyrosine at position 49 and a serine at position 51.

[0095] Within this first aspect of the invention, there are further embodiments in which antibodies are provided that can be modified to deliver cytotoxic agents, immunotoxins, toxoplasma or radioisotopes to IGLV3-21R110-BCR expressing cells, potentially further enhancing efficacy.

[0096] In some embodiments of the first aspect of the invention, the antibody is isolated. An isolated biological component (e.g., a nucleic acid molecule or a protein, e.g., an antibody) is one that has been substantially separated or purified from other biological components, e.g., other chromosomal and extrachromosomal DNA and RNA, proteins and organelles, within the cells of the organism in which it naturally occurs. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods, e.g., as described in Sambrook et al., 1989 (see Sambrook, J. et al. (1989)) and Robert K. Scopes et al., 1994 (Protein Purification, - Principles and Practice, Springer Science and Business Media LLC). The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0097] Second Aspect of the Invention - Therapeutic Uses A second aspect of the invention relates to an antibody according to the first aspect of the invention, R110 Similarly, a second aspect of the invention relates to a method for the treatment of CLL in IGLV3-21 positive patients by administering a therapeutically effective amount of an antibody of the first aspect of the invention. R110 It relates to methods of treating CLL in positive patients.

[0098] Thus, in a first preferred embodiment of the second aspect of the invention, the antibody is characterized by a heavy chain corresponding to SEQ ID NO:1 and a light chain corresponding to SEQ ID NO:2.

[0099] In a second preferred embodiment of the second aspect of the invention the antibody is characterized by a heavy chain corresponding to SEQ ID NO:11 and a light chain corresponding to SEQ ID NO:12.

[0100] In a third preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:16.

[0101] Within the scope of the third preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO:13 and a light chain corresponding to SEQ ID NO:14 is more preferred.

[0102] In a fourth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:17.

[0103] Within the scope of the fourth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 22 is more preferred.

[0104] In a fifth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:18.

[0105] Within the scope of the fifth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 23 is more preferred.

[0106] In a sixth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:15 and a variable light chain region corresponding to SEQ ID NO:19.

[0107] Within the sixth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 24 is more preferred.

[0108] In a seventh preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:16.

[0109] Within the seventh preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:14 is more preferred.

[0110] In an eighth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:17.

[0111] Within the eighth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:22 is more preferred.

[0112] In a ninth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:18.

[0113] Within the ninth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:23 is more preferred.

[0114] In a tenth preferred embodiment of the second aspect of the invention the antibody is characterized by a variable heavy chain region corresponding to SEQ ID NO:20 and a variable light chain region corresponding to SEQ ID NO:19.

[0115] Within the tenth preferred embodiment, an antibody characterized by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:24 is more preferred.

[0116] As mentioned in the description of the first aspect of the invention, all of the above embodiments of the second aspect of the invention rely on antibodies with very high affinity. All of them have a capture speed of about 0.3 nm and a resolution of about 3×10 -10 As such, the second aspect of the invention is directed to IGLV3-21 R110 It allows for highly selective treatment of CLL in positive patients.

[0117] Furthermore, these antibodies (see FIG. 1) inhibited wild-type IGLV3-21 G110 Variants and malignant IGLV3-21R110 The antibodies were able to selectively identify variants of human IGLV3-21 and treatment with these antibodies in a xenograft mouse model (see FIG. 5 and Example 9) demonstrated that the antibodies selectively differentiated human IGLV3-21 variants from human IGLV3-21 variants. R110 It has been demonstrated to result in a significant reduction in positive B cells, leading to amelioration of CLL.

[0118] Thus, surprisingly, IGLV3-21 R110 This antibody has been found to be effective for use in treating CLL in positive patients.

[0119] Furthermore, the antibodies used for such treatment have been shown to exhibit no cross-reactivity to other tissues (see FIG. 4, Example 8).

[0120] Determination of effective dosages of such antibodies is within the capabilities of one of ordinary skill in the art. For any compound, the therapeutically effective dose can be estimated initially in cell culture assays, e.g., tumor cells, or in animal models, usually mice, rabbits, dogs, pigs or monkeys. Animal models are also used to achieve a desired concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

[0121] A therapeutically effective amount refers to an amount of antibody that ameliorates the symptoms or condition to be treated. In the context of the present invention, the condition to be treated is clinically manifested CLL caused by abnormal proliferation of B cells with autonomously active BCR. More specifically, the second aspect of the present invention relates to a method for the treatment of IGLV3-21 R110 The present invention addresses clinically overt CLL, which is characterized by the abnormal proliferation of B cells with an autonomously active BCR that harbors the BCR.

[0122] All of the above embodiments of the second aspect of the invention are R110 The present invention relates to the treatment of CLL in patients who are positive for IGLV3-21. R110 Beyond the definition of IGLV3-21 R110is, in a generally preferred embodiment of this second aspect of the invention, further characterized by greater than 80% sequence identity to the sequence represented by SEQ ID NO:53, said sequence having a lysine at position 16 and an aspartic acid at positions 50 and 52. In a generally more preferred embodiment of this first aspect of the invention, a tyrosine or phenylalanine at position 49 and a serine or threonine at position 51. In an even more generally preferred embodiment of this first aspect of the invention, a tyrosine at position 49 and a serine at position 51.

[0123] Thus, a therapeutically effective amount is an amount of IGLV3-21 that is in the therapeutic range for a patient, either in a single dose or in a multiple dose regimen. R110 This is an amount of antibody that is sufficient to deplete positive CLL cells, and which is a toxicologically acceptable amount.

[0124] In a second aspect of the present invention, IGLV3-21 R110 The antibody for use in treating CLL in positive patients is 0.25 to 25 mg / kg 体重 , more preferably 1 to 20 mg / kg 体重 , and most preferably more than 7 to 15 mg / kg 体重 , particularly preferably 8 to 12 mg / kg 体重 It is preferred to use a dosage of

[0125] These dosages are very low, with a total drug amount hypothetically administered to an average patient weighing approximately 80 kg to achieve a robust reduction in malignant B cells of no more than 640-960 mg being particularly preferred.

[0126] The exact dosage is selected by the individual physician in consideration of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active ingredient or to achieve the desired effect. However, according to the present invention, xenograft mouse models have been shown to be effective already at dosages of 0.3 mg / kg or less twice a week, while a strong reduction in malignant cells was achieved at dosages of 10 mg / kg (twice a week).

[0127] When administering an antibody according to the second aspect of the invention, additional factors may also be taken into consideration, which may include the severity of the condition, e.g., tumor size and location; age, weight and sex of the patient, and diet. Further influencing factors for determining the appropriate dosage may be drug combinations, reaction sensitivities, and tolerance / response to treatment.

[0128] Additionally, multiple doses may be administered, and the time and frequency of administration may further affect the individual dose administered.

[0129] According to the second aspect of the invention, the antibody may be co-administered with known agents. Thus, it may be administered as a single agent or in combination with one or more additional therapeutic agents, provided that the combination does not result in unacceptable side effects. This combination therapy includes administration of a single pharmaceutical dosage formulation comprising an antibody according to the first aspect of the invention and one or more additional therapeutic agents, as well as administration of the antibody of the first aspect of the invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation.

[0130] When separate dosing regimens are used, treatment according to the second aspect of the invention with one or more additional therapeutic agents may be essentially at the same time (e.g., simultaneously) or at different staggered times (e.g., sequentially). In particular, treatment according to the invention may be performed in fixed or separate combination with other antitumor agents, such as alkylating agents, antimetabolites, plant-derived antitumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and / or biological response modifiers, antiangiogenic compounds, and other antitumor agents.

[0131] The antibodies of the invention may also be used in the treatment of cancer in combination with radiation therapy and / or surgical intervention.

[0132] Third aspect - polynucleotides The present invention also relates to a DNA molecule encoding an antibody according to the first aspect of the invention.

[0133] The DNA molecules of the present invention are not limited to the sequences disclosed herein, but also include variants thereof. The DNA variants within the scope of the present invention can be described with reference to physical properties in hybridization. Those skilled in the art will recognize that nucleic acid hybridization techniques can be used to distinguish DNA from its complement, and its equivalent or homologue, since DNA is double-stranded. It will also be recognized that hybridization can occur with less than 100% complementarity. However, hybridization techniques can be used to distinguish DNA sequences based on their structural relatedness to a particular probe, if conditions are appropriately selected. For guidance regarding such conditions, see Sambrook, J., Fritsch, EF, and Maniatis, T. (1989) (Molecular Cloning: A laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, USA) and Ausubel et al., 1995 (Ausubel, FM, Brent, R., Kingston, RE, Moore, DD, Sedman, JG, Smith, JA, & S1ruhl, K. eds. (1995). Current Protocols in Molecular Biology. New York: John Wiley and Sons).

[0134] The structural similarity between two polynucleotide sequences can be expressed as a function of the "stringency" of the conditions under which the two sequences hybridize to each other. As used herein, the term "stringency" refers to the degree to which conditions disfavor hybridization. Stringent conditions are highly disfavoring of hybridization, and only the most structurally related molecules will hybridize to each other under such conditions. Conversely, less stringent conditions favor the hybridization of molecules that exhibit a lower degree of structural relatedness. Thus, the stringency of hybridization directly correlates with the structural relatedness of two nucleic acid sequences. The following relationships are useful in correlating hybridization and relatedness (T m is the melting temperature of the nucleic acid duplex): a.T m =69.3+0.41(G+C)% b. T in double-stranded DNA m However, it decreases by 1°C for every 1% increase in the number of mismatched base pairs. c. (T m )μ2-(T m )μ1=18.5log 10 μ2 / μ1 (where μ1 and μ2 are the ionic strengths of the two solutions).

[0135] The stringency of hybridization is a function of many factors, including total DNA concentration, ionic strength, temperature, probe size, and the presence of agents that disrupt hydrogen bonds. Factors that promote hybridization include high DNA concentration, high ionic strength, low temperature, long probe size, and the absence of agents that disrupt hydrogen bonds. Hybridization is typically performed in two steps: a "binding" step and a "washing" step.

[0136] Yet another class of DNA variants within the scope of the present invention can be described with reference to the products they encode: These functionally equivalent polynucleotides are characterized in that, due to the degeneracy of the genetic code, they encode identical peptide sequences as found in SEQ ID NOs: 1-24.

[0137] It is recognized that variants of the DNA molecules provided herein can be constructed in several different ways. For example, they may be constructed as fully synthetic DNA. Methods for efficiently synthesizing oligonucleotides ranging from 20 to about 150 nucleotides are widely available. See Ausubel et al., section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may be synthesized and constructed as originally reported by Khorana et al. (J. Mol. Biol. 72:209-217 (1971)); see also Ausubel et al., supra, section 8.2. Synthetic DNA is preferably designed with appropriate restriction sites at the 5' and 3' ends of the gene to facilitate cloning into an appropriate vector.

[0138] As mentioned above, a method to generate variants is to start with one of the DNAs disclosed herein and perform site-directed mutagenesis. See Ausubel et al., supra, Chapter 8, Supplement 37 (1997). In a typical method, the target DNA is cloned into a single-stranded DNA bacteriophage vector. The single-stranded DNA is isolated and hybridized with an oligonucleotide containing the desired nucleotide change. The complementary strand is synthesized, and the double-stranded phage is introduced into a host. Some of the resulting progeny contain the desired mutation, which can be confirmed using DNA sequencing. In addition, various methods are available to increase the likelihood that the progeny phage is the desired mutation. These methods are well known to those skilled in the art, and kits for generating such mutations are commercially available.

[0139] As with the first aspect of the invention, there are corresponding preferred embodiments for the third aspect of the invention.

[0140] In a first preferred embodiment of the third aspect of the invention, the invention relates to a DNA molecule encoding the heavy chain of one of the preferred antibodies of the first aspect of the invention, represented by any sequence selected from the list consisting of SEQ ID NO:25, SEQ ID NO:29, SEQ ID NO:33 and SEQ ID NO:38.

[0141] In a second preferred embodiment of the third aspect of the invention, the invention relates to a DNA molecule encoding a light chain of one of the preferred antibodies of the first aspect of the invention, represented by any sequence selected from the list consisting of SEQ ID NO:26, SEQ ID NO:30, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36 and SEQ ID NO:37.

[0142] The nucleic acids of the invention are suitable for the recombinant production of antibodies using standard vectors and host cells containing the nucleic acid sequences of the invention.

[0143] Fourth aspect - diagnostic method The antibody of the present invention is IGLV3-21 R110 It can be used to detect the presence of CLL cells expressing IGLV3-21 in a variety of biological samples, including serum and tissue biopsy specimens. R110 Containing CLL cells or shed IGLV3-21 R110 The presence of expressed CLL BCR can be detected with the antibodies of the invention. Furthermore, the antibodies can be used in a variety of imaging techniques, e.g. 99 It can be used in immunoscintigraphy with Tc (or other isotope)-conjugated antibodies. For example, 111 Imaging protocols similar to those described using In-conjugated anti-PSMA antibodies may be used to detect pancreatic or ovarian cancer (Sodee et al., Clin. Nuc. Med. 21: 759-766, 1997). Another detection method that can be used is positron emission tomography by coupling the antibodies of the invention with a suitable isotope (see Herzog et al., J. Nucl. Med. 34:2222-2226, 1993).

[0144] Thus, according to a fourth aspect, the present invention provides an IGLV3-21 R110 The fourth aspect of the invention also relates to an antibody as described above for use in the diagnosis of CLL characterized by B cells with autonomously active BCRs carrying IGLV3-21, in particular for the diagnosis of "Subset #2 CLL". Similarly, the fourth aspect of the invention relates to a method for the detection of IGLV3-21 by applying said antibody to a patient's tissue or blood sample.R110 In a method for the diagnosis of CLL characterized by B cells with an autonomously active BCR carrying the BCR-1 gene, and thus of subset 2 type CLL, these antibodies are conjugated to a detectable marker or a further antibody which specifically binds to the antibody of the invention and is conjugated to a detectable marker is subsequently applied.

[0145] In a first preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "mAb01-01", characterized by a heavy chain corresponding to SEQ ID NO:1 and a light chain corresponding to SEQ ID NO:2.

[0146] In a second preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC0-LC0" characterized by a heavy chain corresponding to SEQ ID NO:11 and a light chain corresponding to SEQ ID NO:12.

[0147] In a third preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC6-LC6", characterized by a variable heavy chain region corresponding to SEQ ID NO: 15 and a variable light chain region corresponding to SEQ ID NO: 16.

[0148] Within the scope of the third preferred embodiment of the fourth aspect of the invention, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 14 is preferred.

[0149] In a fourth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC6-LC7", characterized by a variable heavy chain region corresponding to SEQ ID NO: 15 and a variable light chain region corresponding to SEQ ID NO: 17.

[0150] Within the fourth preferred embodiment of the fourth aspect of the invention, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 22 is preferred.

[0151] In a fifth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC6-LC8", characterized by a variable heavy chain region corresponding to SEQ ID NO: 15 and a variable light chain region corresponding to SEQ ID NO: 18.

[0152] Within the scope of the fifth preferred embodiment of the fourth aspect of the invention, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 23 is preferred.

[0153] In a sixth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC6-LC9", characterized by a variable heavy chain region corresponding to SEQ ID NO: 15 and a variable light chain region corresponding to SEQ ID NO: 19.

[0154] Within the sixth preferred embodiment of the fourth aspect of the invention, an antibody characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO: 24 is preferred.

[0155] In a seventh preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC7-LC6", characterized by a variable heavy chain region corresponding to SEQ ID NO: 20 and a variable light chain region corresponding to SEQ ID NO: 16.

[0156] Within the seventh preferred embodiment of the fourth aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:14 is preferred.

[0157] In an eighth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC7-LC7", characterized by a variable heavy chain region corresponding to SEQ ID NO: 20 and a variable light chain region corresponding to SEQ ID NO: 17.

[0158] Within the scope of the eighth preferred embodiment of the fourth aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:22 is preferred.

[0159] In a ninth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC7-LC8", characterized by a variable heavy chain region corresponding to SEQ ID NO: 20 and a variable light chain region corresponding to SEQ ID NO: 18.

[0160] Within the ninth preferred embodiment of the fourth aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:23 is preferred.

[0161] In a tenth preferred embodiment of the fourth aspect of the invention, the antibody used for diagnosis is "HC7-LC9", characterized by a variable heavy chain region corresponding to SEQ ID NO: 20 and a variable light chain region corresponding to SEQ ID NO: 19.

[0162] Within the tenth preferred embodiment of the fourth aspect of the invention, an antibody characterised by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:24 is preferred.

[0163] Other aspects of the invention The antibody of the first aspect of the invention for use in the second aspect of the invention may be co-administered with known agents, for example the antibody may be co-administered with any common anti-B cell antibody.

[0164] This combination therapy includes administration of a single pharmaceutical dosage formulation comprising an antibody of the invention and one or more additional therapeutic agents, as well as administration of an antibody of the invention and each additional therapeutic agent in its own separate pharmaceutical dosage formulation, For example, an antibody of the invention and a therapeutic agent may be administered to a patient together in a single injection, or each agent may be administered in a separate dosage formulation.

[0165] When separate dosage formulations are used, the antibody of the present invention and one or more additional therapeutic agents may be administered at essentially the same time (e.g., simultaneously) or at separate staggered times (e.g., sequentially). In particular, the antibody of the present invention may be used in fixed or separate combinations with other anti-tumor agents, such as alkylating agents, antimetabolites, plant-derived anti-tumor agents, hormonal therapy agents, topoisomerase inhibitors, camptothecin derivatives, kinase inhibitors, targeted drugs, antibodies, interferons and / or biological response modifiers, anti-angiogenic compounds, and other anti-tumor agents. In this regard, the following is a non-limiting list of examples of second agents that may be used in combination with the antibody of the present invention: Alkylating agents include, but are not limited to, nitrogen mustard N-oxide, cyclophosphamide, ifosfamide, thiotepa, ranimustine, nimustine, temozolomide, altretamine, apaziquone, brostallicin, bendamustine, carmustine, estramustine, fotemustine, glufosfamide, mafosfamide, bendamustine, and mitolactol; platinum-coordinating alkylating compounds include, but are not limited to, cisplatin, carboplatin, eptaplatin, lobaplatin, nedaplatin, oxaliplatin, and satraplatin; Metabolic inhibitors include, but are not limited to, methotrexate, 6-mercaptopurine riboside, mercaptopurine, 5-fluorouracil alone or in combination with leucovorin, tegafur, doxifluridine, carmofur, cytarabine, cytarabine ocfosfate, enocitabine, gemcitabine, fludarabine, 5-azacytidine, capecitabine, cladribine, clofarabine, decitabine, eflornithine, ethinylcytidine, cytosine arabinoside, hydroxyurea, melphalan, nelarabine, nolatrexed, ocfosfate, premetrexed disodium, pentostatin, perirexol, raltitrexed, triapine, trimetrexate, vidarabine, vincristine, and vinorelbine; Hormonal therapy agents include, but are not limited to, Exemestane, Lupron, Anastrozole, Doxercalciferol, Fadrozole, Formestane, 11-beta hydroxysteroid dehydrogenase I inhibitors, 17-alpha hydroxylase / 17,20 lyase inhibitors such as abiraterone acetate, 5-alpha reductase inhibitors such as finasteride and epristeride, antiestrogens such as tamoxifen citrate and fulvestrant, Trelstar, toremifene, raloxifene, lasofoxifene, relozole, antiandrogens such as bicalutamide, flutamide, mifepristone, nilutamide, Casodex, and antiprogesterones, and combinations thereof; Plant-derived antitumor agents include, for example, mitotic inhibitors, such as those selected from epothilones, e.g., sagopilone, ixabepilone and epothilone B, vinblastine, vinflunine, docetaxel, and paclitaxel; Cytotoxic topoisomerase inhibitors include, but are not limited to, aclarubicin, doxorubicin, amonafide, belotecan, camptothecin, 10-hydroxycamptothecin, 9-aminocamptothecin, diflomotecan, irinotecan, topotecan, edotecarin, epimbicin, etoposide, exatecan, gimatecan, lurtotecan, mitoxantrone, pirambicin, pixantrone, rubitecan, sobuzoxane, tafluposide, and combinations thereof; Immunologic agents include interferons, such as interferon alpha, interferon alpha-2a, interferon alpha-2b, interferon beta, interferon gamma-1a, and interferon gamma-n1, other immune enhancers, such as L19-IL2 and other IL2 derivatives, filgrastim, lentinan, sizofiran, TheraCys, ubenimex, aldesleukin, alemtuzumab, BAM-002, dacaine, and the like. including levadin, daclizumab, denileukin, gemtuzumab, ozogamicin, ibri1umomab, imiquimod, lenograstim, lentinan, melanoma vaccine (Corixa), molgramostim, sargramostim, tasonermin, teculekin, timalasin, tositumomab, Vimlizin, epratuzumab, mitumomab, oregovomab, pemtumomab, and Provenge; Biological response modifiers are agents that modify the body's defense mechanisms or biological responses, such as the survival, growth or differentiation of tissue cells, to have antitumor activity, such agents include, for example, krestin, lentinan, sizofiran, picibanil, ProMune, and ubenimex; Antiangiogenic compounds include, but are not limited to, acitretin, aflibercept, angiostatin, aplidine, acenta, axitinib, bevacizumab, brivanib alaninat, cilengutide, combretastatin, endostatin, fenretinide, halofuginone, pazopanib, ranibizumab, revimastat, rekentin, regorafenib, removab, revlimid, sorafenib, squalamine, sunitinib, telatinib, thalidomide, ukrain, vatalanib, and vitaxin; Antibodies include, but are not limited to, trastuzumab, cetuximab, bevacizumab, rituximab, ticilimumab, ipilimumab, lumiliximab, catumaxomab, atacicept, oregovomab, and alemtuzumab; VEGF inhibitors, such as sorafenib, regorafenib, bevacizumab, sunitinib, rekentin, axitinib, aflibercept, telatinib, brivanib alaninate, vatalanib, pazopanib, and ranibizumab; EGFR (HER1) inhibitors, such as cetuximab, panitumumab, vectibix, gefitinib, erlotinib, and Zactima; HER2 inhibitors, such as lapatinib, ratuzumab, and pertuzumab; mTOR inhibitors, such as temsirolimus, sirolimus / rapamycin, and everolimus; c-Met inhibitors; PI3K and AKT inhibitors; CDK inhibitors such as roscovitine and flavopiridol; Spindle assembly checkpoint inhibitors and targeted mitosis inhibitors, such as PLK inhibitors, Aurora inhibitors (e.g., hesperadin), checkpoint kinase inhibitors, and KSP inhibitors; HDAC inhibitors, such as panobinostat, vorinostat, MS275, belinostat, and LBH589; HSP90 and HSP70 inhibitors; Proteasome inhibitors, such as; bortezomib and carfilzomib; serine / threonine kinase inhibitors, including MEK inhibitors and raffinokinase inhibitors, such as sorafenib; Farnesyltransferase inhibitors, such as tipifarnib; Tyrosine kinase inhibitors, including, for example, dasatinib, nilotibib, regorafenib, bosutinib, sorafenib, bevacizumab, sunitinib, cediranib, axitinib, aflibercept, telatinib, imatinib mesylate, brivanib alaninate, pazopanib, ranibizumab, vatalanib, cetuximab, panitumumab, vectibix, gefitinib, erlotinib, lapatinib, trastuzumab, pertuzumab, and c-Kit inhibitors; Vitamin D receptor agonists; Bcl-2 protein inhibitors, such as obatoclax, oblimersen sodium, and gossypol; cluster of differentiation 20 antagonists, such as rituximab; ribonucleotide reductase inhibitors, such as gemcitabine; 5-hydroxytryptamine receptor antagonists, such as rEV598, zaliprode, palonosetron hydrochloride, granisetron, Zindol, and AB-1001; Integrin inhibitors, such as α5β integrin inhibitors, such as E7820, JSM6425, volociximab, and endostatin; Androgen receptor antagonists, including, for example, nandrolone decanoate, fluoxymesterone, Android, Prostaid, andromustine, bicalutamide, flutamide, apocyproterone, apoflutamide, chlormadinone acetate, Abdricyr, Tabi, cyproterone acetate, and nilutamide; Aromatase inhibitors such as Anastrozole, Letrozole, Testolactone, Exemestane, Aminoglutethimide, and Formestane; Matrix metalloproteinase inhibitors; Other anti-cancer drugs including, for example, alitretinoin, ampligen, atrasentan bexarotene, bortezomib, bosentan, calcitriol, exisulind, fotemustine, ibandronate, miltefosine, mitoxantrone, I-asparaginase, procarbazine, dacarbazine, hydroxycarbamide, pegaspargase, pentostatin, tazarotene, velcade, gallium nitrate, canfosfamide, darinaparsin, and tretinoin.

[0166] In a preferred embodiment, the antibodies of the invention may be used in combination with chemotherapy (i.e., cytotoxic agents), anti-hormones, and / or targeted therapies such as kinase inhibitors, mTOR inhibitors, and angiogenesis inhibitors.

[0167] The antibody according to the first aspect of the invention may itself optionally be modified and thus may, for example, be an ADC (as defined above).

[0168] The antibodies of the invention may also be used in the treatment of cancer in combination with radiation therapy and / or surgical intervention.

[0169] The present invention also relates to a composition comprising an antibody according to any embodiment of the first aspect of the invention, which may be used in the same manner as the second aspect of the invention.

[0170] The present invention therefore includes a pharmaceutical composition comprising an antibody according to the first aspect of the invention, alone or in combination with at least one other pharmaceutical agent and a pharma- ceutically acceptable carrier or excipient.

[0171] The other agent is, for example, a stabilizing compound, and such at least one other agent and the antibody according to the first aspect of the invention may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.

[0172] The pharmaceutical compositions of the present invention may be manufactured in a manner known in the art, for example, by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0173] A preferred pharmaceutical composition is prepared by mixing a lyophilized powder of the antibody according to the first aspect of the invention in 1 mM-50 mM histidine, 0.1%-2% sucrose, 2%-7% mannitol, with a buffer in the pH range 4.5-5.5 prior to use.

[0174] After pharmaceutical compositions containing an antibody of the invention formulated in an acceptable carrier have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition.

[0175] Thus, in a related aspect, the present invention provides IGLV3-21 R110 The present invention also relates to such pharmaceutical compositions for use in CLL in patients with IGLV3-21 positive disease. R110 The present invention relates to a method for treating CLL in a positive patient.

[0176] Such administration is usually performed parenterally. Methods of parenteral administration include topical, intraarterial (directly into the tumor), intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

[0177] The preferred routes of administration are intravenous and intra-arterial (directly to the tumor).

[0178] Pharmaceutical compositions for parenteral administration include aqueous solutions of active compounds. For injection, the pharmaceutical compositions of the present invention may be formulated in aqueous solutions, preferably physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, allowing for the preparation of highly concentrated solutions.

[0179] For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation, and such penetrants are generally known in the art.

[0180] Determining a therapeutically effective amount of an antibody or a pharmaceutical composition comprising such an antibody depends primarily on the specific patient characteristics, the route of administration, and the nature of the disease being treated. General guidance is provided, for example, in publications from the International Conference on Harmonization, and in REMINGTON'S PHARMACEUTICAL SCIENCES, Chapters 27 and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). More specifically, determining a therapeutically effective amount depends on factors such as the toxicity and efficacy of the drug. Toxicity may be determined using methods well known in the art and found in the references cited above. Efficacy is determined using similar guidance.

[0181] The invention further relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention, bearing a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval by the governmental agency of the manufacture, use, or sale of the product for human administration.

[0182] In another embodiment, the kit may comprise a DNA sequence encoding an antibody according to the third aspect of the invention. Preferably, the DNA sequences encoding these antibodies are provided in a plasmid suitable for transfection and expression into a host cell. The plasmid may comprise a promoter (often an inducible promoter) to control expression of the DNA in the host cell. The plasmid may also comprise suitable restriction sites to facilitate the insertion of other DNA sequences into the plasmid to generate different antibodies. The plasmid may also comprise a number of other elements to facilitate cloning and expression of the encoded protein. Such elements are well known to those skilled in the art and include, for example, selectable markers, start codons, stop codons, etc.

[0183] Thus, the present invention further provides IGLV3-21R110 The present invention relates to packs and kits as described above comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention for use in the treatment of CLL in positive patients.

[0184] A preferred embodiment of the present invention is as follows: A. An antibody having a heavy chain amino acid sequence as set forth in SEQ ID NO:1 and a light chain amino acid sequence as set forth in SEQ ID NO:2, or a heavy chain amino acid sequence as set forth in SEQ ID NO:11 and a light chain amino acid sequence as set forth in SEQ ID NO:12, or an antibody comprising a variable heavy chain having a sequence selected from the list consisting of SEQ ID NO:15 and SEQ ID NO:20 and a variable light chain having a sequence selected from the list consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, in any combination. B. The antibody of embodiment A, characterized by a heavy chain corresponding to SEQ ID NO:1 and a light chain corresponding to SEQ ID NO:2. C. The antibody of embodiment A, characterized by a heavy chain amino acid sequence of SEQ ID NO:11 and a light chain amino acid sequence of SEQ ID NO:12. D. The antibody of embodiment A, characterized by a heavy chain corresponding to SEQ ID NO:13 and a light chain corresponding to SEQ ID NO:14. E. The antibody of embodiment A, characterized by a heavy chain corresponding to SEQ ID NO:21 and a light chain corresponding to SEQ ID NO:14. F. The antibody of embodiment A or C which is a chimeric antibody. G. The antibody of embodiment D or E which is a humanized antibody. H. An immunoconjugate comprising an antibody according to any one of the preceding embodiments. I. IGLV3-21 R110 The antibody of any one of the preceding embodiments for use in the treatment of CLL in a positive patient. J. 0.25-25mg / kg 体重 The antibody for use according to embodiment I, wherein the antibody is administered at a dosage of K. 1-20mg / kg 体重 The antibody for use according to embodiment I, wherein the antibody is administered at a dosage of L. 7-15mg / kg 体重The antibody for use according to embodiment I, wherein the antibody is administered at a dosage of M. 8-12mg / kg 体重 The antibody for use according to embodiment I, wherein the antibody is administered at a dosage of N. The antibody for use according to any one of embodiments I to M, wherein the CLL is subset #2 CLL. O. A pharmaceutical composition comprising an antibody according to any one of embodiments A to H. P. A kit comprising the pharmaceutical composition of embodiment O. Q. IGLV3-21 R110 A kit or pharmaceutical composition according to any one of embodiments O to P for use in the treatment of CLL in positive patients. R.IGLV3-21 R110 The antibody of any one of embodiments A to G for use in diagnosing CLL, characterized by a BCR carrying: S. The antibody for use according to embodiment R, wherein the antibody is conjugated to a detectable marker or a further antibody which specifically binds to the antibody and is conjugated to a detectable marker is subsequently applied. [Brief description of the drawings]

[0185] [Figure 1] 1A-1D show FACS FSC-SSC plots (FIGS. 1A, 1C) and gating plots of mAb01-01-APC (FIGS. 1B, 1D, x-axis) against anti-IgM-PE (FIGS. 1B, 1D, y-axis). [Diagram 2] 2A-2F show FACS plots of human PBMCs treated according to Example 6. [Diagram 3] FIG. 3 shows the comparative binding kinetics of antibodies HC0-LC0, HC6-LC6 and HC7-LC6 according to the invention and Example 7. [Figure 4] FIG. 4 shows a comparison of tissue stained with mAb 01-01 and anti-IgG against such mAb 01-01 (sandwich assay) (left column) with the same tissue samples treated with anti-IgG alone according to Example 8 (right column). [Diagram 5] FIG. 5 shows the absolute number of CLL cells in the spleen at the end of the experiment according to Example 9. [Figure 6] FIG. 6 shows a schematic diagram of the BCR-BCR homotypic interactions of the IGLV3-21R110 light chain (described by Minici et al.). [Figure 7] FIG. 7 shows a schematic diagram of IGLV3-21R110. [Figure 8] Array table.

[0186] Description of the drawings 1A-1D show FACS FSC-SSC plots (FIGS. 1A, 1C) and gating plots of mAb01-01-APC (FIGS. 1B, 1D, x-axis) against anti-IgM-PE (FIGS. 1B, 1D, y-axis).

[0187] According to Example 4, the plots in FIG. 1A and FIG. 1B are IGHV3-21 / IGLV3-21 stained with the above-mentioned fluorescently labeled antibody. R110 The plots in Figures 1C and 1D were generated from a 1:1 cell mixture containing BCR-positive TKO mouse cells and BCR-deficient TKO cells (cell mixture A), while the plots in Figures 1C and 1D were generated from a 1:1 cell mixture containing BCR-positive TKO mouse cells and BCR-deficient TKO cells (cell mixture B ... G110 It was generated from a 1:1 cell mixture containing BCR-positive TKO mouse cells and TKO cells lacking BCR (cell mixture B).

[0188] As can be easily seen from Figure 1B and Figure 1D, almost half of the analyzed TKO cells were positively stained with each anti-IgM antibody, indicating that they had BCR, which is consistent with the expression of BCR on the surface of about 50% of the TKO cells in each cell mixture. Comparing Figure 1B and Figure 1D, it is clear that the antibody "mAb01-01" of the present invention recognizes only the malignant variant of IGLV3-21 carrying the R110 mutation.

[0189] Figures 2A-2F show FACS plots of human PBMCs treated according to Example 6. Figures 2A and 2D show FSC-SSC views of the PBMCs, gated on apparently live cells, similar to Figures 1A and 1C. Gated cells are plotted with anti-CD19-VioBright515 in preference to anti-CD5-PE-Cy5 (Figures 2B and 2E), and with anti-CD19-VioBright515 in preference to mAb01-01-APC (Figures 2C and 2F). Figures 2A-2C show IGHV4-39 / IGLV3-21 R110 Figures 2D to 2F show an analysis of human PBMCs from patients diagnosed with B cells expressing IGHV3-21 -BCR. R110 - CLL not characterized by BCR (i.e. non-IGLV3-21 R110 The same analysis of human PBMCs from a patient with CLL is shown. As can be seen by comparing Figures 2B and 2D, the CD5 / CD19 ++ B cells could be isolated, and a comparison of the two patient samples (Figures 2C and 2F) demonstrated that mAb 01-01 inhibited IGLV3-21 R110 We demonstrate that it is possible to selectively identify only positive CLL B cells and differentiate between types of CLL.

[0190] Figure 3 shows the comparative binding kinetics of antibodies HC0-LC0, HC6-LC6 and HC7-LC6 according to the invention and Example 7. As can be seen, the three antibodies show essentially identical binding kinetics and have nearly identical binding specificities at a concentration of 10 μg / ml.

[0191] FIG. 4 shows a comparison of tissue stained with mAb 01-01 and anti-IgG against such mAb 01-01 (sandwich assay) (left column) with the same tissue samples treated with anti-IgG alone according to Example 8 (right column). R110 The respective results are shown for tissue samples from positive CLL patients, and below for healthy donor samples.

[0192] Comparing the top left photo with all the remaining photos in the left column, it can be seen that mAb01-01 reliably and selectively identifies diseased B cells in the spleen of diseased patients, without cross-reactivity with healthy donor samples, either in the same tissue (spleen) or from other tissue types (skin, kidney, heart, brain). This shows that the antibody of the present invention is selective, safe and non-cross-reactive. Further photos in the right column show that the staining achieved in the top left photo and the lack of staining in the bottom photo of the left column is not related to background or other artifactual effects.

[0193] FIG. 5 shows the absolute number of CLL cells in the spleen at the end of the experiment according to Example 9. In this experiment, artificial IGLV3-21 R110 Xenograft mice models affected with positive CLL were treated with mAb 01-01 at doses of (pharmacologically inactive) control (group A), 0.3 mg / kg body weight (group B), 5 mg / kg body weight (group C) and 10 mg / kg body weight (group D). As can be seen from the figure, all treatments lead to a depletion of CLL cells in the spleen, while at 10 mg / kg the depletion is very significant. This indicates that the antibody of the invention is able to inhibit the proliferation and proliferation of IGLV3-21 R110 We show that this indeed facilitates effective treatment of CLL characterized by a positive BCR.

[0194] Figure 6 shows the IGLV3-21 R110 A schematic of BCR-BCR homotypic interactions of light chains is shown (as described by Minici et al.). Two adjacent BCRs are shown with antigen-binding subunits including heavy (HC) and light (LC) chains, transmembrane domains (TM), and signaling subunits (SU) composed of disulfide-linked heterodimers of Igα and Igβ proteins (CD79a / CD79b). A mutated arginine at position 110 (R) of one BCR interacts with a germline-encoded aspartic acid (D) at position 50 of the adjacent BCR. Further interactions between the two BCRs are mediated by germline-encoded amino acid residues lysine (K) at position 16 and aspartic acid (D) at position 52.

[0195] Figure 7 shows the IGLV3-21 R110 FIG. 7A shows a schematic diagram of IGLV3-21 as an example in one-letter code. R110 (SEQ ID NO: 53). The amino acid residues involved in the BCR-BCR homotypic interaction according to Minici et al. are shown in bold. Figure 7B: Line 1: IGLV3-21 R110 Line 2: Amino acid positions of BCR-BCR homotypic interactions according to Minici et al. Lines 3-5: Different variants of the YDSD motif compared. Amino acids are shown in three-letter code.

[0196] The present invention is further illustrated by the following examples, which are provided solely to illustrate the invention with reference to specific embodiments. These exemplifications illustrate certain aspects of the invention, but do not represent limitations or limit the scope of the disclosed invention.

[0197] All examples were carried out using routine standard techniques well known to those skilled in the art unless otherwise detailed. Routine molecular biology techniques in the following examples can be carried out as described in standard laboratory manuals such as Sambrook et al. (1989).

[0198] A preferred embodiment of the present invention is as follows: Working Example Example 1 Generation of mouse antibodies Immunization and generation of hybridoma cell lines IGLV3-21 R110 Murine antibodies against the BCR bearing the BCR were developed by combining immunization of mice with a soluble form of the BCR and selection of appropriate antibodies using a cell system in which intact and functional BCR is displayed membrane-bound.

[0199] First, it was necessary to obtain a soluble BCR in the form of IgG1 for immunization of mice. Thus, a DNA segment encoding IGHV3-21 as an exemplary variable heavy chain (VH) and IGLV3-21 were used. R110Complete light chain (LC) DNA covering the VH, ...

[0200] Mice were then immunized with a recombinantly produced soluble form of BCR (see SEQ ID NOs: 49 and 50).

[0201] Then, immune cells with desired specificity were obtained from these mice and transformed into hybridoma cells by cell fusion. Then, FACS screening was performed using triple knockout cells (gene TKO, Lambda5, RAG2 and SLP65 knockout) expressing various variants of BCR, and IGLV3-21 was identified. R110 Antibodies that specifically target the BCR bearing

[0202] Monoclonal antibodies were produced using standard procedures in mice followed by generation of hybridoma cells.

[0203] This approach allowed the isolation of the unique monoclonal antibody "mAb01-01" (SEQ ID NO: 1 for the heavy chain, SEQ ID NO: 2 for the light chain).

[0204] Selection of monoclonal antibodies Screening of positive clones was not performed by enzyme-linked immunosorbent assay (ELISA) as usual. As the target structure is a membrane-bound receptor, it is very important to validate the binding of potential antibodies in a cellular system, i.e. while more or less maintaining the cellular physiological state specific to this cell type. First, a group of pooled supernatants was tested for binding events using fluorescence-activated cell sorting (FACS) analysis. For this purpose, different BCR variants were expressed on the surface of a triple knockout (TKO) cell line that cannot express the BCR itself.

[0205] The starting point for the generation of TKO cells is formed by transgenic mice with knockouts for the genes Lambda5, RAG1 or RAG2 and SLP65, respectively (Duehren von Minden et al., 2012, Nature 489, p.309-313). The combined knockout of RAG2 or RAG1 and Lambda5 leads to a blockade of the transition from the pro-B cell stage to the pre-B cell stage, which is typically characterized by the onset of rearrangement of the VDJ segments of the heavy chain (HC). These are therefore pro / pre-B cells. The activity of the BCR can be measured by reconstitution with inducible SLP65. The generation of such mice is known to those skilled in the art and belongs to the state of the art. To obtain the cells, the mice were sacrificed and the bone marrow of the femurs was extracted. The obtained cells were cultured under conditions that promote the survival of pro / pre-B cells (37°C, 7.5% CO2, Iscoves medium, 10% FCS, P / S, mouse IL7). After several passages, FACS sorting was performed for control purposes to sort pro / pre-B cells and return them to culture. The markers used for this purpose are known to the expert.

[0206] To reconstitute with the "BCR of interest", the corresponding sequence encoding the VH was fused with a human IgM constant segment by polymerase chain reaction (PCR) and the heavy chain (HC) and light chain (LC) were cloned into their respective expression vectors with a CMV promoter. These were introduced into a packaging cell line (Phoenix cell line) by lipofection. After 36 hours of culture, the viral supernatant was removed and used for spinfektion of TKO cells. BCR expression was measured by FACS using anti-IgM and anti-LC antibodies. For this purpose, some cells were harvested and stained with 5 μl of each antibody in a total volume of 100 μl in PBS. Both the operation of extracting the supernatant and the spinfektion of the TKO are widely known procedures and known to experts. The knockout of RAG2 or RAG1 and Lambda5 ensured that only the "BCR of interest" was expressed on the surface.

[0207] In this way, we generated two different BCR-expressing TKO cell lines, one of which expresses membrane-bound IGHV3-21 / IGLV3-21. R110 To generate the second BCR-expressing TKO cell line, IGLV3-21 R110 The codon for arginine at position 110 of the DNA encoding was reverted to the germline sequence by well-known site-directed mutagenesis techniques (see, e.g., Sambrook et al., 1989, supra). The resulting TKO cells were designated IGLV3-21, which contains a glycine at amino acid position 110 (IGLV3-21 G110 ) was expressed. To generate a third control TKO cell line that does not have BCR expression on its surface, spinfection with an empty expression vector was performed. By reconstituting the cells with inducible SLP65, the functionality of the expressed BCR could be characterized and the cells were then allowed to express IGHV3-21 / IGLV3-21 on the surface prior to selection. R110 The autonomous activation state of the BCR can be verified. The methods chosen here are FACS analysis and the use of Ca2+ receptors such as Indo-1. 2+The aim of this study is to measure Ca flux after induction of SLP65 using a Ca-dependent dye. These methods are known to experts (see M. Duehren von Minden et al., Nature 2012). Using these cells as "targets", FACS was used to isolate IGLV3-21 R110 Identifying antibodies that specifically bind to the harbored BCR. The first step was to identify the supernatants to which the antibodies showed binding. In this first selection round, the supernatants of several clones were combined and tested for binding profile. IGHV3-21 / IGLV3-21 R110 -BCR specific binding, the binding profile is positive. Groups showing such a profile were isolated and the binding profiles of the individual clones were characterized again in a second selection round. Binding of the monoclonal antibodies was verified using a FACS binding assay using a fluorescently labeled anti-mouse IgG antibody.

[0208] This selection approach is IGHV3-21 / IGLV3-21 R110 Binds to BCR-positive TKO mouse cells but not to IGHV3-21 / IGLV3-21 G110 This led to the identification of an antibody, mAb01-01, that does not bind to BCR-positive TKO mouse cells.

[0209] Mouse antibody production After identifying preferred antibodies by selection, mRNA was isolated from individual hybridoma clones, cDNA was generated and amplified by anchored PCR (Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination; Osterroth F, Alkan O, Mackensen A, Lindemann A, Fisch P, Skerra A, Veelken H. J Immunol Methods 1999 Oct 29; 229(1-2):141-53). The sequence of the cDNA encoding monoclonal antibody mAb01-01 was confirmed by Sanger sequencing (SEQ ID NO: 25 for HC nucleotides, SEQ ID NO: 26 for LC nucleotides) and placed into a vector suitable for expression in CHO cells.

[0210] The expression of mAb01-01 as an IgG1 subtype was verified using secondary anti-mouse IgG1-APC and IgG2-APC antibodies. R110 One batch of expressing TKO cells was stained with secondary antibody alone, and the other batch was stained with mAb01-01 and secondary antibody. Subsequent FACS analysis confirmed that the antibody was expressed as IgG1.

[0211] The specific monoclonal antibody mAb01-01 was sequenced. The following amino acid sequences were determined, as shown in Table 1: SEQ ID NO: 1 for HC, SEQ ID NO: 2 for LC, SEQ ID NO: 3 for VH, and SEQ ID NO: 4 for VL. The sequences corresponding to the complementarity determining regions (CDRs) of the heavy chain, H-CDR1, H-CDR2, and H-CDR3, are SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, and the sequences corresponding to the light chain CDRs, L-CDR1, L-CDR2, and L-CDR3, are SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10.

[0212] Example 2 Generation of chimeric antibodies The VH and VL nucleotide sequences of the mouse monoclonal antibody mAb01-01 (SEQ ID NO: 27 for VH nucleotides, SEQ ID NO: 28 for VL nucleotides) were used to synthesize a chimeric antibody. For this purpose, the VH sequence was fused to a human IgG1 isotype constant domain sequence (SEQ ID NO: 31 for IgG1 constant nucleotides) and the VL sequence was fused to a human IgK isotype constant domain (SEQ ID NO: 32 for IgK constant nucleotides) by PCR and expressed using a CHO-based transient expression system. The resulting antibody-containing cell culture supernatant was clarified by centrifugation and filtration. The chimeric antibody was purified from the cell culture supernatant by affinity chromatography. The purity of the antibody was determined to be greater than 95% as judged by reduced denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The antibody was analyzed for protein content and concentration by size exclusion chromatography (SEC) in PBS buffer. All procedures were carried out using state-of-the-art equipment and techniques.

[0213] This approach resulted in the chimeric antibody "HC0-LC0," the sequence of which is summarized in Table 1.

[0214] [Table 1]

[0215] Example 3 Generation of humanized antibodies Humanization of mAb01-01 was performed by in silico grafting of mouse CDRs onto mature human antibody frameworks using standard CDR grafting techniques by Fusion Antibodies Plc (Belfast, N.Ireland). Key residues important for the VH / VL interface and canonical loop structures were maintained as much as possible in the humanized variants using the CDRx platform (Fusion Antibodies Plc, Belfast, N.Ireland). The amino acid sequences of the humanized variants generated by the fusion antibodies were then converted to nucleotide sequences using Geneiouse software (Geneious Prime 2, Auckland, New Zealand). Sixteen pairs of humanized heavy and light chains were generated by fusing the VH sequence with the human IgG1 isotype constant domain sequence (SEQ ID NO: 31 for IgG1 constant nucleotides) and the VL sequence with the human IgK isotype constant domain (SEQ ID NO: 32 for IgK constant nucleotides) by PCR, and the antibody gene sequences were transiently expressed in Chinese Hamster Ovary Cells (CHO). After batch culture, the expressed humanized antibodies were purified from the cell culture supernatant and analyzed for HC0-LC0 as described in Example 2. Eight humanized antibodies shown in Table 2 were successfully obtained.

[0216] [Table 2]

[0217] Example 4 IGLV3-21 R110 -BCR and IGLV3-21 G110 -Binding of mAb01-01 to BCR-expressing TKO cells The specificity of the antibody mAb01-01 observed in the selection (example 1) was verified in a FACS assay using an antibody conjugated to a fluorescent marker.

[0218] For this purpose, two cell mixtures were prepared from three different TKO cell lines according to Example 1, cell mixture A and cell mixture B, and subsequently stained with mAb01-01-APC (mAb01-01 was coupled with the fluorescent marker APC by Immunotools GmbH). As a control, each batch was additionally stained with an anti-IgM antibody (anti-human IgM-PE, clone: ​​MHM-88, BioLegend catalog number: 314508).

[0219] Cell mixture A was used as IGHV3-21 / IGLV3-21 R110 B cell receptor-expressing TKO cells and B cell receptor-negative (empty vector, control) TKO cells were prepared in a 1:1 ratio in PBS buffer (Gibco, pH 7.2, catalog no. 20012-019).

[0220] Cell mixture B was IGHV3-21 / IGLV3-21 G110 B cell receptor-expressing TKO cells and B cell receptor-negative (empty vector, control) TKO cells were prepared at a 1:1 ratio in PBS buffer.

[0221] Cells from cell mixture A or B were suspended in PBS buffer at approximately 106 cells per FACS tube in antibody diluent (5 μg / ml mAb01-01-APC, 2 μg / ml anti-human IgM-PE, total volume 100 μl) and incubated in the dark for 15 min at 4° C. Afterwards, the cells were washed once with 1 ml of cold PBS buffer and resuspended in 200 μL of cold PBS buffer.

[0222] FACS analysis was performed using a MACSQuant Analyzer 10 (Miltenyi Biotec BV&Co.KG, the instrument was calibrated according to the manufacturer's recommendations, flow rate: low, sample mixing: gentle mixing, mode: standard, uptake volume: 50 μL, sample volume: 200 μL). TKO cells of cell mixture A or cell mixture B were gated by side scatter (SSC) vs. forward scatter (FSC), and gated TKO cells were analyzed in a dot plot of anti-IgM-PE vs. mAb01-01-APC to enumerate the different TKO cell populations using quadrant statistics.

[0223] As shown in Figures 1A to 1D, mAb01-01 binds human IGLV3-21 R110 -BCR-expressing TKO mouse cells, whereas human IGLV3-21 G110 -Does not bind to BCR-expressing TKO mouse cells.

[0224] Example 5 IGLV3-21 R110 Affinity of antibodies to the B cell receptor IGLV3-21 R110 To define the binding affinity of the antibody to the B cell receptor carrying the IGLV3-21 antibody, a soluble recombinant version of the BCR (170.5 kDa, SEQ ID NO: 51 for the HC and SEQ ID NO: 52 for the LC) was produced in the 293-HEK cell line as monomeric human IgM by transient expression using the protocol described in Example 1 and bound to immobilized anti-IGLV3-21 antibody. R110 Antibody binding was monitored by biolayer interferometry (BLI) on a Fortebio Octet instrument (Satorius).

[0225] Kinetic assays were performed by first capturing anti-IGLV3-21 antibody via an indirect capture reagent, anti-human IgG Fc antibody. R110 This was carried out by immobilizing the antibody on a biosensor. Anti-IGLV3-21 R110 The antibody was loaded at a concentration of 0.01875 μg / ml, with 0.30–0.34 nm of anti-IGLV3-21 R110Antibody capture levels were generated. A 9 nM BCR fragment solution was prepared in running buffer (PBS, 0.02% Tween 20, 0.1% BSA, 0.05% sodium azide) and serially diluted 1:3 to obtain seven concentrations ranging from 9 to 0.012 nM (9 nM, 3 nM, 1 nM, 0.333 nM, 0.111 nM, 0.037 nM, and 0.012 nM). Then, anti-IGLV3-21 R110 Antibody capture biosensors were immersed in wells containing different concentrations of soluble BCR fragments for 900 seconds (association step), followed by a dissociation step in running buffer for 1200 seconds. The steps were performed at a constant oscillation speed of 1000 rpm. All reagents were used as described by the manufacturer. Capture anti-IGLV3-21 R110 To correct for both the spontaneous dissociation of the antibody and the non-specific binding of the soluble BCR fragment to the sensor surface, the sensorgrams were generated after double reference correction (buffer and blank sensor). The dissociation rate constant (K D ) was calculated from the association rate (k a ) constant and dissociation rate (k d ) constant.

[0226] As shown in Table 3, the chimeric antibody HC0-LC0 has a K D Soluble IGLV3-21 at value R110 The humanized antibodies HC6-LC6 and HC7-LC6 exhibit binding characteristics similar to those of the chimeric antibody HC0-LC0, and show dissociation constants within 2-fold of the chimeric antibody HC01-LC01. D See Table 3 for values.

[0227] [Table 3]

[0228] Example 6 IGLV3-21 R110Binding of mouse antibody mAb01-01 to the surface of -BCR-positive human B-CLL cells IGLV3-21 R110 -B cell receptor positive human CLL cells vs. non-IGLV3-21 R110 To determine the binding characteristics of mAb 01-01 to human B cells, binding was tested by flow cytometry.

[0229] For this purpose, cryopreserved peripheral blood mononuclear cells (PBMCs) from two CLL patients were used. One of these patients had CLL with IGLV3-21 R110 More specifically, CLL cells were characterized by the presence of IGLV3-21 R110 and IGHV4-39 heavy chain combination (IGHV4-39 / IGLV3-21 R110 The other patient expressed BCR with non-IGLV3-21 R110 The patient had been diagnosed with CLL. PBMCs were isolated from heparinized venous blood by Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB) density gradient centrifugation using techniques known in the art, for example according to Boyum A. (Isolation of mononuclear cells and granulocytes from human blood. Scan. J. Clin. Lab. Invest. 1968, 21 (Suppl. 97): 77-89).

[0230] Samples were thawed and resuspended in 5 ml cell culture medium (RPMI, Gib-co, 10% FCS, PAN-Biotec). Cells were centrifuged at 300g (Eppendorf Centrifuge 5425R) and subsequently resuspended in 1 ml RPMI. Cell counts were obtained using a Neubauer Chamber. For staining, 1x10E6 cells were used and transferred to a FACS tube. Cells were stained with 2 μl anti-CD19 VioBright515 (Miltiny Biotech, Klon:REA675), 2 μl anti-CD5-PE-Vio770 (Miltenyi Biotec, Klon:REA782) and 5 μl mAb01-01-APC (mAb01-01 conjugated with the fluorescent marker APC from Immunotools GmbH) in a total volume of 100 μl PBS buffer and incubated for 15 min at 4°C in the dark. The cells were then washed once with 1 ml of cold PBS buffer, resuspended in 200 μL of cold PBS buffer, and transferred to BD LSRFortessa TM Analysis was performed by flow cytometry using a Cell Analyzer (BD bioscience). The instrument was calibrated as recommended by the manufacturer. Analysis of the raw data was performed using FlowJo-Software X (BD bioscience). Analysis gates were set as shown in Figure 2.

[0231] As shown in Figure 2, mAb01-01 inhibited IGLV3-21 R110 IGLV3-21 binds only to IgG1 positive CLL B cells R110 More specifically, mAb01-01 does not bind to the BCR of CLL patients who do not possess IGLV3-21, regardless of the nature of the heavy chain. R110 -Recognize the BCR.

[0232] Example 7 IGLV3-21 R110 -Binding characteristics of chimeric and humanized antibodies to the cell surface of BCR-positive mouse TKO cells IGLV3-21 specific with chimeric antibody and two humanized versions R110-IGLV3-21 to compare BCR binding R110 -BCR mouse TKO cells (see Example 1) were incubated with different concentrations of antibodies HC0-LC0, HC6-LC6 and HC7-LC6 and analyzed by flow cytometry and sandwich assay setup. Controls were performed using the TKO-empty vector cell line (without surface BCR).

[0233] From each cell line, 7.5 x 10E6 cells are transferred to separate 15 ml blue caps, centrifuged for 10 minutes (300 g, 4°C) and resuspended in 1.5 ml PBS (Gibco). Staining was performed in 96-well plates (VWR, U-bottom, untreated). For each reaction, 2x10E5 cells were used. The experimental setup is shown in Table 4.

[0234] [Table 4]

[0235] To characterize the binding properties of the different humanized variants, ten different concentrations of each antibody in PBS (10 μg / ml, 5 μg / ml, 2.5 μg / ml, 0.625 μg / ml, 0.31 μg / ml, 0.16 μg / ml, 0.08 μg / ml, 0.04 μg / ml, 0 μg / ml) were used for staining in a total volume of 200 μl per well. Incubation was performed at 4° C. in the dark for 30 min. Then, the 96-well plates were centrifuged at 300 g for 10 min at 4° C. (VWR, MEGA STAR 1.6R). The supernatant was discarded and the cells were resuspended in 100 μl of ice-cold PBS. For detection, a secondary antibody against human IgG1 labeled with APC was used at a final concentration of 10 μg / ml.

[0236] Incubation was performed in the dark at 4° C. for 15 min in a total volume of 200 μl / well, followed by further washing with ice-cold PBS. Cells were resuspended in 150 μl ice-cold PBS for acquisition. Cells were analyzed on a MACS-Quant10 (Miltenyi Biotec) and calibrated as per the manufacturer's instructions.

[0237] All IGLV3-21 R110 The MFI (median fluorescence intensity) of the -BCR TKO assay was neutralized by subtracting the control cell values ​​and plotted against the antibody concentration. R110 We generated a function showing a concentration-dependent increase in binding of the -BCR. As shown in Figure 3, the two humanized variants have increasingly identical binding properties for the chimeric antibody as the antibody concentration increases, with nearly identical binding specificity at most 10 μg / ml.

[0238] Example 8 Tissue cross-reactivity profile of mAb01-01 To determine the binding characteristics of mAB01-01 to human CLL and healthy tissues in immunohistochemistry (IHC) experiments, IGLV3-21 R110 Immunostaining was performed on sections of -BCR expressing spleen tissue and healthy spleen, skin, kidney, heart, and brain tissue.

[0239] Prior to IHC, tissue sections were deparaffinized and hydrated. To expose antigens, microwave treatment was performed in citrate buffer pH 6.0 (9 ml citric acid (0.1 M) and 41 ml sodium citrate (0.1 M)). For this, sections were boiled for 15 min in foaming citrate buffer, then cooled at room temperature for 30 min and rinsed with PBS for 3 x 5 min. For IHC, the slides were incubated for 2 h at room temperature with the primary antibody diluted 1:200 in a humidity chamber. As a control, all tissue sections were incubated under the same conditions without the primary antibody. Afterwards, slides were washed 3 x 5 min with PBS. As secondary antibody, horseradish peroxidase (HRP)-conjugated anti-IgG antibody (goat anti-mouse IgG (H+L)-HRP, Southern Biotech, Cat. No. 1036-05) was incubated at a dilution of 1:10000 for 1 h at room temperature in a humidity chamber. Then, the sections were washed with PBS for 10 min, and the activity of HRP was detected using a DAB substrate kit (#34065, Thermo Fisher). DAB (3,3'-diaminobenzidine tetrahydrochloride) substrate was incubated for 15 min. Fluoromount-1 was used as a capping agent. Evaluation was performed after 30 min, showing an insoluble brown reaction product at the site where the HRP-conjugated anti-IgG antibody bound to the tissue.

[0240] As shown in Figure 4, the IGLV3-21 R110 - Positive staining was observed for BCR positive spleen sections, therefore mAb01-01 is cross-reactive in binding to human CLL tissue. In contrast, no staining was detected in healthy human tissue sections of spleen, skin, kidney, heart and brain. Thus, mAB01-01 does not show cross-reactivity with healthy human tissues.

[0241] Example 9 Anti-IGLV3-21 in patient-derived xenograft models R110 -BCR antibody testing anti-IGLV3-21 R110To determine the efficacy of the antibodies, a patient-derived xenograft model was chosen. For the dose-finding experiment, four groups including four NOD-scid IL2rg null (NSG) mice (Jackson ImmunoResearch, prepared as described in Qi J et al. (An IgG1-like bispecific antibody targeting CD52 and CD20 for the treatment of B-cell malignancies, Methods 2019, 154:70-76)) were used: Group A: Control group without antibody treatment Group B: Dose 0.3mg / kg body weight Group C: Dose 5mg / kg body weight Group D: Dosage 10mg / kg body weight.

[0242] IGLV3-21 R110 PMBS from -BCR patients were thawed and resuspended in PBS. T cells were isolated using Miltenyi CD3 Beads (Miltenyi Biotec) according to the manufacturer. T cells were purified using CD3 / CD28 Dynabeads (Dynabeads) as previously described (see Qi J et al., Methods, 2019). TM The cells were cultured and expanded for 7 days using Human T-Activator CD3 / CD28 for T Cell Expansion and Activation, Catalog No. 11161D, GIB-CO).

[0243] Seven days later, activated T cells and PBMCs (20 × 10 per mouse) were 6 5 x 10 CLL PBMCs and 5 x 10 5NSG mice were intravenously injected with mAb 01-01 (T cells) at 100x the normal concentration. For treatment, mAb 01-01 was administered intraperitoneally at different doses twice a week for a total of 3 weeks, starting 2 weeks after transplantation. Mice were pretreated with 250 μl human serum at the beginning of each week. Mice were sacrificed 3 weeks after treatment. For analysis, spleens were isolated and analyzed for human IGLV3-21 by flow cytometry using mAb 01-01 and antibodies against human CD45, CD5, and CD19 (CD5 IgG1 UCHT2, BioLegend; CD19 IgG1 HIB19, BD Biosciences; CD45 (human) IgG1 H130 Invitrogen). R110 The presence of positive CLL B cells was analyzed. For flow cytometry, cells were collected by centrifugation and resuspended in ice-cold 0.1% (w / v) BSA in PBS (flow cytometry buffer). 5×10 5 100 μL containing cells were dispensed into V-bottom 96-well plates (Corning). The cells were first blocked with 5% (v / v) goat serum (Jackson ImmunoResearch) for 30 min on ice and then incubated with the indicated antibodies as recommended by the manufacturer. The cells were incubated for 30 min on ice in the dark. The cells were then washed twice with ice-cold flow cytometry buffer, resuspended in 200 μL of flow cytometry buffer, and analyzed using a FACSCanto (BD Biosciences).

[0244] As shown in FIG. 5, treatment with mAb01-01 resulted in a reduction in tumor cell numbers in all treated mice, and significantly reduced tumor growth at 10 mg / kg of mAb01-01.

Claims

1. having a heavy chain amino acid sequence as set forth in SEQ ID NO:1 and a light chain amino acid sequence as set forth in SEQ ID NO:2; or having a heavy chain amino acid sequence of SEQ ID NO:11 and a light chain amino acid sequence of SEQ ID NO:12; or An antibody comprising a variable heavy chain having a sequence selected from the list consisting of SEQ ID NO:15 and SEQ ID NO:20 and a variable light chain having a sequence selected from the list consisting of SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, in any combination.

2. The antibody of claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 1 and a light chain corresponding to SEQ ID NO:

2.

3. The antibody of claim 1, characterized by a heavy chain amino acid sequence of SEQ ID NO:11 and a light chain amino acid sequence of SEQ ID NO:

12.

4. The antibody of claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 13 and a light chain corresponding to SEQ ID NO:

14.

5. The antibody of claim 1, characterized by a heavy chain corresponding to SEQ ID NO: 21 and a light chain corresponding to SEQ ID NO:

14.

6. The antibody of claim 1 or 3, which is a chimeric antibody.

7. The antibody described in claim 4 or 5, which is a humanized antibody.

8. An immune complex comprising an antibody according to any one of claims 1 to 7.

9. IGLV3-21 R110 9. The antibody of any one of claims 1 to 8 for use in the treatment of CLL in positive patients.

10. 0.25-25mg / kg 体重 The antibody for use according to claim 9, which is administered at a dosage of

11. 1-20 mg / kg 体重 The antibody for use according to claim 9, which is administered at a dosage of

12. 7-15 mg / kg 体重 The antibody for use according to claim 9, which is administered at a dosage of

13. 8-12 mg / kg 体重 The antibody for use according to claim 9, which is administered at a dosage of

14. 14. The antibody for use according to any one of claims 9 to 13, wherein the CLL is subset #2 CLL.

15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or excipient.

16. A kit comprising the pharmaceutical composition of claim 15.

17. IGLV3-21 R110 17. A kit or pharmaceutical composition according to claim 15 or 16 for use in the treatment of CLL in a positive patient.

18. IGLV3-21 R110 8. The antibody according to any one of claims 1 to 7 for use in the diagnosis of CLL, characterized by a BCR carrying the

19. 20. The antibody for use according to claim 18, which is conjugated to a detectable marker.

20. 19. The antibody for use according to claim 18, further comprising subsequently applying a further antibody which specifically binds to the antibody according to any one of claims 1 to 7, said further antibody being linked to a detectable marker.

Images