ANTIBODIES AGAINST CLAUDIN 18.2 USEFUL IN CANCER DIAGNOSIS

专利摘要:
patent abstract for: “antibodies against claudin 18.2 useful in the diagnosis of cancer”. the invention relates to antibodies directed against an epitope located within the c-terminal portion of cldn18.2, which are useful, for example, in diagnosing cancer and/or determining whether cancer cells express cldn18.2
公开号:BR112014026755B1
申请号:R112014026755-3
申请日:2013-05-06
公开日:2020-06-16
发明作者:Ugur Sahin；Özlen Türeci；Rita Mitnacht-Kraus；Stefan Wöli
申请人:Ganymed Pharmaceuticals Ag；Tron - Translationale Onkologie An Der Johannes Gutenbert-Universität Mainz Gemeinnützige Gmbh；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: “ANTIBODIES AGAINST CLAUDIN 18.2 USEFUL IN CANCER DIAGNOSIS”.
[001] Claudines are integral membrane proteins located within the tight junctions of the epithelium and endothelium. Claudines are predicted to have four transmembrane segments with two extracellular loops, and N and Cterminals located in the cytoplasm. The claudin (CLDN) of the transmembrane protein family plays a critical role in maintaining the tight epithelial and endothelial junctions and may also play a role in maintaining the cytoskeleton and cell signaling.
[002] The claudin 18 molecule (CLDN 18) is an integral transmembrane protein (tetraspanin) having four membranes spanning hydrophobic regions and two extracellular loops (loopl embraced by a hydrophobic region 1 and hydrophobic region 2; loop2 embraced by hydrophobic regions 3 and 4). CLDN 18 exists in two different processing variants, which are described in the rat and the human (Niimi, Mol Cell Biol 21: 7380 - 90, 2001). Junction variants (Genbank accession number: junction variant 1 (CLDN18.1): NP_057453, NM_016369, and splicing variants (2): 18.2 CLDN NM_001002026, NP_001002026) have a molecular weight of approximately 27.9 / 27.72 kDa. Variants of the CLDN18.1 and CLDN18.2 junctions differ in the N-terminal portion comprising the first transmembrane (TM) and loopl region,
2/118 while the primary C-terminal protein sequence is identical; see Figure 1.
[003] CLDN18.1 is selectively expressed in normal lung cells, while CLDN18.2 is expressed only in gastric cells. However, the expression of CLDN 18.2 in the normal stomach is restricted to differentiated short-lived cells of the stomach epithelium. CLDN 18.2 expression was identified in several tumor tissues. For example, CLDN18.2 has been found to be expressed in pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma and ENT tumors. CLDN 18.2 is a valuable target for the prevention and / or treatment of primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, such as non-small cell lung cancer (NSCLC), ovarian cancer , colon cancer, liver cancer, head-neck cancer and gallbladder cancer, and their metastases, namely, gastric cancer metastasis, such as Krukenberg tumors, peritoneal metastasis and ganglionic metastasis.
[004] The differential expression of CLDN18.2 between cancer and normal cells, its location in the membrane, its absence, in the vast majority of normal tissues corresponding to toxicity, its restriction of the expression of a dispensable cell population in the stomach, the
3/118 differentiated gastric cells, which can be replaced by target-negative stem cells from the stomach, make CLDN18.2 an attractive target for cancer immunotherapy and the use of antibody-based therapies to target CLDN18.2 in cancer therapy promises a high level of therapeutic specificity.
[005] The clinical application of target antibodies CLDN18.2 faces the obstacle that human CLDN18.2 is highly homologous to human CLDN18.1. CLDN18.2 specific antibodies targeting the CLDN18.2 N-terminal extracellular domain could be successfully established by visualizing sequence differences between human CLDN18.2 and human CLDN18.1. Attempts to produce antibodies directed against the N-terminal portion of CLDN18.2 and which have properties making them clinically applicable for diagnostic purposes, for example, for the detection of CLDN18.2 expression in cells of tissue sections cancer, have failed.
[006] Surprisingly, the present inventors have found that antibodies directed against a particular epitope located within the C-terminal portion of CLDN18.2 meet the criteria for the application of antibody diagnostics, in particular, for the detection and identification of cells that express CLDN18.2. Surprisingly, these antibodies, although directed against a sequence that is identical between CLDN18.1 and CLDN18.2, do not target the
4/118 non-cancerous lung cells.
[007] The antibodies of the invention are useful, for example, in the diagnosis of cancer and / or in determining whether cancer cells express CLDN18.2. Preferably, a cancer or tumor cell is characterized by the expression of the CLDN18.2 surface. Cancer cells expressing CLDN18.2 are suitable targets for CLDN18.2 focused therapies such as antibody therapy directed against CLDN18.2. In one embodiment, cancer cells express aberrantly or expressed CLDN18.2 while the corresponding normal cells do not express CLDN18.2 or CLDN18.2 expressed at a lower level. Cells expressing CLDN18.2 are preferably selected from the group consisting of gastric tumorigenic, esophagus, pancreas, lung, ovary, colon, liver, head-neck, and gallbladder cancer cells. SUMMARY OF THE INVENTION
[008] The present invention relates to an antibody or its antigen-binding fragment, which (I) binds to a peptide having an amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6 ) and / or (ii) binds to claudin 18.2 (CLDN18.2), wherein said antibody or its antigen-binding fragment binds to CLDN18.2 by binding at least one epitope within
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CLDN18.2 having the amino acid sequence TEDEVQSYPSKHDYV (SEQ ID NO: 5) or EVQSYPSKHDYV (SEQ ID NO: 6).
[009] In one embodiment, said CLDN18.2 is a cell surface membrane bound to CLDN18.2. In one embodiment, said CLDN18.2 is present in cancer cells, wherein said cancer cells are cancer cells that preferably express CLDN18.2. In one embodiment, said cancer cells are selected from the group consisting of gastric cancer cells, esophagus, pancreas, lung, ovary, colon, liver, head-neck, and gallbladder cells. In one embodiment, an antibody or its antigen-binding fragment of the invention does not bind to non-cancerous cells, except the stomach epithelial cells. In one embodiment, an antibody or its antigen-binding fragment of the invention does not bind to non-cancerous lung cells. In one embodiment, an antibody of the present invention is a chimeric, human or humanized antibody. In one embodiment, an antibody of the present invention is a monoclonal antibody.
[0010] These and other aspects of the present invention refer to an antibody comprising: (I) an antibody heavy chain comprising:
(i) an antibody heavy chain sequence, according to SEQ ID NO: 7 or its variant,
6/118 (ii) at least one, preferably two, more preferably all three CDR sequences of an antibody heavy chain sequence, according to SEQ ID NO: 7 or its variant, or (iii) one sequence of CDR3, according to SEQ ID NO: 10 or its variant, and preferably further comprising a sequence of CDR1, according to SEQ ID NO: 8 or its variant and / or a sequence of CDR2, according to with SEQ ID NO: 9 or its variant, and / or (II) an antibody light chain, comprising:
(i) an antibody light chain sequence, according to SEQ ID NO: 11 or its variant, (ii) at least one, preferably two, more preferably all three CDR sequences of a chain sequence of the antibody, according to SEQ ID NO: 11 or its variant, or (iii) a CDR3 sequence, according to SEQ ID NO: 14 or a variant thereof, and preferably, which further comprises a sequence of CDR1, according to SEQ ID NO: 12 or a variant thereof and / or a sequence of CDR2 according to SEQ ID NO: 13 or its variant.
[0011] In the foregoing and other aspects of the present invention it also relates to an antibody comprising: (I), an antibody heavy chain comprising:
7/118 (i) an antibody heavy chain sequence according to SEQ ID NO: 15 or its variant, (ii) at least one, preferably two, more preferably all three CDR sequences from a sequence of antibody heavy chain according to SEQ ID NO: 15 or its variant, or (iii) a CDR3 sequence, according to SEQ ID NO: 18 or a variant thereof, and preferably further comprises a CDR1 sequence , according to SEQ ID NO: 16 or a variant thereof and / or a CDR2 sequence, according to SEQ ID NO: 17 or a variant thereof, and / or (II) an antibody light chain, comprising:
(i) an antibody light chain sequence according to SEQ ID NO: 19 or a variant thereof, (ii) at least one, preferably two, more preferably all three CDR sequences of a chain sequence of the antibody according to SEQ ID NO: 19 or a variant thereof, or (iii) a sequence of CDR3, according to SEQ ID NO: 22 or a variant thereof, and preferably further comprises a sequence of CDR1, according to SEQ ID NO: 20 or its variant and / or a sequence of CDR2, according to SEQ ID NO: 21 or its variant.
[0012] In preferred embodiments, an antibody from
The invention comprises an antibody heavy chain which comprises a heavy chain of the gamma-1 constant region, preferably a gamma-1 of the heavy chain of the human constant region and / or comprises an antibody light chain which comprises a constant region light chain kappa.
[0013] In the foregoing and other aspects, the present invention relates to an antibody produced by, or obtained from, a hybridoma cell deposited at DSMZ (Inhoffenstr 7B, 38124 Braunschweig, Germany) and having one of the following designations and access numbers:
1. muAB 43-14A, accession number. DSM ACC3144, filed on October 6, 2011; or
2. muAB 35-22A, access number. DSM ACC3143, filed on October 6, 2011.
[0014] The antibodies of the invention are designated herein by reference to the designation of the antibody and / or by reference to the clone producing the antibody, for example, muAB 43-14A.
[0015] Additional preferred antibodies are those that have the specificity of the antibodies produced by, and obtained from, the hybridomas described above and, in particular, those that comprise an antigen or antigen-binding portion, in particular, binding to a region variable, identical or highly homologous to the antibodies produced by and obtainable from
9/118 of the hybridomas described above. Preferred antibodies are contemplated to be those that have the CDR regions or are identical or highly homologous to the CDR regions of antibodies produced by, and obtained from, the hybridomas described above. By highly homologous it is contemplated that from 1 to 5, preferably from 1 to 4, as well as from 1 to 3 or 1 or 2 substitutions can be made in each of the CDR regions. Particularly preferred antibodies are the chimerized and humanized forms of the antibodies produced by, and obtained from, the hybridomas described above.
[0016] Thus, an antibody of the invention can be selected from the group consisting of (i) an antibody produced by, or obtained from a clone deposited under the accession number DSM ACC3144 (muAB 43-14A) or DSM ACC3143 (muAB 35-22A), (ii) an antibody that is a chimerized humanized form or of the antibody in (i), (iii) an antibody that has the specificity of the antibody in (i), and (iv) an antibody that comprises the antigen-binding portion or antigen-binding site of the antibody in (i). The antigen-binding portion or site of the (i) antigen binding antibody may comprise the variable region of the (i) antibody. Further encompassed by the present invention are antigen-binding fragments of the antibodies described herein.
[0017]
An antibody of the present invention is
10/118 preference, capable of binding to CLDN18.2 in its native form, that is, it occurs naturally or in the state of non-denatured, or in its denatured state.
[0018] In one embodiment, an antibody of the present invention is obtained by a method which comprises the step of immunizing an animal with a peptide which preferably comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO : 6, or an immunologically equivalent peptide, or a nucleic acid or host cell that expresses said peptide. Preferably, said peptide does not comprise more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 contiguous amino acids of CLDN18.2.
[0019] In one embodiment, an antibody of the present invention is obtained by a method which comprises the step of immunizing an animal with a peptide which preferably comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO : 25, or an immunologically equivalent peptide, or a nucleic acid or host cell that expresses said peptide. Preferably, said peptide does not comprise more than 110, 100, 90, 80, or 75 contiguous amino acids of CLDN18.2.
[0020] Antibodies or antigen-binding fragments of the invention can be coupled, i.e., covalently or non-covalently, to other radicals, such as detectable markers.
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The present invention also relates to a cell, such as a hybridoma cell that produces an antibody as described herein.
[0022] Hybridoma cells are preferred those deposited at DSMZ (Inhoffenstr 7B, 38124 Braunschweig, Germany) and having one of the following designations and access numbers:
1. muAB 43-14A, DSM accession number ACC3144, deposited on October 6, 2011; or
2. muAB 35-22A, DSM accession number ACC3143, deposited on October 6, 2011.
[0023] The present invention also relates to a peptide which preferably comprises the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or an immunologically equivalent peptide. Preferably, said peptide does not comprise more than 110, 100, 90, 80, 70, 60, 50, 40, 30, or 20 contiguous amino acids of CLDN18.2.
[0024] The present invention also relates to a peptide which preferably comprises the amino acid sequence of SEQ ID NO: 24 or SEQ ID NO: 25, or an immunologically equivalent peptide, or a nucleic acid or a host cell which expresses said peptide. Preferably, said peptide does not comprise more than 110, 100, 90, 80, or 75 contiguous amino acids of
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CLDN18.2.
[0025] The present invention also relates to nucleic acids that comprise the genes or nucleic acid sequences that encode the antibodies, or parts thereof, for example, an antibody chain, or its antigen-binding fragments, or peptides, such as described here. Preferably, the nucleic acid of the invention is operably linked to the expression control elements allowing expression in eukaryotic or prokaryotic cells. The control elements that ensure expression in eukaryotic or prokaryotic cells are well known to those skilled in the art.
[0026] The nucleic acids of the invention can be included in a vector, for example, a plasmid, cosmid, virus, bacteriophage, or another vector used, for example, conventionally, in genetic engineering. The vector can comprise other genes, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector can comprise expression control elements that allow for the correct expression of coding regions in suitable hosts. Such control elements are known to those skilled in the art and may include a promoter, a junction cassette, and a translation initiation codon.
[0027]
Methods for building molecules of
Nucleic acids, for the construction of vectors comprising the nucleic acid molecules, for the introduction of vectors into appropriately chosen host cells, to cause or achieve the expression of nucleic acid molecules are well known in the art.
[0028] Another aspect of the present invention relates to a host cell comprising a nucleic acid or vector, as disclosed herein.
[0029] Another aspect of the present invention relates to the detection of CLDN18.2 or cells expressing CLDN18.2 or determining the amount of CLDN18.2 or cells expressing CLDN18.2 using an antibody or antigen binding fragment of the invention . CLDN18.2 or cells that express CLDN18.2 are detected or the amount of CLDN18.2 or cells that express CLDN18.2 is determined by detecting or determining the amount of a complex between CLDN18.2 and an antibody or antigen fragment of the invention. The formation of a complex indicates the presence of CLDN18.2 or cells that express CLDN18.2. Such detection or quantity determination can be carried out in a number of ways, including, but not limited to immunodetection using an antibody or antigen binding fragment of the invention. Methods for using antibodies to detect peptides or proteins are well known and include ELISA, competitive binding assays, and others
Similar 14/118. In general, such assays use an antibody fragment or antibody that specifically binds to the target peptide or protein directly or indirectly linked to a marker that provides for detection, for example, enzymes, radioactive markers indicating fluorophores or paramagnetic particles. The methods of the invention allow quantitative and / or qualitative assessments, for example, absolute and / or relative assessments, either of CLDN18.2 levels or of levels of cells that express CLDN18.2.
[0030] In one aspect, the present invention relates to a method for detecting CLDN18.2 or determining the amount of CLDN18.2 in a sample, comprising the steps of:
(i) contacting a sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention; and (ii) detecting the formation of a complex or determining the amount of a complex between the antibody, its antigen-binding fragment or the conjugate and CLDN18.2.
[0031] In one embodiment, the sample is a cell sample, that is, a sample comprising cells, such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, its antigen-binding fragment or the conjugate and expressed CLDN18.2
15/118 by the cells in that sample.
[0032] In one aspect, the present invention relates to a method for determining whether cells express CLDN18.2, in which it comprises the steps of:
(i) contacting a cell sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention and (ii) detecting the formation of a complex between the antibody, its antigen-binding fragment or the conjugate and CLDN18. 2 expressed by the cells in said sample.
[0033] In one embodiment, the cells in the sample are cancer cells. The complex is preferably formed between the antibody, its antigen-binding fragment or the conjugate and CLDN18.2 expressed by the cells in that sample.
[0034] Other aspects of the present invention refer to methods of diagnosis or classification of diseases by targeting CLDN18.2 using an antibody or antigen-binding fragment of the invention. These methods provide for the selective detection of cells that express CLDN18.2, thus differentiating these cells from normal cells that do not express CLDN18.2 or diseased cells not expressing CLDN18.2. Diseases characterized by diseased cells expressing CLDN18.2 are treatable by therapy
16/118 target of CLDN18.2, such as therapy with therapeutic antibodies directed against CLDN18.2. Preferred diseases for therapy or diagnosis are those in which CLDN18.2 is expressed or expressed aberrantly, in cancerous diseases, in particular, such as those described herein.
[0035] In one aspect, the present invention relates to methods for diagnosis, detection and monitoring, that is, determining the regression, progression, course and / or appearance, of a cancer disease comprising the detection of CLDN18.2 or cells that express CLDN18.2 and / or determining the amount of CLDN18.2 or cells that express CLDN18.2 in a biological sample isolated from a patient using an antibody or antigen binding fragment of the invention. Such methods can be used to detect whether a subject has a cancer disease or is at (increased) risk of developing a cancer disease, for example, if a treatment regimen is effective.
[0036] Thus, in one aspect, the present invention relates to a method for diagnosis, detection and monitoring of cancer, comprising the steps of:
(i) contacting a biological sample with an antibody or antigen-binding fragment of the invention or a conjugate of the invention and (ii) detecting the formation of a complex and / or determining the amount of a complex between the antibody, the
17/118 its antigen-binding fragment or the conjugate and CLDN18.2.
[0037] In one embodiment, the biological sample is a cell sample, that is, a sample comprising cells, such as cancer cells. In this embodiment, the complex is preferably formed between the antibody, its antigen-binding fragment or the conjugate and CLDN18.2 expressed by the cells in said sample.
[0038] The control methods according to the invention preferably comprise a detection and / or determination of the amount of CLDN18.2 or cells that express CLDN18.2 in a first sample with a first point in time and in a another sample at a second point in time, in which the regression, progression, course and / or appearance of a tumor disease can be determined by comparing the two samples.
[0039] Typically, the level of CLDN18.2, or level of cells expressing CLDN18.2 in a biological sample, is compared to a reference level, where a deviation from said reference level is indicative of the presence and / or stage of a cancer in an illness of the subject. The reference level can be a level, as determined in a control sample (for example, from a healthy tissue or subject, in particular, a patient without a cancer disease) or for an average level of individuals
18/118 healthy. A deviation from said reference level designates a significant change, such as an increase of at least 10%, 20%, or 30%, preferably by at least 40% or 50%, or even more.
[0040] Preferably, the presence of CLDN18.2 or cells that express CLDN18.2 and / or an amount of CLDN18.2 or cells that express CLDN18.2 that is increased over a reference level, for example, in relation to a patient, without the cancer disease, indicates the presence or risk of (ie, a potential for the development of) a cancer disease in the patient.
[0041] An amount of CLDN18.2 or cells that express CLDN18.2 which is decreased compared to a biological sample taken earlier from a patient can indicate a regression, a positive course, for example, a successful treatment, or a reduced risk of an onset of cancer disease in a patient.
[0042] An amount of CLDN18.2 or cells that express CLDN18.2 that is increased compared to a biological sample taken earlier from a patient may indicate a progression, a clear negative, for example, a successful treatment , recurrence or metastatic behavior, an onset or a risk for an onset of a cancer disease in said patient.
[0043] In one aspect, the present invention relates to
19/118 a method for determining whether a cancer can be treated by a targeted cancer therapy CLDN18.2 comprising the steps of:
(I) contacting a sample comprising cancer cells, with an antibody or antigen-binding fragment of the invention or a conjugate of the invention, and (ii) detecting the formation of a complex between the antibody, its antigen-binding fragment or the conjugate and CLDN18.2.
[0044] The complex is preferably formed between the antibody, its antigen-binding fragment or the conjugate and CLDN18.2 expressed by cancer cells in which said sample.
[0045] Such methods can be used to detect whether the patient is suitable for therapy that involves targeting cells that express CLDN18.2, such as antibody therapy exercising one or more immune effector functions, such as specific CLDN18.2 antibodies. cytotoxic, for example, antibodies labeled with a cytotoxic substance, such as a toxin or a radioactive marker or a cell death inducing mechanism, such as CDC or ADCC. Diseases characterized by diseased cells that express CLDN18.2 are treatable by the target therapy CLDN18.2, such as cancer diseases, in particular, those described here.
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[0046] In one embodiment of any of the above, the sample, cell sample or biological sample, is from a patient with a cancer disease, being suspected of having or falling ill with cancer or who has a potential for a cancer disease. In one embodiment, the sample, cell sample or biological sample, is from a tissue or organ, in which the cells, when the tissue or organ is cancer-free, do not substantially express CLDN18.2. Preferably, said tissue is a tissue other than stomach tissue. Preferably, said tissue is the tissue of the lung, esophagus, pancreas or breast and the tissue or organ, optionally, has already been diagnosed as being affected by a cancer disease, for example, by visual inspection or culture tests of cells of said tissue or organ. In this embodiment, the presence of CLDN18.2 or cells that express CLDN18.2 and / or an amount of CLDN18.2 or cells that express CLDN18.2 that is improved compared to a reference level, for example, in relation to a patient, without a disease tumor, may indicate that the patient is suitable for a therapy that involves targeting cells that express CLDN18.2.
[0047] In one aspect, the invention provides compositions, for example, diagnostic compositions, or kits, comprising an antibody or antigen binding fragment, or a combination of antibodies and / or theirs
21/118 antigen-binding fragments described herein. Such diagnostic compositions or test kits are useful in the methods of the invention, such as the methods for diagnosing, detecting and monitoring the invention. Such kits may optionally comprise a detectable marker, for example, enzymes, radioactive markers, indicators, fluorophores or paramagnetic particles. Kits can include informational manuals, for example, manuals telling you how to use reagents to practice a process described here.
[0048] Other features and advantages of the present invention will be apparent from the following detailed description and claims.
DETAILED DESCRIPTION OF THE INVENTION
[0049] Although the present invention is described in detail below, it is to be understood that this invention is not limited to particular methodologies, protocols and reagents described here, as these may vary. It is also to be understood that the terminology used here is for the purpose of describing only particular embodiments, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly understood by a skilled technician
22/118 on the subject.
[0050] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they can be combined in any form and in any number to create additional embodiments. The embodiments variously describe preferred examples and are not to be construed to limit the present invention to only explicitly described embodiments. This description must be understood to support and encompass embodiments that combine the embodiments explicitly described, with any number of the elements described and / or preferred. In addition, any changes and combinations of all elements described in this application should be considered disclosed by the description of this application, unless the context indicates otherwise.
[0051] Preferably, the terms used here are defined as described in A multilingual glossary of biotechnological terms: (IUPAC Recommendations), HGW Leuenberger, B. Nagel, and H. Kolbl, Eds, Helvetica Chimica Acta, CH-4010 Basel , Switzerland., (1995).
[0052] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques that are explained in the literature of the area
23/118 (cf., for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al eds, Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989..).
[0053] Throughout this specification and the claims that follow, unless the context otherwise requires, the word understand, and variations, such as understand and understand, will be understood to imply the inclusion of a declared, integer element or step or group of members, integers or steps, but not the exclusion of any other member, integer or step or group of members, integers or steps, although in some modalities, such another member, integer or step or group of members, whole or stages can be excluded, that is, the object consists of the inclusion of a indicated member, whole or stage or group of members, integers or stages. The terms one and one eoe, of reference, similar to that used in the context of the description of the invention (especially, in the context of the claims) are to be understood to cover both the singular and the plural, unless otherwise indicated here or clearly contradicted by context. Recitation of value ranges here is only intended to serve as an abbreviated method of individually referring to each separate value that falls within the range. Unless otherwise indicated here, each individual value is incorporated into the specification as if described here
24/118 individually. All of the methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples, or exemplary language (for example, how), provided herein is intended only to better illustrate the invention and does not represent a limitation on the scope of the otherwise claimed invention. No language in the specification should be understood to indicate any unclaimed element essential to the practice of the invention.
[0054] Several documents are cited throughout the text of this specification. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), either above or below, are hereby incorporated by reference in their entirety. Nothing contained herein should be construed as an admission that the invention has no right to anticipate this disclosure by virtue of the previous invention.
[0055] The term recombinant in the context of the present invention means made by means of genetic engineering. Preferably, a recombinant object, such as a recombinant cell, within the scope of the present invention is not naturally occurring.
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[0056] The term that occurs naturally as used here refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), and can be isolated from a source in nature and that was not intentionally modified by man in the laboratory is naturally occurring.
[0057] The term antigen refers to an agent that comprises an epitope against which an immune response is directed and / or is being generated. Preferably, an antigen in the context of the present invention is a molecule that, optionally, after processing, induces an immunological reaction, which is preferably specific for the antigen. The term antigen includes, in particular, proteins, peptides, polysaccharides, nucleic acids, especially, RNA and DNA, and nucleotides.
[0058] The term epitope refers to an antigenic determinant of a molecule, that is, to the part of a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are discrete, three-dimensional sites on an antigen, which are recognized by the immune system. Epitopes normally consist of chemically active surface clusters of molecules, such as amino acids or sugar side chains, usually have
26/118 specific three-dimensional structural characteristics, as well as specific load characteristics. Conformational and non-conformational epitopes are distinguished by the fact that the bond to the former, but not the latter, is lost in the presence of denaturing solvents. An epitope of a protein, such as a CLDN, preferably comprises a continuous or discontinuous portion of said protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, more preferably, between 10 and 25 amino acids in length, for example, the epitope can be preferably 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.
[0059] The term discontinuous epitope, as used herein, means a conformational epitope on a protein antigen, which is formed from at least two separate regions in the primary protein sequence.
[0060] In a preferred embodiment, an antigen is an antigen associated with a tumor, such as CLDN 18.2, that is, a component of cancer cells that can be derived from the cytoplasm, the cell surface and the nucleus of the cell, in particular, antigens that are preferably produced in large quantities, or intracellular like the surface antigens on cancer cells.
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[0061] In the context of the present invention, the terms tumor-associated antigen or tumor antigen refer to proteins that are in normal conditions specifically expressed in a limited number of tissues and / or organs or at specific stages of development, for example, the tumor-associated antigen can be under normal conditions specifically expressed in the stomach tissue, preferably in the gastric mucosa, in reproductive organs, for example, in testicles, in trophoblastic tissue, for example, in the placenta, or in germ cells, and are expressed or expressed aberrantly in one or more tumor or cancer tissues. In this context, a limited number preferably means no more than 3, more preferably, no more than 2 tumor-associated antigens, in the context of the present invention include, for example, cell type differentiation antigens, preferably antigens of specific differentiation, that is, proteins that are expressed under normal conditions, especially, in a certain type of cell at a certain stage of differentiation, cancer / testis antigens, that is, proteins that are in normal conditions specifically expressed in testicles and sometimes in the placenta, and specific germ line antigens. In the context of the present invention, the antigen associated with a tumor is preferably associated with the cell surface of a cell
28/118 cancerous and preferably is not or only rarely expressed in normal tissues. Preferably, the tumor-associated antigen or the aberrant expression of the tumor-associated antigen identifies cancer cells. In the context of the present invention, the antigen associated with a tumor that is expressed by a cancer cell in a subject, for example, a patient suffering from a cancer disease, is preferably an auto-protein in said individual. In preferred embodiments, the tumor-associated antigen in the context of the present invention is expressed under normal conditions specifically to a tissue or organ that is nonessential, that is, tissues or organs that when damaged by the immune system do not lead to the death of the subject, or in organs or structures of the body, which are not or only difficultly accessible by the immune system. Preferably, the amino acid sequence of the tumor-associated antigen is identical between the tumor-associated antigen that is expressed in normal tissues and the tumor-associated antigen that is expressed in cancer tissues.
[0062] Examples of differentiating antigens that ideally meet the criteria for tumor-associated antigens as target structures in tumor immunotherapy, in particular, in tumor vaccination are the cell surface proteins of the claudin family, such as CLDN18.2. Claudines are a family of proteins that are
29/118 the most important components of tight junctions, where they establish the paracellular barrier that controls the flow of molecules in the intercellular space between the cells of the epithelium. Claudines are transmembrane proteins that span the membrane 4 times with the N-terminus and the C-terminus, both located in the cytoplasm.
[0063] The term claudin 18 or CLDN18 preferably refers to human CLDN18 and includes any processing variants, such as CLDN18.1 and CLDN18.2 of CLDN18. CLDN18.1 and CLDN18.2 differ in the N-terminal portion comprising the first transmembrane (TM) and loopl region, while the primary C-terminal protein sequence is identical.
[0064] The term CLDN18.1 refers preferably to human CLDN18.1, and in particular to a protein comprising the amino acid sequence, according to SEQ ID NO: 1 of the amino acid sequence listing or a variant of that sequence.
[0065] The term CLDN18.2 refers preferably to human CLDN18.2, and, in particular, to a protein comprising the amino acid sequence according to SEQ ID NO: 2 of the amino acid sequence listing or a variant of that sequence.
[0066] The term variant, according to the invention refers, in particular, to mutants, variants of
30/118 splicing, isoforms, conformations, allelic variants, species variants and homologous species, in particular, those that are naturally present. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often unclear. Complete genetic sequencing often identifies numerous allelic variants of a given gene. A homologous species is a nucleic acid or amino acid sequence with a different species of origin than a given nucleic acid or amino acid sequence.
[0067] The terms CLDN, CLDN18, CLDN18.1 and CLDN18.2 cover all post-translationally modified variants and conformation variants.
[0068] CLDN18.2 is selectively expressed in normal tissues in epithelial cells differentiated from the gastric mucosa. CLDN18.2 is expressed in cancers of various origins, and is particularly suitable as the target structure for the development of antibody-mediated cancer immunotherapy, due to its selective expression (without expression in the toxicity in a corresponding normal tissue) and location to the plasma membrane. For example, CLDN18,2 was found to be expressed in pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, tumors and otorhinolaryngology. CLDN18.2 is a valuable target for the prevention and / or treatment of
11/31 primary tumors, such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer, such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, head-neck cancer and gallbladder cancer, and metastases thereof, in particular, gastric cancer metastasis, such as Krukenberg tumors, peritoneal metastasis and ganglionic metastasis. The cells that express CLDN18.2 are, preferably, cancer cells and are, in particular, selected from the group consisting of gastric tumorigenic, esophagus, pancreas, lung, ovary, colon, liver, head-neck, and cells of gallbladder cancer.
[0069] According to the invention, a cell expressing CLDN18.2 is preferably characterized by CLDN18.2 attached to the cell surface membrane, that is, CLDN18.2 is associated with the cell surface. In addition, according to the invention, cellular CLDN18.2 is preferably CLDN18.2 attached to the cell surface membrane. A cell expressing CLDN18.2 or a cell characterized by the association of CLDN18.2 with its cell surface is preferably a cancer cell, preferably a cancer cell from a cancer described herein.
[0070] The term associated with the cell surface means that an antigen associated with a tumor, such as
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CLDN18.2 is associated with and located on the plasma membrane of a cell, in which at least part of the tumor-associated antigen faces the extracellular space of that cell and is accessible from the outside of that cell, for example, by means of antibodies located outside the cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be made by one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that can be found in the outer leaflet of a plasma membrane. cell. For example, an antigen associated with a tumor associated with the surface of a cell may be a transmembrane protein having an extracellular portion, or it may be a protein associated with the surface of a cell through interaction with another protein, which is a transmembrane protein .
[0071] Cell surface or cell surface are used according to their normal meaning in the state of the art, and thus include the outer part of the cell that is accessible for binding proteins and other molecules.
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[0072] According to the invention, CLDN18.2 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression and association exceeds the level of expression and association in non-cancerous tissue except stomach not more than 2 times, preferably 1, 5 times, and preferably not to exceed the level of expression and association in which said non-cancerous tissue. Preferably, CLDN18.2 is not substantially expressed in a cell and is not substantially associated with a cell surface if the level of expression or association is below the detection limit and / or if the level of expression or association is very low to allow binding by CLDN18.2 specific antibodies added to cells.
[0073] According to the invention, CLDN18.2 is expressed in a cell and is associated with a cell surface if the level of expression and association exceeds the level of expression and association in non-cancerous tissue except stomach, preferably more than twice, preferably 10 times, 100 times, 1000 times or 10,000 times. Preferably, CLDN18.2 is expressed in a cell and is associated with a cell surface if the level of expression and association is above the detection limit and / or if the level of expression and association is high enough to allow binding by antibodies
34/118 specific - CLDN18.2 added to the cells.
[0074] The term antibody refers to a glycoprotein comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, and includes any molecule comprising an antigen-binding portion thereof. The term antibody includes monoclonal antibodies and antibody fragments or derivatives, including, without limitation, human antibodies, humanized antibodies, chimeric antibodies, single chain antibodies, for example, scFv and antigen-binding antibody fragments such as Fab and Fab 'also includes all recombinant forms of antibodies, for example, antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives as described herein. Each heavy chain is comprised of a variable region of the heavy chain (here abbreviated as VH) and a constant region of the heavy chain. Each light chain consists of a variable region of the light chain (here abbreviated as VL) and a constant region of the light chain. The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDR), interspersed with regions that are more conserved, called structural regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminal
35/118 for carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the light and heavy chains contain a binding domain that interacts with an antigen. The antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classic complement system.
[0075] The antibodies described herein can be human antibodies. The term human antibody, as used herein, is intended to include antibodies with variable and constant regions derived from immunoglobulin sequences of the human germline. The human antibodies of the invention can include amino acid residues not encoded by the immunoglobulin sequences of the human germline (for example, mutations introduced by random mutagenesis, or site specific in vitro or by somatic mutation in vivo).
[0076] The term humanized antibody refers to a molecule containing an antigen-binding site that is substantially derived from an immunoglobulin of a non-human species, in which the remaining immunoglobulin structure of the molecule is based on the structure and / or the following a human immunoglobulin. The antigen-binding site can include either complete variable domains
36/118 fused to the constant domains or only the complementarity determining regions (CDR) grafted into suitable structural regions in the variable domains. Antigen binding sites can be wild-type or modified by one or more amino acid substitutions, for example, modified to more closely resemble human immunoglobulins. Some forms of humanized antibodies preserve all CDR sequences (for example, a humanized mouse antibody that contains all six mouse antibody CDRs). Other forms have one or more CDRs that are altered with respect to the original antibody.
[0077] The term chimeric antibody refers to these antibodies, where a portion of each of the amino acid sequences of the light and heavy chains is homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a certain corresponding class, while that the rest of the chain segment is homologous to the corresponding sequences in another corresponding one. Typically, the variable region of both light and heavy chains, to mimic the variable regions of antibodies derived from one species of mammal, while the constant portions are homologous to antibody sequences derived from another. A clear advantage of such chimeric forms is that the region
37/118 variable can be conveniently derived from presently known sources using readily available B cells or hybridomas from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of ease of preparation and specificity is not affected by the source, the constant region being human, is less likely to elicit an immune response to a human individual when antibodies are injected than the constant region from from a non-human source. However, the definition is not limited to this particular example.
[0078] The terms of an antibody (or simply binding portion) or antigen-binding fragment of an antibody (or simply binding fragment) refer to one or more fragments of an antibody that retain the ability of the binding portion to antigen specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term antibody of an antigen binding portion include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) F (ab ') 2 fragments, divalent fragments comprising two Fab fragments linked by a
38/118 disulfide bridge in the hinge region; (iii) Fd fragments consisting of the VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody, (v) dAb fragments (Ward et al, (1989) Nature 341: 544 - 546), which consists of a VH domain; (vi) the isolated complementarity determining regions (CDR), and (vii) combinations of two or more CDR isolates that can optionally be joined by a synthetic linker. In addition, although the two Fv fragment domains, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows them to be made up of a single protein chain, in which the pairs from VL and VH regions form monovalent molecules (known as single chain Fv (scFv), see for example, Bird et al (1988) Science 242: 423 - 426, and Huston et al (1988) Proc Natl Acad Sci USA. USA 85: 5879 5883). Such single chain antibodies are also intended to be encompassed by the term antigen binding fragment of an antibody. Another example is immunoglobulin fusion proteins that bind to a domain comprising (i) a polypeptide binding domain that is fused to a polypeptide from the immunoglobulin chRNA region, (ii) a CH2 immunoglobulin heavy chain from constant region fused to the hinge region, and (iii) a heavy chain CH3 immunoglobulin from the constant region fused to the region
39/118 constant in CH2. The polypeptide of the binding domain can be a variable region of the heavy chain or a variable region of the light chain. The immunoglobulin fusion proteins that bind to the domain are further described in US 2003/01 18592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are searched for usefulness in the same way as antibodies are intact.
[0079] The antibodies described herein can be monoclonal antibodies. The term monoclonal antibody, as used herein, refers to a preparation of antibody molecules of the single molecular composition. A monoclonal antibody has a unique binding and affinity specificity. In one embodiment, monoclonal antibodies are produced by a hybridoma that includes a B cell obtained from a non-human animal, for example, a mouse fused to an immortalized cell.
[0080] The antibodies described herein can be recombinant antibodies. The term recombinant antibody, as used herein, includes all antibodies that are prepared, expressed, raised or isolated by recombinant means, such as: (a) antibodies isolated from an animal (for example, a mouse) that is transgenic or transcromosomal with respect for immunoglobulin genes
40/118 or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, for example, from a transfectome, (c) antibodies isolated from a recombinant, library combinatorial antibodies, and (d) antibodies prepared, expressed, raised or isolated by any other means that involve splicing the immunoglobulin gene sequences from other DNA sequences.
The term transfectoma, as used herein, includes recombinant eukaryotic host cells that express an antibody, such as CHO, NS / 0 cells, HEK293 cells, HEK293T cells, plant cells or fungi, including yeast cells.
[0082] As used herein, a heterologous antibody is defined in relation to a transgenic organism producing such an antibody. This term refers to an antibody having an amino acid sequence or a nucleic acid sequence encoding corresponding to that found in an organism that is not constituted, the transgenic organism, and being generally derived from an exception, the organism transgenic species.
[0083] As used herein, a heterohybrid antibody refers to an antibody having light and heavy chains of different organismic origins. For example, an antibody having a human heavy chain
41/118 associated with a murine light chain is a heterohybrid antibody.
[0084] The invention includes all antibodies and antibody derivatives, as described herein, which for the purposes of the present invention are encompassed by the term antibody. The term antibody derivatives refers to any modified form of an antibody, for example, an antibody conjugate and the other agent or antibody, or an antibody fragment.
[0085] The antibodies described herein are preferably isolated. An isolated antibody, as used herein, is intended to refer to an antibody that is substantially free of other antibodies that have different antigen specificities. In addition, an isolated antibody can be substantially free of other cellular material and / or chemicals.
[0086] According to the present invention, an antibody is able to bind to a predetermined target, if it has significant affinity for said predetermined target and binds to said predetermined target in conventional assays. Affinity or binding affinity is often measured by the dissociation equilibrium constant (KD). Preferably, the term significant affinity refers to binding to a predetermined target, with a dissociation constant (KD) of 10 ^-5 M or less,
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10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less, 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less .
[0087] An antibody is (substantially) unable to bind to a target that has no significant affinity for said target and does not bind significantly, in particular, it cannot detectably bind to said target in standard tests. Preferably, the antibody will not detectably bind to said target if it is present in a concentration of up to 2, preferably 10, more preferably 20, in particular 50 or 100 pg / ml or greater. Preferably, an antibody has no significant affinity for a target that binds to that target with a KD that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times greater than a Kd for binding to the predetermined target to which the antibody is capable of binding. For example, if the KD for binding an antibody to the target to which the antibody is capable of binding is 10 ^-7 M, the KD for binding to a target for which the antibody has no significant affinity would be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.
[0088] An antibody is specific for a predetermined target, if it is able to bind to that predetermined target, although it is not able to bind to others
43/118 targets, that is, it has no significant affinity for the other objectives and does not bind significantly to other targets in conventional assays. According to the invention, an antibody is specific for CLDN18.2 if it is capable of binding to CLDN18.2 but is not (substantially) capable of binding to other targets, in particular, proteins other than claudin proteins, of preferably proteins from other CLDN18, where other CLDN18.2 particular proteins. Preferably, an antibody is specific for CLDN18.2 if the affinity for binding and any other targets does not significantly exceed the affinity for binding proteins or claudin-independents, such as bovine serum albumin (BSA), casein, human serum (HSA) or non-claudin transmembrane proteins, such as MHC molecules or transferrin receptor or any other specified polypeptide. Preferably, an antibody is specific to a predetermined target, it binds to that target with a Kd that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 105 times, or 106 times less than than KD for binding to a target for which it is not specific. For example, if the KD for binding an antibody to the target for which it is specific is 10-7 M, the KD for binding to a target for which the antibody has no significant affinity would be at least 10 ^-6 M, 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M, or 10 ^-1 M.
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[0089] The binding of an antibody to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., antigen-antibody Interactions In Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY (1992), and the methods described here. Affinities can be easily determined using conventional techniques, such as by equilibrium dialysis; using the BIAcore 2000 instrument, through general procedures indicated by the manufacturer; by radioimmunoassay using the radiolabeled target antigen; or by any other method known to the person skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. SCL, 51: 660 (1949). The measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, for example, the concentration of salt, pH. Thus, measurements of affinity and other antigen binding parameters, for example, KD, IC50, are preferably made with standardized antibody and antigen solutions, and a standardized buffer.
[0090] As used herein, isotype refers to the class of antibodies (for example, IgM or IgGl) that is encoded by genes from the heavy chain constant regions. Antibodies according to the invention include antibodies
45/118 polyclonal and monoclonal antibodies and include antibodies IgG2a (e.g., IgG2a, κ, ã), IgG2b (e.g., IgG2b, K, ã), IgG3 (e.g., IgG3, κ, ã) and IgM. However, other antibody isotypes are also encompassed by the invention, including IgG1, igal, IgA2, secreted IgA, IgD, and IgE.
[0091] As used herein, isotype switching refers to the phenomenon by which the class, or isotype, of an antibody changes from an Ig class to one of the other Ig classes.
[0092] The term rearranged as used herein refers to a configuration of an immunoglobulin heavy or light chain in which the locus of a V segment is positioned immediately adjacent to a J or DJ segment in a conformation encoding essentially a VH domain or full VL, respectively. A rearranged immunoglobulin (antibody) locus of the gene can be identified by comparison with germline DNA; a rearranged locus will have at least one recombined heptamer / nonamer homology element.
[0093] The term germline configuration or not rearranged, as used herein in reference to a segment V refers to the configuration in which the segment V is not recombined so as to be immediately adjacent to a segment D or J.
[0094]
According to the invention, antibodies can
46/118 be derived from different species, including, but not limited to mice, rat, rabbit, guinea pig and human. Antibodies also include chimeric molecules, in which an antibody constant region derived from one species, preferably human, is combined with the antigen-binding site derived from another species. In addition, humanized antibodies include molecules in which the sites of an antibody derived from a non-human antigen-binding species are combined with constant and skeleton regions of human origin.
[0095] Antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, for example, the Kohler and Milstein standard somatic cell hybridization technique, Nature 256: 495 (1975). While somatic cell hybridization procedures are preferred, in principle, other techniques can be used for the production of monoclonal antibodies, for example, viral or B-lymphocyte transformation, or phage display techniques using antibody gene libraries .
[0096] The preferred animal system for the preparation of hybridomas that secrete monoclonal antibodies is the murine system. Hybridoma production in the rat is a very well established procedure. Immunization protocols and techniques for the isolation of immunized splenites for
47/118 fusion are known in the prior art. Fusion partners (for example, murine myeloma cells) and fusion procedures, are also known.
[0097] Other preferred animal systems for the preparation of hybridomas that secrete monoclonal antibodies are the rat and the rabbit system (for example, described in Spieker-POLET et al, Proc Natl Acad Sci USA 92: 9348 (1995), see also Rossi et al., Am J. Clin Pathol 124: 295 (2005)).
[0098] In yet another preferred embodiment, human monoclonal antibodies directed against CLDN18.2 can be generated using transgenic mice that carry transcromosomal or parts of the human immune system instead of the mouse system. These transgenic and transchromosomal mice include mice, known as HuMAb KM mice and rats, respectively, and are collectively referred to here as transgenic mice. The production of human antibodies in such transgenic mice can be performed as described in detail by CD20 in WO 2004 035607
[0099] However, another strategy for the production of monoclonal antibodies is to directly isolate genes that encode lymphocyte antibodies that produce antibodies of defined specificity; see, for example Babcock et al, 1996 .; An innovative strategy for the production of individual lymphocyte monoclonal antibodies
48/118 isolates produce antibodies of defined specificities. For more details on recombinant antibody engineering see also Welschof and Kraus, recombinant antibodies for cancer therapy ISBN-0-89603-918-8 and Benny KC Lo Antibody Engineering ISBN 1-58829-092-1.
[00100] To generate antibodies to CLDN18.2, mice can be immunized with peptides conjugated to carriers derived from the CLDN18.2 sequence, an enriched preparation of the CLDN18.2 antigen recombinantly expressed or its fragments and / or cells that express CLDN18.2 or fragments thereof, as described. Alternatively, mice can be immunized with DNA encoding human CLDN18.2 full length or fragments thereof. In the event that immunizations using a purified or enriched preparation of the CLDN18.2 antigen do not result in antibodies, mice can be immunized with cells that express CLDN18.2, for example, a cell line to promote immune responses.
[00101] The immune response can be monitored throughout the course of the immunization protocol with plasma and serum samples to be obtained through the tail vein or retro-orbital hemorrhages. Mice with sufficient anti-CLDN18.2 immunoglobulin titers can be used for fusions. Mice can be driven intraperitoneally or intravenously with cells expressing CLDN18.2 3-5 days
49/118 before sacrifice and removal of the spleen to increase the rate of specific antibody-secreting hybridomas.
[00102] To generate hybridomas that produce monoclonal antibodies to CLDN18.2, lymph node cells, spleen or bone marrow obtained from immunized mice can be isolated and fused with an appropriate immortalized cell line, such as a myeloma cell line of mouse. The resulting hybridomas can then be screened for the production of antibodies specific for the antigen. Individual wells can then be screened by ELISA for antibodies that secrete hybridomas. By immunofluorescence and FACS analysis using cells expressing CLDN18.2, antibodies with specificity for CLDN18.2 can be identified. The hybridoma-secreting antibody can be seeded again, sieved again and if still positive for anti-CLDN18.2 monoclonal antibodies can be subcloned by limiting dilution. The stable subclones can then be cultured in vitro to generate the antibody in tissue culture medium for characterization.
[00103] The antibodies of the invention can also be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods, as are well known in the art (Morrison, S. (1985)
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Science 229: 1202).
[00104] For example, in one embodiment, the gene (s) of interest, for example, antibody genes can be linked in an expression vector, such as a eukaryotic expression plasmid, as used by the gene expression system of GS disclosed in WO 87/04462, WO 89/01036 and EP 338 841 or other expression systems well known in the art. The plasmid purified with the cloned antibody genes can be introduced into eukaryotic host cells, such as CHO, NS / 0 cells, HEK293T cells or HEK293 cells or, alternatively, other eukaryotic cells, such as plant, fungal or yeast cells . The method used to introduce these genes can be those described in the prior art, such as electroporation, lipofectin, lipofectamine or others. After the introduction of these antibody genes into host cells, cells that express the antibody can be identified and selected. These cells represent the transfectomas that can then be amplified to their level of expression and then scaled up to produce antibodies. Recombinant antibodies can be isolated and purified from these culture supernatants and / or cells.
[00105] Alternatively, the cloned antibody genes can be expressed in other expression systems,
51/118 including prokaryotic cells, such as microorganisms, for example, E. coli. In addition, antibodies can be produced in transgenic animals, such as in sheep and rabbit milk or in chicken eggs, or in transgenic plants; for example, see Verma, R., et al. (1998) J. Immunol. Meth. 216: 165 - 181; Pollock et al. (1999) J. Immunol. Meth. 231: 147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825 - 839.
[00106] Chimeric antibodies are antibodies, the different parts of which are derived from different animal species, such as those that have a variable region derived from a murine antibody and a human immunoglobulin constant region. Chimerization of antibodies is achieved by joining the variable regions of the murine heavy and light chain of antibodies with the human heavy and light chain constant region (for example, as described by Kraus et al., In Methods in Molecular Biology, series of antibodies recombinants for cancer ISBN 0-89603-918-8therapy). In a preferred embodiment, chimeric antibodies are produced by uniting the human kappa-constant light chain to the murine light chain variable region. In a also preferred embodiment, chimeric antibodies can be produced by joining the lambda light chain from the human constant region to the murine light chain variable region. The heavy chain regions
52/118 preferred for the production of chimeric antibodies are IgGl, IgG3 and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD and IgM.
[00107] Antibodies interact with target antigens predominantly through amino acid residues that are located in the complementarity determining regions of six heavy and light chains (CDR). For this reason, the amino acid sequences in the CDR are more diverse between individual antibodies than the sequences of the outside CDRs. Since CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of naturally occurring specific antibodies by constructing expression vectors that include CDR sequences from the naturally occurring antibody grafted to specific structural sequences of a different antibody with different properties (see, for example, Riechmann, L. et al (1998) Nature 332: 323 - 327; Jones, P. et al (1986) Nature 321: 522-525; and Queen, C. et (1989) Proc Natl Acad Sci USA 86: 10029-10033). Such framing sequences can be obtained from Public DNA databases that include germline antibody gene sequences. These germline sequences will be different from mature antibody gene sequences, because they will not include the genes
53/118 fully assembled variables, which are formed by V (D) J during binding to B cell maturation. Sequences of germline genes will also differ from the sequences of a secondary repertoire of high affinity antibody at individual positions uniformly across the variable region. For example, somatic mutations are relatively rare in the amino-terminal part of structure region 1 and the carboxy terminal portion of structure region 4. In addition, many somatic mutations do not significantly alter the binding properties of the antibody. For this reason, it is not necessary to obtain the entire DNA sequence of a particular antibody in order to recreate an intact recombinant antibody having binding properties similar to that of the original antibody (see WO 99/45962). Partial light and heavy chain sequences spanning the CDR regions are generally sufficient for this purpose. The partial sequence is used to determine which variable and segments of the germline gene they unite contributed to the recombinant antibody variable genes. The germline sequence is then used to fill, in portions of the missing variable regions. Leading heavy and light chain sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody. To add the missing strings, the cDNA strings
54/118 clones can be combined with synthetic oligonucleotides binding or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of overlapping shorts, oligonucleotides and combined through PCR amplification to create a fully synthetic variable region clone. This process has certain advantages, such as the elimination or inclusion or particular restriction sites, or optimization of specific codons.
[00108] The nucleotide sequences of the heavy and light chain transcripts from hybridomas can be used to design an overlapping set of synthetic oligonucleotides to create synthetic V sequences with the ability to encode amino acids identical to natural sequences. The sequences of the synthetic heavy and kappa chains can differ from the natural sequences in three ways: repeated nucleotide base chains are disrupted to facilitate oligonucleotide synthesis and PCR amplification; ideal translation initiation sites are incorporated according to Kozak rules (Kozak, 1991, J. Biol Chem 266: 19867 - 19870); and Hind II locations! they are designed upstream of the translation initiation sites.
[00109] For both variable regions of the heavy and light chain, the corresponding optimized coding and not coding, chain strings are divided into 30 - 50
55/118 nucleotides, approximately, at the center point of the corresponding non-coding oligonucleotide. Thus, for each chain, the oligonucleotides can be assembled in double overlapping sets that cover unrecoverable segments of 150 - 400 nucleotides. The mixture is then used as templates to produce 150 - 400 nucleotide PCR amplification products. Typically, a single set of variable region oligonucleotides will be divided into two sets that are amplified separately to generate two overlapping PCR products. These overlapping products are then combined through PCR amplification to form the complete variable region. It may also be desirable to include an overlapping fragment of the heavy or light chain constant in the PCR amplification region to generate fragments that can be easily cloned into the expression vector constructs.
[00110] The variable regions of the reconstructed chimerized or humanized light and heavy chain are then combined with the cloned, leader promoter, translation initiation, constant region, 3 'untranslated, polyadenylation, and transcription termination sequences for form the expression constructs of the vector. The heavy and light chain expression constructs can be combined into a single vector, co-transfected, serially transfected, or separately transfected into cells
56/118 hosts that are then fused to form a host cell that expresses both chains. Plasmids for use in the construction of expression vectors for human IgGK are described. Plasmids can be constructed so that amplified PCR V kappa and heavy V light chain cDNA sequences can be used to reconstruct complete heavy and light chain minigenes. These plasmids can be used to express completely human, or chimeric IgGl, IgG4 or Kappa, Kappa antibodies. Similar plasmids can be constructed for the expression of other heavy chain isotypes, or for the expression of antibodies comprising lambda light chains.
[00111] Thus, in another aspect of the invention, the structural characteristics of the anti-CLDN18.2 antibodies described herein, are used to create structurally related humanized anti-CLDN18.2 antibodies that retain at least one functional property of the antibodies of the invention, such as connection to CLDN18.2. More specifically, one or more CDR regions of mouse monoclonal antibodies can be combined recombinantly with known human framework regions and CDRs to recombinantly engineer additional, humanized anti-CLDN18.2 antibodies.
[00112] The ability of an antibody to bind to CLDN18.2 can be determined using binding assays
57/118, for example, ELISA, Western Blot, immunofluorescence and flow cytometric analysis. ELISA can be used to demonstrate the presence of antibodies in the serum of immunized mice or the binding of monoclonal antibodies to CLDN18.2 proteins or peptides. The peptides or proteins used for immunization can be used to determine the specificity of hybridoma supernatants or to analyze serum titers.
[00113] In order to demonstrate the presence of antibodies in immunized mice sera or monoclonal antibody binding to living cells, flow cytometry can be used. Cell lines that express naturally or after transfection antigens and negative controls lack antigen expression (grown under normal growth conditions) can be mixed with various concentrations of monoclonal antibodies in the hybridoma supernatant or in PBS containing 1% FBS, and can be incubated at 4 ° C for 30 min. After washing, the anti-Alexa647 or APC-labeled IgG antibody can bind to the monoclonal antibody bound to the antigen according to the same conditions as the staining of the primary antibody. The samples can be analyzed by flow cytometry with a FACS instrument using light and lateral dispersion properties, the door in individual living cells. In order to distinguish monoclonal antibodies specific for the
58/118 antigen from non-specific ligands from a single measurement, the co-transfection method can be employed. Cells transiently transfected with plasmids encoding the antigen and a fluorescent marker can be stained, as described above. The transfected cells can be detected in a different fluorescence channel than cells labeled with the antibody. Since most transfected cells express both transgenes, antigen-specific monoclonal antibodies preferentially bind to cells that express the fluorescence marker, whereas non-specific antibodies bind in a comparable proportion of non-transfected cells. An alternative assay using fluorescence microscopy can be used in addition to or instead of the flow cytometry assay. The cells can be stained exactly as described above and examined by fluorescence microscopy.
[00114] In order to demonstrate the presence of anti-CLDN18.2 antibodies in immunized mouse sera or monoclonal antibody binding to living cells that express CLDN18.2, immunofluorescence microscopy analysis may be used. For example, cell lines that express either spontaneously or after CLDN18.2 transfection and negative controls lacking CLDN18.2 expression are grown on growth chamber slides, under
59/118 standard conditions in DMEM F12 medium, supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 IU / ml penicillin and 100 pl streptomycin. The cells can then be fixed with methanol or paraformaldehyde or left untreated. The cells can then react with the monoclonal antibodies against CLDN18.2 for 30 min. at 25 ° C. After washing, cells can react with a secondary Alexa555-labeled anti-mouse IgG antibody (Molecular Probes), under the same conditions. The cells can then be examined by fluorescence microscopy.
[00115] The total levels of CLDN18.2 in cells can be observed when the cells are fixed methanol or paraformaldehyde fixed and permeabilized with Triton X-100. In living, non-permeabilized cells, fixed cells paraformaldehyde surface location of CLDN18.2 can be examined. Additionally CLDN18.2 segmentation of tight junctions can be analyzed by co-staining with tight junction markers such as ZO-1. In addition, the effects of antibody binding and CLDN18.2 location within the cell membrane can be examined.
[00116] Anti-IgG CLDN18.2 can be further tested for reactivity with the CLDN18.2 antigen by Western Blotting. Briefly, cell extracts from cells expressing CLDN18.2 and appropriate negative controls can be prepared and subjected to
60/118 sodium dodecyl sulfate electrophoresis (SDS) in polyacrylamide gel. After electrophoresis, the separated antigens will be transferred to nitrocellulose membranes, blocked, and probed with the monoclonal antibodies to be tested. Binding IgG can be detected using anti-mouse IgG peroxidase and developed with ECL substrate.
[00117] Anti-mouse IgG CLDN18.2 can be further tested for reactivity with the CLDN18.2 antigen by immunohistochemistry in a manner well known to a person skilled in the art, for example, using acetone or fixed paraformaldehyde cryosections or cuts of paraffin-embedded tissue fixed with paraformaldehyde from non-cancer tissue or tissue samples obtained from cancer patients during routine surgical procedures or from mice bearing xenografted tumors inoculated with cell lines that spontaneously express or after transfection with CLDN18.2. For immunostaining, antibodies reactive to CLDN18.2 can be incubated followed by conjugated goat horseradish peroxidase or goat anti-rabbit antibodies according to the manufacturer's instructions.
[00118] A particularly preferred methodology for testing CLDN18.2 in the methods of the invention is immunohistochemistry or IHC. Immunohistochemistry or IHC refers to the process of detecting antigens (for example, proteins) in the cells of
61/118 a tissue cut, for example, the tissue cells mentioned here. Immunohistochemical staining is widely used in the diagnosis of abnormal cells, such as those found in cancerous tumors. Visualization of an antibody-antigen interaction can be accomplished in a number of ways. In the most common case, the antibody is conjugated to an enzyme, such as peroxidase, which can catalyze a color-producing reaction. Alternatively, the antibody can also be labeled with a fluorophore, such as fluorescein or rhodamine.
[00119] Sample preparation is critical to maintain cell morphology, tissue architecture and antigenicity of target epitopes. This requires adequate tissue collection, fixation and cutting. Paraformaldehyde is generally used with fixation. Depending on the purpose and thickness of the experimental sample, (about 4-40 μπι) thin sections are cut from the fabric of interest, or if the fabric is not very thick and is penetrable it is used all over. The cut is usually carried out using a microtome, and the slices are mounted on blades.
[00120] The sample may require additional steps for the available epitopes for antibody binding, including dewaxing and antigen recovery. Detergents such as Triton X-100 are generally used in immunohistochemistry to reduce surface tension,
62/118 allowing less of the reagent to be used to achieve better and more uniform coverage of the sample.
[00121] The direct immunohistochemical staining method uses a labeled antibody, which binds directly to the antigen to be stained for. The most common indirect method of immunohistochemical staining uses an antibody against the antigen to be investigated for, and a second, labeled, antibody against the first.
[00122] To reduce background staining in the IHC, samples are incubated with a buffer that blocks the reactive sites to which the primary or secondary antibodies can otherwise bind. Primary antibodies are raised against an antigen of interest and are generally unconjugated (unlabeled), while secondary antibodies are raised against immunoglobulins of the primary antibody species. The secondary antibody is normally conjugated to a binding molecule, such as biotin, which then recruits reporter molecules, or the secondary antibody is bound directly to the reporter molecule itself. Common blocking buffers include normal whey, non-fat milk powder, BSA or gelatin, and commercial blocking buffers.
[00123] Reporter molecules vary based on the nature of the detection method, and the most popular methods of detection are with chromogenic and fluorescence detection and
63/118 fluorophore-mediated enzyme, respectively. With chromogenic reporters, an enzyme tag reacts with a substrate to produce an intensely colored product that can be analyzed with a standard light microscope. Although the list of enzyme substrates is long, alkaline phosphatase (AP) and horseradish peroxidase (HRP) are the two enzymes most widely used as markers for protein detection. A variety of chromogenic, fluorogenic and chemiluminescent substrates are available for use with any of the enzymes, including DAB or BCIP / NBT. Fluorescent reporters are small organic molecules used for the detection of HCI. For chromogenic and fluorescent detection methods, densitometric signal analysis can provide semi-quantitative and fully data, respectively, to correlate the reporter signal level to the level of protein expression or localization.
[00124] After immunohistochemical staining of the target antigen, a second staining is often applied to provide contrast that helps the main stain to stand out. Many of these stains show specificity for discrete cell compartments or antigens, while the others will color the entire cell. Both chromogenic and fluorescent dyes are available for IHC to provide a wide range of reagents to
64/118 adjust each experimental design. Hematoxylin, Hoechst staining and DAPI are commonly used.
[00125] Mapping of epitopes recognized by antibodies can be performed, as described in detail in Epitope Mapping Protocols (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in Epitope Mapping: A Practical Approach Practical Approach Series, 248 by Olwyn MR Westwood, Frank C. Hay.
[00126] The term immune effector functions in the context of the present invention includes all functions mediated by components of the immune system that result in inhibition of tumor growth and / or inhibition of tumor development, including inhibition of the spread of tumors and metastases. Preferably, immune effector functions result in the death of cancer cells. Preferably, the immunological effector functions in the context of the present invention are antibody-mediated effector functions. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells that carry the tumor-associated antigen, for example, by binding the antibody to a surface antigen, inhibition of CD40L-mediated signal transduction, for example, by
65/118 binding of the antibody to the CD40 or CD40 receptor (CD40L), and / or inhibiting the proliferation of cells that carry the tumor-associated antigen, preferably from ADCC and / or CDC. Thus, antibodies that are able to mediate one or more immune effector functions are preferably capable of mediating cell death by inducing lysis, ADCC-mediated lysis, apoptosis, homotypic adhesion, and / or phagocytosis CDC-mediated, preferably by inducing CDC-mediated lysis and / or ADCC-mediated lysis. Antibodies can also have an effect simply by binding to tumor-associated antigens on the surface of a cancer cell. For example, antibodies can block the function of a tumor-associated antigen or induce apoptosis by binding only to the tumor-associated antigen on the surface of a cancer cell.
[00127] ADCC describes the ability to kill cells from effector cells, in particular lymphocytes, which preferably requires the target cell to be labeled by an antibody. ADCC preferably occurs when antibodies bind to antigens in cancer cells and the antibody Fe domains involve Fc receptors (FcR) on the surface of immune effector cells. Several families of Fe receptors have been identified, and specific cell populations characteristically express defined Fc receptors. ADCC can be seen as a mechanism to directly induce a
66/118 variable degree of destruction of the immediate tumor that also leads to the presentation of antigen and the induction of T cell responses directed to the tumor. Preferably, in vivo, induction of ADCC will lead to tumor-directed T cell responses and other host-derived antibody responses.
[00128] CDC is another method of killing cells that can be driven by antibodies. IgM is the most effective isotope for complement activation. IgGl and IgG3 are also very effective in the CDC direction through the classic complement activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in the unveiling of CLQ multiple binding sites in close proximity in the CH2 domains of participating antibody molecules, such as IgG molecules (Clq is one of the three subcomponents of the complement Cl ). Preferably, these uncovered Clq binding sites convert the previously low affinity Clq-IgG interaction to one of high avidity, which triggers a cascade of events involving a series of other complement proteins and leads to the proteolytic release of cell chemotactics effector / activation agents C3a and C5a. Preferably, the complement cascade ends in the formation of a membrane attack complex, which creates pores in the cell membrane that facilitate the free passage of water and solutes into and out of the cell and can
67/118 lead to apoptosis.
[00129] The term immunological effector cells, in the context of the present invention refers to cells that exercise effector functions during an immune reaction. For example, such cells secrete cytokines and / or chemokines, kill microbes, secrete antibodies, recognize cancer cells, and optionally, eliminate those cells. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages and dendritic cells.
[00130] A nucleic acid is, according to the invention, preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), more preferably, RNA, more preferably, in RNA transcribed in vitro (RNA IVT). Nucleic acids include, according to the invention, genomic DNA, cDNA, mRNA, prepared recombinantly and chemically synthesized molecules. A nucleic acid can, according to the invention, be in the form of a molecule that is single or double stranded and linear or covalently closed to form a circle. A nucleic acid can be used for introduction, that is, the transfection of cells, for example, in the form of RNA, which can be prepared by in vitro transcription from a DNA template. The RNA can also be modified before application through
68/118 sequence stabilization, capping and polyadenylation.
[00131] The nucleic acids described herein can be included in a vector. The term vector, as used herein, includes any vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phages, viral vectors, such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), artificial or PI chromosomes (PAC). Said expression vectors include, as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and generally contain a desired coding sequence and DNA sequences necessary for the proper expression of the operably linked coding sequence in a particular host organism (eg, bacteria, yeast, plants , insects, or mammalian) or in vitro expression systems. Cloning vectors are generally used for engineering and amplifying a particular desired DNA fragment and may not have the necessary functional sequences for the expression of the desired DNA fragments.
[00132] Like the vector for the expression of an antibody, or a type of vector, in which the antibody chains are present in different vectors or a type of vector, in which
69/118 the antibody chains are present in the same vector can be used.
[00133] As used herein, the term RNA means a molecule that comprises at least one ribonucleotide residue. By ribonucleotide is meant a nucleotide with a hydroxyl group at the 2 'position of a beta-D-ribo-furanose radical. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as altered RNA that differs from naturally occurring RNA by adding , deletion, substitution and / or alteration of one or more nucleotides. Such modifications may include the addition of non-nucleotide material, such as to the end (s) of an RNA or internally, for example, in one or more nucleotides of the RNA. Nucleotides of RNA molecules can further comprise unconventional nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as naturally occurring RNA analogues or analogues.
[00134] According to the present invention, the term RNA includes, and preferably refers to, mRNA, which means messenger RNA and refers to a transcription, which can be produced using as a DNA template and encodes a
70/118 peptide or protein. MRNA typically comprises an untranslated region, a protein or a peptide and a 3 'coding region of an untranslated region 5. mRNA has a limited half-life in cells and in vitro.
[00135] In the context of the present invention, the term transcription refers to a process, in which the genetic code in a DNA sequence is transcribed within the RNA.
[00136] The described nucleic acids according to the invention have preferably been isolated. The term isolated nucleic acid means, according to the invention, that the nucleic acid has been (i) amplified in vitro, for example, by polymerase chain reaction (PCR), (ii) produced recombinantly by cloning, (iii) purified, for example, by cleavage and fractionation by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation using recombinant DNA techniques.
[00137] Nucleic acids can, according to the invention, be present alone or in combination with other nucleic acids, which can be homologous or heterologous. In preferred embodiments, a nucleic acid that is functionally linked to expression control sequences, can be homologous or heterologous to said nucleic acid. The term homologous means that acids
71/118 nucleic acids are also functionally linked naturally and the term heterologous indicates that nucleic acids are not functionally linked naturally.
[00138] A nucleic acid and an expression control sequence are functionally linked to each other, if they are covalently linked to each other, in such a way that the expression or transcription of said nucleic acid is under control or under the influence of said expression control sequence. If the nucleic acid is to be translated into a functional protein, then with an expression control sequence functionally linked to a coding sequence, the induction of said expression control sequence results in the transcription of said nucleic acid, without cause a change of frame in the coding sequence or said coding sequence from not being able to be translated into the desired protein or peptide.
[00139] The term expression control sequence or expression control element comprises, according to the invention, promoters, ribosomes, enhancer sites and other control elements that regulate the transcription of a gene or translation of a mRNA from Link. In particular embodiments of the invention, expression control sequences can be regulated. The exact structure of expression control sequences can vary depending on
72/118 function of species or cell type, but in general comprises 5'-transcribed and 5'-and 3 'untranslated sequences that are involved in initiating transcription and translation, respectively, such as TATA BOX, capping sequence; CAAT string, and the like. More specifically, the 5 'non-transcribed expression control sequences comprise a promoter region that includes a promoter sequence for controlling functionally linked nucleic acid transcription. Expression control sequences can also include enhancer sequences or upstream activator sequences.
[00140] According to the invention, the term promoter or promoter region refers to a nucleic acid sequence, which is located upstream (5 ') with the nucleic acid sequence to be expressed and controls the expression of the sequence of providing a recognition and binding site for RNA polymerase. The promoter region can also include recognition and binding sites for other factors that are involved in regulating the transcription of a gene. A promoter can control the transcription of a prokaryotic or eukaryotic gene. In addition, a promoter may be inducible and may initiate transcription in response to an inducing agent or may be constitutive if the transcription is not controlled by an inducing agent. A gene that is under the control of an inducible promoter is not expressed, or
73/118 expressed only to a small extent, if an inducing agent is absent. In the presence of the gene-inducing agent it is turned on or the level of transcription is increased. This is mediated, in general, through the binding of a specific transcription factor.
[00141] Promoters that are preferred according to the invention include the SP6, T3 and T7 polymerase promoters, U6 Human RA promoter, the CMV promoter, and artificial hybrid promoters thereof (for example, CMV), where one or further parts are fused with a part or parts of promoters from the genes of other cellular proteins, such as, for example, from human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and whether or not they include (an) additional intron (s).
[00142] The term expression is used here in its broadest sense and includes the production of RNA or RNA and protein or peptide. With regard to RNA, the term expression or translation refers, in particular, to the production of peptides or proteins. The expression can be transient or it can be stable. According to the invention, the term expression also includes an aberrant expression or an abnormal expression.
[00143] Aberrant expression or abnormal expression means, according to the invention, that the expression is preferably changed, increased, in comparison with a reference, for example, a state in a subject does not have a
74/118 disease associated with the aberrant or abnormal expression of a certain protein, for example, an antigen associated with a tumor. An increase in expression refers to an increase of at least 10%, in particular, at least 20%, at least 50% or at least 100%, or more. In one embodiment, the expression is found only in diseased tissue, while the expression of healthy tissue is suppressed.
[00144] The term specifically expressed means that a protein is essentially expressed only in a specific tissue or organ. For example, an antigen associated with a tumor specifically expressed in the gastric mucosa, that protein is mainly expressed in the gastric mucosa, and is not expressed in other tissues or is not significantly expressed in other types of tissues or organs . Thus, a protein that is exclusively expressed in cells of the gastric mucosa and a significantly smaller proportion in any other tissue is specifically expressed in cells of the gastric mucosa. In some embodiments, an antigen associated with a tumor can also be expressed specifically under normal conditions, in more than one type of tissue or organ, such as in two or three types of tissues or organs, but preferably not more than three different types of tissues or organs. In this case, the tumor-associated antigen is then specifically expressed in these organs.
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[00145] The term conversion, according to the invention relates to the process in the ribosomes of a cell, through which a messenger RNA chain directs the assembly of an amino acid sequence to produce a protein or peptide.
[00146] According to the invention, the term nucleic acid which encodes means that the nucleic acid, if present in the appropriate medium, preferably within a cell can be expressed to produce a protein or a peptide which it encodes.
[00147] The term peptide includes oligo- and polypeptides and refers to substances that comprise two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and preferably up to 8, 10, 20, 30, 40 or 50, in particular, 100 amino acids covalently linked via peptide bonds. The term protein refers to large peptides, preferably for peptides with more than 100 amino acid residues, but, in general, the terms peptides and proteins are synonymous and are used here interchangeably.
[00148] Preferably, the proteins and peptides described according to the invention have been isolated. The terms
76/118 isolated protein or peptides alone means that the protein or peptide has been separated from its natural environment. An isolated protein or peptide can be in an essentially purified state. The term essentially purified means that the protein or peptide is essentially free of other substances with which it is associated in nature or in vivo.
[00149] The teaching given here with respect to specific amino acid sequences, for example, those shown in the sequence listing, is to be interpreted so as to also relate to changes, that is, variants, of said specific sequences that result in sequences that are functionally equivalent to diats specific sequences, for example, amino acid sequences that exhibit properties identical or similar to those of specific amino acid sequences. An important property is to retain the binding of an antibody to its target. Preferably, a modified sequence with respect to a specific sequence, when replacing the specific sequence of an antibody retains the binding of said antibody to the target.
[00150] It will be appreciated by experts versed in the subject that, in particular, the sequences of the CDR sequences, hypervariable and variable regions can be modified without losing the ability to bind to a target. For example,
77/118 CDR sequences will be identical or highly homologous to the CDR sequences specified herein.
[00151] By highly homologous it is contemplated that from 1 to 5, preferably from 1 to 4, such as from 1 to 3, or 1 or 2 can be substituted.
[00152] The term variant according to the invention also includes mutants, splicing variants, isoforms, conformations, allelic variants, species variants and species homologs, in particular, those that are naturally present. An allelic variant refers to a change in the normal sequence of a gene, the meaning of which is often unclear. Complete genetic sequencing often identifies numerous allelic variants of a given gene. A homologous species is a nucleic acid or amino acid sequence with a different species of origin than a given nucleic acid or amino acid sequence.
[00153] For the purposes of the present invention, variants of an amino acid sequence comprise amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. Variants of amino acid deletion that comprise the N-terminal and / or C-terminal deletion of the protein are also called C-terminal and / or N-terminal truncation variants.
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[00154] Amino acid insertion variants comprise single or two or more amino acid insertions in a particular amino acid sequence. In the case of amino acid sequence insertion variants, one or more amino acid residues are introduced at a particular site in an amino acid sequence, although random insertion, with appropriate screening of the resulting product is also possible.
[00155] Amino acid addition variants comprise amino and / or carboxy-terminal fusions of or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.
[00156] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, such as by removing 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Deletions can be in any position of the protein.
[00157] The amino acid substitution variants are characterized by at least one residue in the sequence to be removed and another residue to be inserted in its place. Preference is given to modifications being in positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or the replacement of amino acids with others that have similar properties. Preferably, changes in amino acids in protein variants are changes in conservative amino acids,
79/118 that is, similarly charged or unloaded amino acid substitutions. A conservative amino acid change involves replacing one of a family of amino acids that are related in its side chains. Naturally occurring amino acids are generally divided into four families: acid (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and not polar charged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan and tyrosine are sometimes classified together as aromatic amino acids.
[00158] Preferably, the degree of similarity, preferably identity between a given amino acid sequence and a sequence of amino acids that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or 99%. The degree of similarity or identity is given preference for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50 %, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the
80/118 reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is given preference, by at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably the continuous amino acids. In preferred embodiments, the degree of similarity or identity is determined by the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity can be performed with the tools of the known art, preferably using the best sequence alignment, for example, using Align, using standard settings, preferably EMBOSS :: needle, Matrix: Blosum62, Open Gap 10.0, Gap Extend 0.5.
[00159] Sequence similarity indicates the percentage of amino acids that are either identical or that represent conservative amino acid substitutions. Sequence identity between two amino acid sequences indicates the percentage of identical amino acids between the sequences.
[00160] The term percent identity is intended to denote a percentage of amino acid residues that are identical between the two sequences to be compared, obtained
81/118 after the best alignment, this percentage being purely statistical and the differences between the two sequences to be distributed randomly and along its entire length. The sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences, after having optimally aligned them, said comparison being made by the segment or by the comparison window to identify and compare local regions of sequence similarity. The ideal alignment of the strings for comparison can be produced, in addition manually, using the local homology algorithm of Smith and Waterman, 1981, App ads. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, using Pearson and Lipman's similarity search method, 1988, Proc. Natl Acad. Know. USA 85, 2444, or through computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin).
[00161] The percentage of identity is calculated by determining the number of identical positions between the two sequences to be compared, dividing this value by the number of comparison positions and multiplying the result obtained by 100, in order to obtain the percentage of identity between these two strings.
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[00162] Homologous amino acid sequences exhibit, according to the invention, at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98, or at least 99% identity of the amino acid residues.
[00163] The amino acid sequence variants described herein can be readily prepared by the person skilled in the art, for example, by manipulating the recombinant DNA. The manipulation of DNA sequences for the preparation of proteins and peptides that have substitutions, additions, insertions or deletions, is described in detail in Sambrook et al. (1989), for example. In addition, the peptides and amino acid variants described herein can be easily prepared with the aid of known peptide synthesis techniques, such as, for example, by solid phase synthesis and similar methods.
[00164] The invention covers derivatives of the peptides or proteins described herein, which are composed of the terms peptide and protein. According to the invention, protein and peptide derivatives are forms of modified proteins and peptides. Such modifications include any chemical modification and comprise single or multiple substitutions, deletions and / or additions of any molecule associated with the protein or peptide, such as
83/118 carbohydrates, lipids and / or proteins or peptides. In one embodiment, protein or peptide derivatives include the modified analogs resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of blocking / protecting groups, proteolytic or binding cleavage to an antibody or another cell ligand. The derived term also extends to all functional chemical equivalents of said proteins and peptides. Preferably, a modified peptide has greater stability and / or increased immunogenicity.
[00165] According to the invention, a variant thereof, derivative, modified form, fragment, a part or portion of an amino acid, peptide or protein sequence, preferably has a functional property of the amino acid, peptide or protein sequence, respectively , from which it was derived, that is, which is functionally equivalent. In one embodiment, a variant, derivative, modified form, fragment, a part or portion of an amino acid, peptide or protein sequence is immunologically equivalent to the amino acid, peptide or protein sequence, respectively, from which it was derived. In one embodiment, the functional property is an immunological property.
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[00166] The term derivative means according to the invention that a particular entity, in particular a particular sequence, is present in the object from which it is derived, in particular an organism or molecule. In the case of amino acid sequences, in particular the regions of specific sequences, derivatives, in particular, means that the relevant amino acid sequence is derived from an amino acid sequence in which it is present.
[00167] The term cell or host cell is preferably an intact cell, that is, a cell with an intact membrane that does not release its normal intracellular components, such as enzymes, organelles, or genetic material. An intact cell is preferably a viable cell, that is, a living cell capable of carrying out its normal metabolic functions. The term cell includes according to the invention prokaryotic cells (for example, E. coli) or eukaryotic cells (for example, dendritic cells, B cells, CHO cells, COS cells, K562 cells, HEK293 cells, HeLa cells, yeast, and insect cells). Mammalian cells are particularly preferred, such as the cells of humans, mice, hamsters, pigs, goats and primates. Cells can be derived from a large number of tissue types and include primary cells and cell lines. The term cell includes cells and cells
85/118 non-cancerous carcinogens, such as cells of the types of cancer disclosed herein.
[00168] A cell comprising a nucleic acid molecule, preferably expressing the peptide or protein encoded by the nucleic acid.
[00169] Target cell, a cell that is the target of an immune response, such as an antibody. The target cells include any undesirable cells such as a cancer cell, as described herein. In preferred embodiments, the target cell is a cell that expresses CLDN18.2. Cells that express CLDN18.2 typically include cancer cells.
[00170] The terms transgenic animal refers to an animal with a genome that comprises one or more transgenes, preferably heavy and / or light transgenic antibodies, or transchromosomes (either integrated or not integrated into the animals' natural genomic DNA) and which is preferably capable of expressing the transgenes. For example, a transgenic mouse may have a human heavy and light chain human transgene, either a human chain transgene or heavy chain transchromosome, such that the mouse produces human anti-CLDN18.2 antibodies when immunized with CLDN18 antigen. 2 and / or cells that express CLDN18.2. The human heavy chain transgene can be integrated into the mouse chromosomal DNA, as is the case for mice
86/118 transgenics, for example, HuMAb mice, such as mice or HCo7 HCol2, or the human heavy chain transgene can be maintained extrachromosomally, as is the case for transchromosomal (eg, KM) mice, as described in WO 02/43478. Such transgenic and transchromosomal mice may be able to produce various isotypes of human monoclonal antibodies to CLDN18.2 (for example, IgG, IgA and / or IgE) by undergoing a VDJ and isotype recombination.
[00171] The term immunologically equivalent means that the immunologically equivalent molecule, such as the immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effects, for example, as regards with respect to the type of the immune effect, such as the induction of a humoral immune reaction, the strength and / or duration of the induced immune reaction, or the specificity of the immune reaction. In the context of the present invention, the term immunologically equivalent is used preferably in connection with the immunological effects or properties of a peptide or peptide variant used for immunization, or an antibody. A specific immunological property is the ability to bind to antibodies and, if necessary, produce an immune response, preferably through
87/118 stimulation of antibody production. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence, if that amino acid sequence when exposed to an individual's immune system induces an immune reaction, preferably antibodies, which have a specific reaction, with the reference amino acid sequence, such as the amino acid reference sequence forming part of CLDN18.2.
[00172] The invention provides methods for detecting the presence of the antigen in a CLDN18.2 sample, or by measuring the amount of CLDN18.2 antigen, comprising contacting the sample, and, optionally, a control sample, with an antibody of the invention that binds to CLDN18.2, under conditions that allow the formation of a complex between the antibody and CLDN18.2. The formation of a complex is then detected, in which a difference in complex formation between the sample compared to the control sample, is indicative of the presence of antigen in the sample CLDN18.2.
[00173] Methods as described above are useful, in particular, for the diagnosis of CLDN18.2 related diseases such as cancerous diseases. Preferably, an amount of CLDN18.2 in a sample, which is higher than the amount of CLDN18.2 in a reference or control sample, is indicative of the presence of a related disease
88/118 with CLDN18.2 in a subject, in particular a human, from which the sample is derived.
[00174] When used in methods as described above, an antibody described herein, can be provided with a label that works to: (i) provide a detectable signal; (Ii) interact with a second marker to modify the detectable signal provided by the first or second marker, for example, TERF (fluorescence resonance energy transfer); (iii) affect mobility, for example, electrophoretic mobility, by charge, hydrophobicity, shape, or other physical parameters, or (iv) provide a capture unit, for example, affinity, antibody / antigen, or ionic complexation . Indicated as a marker are structures such as fluorescent markers, luminescent markers, chromophoric markers, radioisotope markers, isotopic markers, stable isotopic markers, preferably isobaric markers, enzymatic markers, particle markers, in particular, metal particle markers, markers magnetic particles, polymer particle markers, small organic molecules, such as biotin, receptor ligands or binding molecules, such as cell adhesion proteins or lectins, marker sequences comprising nucleic acids and / or amino acid residues that can be detected by using
89/118 connection, etc. Markers include, but are not limited to, barium sulfate, iocetamic acid, iopanic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium thyropanoate and radio-diagnosis, including positron emitters such as fluorine-18 and carbon 11 , gamma emitters, such as iodine 123, technetium-99m, iodine-131 and indio-111, for nuclear magnetic resonance nuclides, such as fluorine and gadolinium.
[00175] According to the invention, a reference, such as a reference sample or reference organism can be used to correlate and compare the results obtained in the methods of the invention from a test sample or test organism. Typically, the reference organism is a healthy organism, in particular, from an organism that does not suffer from a disease such as a cancer disease. A reference value or reference level can be determined empirically from a reference by measuring a sufficiently large number of references. Preferably, the reference value is determined by measuring at least 2, preferably at least 3, preferably at least 5, preferably at least 8, preferably at least 12, preferably at least 20, preferably at least 30, preferably at least 50, or preferably at least 100 references.
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[00176] Reducing or inhibiting, as used herein, means the ability to cause an overall decrease, preferably by 5% or greater, 10% or more, 20% or more, more preferably, 50% or greater, and with greater preference of 75% or higher, of the level. The term inhibition or similar phrases includes complete or essentially complete inhibition, that is, a reduction to zero or essentially zero.
[00177] Terms such as increasing or improving refer, preferably, to an increase or improvement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably, at least 40%, more preferably, at least 50%, even more preferably, at least 80%, and most preferably, at least 100%.
[00178] The agents, compositions and methods described herein can be used to diagnose a patient with a disease. The diseases that can be diagnosed encompass all diseases that express CLDN18.2. Particularly preferred diseases are cancerous diseases, such as the cancerous diseases described herein.
[00179] According to the invention, the term sick refers to any pathological condition, including cancer diseases, in particular, the forms of cancerous diseases described herein.
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[00180] The term normal, as used in terms of normal tissue or normal conditions refers to healthy tissue or conditions in a healthy individual, that is, non-pathological situations, in which healthy, preferably, means non-cancerous .
[00181] The disease involving cells expressing CLDN18.2 means, according to the invention that the expression of CLDN18.2 in cells of a diseased tissue or organ is preferably an increase over the state in a healthy tissue or organ. An increase refers to an increase of at least 10%, in particular, at least 20%, at least 50%, at least 100%, at least 200%, At least 500%, at least 1000% o, at least , 10000%) or even more. In one embodiment, the expression is found only in diseased tissue, while the expression of healthy tissue is suppressed. According to the invention, diseases involving or being associated with cells expressing CLDN18.2 include cancerous diseases, in particular the forms of cancer described herein.
[00182] According to the invention, the term tumor or tumor disease refers to a swelling or injury formed by an abnormal growth of cells (called neoplastic cells and tumor cells). Tumor cells are understood as an abnormal cell that grows by rapid, uncontrolled cell proliferation and continues to grow after
92/118 the stimuli that started the new growth to cease. Tumors show partial or complete lack of structural organization and functional coordination with normal tissue, and generally form a distinct mass of tissue, which can be benign, premalignant or malignant.
[00183] A benign tumor is a tumor that lacks all three malignant properties of a cancer. Thus, by definition, a benign tumor does not grow in an unlimited, aggressive manner, does not invade surrounding tissues, and does not spread to non-adjacent tissues (metastases). Common examples of benign tumors include warts and uterine fibroids.
[00184] The term benign implies a mild and non-progressive disease, and in fact, many types of benign tumors are harmless to health. However, some neoplasms that are defined as benign tumors, because they do not have the invasive properties of cancer, can still have negative health effects. Examples of this include tumors that produce a mass effect (compression of vital organs, such as blood vessels), or functional tumors of the endocrine tissues, which may produce more certain hormones (examples include thyroid adenomas, adrenocortical adenomas and pituitary adenomas).
[00185] Benign tumors are usually surrounded by an outer surface that inhibits their ability to
93/118 behave in an evil way. In some cases, certain "benign" tumors can later give rise to malignant cancers, which result from additional genetic alterations in a subpopulation of tumor neoplastic cells. A prominent example of this phenomenon is tubular adenoma, a common type of colon polyp, which is an important precursor to colon cancer. The cells in tubular adenomas, like most tumors that often progress to cancer, show certain abnormalities of cell maturation and appearance collectively known as dysplasia. These cellular abnormalities are not seen in benign tumors that rarely or never become cancerous, but are seen in other abnormalities of precancerous tissue that do not form discrete masses, such as precancerous lesions of the cervix. Some authorities prefer to refer to dysplastic tumors as premalignant, and reserve the term benign for tumors that rarely or never give rise to cancer.
[00186] Neoplasm is an abnormal mass of tissue, as a result of the neoplasm. Neoplasia (new growth in Greek) is the abnormal proliferation of cells. Cell growth exceeds and is uncoordinated with that of normal tissue around it. The growth persists in the same excessive way, even after the stimulus ceases. It usually causes a lump or tumor. Neoplasms can be benign, premalignant or malignant.
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[00187] Tumor growth or tumor growth, according to the invention refers to the tendency of a tumor to increase its size and / or the tendency of tumor cells to proliferate.
[00188] Cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells show uncontrolled growth (division beyond normal limits), invasion (intrusion and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancer differentiate it from benign tumors, which are self-limiting and do not invade or metastasize. Most cancers form a tumor, but some, like leukemia, do not. According to the invention, the terms cancer and tumor or cancer disease and tumor disease are generally used interchangeably here to refer to diseases in which cells exhibit uncontrolled growth and invasion and / or metastasis, optionally.
[00189] Preferably, a cancer disease according to the invention is characterized by cells that express CLDN18.2. A cell that expresses CLDN18.2, preferably, is a cancer cell, preferably tumors and cancers described here. Preferably, such a cell is a cell other than a stomach cell.
[00190] Cancers are classified by the type of
95/118 cell that resembles the tumor and therefore the tissue that is assumed to be the origin of the tumor. These are histology and location, respectively.
[00191] The term cancer, according to the invention includes leukemias, seminomas, melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, kidney cancer, colon cancer, stomach cancer, bowel cancer, head and neck cancer, gastrointestinal cancer, lymphatic system cancer, esophageal cancer, colorectal cancer , pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and their metastases. Examples of these are lung carcinomas, mamma carcinomas, prostate carcinomas, colon carcinomas, kidney cell carcinomas, cervical carcinomas, or cancer metastases or the types of tumors described above. The term cancer according to the invention also includes cancerous metastases.
[00192] The main types of lung cancer are small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). There are three main subtypes of non-small cell lung carcinoma:
96/118 squamous cell lung carcinoma, adenocarcinoma and large cell lung carcinoma. Adenocarcinomas represent about 10% of lung cancers. This type of cancer is usually seen on the periphery of the lungs, unlike small cell lung cancer and squamous cell lung cancer, which both tend to be more central.
[00193] According to the invention, a carcinoma is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including the most common forms of breast, prostate, lung and colon cancer.
[00194] By metastases is meant the spread of cancer cells from their place of origin to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, the penetration of the endothelial basement membranes to enter the body cavity and vessels, and then, after being transported by the blood , the infiltration of target organs. Finally, the growth of a new tumor, that is, a secondary tumor, or tumor metastasis, at the target site depends on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, because the tumor cells or components can remain and develop metastatic potential. In one embodiment, the term metastasis, from
97/118 according to the invention refers to metastasis to which a remote metastasis that is from the primary tumor and the regional lymph node system.
[00195] The cells of a secondary or metastatic tumor are those in which the original tumor. This means, for example, that in the case of ovarian cancer metastases to the kidney, the secondary tumor consists of abnormal ovary cells, not liver cells. The kidney tumor is then called metastatic ovarian cancer and not kidney cancer.
[00196] Relapse or recurrence occurs when a person is affected again by a condition that has affected them in the past. For example, if a patient has suffered from a cancer disease, received successful treatment of said disease and again develops said disease, the newly developed said disease can be considered as a relapse or a new occurrence. However, according to the invention, a relapse or recurrence of a cancer disease may, but not necessarily, occur at the site of the initial cancer disease. So, for example, if a patient suffered from an ovarian tumor and received successful treatment, a relapse or a new occurrence could be the occurrence of an ovarian tumor or the occurrence of a tumor in a location other than the ovary. The relapse or recurrence of a tumor also includes situations in which a tumor occurs in a
98/118 site different from the site of the original tumor, as well as the site of the original tumor. Preferably, the original tumor for which the patient has received treatment for a primary tumor and the tumor at a site other than the original tumor site is a secondary tumor, or metastasis.
[00197] By treating is meant administering a compound or composition to a patient in order to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in a subject; stop or delay a disease in a subject; inhibit or delay the development of a new disease in a subject; decrease the frequency or severity of symptoms and / or recurrences in a subject that you currently have or have had an illness; and / or prolong, that is, increase the useful life of the subject.
[00198] In particular, the term treatment of a disease includes healing, shortening the duration, improving, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms.
[00199] Being at risk means a subject, that is, a patient, who is identified as having a greater than normal probability of developing a disease, in particular cancer, compared to the general population. Furthermore, a subject who has had, or currently has, a disease, in particular cancer is a subject
99/118 who has an increased risk for the development of a disease, since such a subject can continue to develop the disease. Individuals who currently have, or have had, cancer also have an increased risk of cancer metastases.
[00200] The term immunotherapy refers to a treatment that involves a specific immunological reaction. In the context of the present invention, the terms how to protect, prevent, prophylactic, prevention or protective refer to the prevention or treatment or both of the occurrence and / or the spread of a disease in a subject and, in particular, to minimize the possibility that an individual will develop a disease or to delay the development of a disease. For example, a person at risk for a tumor, as described above, would be a candidate for therapy to prevent a tumor. Immunotherapy can be performed using any of a variety of techniques, in which the agents have the function of removing cells that express the antigen from a patient.
[00201] Within certain embodiments, immunotherapy can be active immunotherapy, in which treatment is based on in vivo stimulation of the immune system of the endogenous host to react against diseased cells with the administration of immune response modifying agents (for example, peptides immunoreactives and nucleic acids).
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[00202] Within other embodiments, immunotherapy can be passive immunotherapy, in which the treatment involves the administration of agents with established tumor immunological reactivity (such as antibodies) that can directly or indirectly mediate antitumor effects and does not necessarily depend on of an immune system of the intact host.
[00203] The term in vivo is related to the situation of a subject.
[00204] The terms subject, individual, organism or patient are used interchangeably and relate to vertebrates, preferably mammals. For example, mammals in the context of the present invention are humans, non-human primates, domestic animals, such as dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals, such as mice, rats , rabbits, guinea pigs, etc., as well as animals in captivity such as zoo animals. The term animal, as used herein, also includes humans. The term subject may also include a patient, that is, an animal, preferably a human being with a disease, preferably a disease, as described herein.
[00205] According to the invention, a sample can be any useful sample according to the present invention, in particular, a biological sample from a sample of that
101/118 tissue, including body fluids, and / or a cell sample and can be obtained in the conventional manner, such as by tissue biopsy, including biopsy, and taking blood, bronchial aspirate, sputum, urine, feces and others body fluids. According to the invention, the term sample also includes samples or fractions such as isolates from biological samples, for example, peptide nucleic acid and processed protein / isolates. Preferably, it contains a sample of cells or tissue from the organ being examined, for example, what is being diagnosed by cancer. For example, if the cancer to be diagnosed lung cancer is a sample it may contain cells or tissues obtained from the lungs.
[00206] According to the invention, a sample can be a sample, such as a body sample from a patient, containing or expected to contain tumor or cancer cells. The body sample can be any tissue sample, such as blood, a tissue sample obtained from the primary tumor or from tumor metastases or other cells that contain tumor or cancer samples.
[00207] The present invention is described in detail by the following figures and examples, which are used for illustration purposes only and are not intended to be limiting. Given the description and examples, other embodiments that are
102/118 also included in the invention are available to the person skilled in the art.
BRIEF DESCRIPTION OF THE FIGURES
[00208] Figure 1 shows the alignment of claudin 18 protein sequences (human / murine). The sequence alignment shows the homology between human and rat claudin 18.2 and 18.1 human claudin and Claudin 18.2.
[00209] Figure 2 shows the recombinant protein, including the C-terminal portion of CLDN18.2 (aal91-261) used for the immunization of mice
[00210] Figure 3 shows the analyzes of Antibody Sequences 43-14A and 35-22A EXAMPLES
[00211] The techniques and methods used herein are described or performed here in a manner known per se and as described, for example, in Sambrook et al, Molecular Cloning :. A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. All methods, including the use of kits and reagents, are performed according to the manufacturer's information, unless specifically indicated.
Example 1: Generation of monoclonal antibodies
[00212] The objective of this project was to generate monoclonal antibodies specific for murine CLDN18 capable of detecting CLDN18.2 that express tumor cells
103/118 in CA stomach, CA esophagus, CA pancreas and CA lung and FFEP CA tissues.
[00213] To generate a high affinity for highly specific diagnostic CLDN18.2 antibodies that was essential to initiate immunization protocols with a wide range of different immunogens and adjuvants. During the project, about 100 rats (C57B1 / 6 and Balb / c) were inoculated, using various immunization strategies to trigger an immune response to -CLDN18.
[00214] To activate the rat's immune system and to overcome immune tolerance we use virus-like particles (VLP), de-conjugated Pepti and recombinant proteins that code for different parts of human CLDN18.2 expressed as recombinant fusion proteins, with different expression partners (tags).
[00215] Of the 13 different immunization strategies, the best results were obtained by treating mice with the thermally labeled His C-recombinant CLDN18 protein (see Figure 2; Immunization # 20), in combination with various adjuvants (see Table 1, below, merger 35).
[00216] One candidate (35 - 22A) resulted from a 4-step immunization strategy (30 days). Another candidate (43 - 14A) was generated following a 7-step immunization protocol (79 days) (see Table 1, below, fusion 43).
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[00217] Two days before the splenectomy, the mice were boosted to activate the targeted B cells.
[00218] On the day of the rat splenocyte fusion they were isolated and fused with an Ag8.653 mouse myeloma cell line. For the fusion of mouse cells for myeloma the standard protocol published by Ohler and Milstein 1975 was followed. After HAT selection supernatants were tested in ELISA for the secretion of antibodies that recognize the antigen used for immunization.
[00219] ELISA positive supernatant hybridoma cells were subcloned to generate monoclonal hybridomas and subcloned hybridoma cell supernatants were retested in ELISA. Hybridoma cells from positive clones were expanded and supernatants analyzed below.
Example 2: Western blot selection of supernatants from monoclonal hybridomas
[00220] To answer the question whether ELISA-positive antibodies in the supernatants are able to bind to any recombinant claudin 18 or transfected claudin 18 stable protein lysates expressing HEK293 cells by Western Blot analysis was performed. The antibodies that were able to specifically bind to claudin 18 in a Western Blot analysis were expanded.
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The cells were cryopreserved and antibodies purified using MabSelect (FPLC). The antibodies selected by Western Blot screening were purified and evaluated for their ability to bind to their antigen in tissues fixed in formalin and embedded in paraffin (FFPE) by immunohistochemistry.
Example 3: Histological analysis - first selection of positive antibodies by Western Blot
[00221] The objective of this work was to verify the CLDN18 specificity and sensitivity of antibodies. This was done using CLDN18 expressing normal FFPE tissue from the stomach.
[00222] In a first experiment, the purified antibodies tested by Western Blot were analyzed at a concentration of 0.5 pg / ml in FFPE sections in the human stomach. Antibodies that performed well and do not produce large amounts of background were further titrated to 0.2 and 0.1 pg / ml in various normal stomach tissues to test for sensitivity and specificity. In the subsequent development stages the newly generated antibodies were tested directly at a concentration of 0.2 pg / ml, as the best antibody has already been performed very well at 0.2 pg / ml and served as a reference point. Antibodies generating strong signals in the mucosal epithelium of the tested human stomach tissues and no background on the tissues of the
106/118 adjacent mucosa were selected for further titration experiments and specificity analysis. Two antibodies had excellent performance: 35 - 22A and 43 - 14A; see Tables 2 and 3, below.
[00223] The antibodies that produce strong signals in the normal stomach tissue tested were analyzed in cancer tissues. The corresponding hybridoma cells were adapted to serum-free media. The signals produced using muMAb 43 - 14A were slightly stronger than the signals produced using muMAb 35-22A; see Table 4, below. Example 4: In-depth histological analysis and antibody characterization
[00224] Antibodies produced free of serum were used to color microarrays of stomach tissue CA (TMA). The number of stained cases was analyzed, the intensity of the signal and the number of positive tumor cells.
[00225] The staining intensities of the mumAbs 35 -22 A and 43-14A were excellent. There were no significant differences in the staining pattern and only small differences in staining intensities between the tested antibodies 35-22A and 43-14A.
Example 5: Analysis of antibody specificity using a panel of normal tissues
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[00226] The selected antibodies were tested on various normal tissues needed to ensure the high specificity of target CLDN18; see Tables 5A and 5B, below.
[00227] There were no significant differences in the staining pattern and staining intensity of antibodies 35-22A and 43-14A were visible in previous experiments. Therefore, the antibodies were subjected to staining experiments with a more clinically oriented protocol. To simulate the staining processes applied in standard pathology laboratories a One-Day-Protocol with a short (1 hour) primary antibody incubation step was established.
[00228] In all cases analyzed muMAb 43-14A performed extremely good and even better compared to muMAb 35-22A; see Table 6, below.
Example 6: In-depth analysis on relevant respiratory epithelial tissue
[00229] muMAb 43-14A was further analyzed in several relevant respiratory tissues to ensure its specificity, especially in target tissues of the lungs / bronchial tract. For these tissues, CLDN18.1 expression has been reported. To analyze whether the diagnostic antibody crosses reacts with the lung / bronchi expressing the CLDN18.1 isoform all available lung / bronchial tissues were selected. There are no signs that were detected with lung and bronchial tissues; see Table 7, below. The isoform
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CLDN18 expressed in these respiratory tissues is not recognized by antibody 43-14A.
Example 7: Mapping epitopes of mumAbs 43 - 14A and 35 22A
[00230] ELISA peptide was performed to identify the antibody binding epitopes on CLDN18.2. Each purified antibody was tested on overlapping peptides covering the C-terminal sequence of CLDN18.2. 35-22A and 4314A. Both showed specific binding to an epitope mapping for the TEDEVQSYPSKHDYV peptide. The following sequence was determined as the reactive sequence: EVQSYPSKHDYV.
Example 8: Sequence Analysis of mumAbs 43 - 14A and 35 - 22A
[00231] Analysis of the sequence of antibodies 43-14A and 35-22A is shown in Figure 3.
Example 9: Coloring of different cancer tissues
[00232] Immunohistochemistry (IHC) was performed on slides of 4% of the paraffin samples fixed in buffered formalin. Paraffin was performed according to standard protocols.
[00233] After deparaffinization, the slides were subjected to antigen recovery by boiling in 10 mM citric acid supplemented with 0.05% Tween-20 (pH 6.0) at 120 ° C for 10 minutes, subsequently quenched (by 2% H202) and incubated blocked at 4
109/118 ° C, with 0.2 to 0.5 pg / ml of monoclonal anti-antibody CLNDN18.2 diagnostic 43-14A or 35-22A. Antibody binding was visualized with horseradish-peroxidase-labeled secondary antibodies, using the polymer-based Powervision antibody (Goat-arate Power Vision HRP; Immunological, Duiven, The Netherlands) and a substrate-chromogen solution (VetorRed; Vector Labs, Burlingame, USA). The cuts were subsequently counterstained with Mayer's hematoxylin (Carl Roth GmbH, Karlsruhe, Germany) and subjected to evaluation by the evaluators.
Histological evaluation
[00234] All samples were analyzed for the relative proportion of positive stained tumor cells in relation to all visible tumor cells for each section. The staining intensity was classified as negative (), weakly positive (1 +), positive (2 +) and strongly positive (3 +). Only the staining of the membranes was considered positive. Human stomach tissue served as a positive control for each stain. Since PanIN (pancreatic intraepithelial neoplasia) are often found to be strongly positive, these areas have also been considered as reference internal staining intensity for strong positivity (3 +).
[00235] Strong membranous signals were generated by both antibodies in the cancerous tissues of the pancreas,
110/118 stomach and esophageal (Table 8) or with antibody to cancerous lung tissue 43-14A (Table 9). The number of positive tumor cells varied interindividually between different tumor cases. Most of the samples analyzed were from 2 + to 3 + positive.
[00236] Table 1: Immunization Schemes for Antibodies
Mouse 5 Immunization # 20 - Fusion 35
Date Day Event Strain Mouse ID Antigen Adjuvant Administration [μΐ] [gg] Code [μ1] / [μδ] Code Route Volume 27. Oct. 2010 0 1. Immunization C57BL / 6 M5 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 4. Nov. 2010 7 2. Immunization C57BL / 6 M5 100 100 C-terminalGC182 -HIS 50/50 CpG-PTO i.p. 200ul 10. Nov. 2010 14 3. Immunization C57BL / 6 M5 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 17. Nov. 2010 21 4. Immunization C57BL / 6 M5 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 24. Nov. 2010 28 Reinforcement C57BL / 6 M5 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 26. Nov. 2010 30 Fusion # 35 C57BL / 6 M5
Mouse 4 - Immunization # 20 - Fusion 43
Date Day Event Strain Mouse ID Antigen Adjuvant Administration [μΐ] [μδ] Code [μ1] / [μδ] Code Route Volume 27. Oct. 2010 0 1. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 10. Nov. 2010 14 2. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 17. Nov. 2010 21 3. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 24. Nov. 2010 28 4. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 08. Dec. 2010 42 5. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 22. Dec. 2010 56 6. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 5. Jan. 2011 70 7. Immunization BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 12. Jan. 2011 77 Reinforcement BALB / c M4 100 100 C-terminalGC182 -HIS 100 Gerbu MM i.p. 200ul 14. Jan. 2011 79 Fussion # 43 BALB / c M4
Table 2: positive mumAbs selected by Western Blot analysis
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Number of sliede Fabric Antibody conc. of the antibody Cryo- / Paraffin Mucous epithelium Subcellular pattern % positive cells bg on own blade Lymphocytes Vases Smooth muscle 11_413 stomach 35-22A 0.5pg / ml Paraffin +++ m 90 - - - - ll_910 human stomach 43-14A 0.2pg / ml Paraffin +++ m 90 - - - -
Table 3: Comparison of two antibodies 35-22A and 43-14A in the FFPE tissue of the normal human stomach
Slide ID Fabric details Antibody conc. antibody Development Epithelial mucisa Subscapular pattern % positive cells bg & cells in wool, mine mine Lymphoocytes Fibrous tissue Vases Smooth muscle comments 11_413 stomach 5 35-22A 0.5pg / ml 1:30 min +++ m 90 - - - - - very strong staining of mucous epithelial membranes, not bg in the stroma, vessels and muscles The 43-14A has not been tested at 0.5pg / ml, because ο 35-22A has not performed very well at 0.2 Lig / ml does not serve as a rule.11-975 stomach 1 35-22A 0.1 pg / nil 2:30 min +++ m 90 - - - - - strong membranous staining of the epithelium mucosa, no bg on stroma, muscles, vessels or lamina propria 11-975 stomach 1 43-14A 0.1 pg / nil 2:30 min +++ m 90 - - - - - strong staining of mucous epithelial membranes, not bg in the stroma, muscles, vessels or lamina propria 11-907 stomach 5 35-22A 0.2 pg / iiil 2:30 min +++ m 90 - - - - - strong staining of mucous epithelial membranes, not bg in the stroma, muscles, vessels or lamina
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own ll_910 stomach 5 43-14A 0.2pg / ml 2:30 min +++ m 90 - - - - - strong staining of mucous epithelial membranes, not bg in the stroma, muscles, vessels or lamina propria
Table 4: Analysis of cancerous tissues - TMA127A
slide in umber Fabric details Tissue id Antibody conc. antibody Cryo / Paraffin development Tumor cells Subcellular Pattern % positive cells Normal epithelial cells Fibrous tissue Vases Smooth muscle 11-474 Stomach CA (+++) B04 / 01221 II 35-22A 0, lpg / ml Paraffin 2:30 min ++ / +++ m 90 at. - - - 11-474 Stomach CA (++) B06 / 09514 (5 35-22A 0, lpg / ml Paraffin 2:30 min (+) / + w / m 10 at. - - - 11-474 Stomach AC (+) B05 / 09809 (4 35-22A 0, lpg / ml Paraffin 2:30 min - at. 0 at. - - - 11-474 Renal CA B08 / 13471 (3 35-22A 0, lpg / ml Paraffin 2:30 min - at. 0 at. - - at. 11-475 Stomach CA (+++) B04 / 01221 II 43-14A 0, lpg / ml Paraffin 2:30 min +++ m 90 at. - - - 11-475 Stomach CA (++) B06 / 09514 (5 43-14A 0, lpg / ml Paraffin 2:30 min (+) / + m / c 10 at. - - - 11-475 Stomach AC (+) B05 / 09809 (4 43-14A 0, lpg / ml Paraffin 2:30 min - - 0 at. - - - 11-475 Renal CA B08 / 13471 (3 43-14A 0, lpg / ml Paraffin 2:30 min - at. 0 at. - - at.
Table 5A: Normal tissue analysis
slide in umber Fabric Tissue id Antibody Conc. Cryo / Paraffin development Normal and epithelial cellsFunctional tissue Subcellular pattern % positive cells Lymphocytes Fibrous tissue Vases Smooth muscle Fatty fabrics 11-577 Stomach Stomach 9 35-22A 0.2 μg / m 1 Paraffin 00:50 ++ / +++ m 90 - - - - at. 11-1756 Stomach Stomach 9 43-14A 0.2 μg / m 1 Paraffin 00:50 +++ m > 90 - - - - at. 11-580 Colon Colon 2 35-22A 0.2 μg / m 1 Paraffin 02:15 - at. at. - - - - at. 11-1753 Colon Colon 2 43-14A 0.2pg / ml Paraffin 02:15 - at. at. - - - - - 11-586 Kidney Kidney 2 35-22A 0.2 μg / m 1 Paraffin 02:15 - at. at. - - - - at. 11-1754 Kidney Kidney 2 43-14A 0.2pg / ml Paraffin 02:15 - at. at. at. - - - - 11-589 Lung Lung 2 35-22A 0.2 μg / m 1 Paraffin 02:15 - at. at. - - - - at. 11-1663 Lung Lung 2 43-14A 0.2 μg / m 1 Paraffin 03:00 - at. at. - - - - at. 11-595 Pancreas Pancreas 3 35-22A 0.2 μ g / m 1 Paraffin 02:15 - at. at. - - - - - 11-1749 Pancreas Pancreas3 43-14A 0.2pg / ml Paraffin 02:15 - at. at. at. - - - - 11-599 Liver Figado 1 35-22A 0.2 μg / m 1 Paraffin 02:15 - at. at. - - - - - 11-1751 Liver Figado 1 43-14A 0.2pg / ml Paraffin 02:15 - at. at. at. - - - -
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Table 5B: Analysis of normal tissue - 43-14 A
slide in umber Fabric Fabric id Antibody Conc. Cryo / Paraffin Development Normal epithelial cells and functional tissue Subcellular pattern % positive cells Lymphocytes Fibrous tissue Vases Smooth muscle Fatty tissue 11_1748 Pancreas Pancreas 5 43-14A SF 0.2ug / ml Paraffin 02:15 - at. at. at. - - - - 11_1750 Liver Figado 4.5 43-14A SF 0.2ug / ml Paraffin 02:15 - at. at. at. - - - - 11_1755 Kidney Kidney 3 43-14A SF 0.2ug / ml Paraffin 02:15 - at. at. at. - - - - 11_1757 Stomach Stomach 12 43-14A SF 0.2ug / ml Paraffin 02:15 +++ m > 90 - - - - at. 11_1758 Heart Heart 1 43-14A SF 0.2ug / ml Paraffin 02:15 - at. at. at. - - - - 11_1759 Heart Heart 2 43-14A SF 0.2pg / ml Paraffin 02:15 - at. at. at. - - - -
Table 6: Analysis of normal tissue - One-day protocol
slide in umber Fabric Fabric id Antibody Conc. development Functional tissue tissue tumor! Subcellular Standard % positive cells Lymphocytes Fibrous tissue Vases Smooth muscle Fatty tissue 11_2092 Stomach Stomach 9 43-14 0.1pg / ml 3:30 ++ / +++ at. m > 90 - - - - - 11_2092 Stomach Stomach 9 35-22 A 0.1pg / ml 3:30 + / ++ at. m > 90 - - - - - 11_2093 Stomach Stomach 9 43-14 The SF 0.1pg / ml 3:30 +++ at. m > 90 - - - - - 11_2093 Stomach Stomach 9 35-22 SF 0.1pg / ml 3:30 + at. m 7080 - - - - - 11_2094 TMA 127 A TMA 127 A 43-14 0.1pg / ml 3:30 at. + / ++ m 90 - - - at. at. 11_2094 TMA 127 A TMA 127 A 35-22 A 0.1pg / ml 3:30 at. + m <5 - - - at. at. 11 2095 TMA 127 A TMA 127 A 43-14 The SF 0.1gg / ml 3:30 at. + / ++ m 90 - - - at. at. 11_2095 TMA 127 A TMA 127 A 35-22 SF 0.1pg / ml 3:30 at. - / (+) ç <5 - - - at. at.
Table 7: Analysis of normal respiratory tissues
slide in umber Fabric id Antibody Conc. Normal epithelial cells Subcellular Standard % positive cells Lymphocytes Fibrous tissue Vases Smooth muscle 11_1660 lung 1 muAb 43-14A 0.2pg / ml - - - at. - - - 11_1660 lung 1 muAb 43-14A 0.5pg / ml - - - at. - - - 11_1663 lung 2 muAb 43-14A 0.2pg / ml - - - - - - - 11_1663 lung 2 muAb 43-14A 0.5pg / ml - - - - - - - 11_1666 lung 3 muAb 43-14A 0.2pg / ml - - - - - - - 11_1666 lung 3 muAb 43-14A 0.5pg / ml - - - - - - - 11_1669 lung 4 muAb 43-14A 0.2pg / ml - - - - - - - 11_1669 lung 4 muAb 43-14A 0.5pg / ml - - - - - - - 11_1672 lung 5 muAb 43-14A 0.2pg / ml - - - - - - -
114/118
11_1672 lung 5 muAb 43-14A 0.5pg / ml - - - - - - - 11_1675 bronchi 1 muAb 43-14A 0.2pg / ml - - - - - - - 11_1675 bronchi 1 muAb 43-14A 0.5pg / ml - - - - - - - 11_1678 bronchi 2 muAb 43-14A 0.2pg / ml - - - - - - - 11_1678 bronchi 2 muAb 43-14A 0.5pg / ml - - - - - - - 11_1681 bronchi 3 muAb 43-14A 0.2pg / ml - - - - - - - 11_1681 bronchi 3 muAb 43-14A 0.5pg / ml - - - - - - - 11_1684 stomach 9 muAb 43-14A 0.2pg / ml +++ m > 90 - at. - -
Table 8: Analysis of CLDN18.2 expression in esophageal, pancreatic and stomach cancer tissues using murine monoclonal antibodies 43-14A and 35-22A
Fabric ID Ab Fabric Subtype details Tumor cells % positive cells H / 2010/10869IVG 43-14A pancreas CA cell carcinomaAcinar - 0 1125005.22 43-14A pancreas CA CarcinomaNeuroendocrine + + 70 H / 2006/22797 **IA 43-14A pancreas CA CarcinomaNeuroendocrine - 0 B08 / 6284-VC 3522A pancreas CA Neuroendocrine Carcinoma, NET G1 - 0 B08 / 8549-4 3522A pancreas CA Neuroendocrine Carcinoma, NET G1 - 0 B05 / 8523-3 3522A pancreas CA PDAC + + 1 B06 / 16136-NP2 3522A pancreas CA PDAC + + 50 B07 / 14168 3522A pancreas CA PDAC + + 80 B07 / 14935 3522A pancreas CA PDAC - 0 B07 / 2633-III3 3522A pancreas CA PDAC + + + 90 B07 / 7430 3522A pancreas CA PDAC + + 90 B08 / 5618-2 3522A pancreas CA PDAC + + + 80 B10 / 14198-VC 3522A pancreas CA PDAC + + + 80 B10 / 706-VC3 3522A pancreas CA PDAC + + + 60 B11 / 2059-D 3522A pancreas CA PDAC + + + 80 B11 / 4084 3522A pancreas CA PDAC + + + 90 1125005.30 43-14A pancreas CA PDAC - 0 1125005.27 43- pancreas PDAC + + 10
115/118
14A HERE 1125005.24 43-14A pancreas CA PDAC + + 50 1125005.23 43-14A pancreas CA PDAC + + 100 1125005.25 43-14A pancreas CA PDAC + + + 30 1125005.28 43-14A pancreas CA PDAC + + + 80 H / 2008/13074 43-14A pancreas CA PDAC + + + 40 H / 2008/13194 43-14A pancreas CA PDAC + + + 50 H / 2008/380 43-14A pancreas CA PDAC + + + 90 H / 2009/11847 43-14A pancreas CA PDAC + + + 15 H / 2009/20336 43-14A pancreas CA PDAC + + + 90 H / 2009/23598VII B 43-14A pancreas CA PDAC + + + 40 H / 2010/11569 43-14A pancreas CA PDAC + + + 80 H / 2011/17191GO 43-14A pancreas CA PDAC + + + 60 H / 2009/4917 43-14A pancreas CA PDAC + + + 70 H / 2010/15941 43-14A pancreas CA PDAC + + + 50 H / 2010/6709 43-14A pancreas CA PDAC + + + 70 1125005.19 43-14A esophagus CA adenocarcinoma + + 50 B09 / 1491-III-2 43-14A esophagus CA adenocarcinoma - 0 06 / 14957-2 43-14A esophagus CA adenocarcinoma + + 30 1125005.17 43-14A esophagus CA adenocarcinoma + + + 70 1083435B 43-14A esophagus CA adenocarcinoma + + 70 10b06684-II-3 43-14A stomach AC adenocarcinoma + + + 80 1125005.10 43-14A stomach AC adenocarcinoma + + 30 1125005.6 43-14A stomach AC adenocarcinoma + + 90
PDAC = pancreatic duct adenocarcinoma
116/118
Table 9: Analysis of CLDN18.2 expression in cancerous lung tissues using murine monoclonal antibody 43-14A
Tumor cellsB09 / 147583 0.2pg / ml bronchialalveolar type - - B09 / 18323IV5 0.2pg / ml bronchialalveolar type - - B10 / 13211-VC4 0.2pg / ml bronchialalveolar type +++ 80% B07 / 4771-3 0.2pg / ml carcinoid - - B07 / 5358II2 0.2pg / ml carcinoid - - B08 / 3382-1 0.2pg / ml carcinoid - - B08 / 8898-II6 0.2pg / ml clear cell carcinoma + / 12-<1st B09 / 12293 II2 0.2pg / ml carcinoma, spinocellular - - B06 / 12562-III3 0.2pg / ml Large cell carcinoma - - B06 / 10820II2 0.2pg / ml carcinoma, adeno - - B06 / 108762 0.2pg / ml carcinoma, adeno - - B06 / 16831I3 0.2pg / ml carcinoma, adeno +++ 80% B07 / 03255 IV5 0.2pg / ml carcinoma, adeno +++ 5% B07 / 2296-3 0.2pg / ml carcinoma, adeno - - B07 / 709 II4 0.2pg / ml carcinoma, adeno ++ <5% B10 / 12713II1 0.2pg / ml carcinoma, adeno ++ <5% B10 / 141972 0.2pg / ml carcinoma, adeno - - B10 / 16367-V2 0.2pg / ml carcinoma, adeno + 1 21% B09 / 1743 II3 0.2pg / ml carcinoma, adeno, squamous - - B09 / 179161 0.2pg / ml carcinoma, large cells - - B10 / 108143 0.2pg / ml carcinoma, large cells + 1 21%
117/118
neuro endocrine B010 / 10646 0.2pg / ml carcinoma, squamous cell - - B06 / 3204-2 0.2pg / ml carcinoma, squamous cell - - B08 / 292-2 0.2pg / ml carcinoma, squamous cell ++ <5% B08 / 7434IV 0.2pg / ml carcinoma, squamous cell - - B09 / 45-2 0.2pg / ml carcinoma, squamous cell - - B10 / 10-2 0.2pg / ml carcinoma, squamous cell + / 12-<1st B10 / 11714 0.2pg / ml carcinoma, squamous cell - - B10 / 15779 0.2pg / ml carcinoma, squamous cell - - B10 / 17043 0.2pg / ml carcinoma, squamous cell - - B10 / 18106 0.2pg / ml carcinoma, squamous cell - - B07 / 6782-1 0.2pg / ml carcinoma;adeno, clear cells ++ / 12-<1st B07 / 9741-5 0.2pg / ml carcinoma;adeno, clear cells - - B08 / 164252 0.2pg / ml large-cell neuroendocrine ++ <5% B08 / 3019-V3 0.2pg / ml non-small cell CA - - B08 / 1099V3 0.2pg / ml nt small cell long CA - / 12-<1st B08 / 120102 0.2pg / ml nt small cell long CA ++ / 12-<1st
118/118
B10 / 17662-III3 0.2pg / ml Small cell carcinoma - -
1/6

权利要求:
Claims (21)
[1]
1. Antigen or antigen binding fragment characterized by the fact that (i) it binds to a peptide having the amino acid sequence SEQ ID NO: 5 or SEQ ID NO: 6 and / or (ii) binds to claudin 18.2 (CLDN18.2), wherein said antibody or antigen binding fragment binds to CLDN18.2 by binding at least one epitope within CLDN18.2 having the amino acid sequence SEQ ID NO: 5 or SEQ ID NO: 6, wherein the antibody comprises (A) a heavy chain antibody comprising: a sequence of CDR1 according to SEQ ID NO: 8, a sequence of CDR2 according to SEQ ID NO: 9, and a sequence of CDR3 according to SEQ ID NO: 10, and a light chain antibody comprising: a sequence of CDR1 according to SEQ ID NO: 12, a sequence of CDR2 according to SEQ ID NO: 13, and a sequence of CDR3 according to SEQ ID NO: 14, or (B) a heavy chain antibody comprising:
a sequence of CDR1 according to SEQ ID NO: 16, a sequence of CDR2 according to SEQ ID NO: 17, and a sequence of CDR3 according to SEQ ID NO: 18,
Petition 870200031947, of 03/09/2020, p. 14/26
[2]
2/6 and
a light chain antibody comprising:
a CDR1 sequence according to SEQ ID NO: 20, a CDR2 sequence according to SEQ ID NO: 21, and a CDR3 sequence according to SEQ ID NO: 22.
2. Antigen or antigen-binding fragment according to claim 1, characterized in that said CLDN18.2 is bound to the cell surface membrane of CLDN18.2.
[3]
3. Antigen or antibody-binding fragment according to claim 1 or 2, characterized by the fact that said CLDN18.2 is present in cancer cells.
[4]
4. Antibody or antigen-binding fragment according to claim 3, characterized by the fact that said cancer cells are cancer cells expressing CLDN18.2.
[5]
5. Antigen or antigen-binding fragment according to claim 3 or 4, characterized by the fact that said cancer cells are selected from the group consisting of gastric cancer cells, from the esophagus, pancreas, lung, ovary, colon, liver, head-neck, and gallbladder.
[6]
An antigen-binding antibody or fragment according to any one of claims 1 to 5, characterized in that it does not bind to non-cancer cells except stomach epithelial cells.
Petition 870200031947, of 03/09/2020, p. 15/26
3/6
[7]
7. Antigen or antibody-binding fragment according to any one of claims 1 to 6, characterized in that it does not bind to non-cancerous lung cells.
[8]
8. Antibody according to any one of claims 1 to 5, characterized in that it is a chimeric, humanized or human antibody.
[9]
Antibody according to any one of claims 1 to 8, characterized in that it is a monoclonal antibody.
[10]
10. Antibody characterized by the fact that it is selected from the group consisting of:
(i) an antibody produced by or obtainable from a clone deposited under accession number DSM ACC3144 (muAB 43-14A) or DSM ACC3143 (muAB 35-22A), (ii) an antibody that is a humanized or chimerized form of the antibody in (i), (iii) an antibody that has the specificity of the antibody under (i), and (iv) an antibody comprising the antigen binding portion or antigen binding site of the antibody in (i), or an antigen-binding fragment of the antibody in any of items (i) to (iv).
[11]
11. Antibody according to claim 10, characterized by the fact that the antigen-binding portion or antigen-binding site of the antibody in
Petition 870200031947, of 03/09/2020, p. 16/26
4/6 (i) comprises the variable region of the antibody in (i).
[12]
12. Conjugate characterized by the fact that it comprises an antibody or antigen-binding fragment as defined by any one of claims 1 to 11, together with at least one detectable marker.
[13]
13. Hybridoma characterized by the fact that it is deposited under the accession number DSM ACC3144 (muAB 43-14A) or DSM ACC3143 (muAB 35-22A).
[14]
14. Method for detecting CLDN18.2 or determining the amount of CLDN18.2 in a sample characterized by the fact that it comprises the steps of:
(i) contacting a sample with the antibody or antigen-binding fragment as defined by any one of claims 1 to 11 or the conjugate as defined by claim 12 and (ii) detecting the formation of a complex or determining the amount of a complex between the antibody, the antigen binding fragment or the conjugate and CLDN18.2.
[15]
15. Method for determining whether cells express CLDN18.2 characterized by the fact that it comprises the steps of:
(i) contacting a cell sample with the antibody or antigen binding fragment as defined by any one of claims 1 to 11 or the conjugate as defined by claim 12 and (ii) detecting the formation of a complex between the
Petition 870200031947, of 03/09/2020, p. 17/26
5/6 antibody, antigen-binding fragment or the conjugate and CLDN18.2 expressed by the cells in said sample.
[16]
16. Method for the diagnosis, monitoring or detection of cancer characterized by the fact that it comprises the steps of:
(i) contacting a biological sample with the antibody or antigen-binding fragment as defined by any one of claims 1 to 11 or the conjugate as defined by claim 12 and (ii) detecting the formation of a complex and / or determining the amount of a complex between the antibody, the antigen binding fragment or the conjugate and CLDN18.2.
[17]
17. Method for determining whether a cancer is treatable by cancer therapy targeting CLDN18.2 characterized by the fact that it comprises the steps of:
(i) contacting a sample comprising the cancer cells with the antibody or antigen binding fragment as defined by any one of claims 1 to 11 or the conjugate as defined by claim 12 and (ii) detecting the formation of a complex between the antibody , the antigen-binding fragment or the conjugate and CLDN18.2.
[18]
18. Diagnostic test kit characterized by the fact that it comprises the antibody or antigen binding fragment as defined by any one of claims 1 to 11, or the conjugate as defined
Petition 870200031947, of 03/09/2020, p. 18/26
6/6 by claim 12.
[19]
19. The antigen-binding antibody or fragment as defined by claims 1 to 11, the conjugate as defined by claim 12, the hybridoma as defined by claim 13, or the method as defined by any of claims 14 to 17, characterized by the fact that said CLDN18.2 comprises the amino acid sequence set forth in SEQ ID NO: 2 of the sequence listing or a variant of said amino acid sequence.
[20]
20. Use of the antibody as defined by any of claims 1 to 11, or the conjugate as defined by claim 12 characterized by the fact that it is for the preparation of a diagnostic composition for a method of diagnosing, monitoring or detecting cancer.
[21]
21. Use of the antibody as defined by any one of claims 1 to 11, or the conjugate as defined by claim 12 characterized by the fact that it is for the preparation of a pharmaceutical composition to determine whether a patient's cancer can be treated by a cancer therapy targeting CLDN18.2.

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法律状态:
2018-12-11| B65X| Notification of requirement for priority examination of patent application|

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2019-01-15| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|

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2019-02-05| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

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2019-08-13| B07G| Grant request does not fulfill article 229-c lpi (prior consent of anvisa) [chapter 7.7 patent gazette]|Free format text: NOTIFICACAO DE DEVOLUCAO DO PEDIDO POR NAO SE ENQUADRAR NO ART. 229-C DA LPI. |

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2019-12-10| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-06-16| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 06/05/2013, OBSERVADAS AS CONDICOES LEGAIS. |

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2020-07-07| B16C| Correction of notification of the grant [chapter 16.3 patent gazette]|Free format text: REF. RPI 2580 DE 16/06/2020 QUANTO AO INVENTOR. |

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2021-06-08| B25D| Requested change of name of applicant approved|Owner name: TRON - TRANSLATIONALE ONKOLOGIE AN DER JOHANNES GUTENBERG-UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) ; GANYMED PHARMACEUTICALS GMBH (DE) |

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2021-06-29| B25D| Requested change of name of applicant approved|Owner name: GANYMED PHARMACEUTICALS GMBH (DE) ; TRON - TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) |

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2021-07-20| B25A| Requested transfer of rights approved|Owner name: TRON - TRANSLATIONALE ONKOLOGIE AN DER UNIVERSITAETSMEDIZIN DER JOHANNES GUTENBERG UNIVERSITAET MAINZ GEMEINNUETZIGE GMBH (DE) ; ASTELLAS PHARMA INC. (JP) |

优先权:

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申请号 | 申请日 | 专利标题

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EPPCT/EP2012/001991|2012-05-09|

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PCT/EP2012/001991|WO2013167153A1|2012-05-09|2012-05-09|Antibodies useful in cancer diagnosis|

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PCT/EP2013/001331|WO2013167259A1|2012-05-09|2013-05-06|Antibodies against claudin 18.2 useful in cancer diagnosis|

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