US20110123447A1 - Method of providing human tumor-specific antibodies - Google Patents

Method of providing human tumor-specific antibodies Download PDF

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US20110123447A1
US20110123447A1 US12/450,101 US45010108A US2011123447A1 US 20110123447 A1 US20110123447 A1 US 20110123447A1 US 45010108 A US45010108 A US 45010108A US 2011123447 A1 US2011123447 A1 US 2011123447A1
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antibody
tumor
cells
antigen
antibodies
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Christoph Esslinger
Sandra Kuenzle
Irene Abela
Roger Nitsch
Holger Moch
Norbert Goebels
Dirk Jaeger
Alfred Zippelius
Alexander Knuth
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Universitaet Zuerich
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Assigned to ZURICH, UNIVERSITY OF reassignment ZURICH, UNIVERSITY OF ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: GOEBELS, NORBERT, ABELA, IRENE, ZIPPELIUS, ALFRED, JAGER, DIRK, KUENZLE, SANDRA, ESSLINGER, CHRISTOPH, MOCH, HOLGER, KNUTH, ALEXANDER, NITSCH, ROGER
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3053—Skin, nerves, brain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man

Definitions

  • the present invention generally relates to novel tumor-specific binding molecules of human origin, particularly human antibodies as well as fragments, derivatives and variants thereof that recognize tumor antigens and tumor-associated antigens, respectively.
  • the present invention relates to compositions comprising such binding molecules, antibodies and mimics thereof, and to methods of screening for novel binding molecules, which may or may not be antibodies, targets and drugs in the treatment of various tumors, in particular melanoma, breast cancer and metastasis.
  • pathological conditions such as infections, cancer, autoimmune disorders, etc.
  • these molecules thus serve as “markers” for a particular pathological or abnormal condition.
  • diagnostic “targets” i.e., materials to be identified to diagnose these abnormal conditions
  • the molecules serve as reagents which can be used to generate diagnostic and/or therapeutic agents.
  • cancer markers such as tumor-associated antigens to produce antibodies specific to a particular marker.
  • Humoral immune responses to tumors occur in a relatively high frequency in (1, 2). This phenomenon was exploited to identify a variety of tumor-associated antigens (taa) by screening autologous expression libraries with serum from cancer patients (1). Several of these taa now serve as T cell antigens for the induction of anti-tumor CTL-responses in patients (3, 4). This preference for the cellular-, in most cases cytotoxic immune response as therapeutic strategy is now being reconsidered and novel vaccines are designed to also induce antibody responses. In part, this change of concept may have been influenced by the recent success of various monoclonal antibodies for tumor therapy such as trastuzumab (Herceptin) and bevacizumab (Avastin) (5).
  • trastuzumab Herceptin
  • bevacizumab Avastin
  • Object of the present invention is a method for identifying, validating and producing tumor-specific diagnostically and therapeutically useful binding molecules, in particular antibodies that are directed against antigens of and/or associated with tumor cells and cancerous tissue, and which overcome the drawbacks of murine derived antibodies such as HAMA (human anti-murine antibody) response. More specifically, the present invention relates to consistent further development and thus confirmation of the general method of providing disease-specific binding molecules and targets as disclosed in applicant's co-pending international application PCT/EP2008/000053, the disclosure content of which is incorporated herein by reference. Thus, in one aspect the present invention relates to a method for isolating a human binding molecule specific for a tumor antigen or tumor-associated antigen comprising:
  • the present invention makes use of the tumor-specific immune response of cancer patients for the isolation of tumor antigen and tumor-associated antigen (taa) specific human monoclonal antibodies.
  • tumor antigen and tumor-associated antigen (taa) specific human monoclonal antibodies were successful in the isolation of a monoclonal antibody specific for the taa NY-ESO-1 from a melanoma patient who showed a serum titer to NY-ESO-1 and a partial clinical response.
  • the method of the present invention makes use of immunohistochemistry (IHC) using tissue microarrays (TMA).
  • the present invention is thus directed to human antibodies, antigen-binding fragments and similar antigen binding molecules which are capable of recognizing tumor antigen and tumor-associated antigen (taa), respectively. Furthermore, the present invention relates to compositions comprising said antibodies and to immunotherapeutic and immunodiagnostic methods using the same.
  • a further embodiment concerns novel tumor antigens and tumor associated antigens, which are characterized by their property of being bound by the human tumor-specific antibody of the present invention with high affinity.
  • the present invention extends to the immortalized human B memory lymphocyte and B cell, respectively, that has been isolated in the course of performing the method of the present invention and produces the human antibody.
  • the present invention also relates to polynucleotides encoding at least a variable region of an immunoglobulin chain of the antibody of the invention. Accordingly, the present invention also encompasses vectors comprising said polynucleotides and host cells transformed therewith as well as their use for the production of an antibody and equivalent binding molecules which are specific for antigens that are indicative and/or causative for a tumor, in particular for melanoma or breast cancer.
  • the antibody, immunoglobulin chain(s), binding fragments thereof and antigen binding to said antibody can be used in pharmaceutical and diagnostic compositions for tumor immunotherapy and diagnosis, respectively.
  • the use of the foregoing compositions in the preparation of a medicament is however preferred.
  • the present invention to provide methods for treating or preventing a cancerous disease such as primary breast carcinoma and metastases.
  • the methods comprise administering an effective concentration of an antibody or antibody derivative to the subject where the antibody targets tumor tissue and cells.
  • FIG. 1 Memory B cell culture well 12 D7 contains antibodies specific for NY-ESO-1. Medium conditioned by memory B cell cultures was assayed for the presence of NY-ESO-1-specific human antibodies A) In ELISA displaying full length recombinant NY-ESO-1. B) In immunohistochemistry on NY-ESO-1-positive mamma carcinoma (mc) and on NY-ESO-1-negative control tissue (ct). Shown is the staining obtained with conditioned medium of two ELISA-positive memory B cell culture wells (9D1, 12D7).
  • mc mamma carcinoma
  • ct NY-ESO-1-negative control tissue
  • NY-ESO-1-specific antibody contained in well 12D7 is of the IgG1 subclass as demonstrated by the staining of NY-ESO-1-positive tissue with B cell conditioned medium from culture well12D7 followed by secondary antibodies against IgG subclasses IgG1-4.
  • FIG. 2 Recombinant human antibody 12D7 clone number 4 obtained by single cell RT-PCR of cultured memory B cells specifically recognizes NY-ESO-1 in ELISA and on tissue sections.
  • ELISA values are indicated for undiluted SN (1:12 D7.4 SN) a 1/10 dilution (2:12 D7.4 SN) and a 1/100 dilution (3:12 D7.4 SN).
  • the ELISA signal obtained with plasma of the patient from which the memory B cell cultures were derived used as a 1/100 dilution is also shown (4).
  • the absence of binding to NY-ESO-1 coated ELISA plates of SN obtained upon transfection of an irrelevant recombinant antibody produced in the same way as 12D7.4 is shown (5) as well as the absence of binding of 12D7 clone No. 4 to ELISA plates coated with an irrelevant antigen.
  • FIG. 3 Characteristics of human monoclonal antibody Manhattan. Epitope mapping was performed using overlapping peptides spanning the entire NY-ESO-1 protein coated onto ELISA plates.
  • A) Manhattan specifically binds to a peptide spanning amino acids 11 to 30 at the N-terminus of the NY-ESO-1 protein.
  • B) serum of patient C1 recognizes various peptide fragments at the N-terminus and the mid-region of NY-ESO-1.
  • FIG. 4 Amino and nucleotide sequences of the variable region, i.e. heavy chain and kappa light chain of antibody 12D7. Complementarity determining regions (CDRs) are underlined.
  • CDRs Complementarity determining regions
  • the present invention generally relates to means and methods for discovering therapeutically efficient tumor (associated) antigen binding molecules, i.e. antibodies from clinically pre-selected cancer patients.
  • therapeutically efficient tumor (associated) antigen binding molecules i.e. antibodies from clinically pre-selected cancer patients.
  • antibodies and antigen-binding fragments thereof are provided, which demonstrate the immunological binding characteristics and/or biological properties as outlined for the antibody illustrated in the Examples.
  • the present invention extends to the antibody producing cell lines and recombinant cells as well.
  • the present invention further relates to diagnostic assays and kits that comprise the binding molecule of the present invention and to therapeutic methods based thereon.
  • the present invention is based on a novel approach for the isolation and cloning of cancer patient-derived antibodies by using a method for identifying, validating and producing tumor-specific diagnostically and therapeutically useful binding molecules essentially as disclosed in applicant's co-pending international application, serial number PCT/EP2008/000053 “Method of providing disease-specific binding molecules and targets”, filed on Jan. 7, 2008, the disclosure content of which is incorporated herein by reference.
  • a method for the isolation, molecular cloning and recombinant production of patient-derived human antibodies to tumor-associated antigens could be established.
  • oligoclonal memory B cell cultures established from patient peripheral blood lymphocytes (PBLs) combined with a molecular cloning step using single cell RT-PCR and the re-screening of recombinant antibody clones with tissue micro-sections.
  • PBLs peripheral blood lymphocytes
  • a human antibody specific for the tumor-associated antigen NY-ESO-1 was cloned from a melanoma patient who was seropositive for NY-ESO-1 in ELISA and on autologous tumor sections. The screening was performed on ELISA and on tumor tissue using an adaptation of the tissue microarray technology. The obtained tissue-reactive human monoclonal antibody was shown to bind to the N-terminus of NY-ESO-1 that is also shared by the tumor-associated antigen LAGE-1; see Example 3.
  • This method greatly facilitates the cloning of patient-derived antibodies; applied to selected clinical responders it is expected to lead to the identification and isolation of novel candidate antibodies for the immunotherapy of cancer as well as a means for the isolation of novel tumor (associated) antigens which because of the selectivity and specificity of the method of the present invention may be more reliable for use as tumor markers and targets for therapeutic intervention.
  • the present invention relates to a method of isolating a human binding molecule specific for a tumor antigen or tumor-associated antigen comprising:
  • Associated is used herein because while it is clear that the relevant molecule was specifically and aberrantly, respectively, expressed by a tumor or cancer such as melanoma and breast, other cancers such as prostate and lung may also express the antigen and/or non-cancerous cells and tissue.
  • cancer and “tumor” are used interchangeably herein.
  • antibody and fragment thereof, may also refer to other non-antibody binding molecules that bind to a human derived tumor (associated) antigen including but not limited to hormones, receptors, ligands, major histocompatibility complex (MHC) molecules, chaperones such as heat shock proteins (HSPs) as well as cell-cell adhesion molecules such as members of the cadherin, integrin, C-type lectin and immunoglobulin (Ig) superfamilies.
  • MHC major histocompatibility complex
  • HSPs heat shock proteins
  • Ig immunoglobulin
  • Whether a given structure, for example cell or tissue displays a tumor (associated) antigen can be verified by reversing the method described below for isolating and characterizing a tumor (associated) antigen specific binding molecule in that a binding molecule, for example antibody identified by said method is used to screen a sample for binding to the antibody, thereby determining the presence of a tumor (associated) antigen.
  • a liquid sample obtained from the patient can be passed through a first aperture of a duct which is in contact with the specimen target structure firmly held in an object holder, thereby allowing putative binding molecules present in the sample, either in a soluble form or expressed on the cell surface and membrane, respectively, to bind to said target structure.
  • the liquid sample may contain for example lymphocytes and/or antibodies while the specimen may be a tissue section or a membrane coated with molecules or molecular combinations which are distinct for a pathological target structure.
  • the temperature of the object holder may be controlled by an object holder thermostat, for example at a temperature at which natural binding of the putative binding molecule to the tumor (associated) antigen specific for the specimen takes place in the human body.
  • an object holder thermostat for example at a temperature at which natural binding of the putative binding molecule to the tumor (associated) antigen specific for the specimen takes place in the human body.
  • the flowing motion i.e. passing the liquid sample containing binding molecules, preferably at body temperature over the target structure natural systems of binding interactions can be simulated.
  • other methods of incubating the sample with the specimen such as by means of a shaker or rotating table may be used as well.
  • a particular advantage of the above-mentioned system is that it allows an interruption of metabolic processes at any time by decreasing the temperature of the object holder by means of the object holder thermostat.
  • the temperature of the object holder can be decreased to for example 2-10° C., in particular 4° C.
  • a corresponding device that can be used in accordance with the method of the present invention is described in European patent application EP 1 069 431 A2.
  • the method of the present invention will allow identification and characterization of the binding partners as well as at the same time to identify and characterize the molecular classes, molecular groups and/or molecular parts required for the binding process, i.e. the target structures of the specimen, which hitherto may be unknown. This will not only open up new possible ways of diagnosis, but will also provide a new test system for therapeutic approaches on a molecular level.
  • a patient may qualify in accordance with the present invention a pool of healthy volunteers if specific tumor markers, genotype and/or cancerous phenotype predict a high probability of a status of a tumor, which has surprisingly—and possibly due to a specific endogenous immune response—not become clinically manifest, however, by means of early intervention of the humoral immune system with or without involvement of cellular components of the immune system.
  • samples from patients may be used, who have undergone an active immunization with a tumor (associated) antigen, wherein the antibody development has been boosted by the immunization.
  • samples from volunteers which have not received such immunization or corresponding tumor medication may be used as well.
  • samples of a cancer patient i.e. of individuals that have been clinically pre-selected are analyzed for the presence of binding molecules specifically recognizing specimen of pathologically conspicuous structures, for example in ex vivo tissue from clinico-pathologically characterized human patients or animal models like, for example, transgenic mice, or in vitro cell structures, or in pathological allogenic or xenogenic tissue.
  • said patient and/or as said subject providing the specimen are human, most preferably both.
  • said patient has been determined to be affected with a not yet manifested tumor or at risk to develop a tumor by the presence or absence of a tumor marker, or by an unusually stable clinical course.
  • the characteristic cancerous altered sample, cell or tissue specimen is preferably displayed by optical detection after reaction with a binding molecule, i.e. antibody of the present invention.
  • the specimen may be obtained as/from a cell sample, tissue section, cellular smear test, cell or tissue sample of an animal model of a human tumor or in vitro cultured cell and tissue material. Histopathological grading can be performed for example according the modified Bloom and Richardson system (Ellis et al., Tumors of the breast. In: Tavassoli F A, Devilee P. Genetics Tumors of the Breast and the Female Genital Organs. Lyon: IARC Press, (2003), 9-110).
  • the method of the present invention employs a multiple tissue microarray (TMA) as outlined in the Examples; for tissue microarray (TMA) technology comprising miniaturized pathology archives for high-throughput in situ studies see also, e.g., Bubendorf et al., J. Pathol., 195 (2001), 72-79, the disclosure content of which is incorporated herein by reference.
  • TMA tissue microarray
  • the method of the present invention preferably employs immunohistochemistry (IHC) using tissue microarrays (TMA).
  • TMAs and large paraffin embedded tissue sections can be analyzed with the Ventana Benchmark automated staining system (Ventana Medical Systems S.A., Illrich, CEDEX, France) using Ventana reagents for the entire procedure.
  • Ventana Benchmark automated staining system Ventana Medical Systems S.A., Illrich, CEDEX, France
  • Four-micron thick formalin-fixed, paraffin-embedded large tissue sections or TMA sections can be used. Paraffin sections are deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol. Following a rinse with distilled water endogenous peroxidase can be blocked using the Ventana endogenous peroxidase blocking kit.
  • slides are heated with cell conditioning solution (CC1) in a standard protocol.
  • CC1 cell conditioning solution
  • the human antibody candidate and sample containing the same can be adjusted to the Ventana Benchmark system by performing titrations and initial reactivity assessment, for example using spermatogonia of the testis as a positive control in case of supposed anti NY-ESO-1-antibodies.
  • Antigen staining can usually be accepted as positive when there is nuclear and/or cytoplasmic and/or cell surface staining.
  • the immunohistochemical reactivity for the tumor antigen and taa such as NY-ESO-1 can be measured as percentage of positive cells per spot regardless of staining intensities.
  • multiple tissue cores from a variety of patients and tumors can be used.
  • the sample to be analyzed may comprise a body fluid, a cell sample or the supernatant of a cell sample or a derivative thereof.
  • Body fluids such as plasma or peripheral blood can be collected following standard clinical procedures after informed consent of the patients.
  • the sample comprises or is derived from B-cells or memory B-cells and/or comprises antibodies.
  • a particular approach in employing the method according to the present invention is testing samples of B cells and B memory cells from clinically pre-selected volunteers against arrays of specimen of tumor conspicuous tissues expressing a differentiation and cancer testis (CT) antigen, respectively, selected from the group consisting of SCP-1, SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2, NY-BR-1, LAGE-1 and NY-ESO-1.
  • CT differentiation and cancer testis
  • breast cancer specific differentiation antigen is NY-BR-1, which is selectively expressed in normal breast epithelium and about 60% of primary breast cancers on protein level making it a potential target for immunotherapy (Varga et al., Clin Cancer Res. 12 (2006), 2745-2751).
  • NY-ESO-1 may represent the most immunogenic CT antigen, because spontaneous cellular and humoral immune responses can be observed in a high percentage of patients with NY-ESO-1 expressing tumors (Gnjatic et al., Proc. Natl. Acad. Sci. USA 100 (2003), 8862-8867; Jager and Knuth, Breast 14 (2005), 631-635).
  • CT antigens are selectively expressed in human tumor cells and in spermatogonias of the testis, they represent a promising group of target antigens for an immunotherapeutic approach in cancer patients.
  • NY-ESO-1 appears to be strongly immunogenic and is known to induce an efficient humoral and cellular immune response in patients with melanoma and ovarian, breast, lung, as well as bladder cancer making it an ideal target for active cancer immunotherapy. Therefore, in a particular preferred embodiment, the tumor conspicuous tissue employed in the method of the present invention expresses tumor-associated antigen NY-ESO-1.
  • tumor antigens and tumor associated antigens see appropriate databases such as UniProtKB/Swiss-Prot hosted by EMBL, in which an entry for, e.g., NY-ESO-1 may be found under primary accession number P78358.
  • said specimen comprises autologous tumor tissue or cells of said patient, most preferably said patient is a melanoma patient and said specimen comprises autologous sections derived from a lymph node metastasis; see also the examples.
  • the method of the present invention further comprises the steps of:
  • This international application also describes methods for obtaining a nucleic acid sequence that encodes an antibody of interest, comprising the steps of preparing an immortalized B cell clone and obtaining/sequencing nucleic acid from the B cell clone that encodes the antibody of interest and further inserting the nucleic acid into or using the nucleic acid to prepare an expression host that can express the antibody of interest, culturing or sub-culturing the expression host under conditions where the antibody of interest is expressed and, optionally, purifying the antibody of interest.
  • the nucleic acid may be manipulated in between to introduce restriction sites, to change codon usage, and/or to add or optimize transcription and/or translation regulatory sequences. All these techniques are state of the art and can be performed by the person skilled in the art without undue burden.
  • RT-PCR of single sorted cells is preferably employed for obtaining the immunoglobulin gene repertoire for said antibody; see also the Examples.
  • the present invention relates to a binding molecule which is capable of selectively recognizing a tumor antigen or tumor-associated antigen, which preferably can be obtained or validated by the method of the present invention described hereinbefore and illustrated in the Examples.
  • the binding molecule of the present invention recognizes an antigen selected from cancer testis (CT) antigens, such as selected from the group consisting of SCP-1, SSX-4, HOM-TES-85/CT-8, GAGE, SSX-1, SSX-2, NY-BR-1, LAGE-1 and NY-ESO-1; see also supra.
  • CT cancer testis
  • said antigen is tumor-associated antigen NY-ESO-1.
  • NY-ESO-1 is a cancer testis antigen expressed in various malignancies and testicular germ cells; see also supra. Because of its capacity to induce specific humoral and cellular immunity in patients with NY-ESO-1-positive carcinomas, it represents a promising target for cancer immunotherapy.
  • said antibody binds to an epitope defined by an amino acid sequence set forth in SEQ ID NO: 11 representing the amino acid residues 11 to 30 of the NY-ESO-1 protein.
  • the antibody identified in accordance with the method of the present invention to specifically bind to the tumor tissue specimen but not to corresponding healthy tissue does not necessarily recognize the predetermined antigen but a different antigen, hitherto unknown to be specifically or aberrantly expressed in tumor tissue. Therefore, the method of the present invention is also suitable to identify and isolate novel tumor antigens and tumor associated antigens, respectively, and their cognate antibodies; see also infra.
  • the antibody of the present invention is a human antibody in the sense that application of said antibody is substantially free of a HAMA response otherwise observed for chimeric and even humanized antibodies.
  • a binding molecule i.e. antibody has been identified and cloned, which displays particularly high binding affinity with a equilibrium dissociation constant (KD) of the interaction with its cognate antigen in the lower nanomolar range.
  • KD equilibrium dissociation constant
  • the binding affinity of the binding molecule of the present invention with its cognate antigen is about at least 10 ⁇ 7 M, more preferably at least 10 ⁇ 8 M, particularly preferred 10 ⁇ 9 M and still more preferred at least 10 ⁇ 10 M.
  • the present invention exemplifies such binding molecule, i.e. antibody and binding fragments thereof, which may be characterized by comprising in their variable region, i.e. binding domain at least one complementarity determining region (CDR) of the VH and/or VL of the variable region comprising the amino acid sequence depicted in FIG. 4 of (V H ) (SEQ ID NO: 2) and (V L ) (SEQ ID NO: 4).
  • CDR complementarity determining region
  • An exemplary set of CDRs of the above amino acid sequences of the V H and/or V L region as depicted in FIG. 4 are given in SEQ ID NOs: 5 to 10.
  • CDRs may be used, which differ in their amino acid sequence from those set forth in SEQ ID NOs: 5 to 10 by one, two, three or even more amino acids in case of CDR2 and CDR3.
  • the antibody of the present invention is an antibody or antigen-binding fragment thereof, which competes for binding to the NY-ESO-1 with at least one of the antibodies having the V H and/or V L region as depicted in FIG. 4 .
  • Those antibodies may be murine as well, however, humanized, xenogeneic, or chimeric human-murine antibodies being preferred, in particular for therapeutic applications.
  • An antigen-binding fragment of the antibody can be, for example, a single chain Fv fragment (scFv), a F(ab′) fragment, a F(ab) fragment, or an F(ab′) 2 fragment.
  • variable regions of the antibodies are required, which can be obtained by treating the antibody with suitable reagents so as to generate Fab′, Fab, or F(ab′′) 2 portions.
  • suitable reagents so as to generate Fab′, Fab, or F(ab′′) 2 portions.
  • Such fragments are sufficient for use, for example, in immunodiagnostic procedures involving coupling the immunospecific portions of immunoglobulins to detecting reagents such as radioisotopes.
  • the immortalized cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation.
  • Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA.
  • the heavy chain constant region can be exchanged for that of a different isotype or eliminated altogether.
  • the variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed.
  • the coding sequences can be inserted into expression systems contained on vectors which can be transfected into standard recombinant host cells.
  • a variety of such host cells may be used; for efficient processing, however, mammalian cells are preferred.
  • Typical mammalian cell lines useful for this purpose include CHO cells, HEK 293 cells, or NSO cells.
  • the production of the antibody or analog is then undertaken by culturing the modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences.
  • the antibodies are then recovered by isolating them from the culture.
  • the expression systems are preferably designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.
  • the method of the present invention can be extended to also identify and isolate the cognate antigen bound by the antibody of the present invention identified and cloned beforehand.
  • the method of the present invention serves and is used for the identification and isolation of novel tumor (associated) antigens.
  • the method involves the expression of cDNA libraries in a prokaryotic host, said libraries being secured from a tumor sample.
  • the expressed libraries are then immuno-screened with absorbed and diluted sera, in order to detect those antigens which elicit high titer humoral responses.
  • This methodology is known as the SEREX method (“Serological identification of antigens by Recombinant Expression Cloning”).
  • serological identification of tumor-specific antigens is accomplished by use of a cognate antibody that has been screened against tumor tissue derived from cancer patients who show a clinical response and have developed antibodies against their autologous tumor tissue.
  • the antigen and truncated forms thereof can than be validated to be reactive with antibodies in the serum of said cancer patient and other cancer patients.
  • the target structure i.e. the tumor-associated antigen
  • the sample and respective binding molecule therein it may be identified and its encoding nucleic acid molecule isolated and cloned.
  • Identification of the amino acid sequence of the antigen can be achieved by means and methods well known in the art, for example using mass spectrometric (MS) techniques such as those described in international application WO00/11208 and specifically those described in Hock et al., Nat Med 8 (2002), 1270-1275; Hock et al., Neuron 38 (2003), 547-554.
  • MS mass spectrometric
  • the antibody identified in accordance with present invention produced in vitro binds to pathological structures, for example to tumor tissue sections, but not significantly to healthy tissues
  • a promising antibody candidate has been identified whose molecular target structure can subsequently be enriched and purified via its binding properties to the antibody from pathological tissues and, as a result, can be identified and characterized by means of protein analytical and mass spectrometric methods like, for example, MALDI/TOF (Williams, Methods Cell. Biol. 62 (2000), 449-453; Yates, J. Mass. Spectrom. 33 (1998), 1-19).
  • the present invention relates to a tumor antigen and tumor-associated antigen, respectively, which is recognized by the binding molecule, especially antibody of the present invention described hereinbefore, both in peptide form and in post translational modified form, wherein the antigen is preferably a peptide consisting of least 6-50, and preferably no more than 10-100 amino acids in length, which contain the cognate epitope.
  • the antigen of the present invention comprises the amino acid sequence of SEQ ID NO: 11 and consists of about 10 to 30 amino acids, and preferably is no more than about 20 amino acids in length.
  • the molecule is large enough to be antigenic without any posttranslational modification, and hence it is useful as an immunogen, when combined with an adjuvant (or without it), in both precursor and post-translationally modified forms.
  • These antigens and peptides can be used to determine whether or not antibodies are present in a sample, such as serum or blood.
  • the antigen of the present invention is capable of eliciting a humoral response in human.
  • the present invention relates to novel tumor antigens and tumor-associated antigens detectable with the antibody of the present invention, which hitherto have not been disclosed in the prior art and/or subject of a patent or patent application.
  • the present invention also relates to a polynucleotide encoding the antigen or binding molecule of the present invention, in case of the antibody preferably at least a variable region of an immunoglobulin chain of the antibody described above.
  • said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the V H and/or V L of the variable region of the said antibody.
  • each variable domain (the heavy chain V H and light chain V L ) of an antibody comprises three hypervariable regions, sometimes called complementarity determining regions or “CDRs” flanked by four relatively conserved framework regions or “FRs” and refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • CDRs complementarity determining regions
  • FRs relatively conserved framework regions
  • the hypervariable regions or CDRs of the human IgG subtype of antibody comprise amino acid residues from residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues from a hypervariable loop, i.e.
  • the antibody binds with a dissociation constant (K D ) of 10 ⁇ 7 M or less, and binds to the predetermined antigen with a K D that is at least twofold less than its K D for binding to a nonspecific antigen (e.g., BSA, casein, or any other specified polypeptide) other than the predetermined antigen.
  • K D dissociation constant
  • an antibody recognizing an antigen and an antibody specific for an antigen are used interchangeably herein with the term “an antibody which binds specifically to an antigen”.
  • “highly specific” binding means that the relative K D of the antibody for the specific target epitope, i.e.
  • tumor antigen or taa is at least 10-fold less than the K D for binding that antibody to other ligands.
  • the antibody binds its cognate tumor antigen and taa, respectively, with a dissociation constant (K D ) of 10 ⁇ 9 M or less.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Berzofsky et al., “Antibody-Antigen Interactions” In Fundamental Immunology, Paul, W. E., Ed., Raven Press New York, N Y (1984), Kuby, Janis Immunology, W. H. Freeman and Company New York, N Y (1992), and methods described herein.
  • the measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions, e.g., salt concentration, pH.
  • affinity and other antigen-binding parameters e.g., K sub D, IC50, are preferably made with standardized solutions of antibody and antigen, and a standardized buffer.
  • variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function.
  • present invention also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and which advantageously have substantially the same or similar binding properties as the antibody described in the appended examples.
  • variable domains or CDRs described herein antibodies can be constructed according to methods known in the art, e.g., as described in European patent applications EP 0 451 216 A1 and EP 0 549 581 A1.
  • the present invention also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more, preferably not more than two amino acid substitutions.
  • the antibody of the invention comprises in one or both of its immunoglobulin chains two or all three CDRs of the variable regions as set forth in SEQ ID NOs: 5 to 10.
  • the polynucleotide of the invention encoding the above described antibody may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • said polynucleotide is part of a vector.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the polynucleotide of the invention is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions.
  • polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or only one.
  • said polynucleotides may be under the control of the same promoter or may be separately controlled for expression.
  • Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the P L , lac, trp or tac promoter in E.
  • coli and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter, CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • transcription termination signals such as the SV40-poly-A site or the tk-poly-A site
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (Invitrogen), or pSPORT1 (GIBCO BRL).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the immunoglobulin light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms may follow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979).
  • the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding the antigen or preferably a variable domain of an immunoglobulin chain of an antibody of the invention; optionally in combination with a polynucleotide of the invention that encodes the variable domain of the other immunoglobulin chain of the antibody of the invention.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.
  • viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus.
  • Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides of the invention can be transferred into the host cell by well known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts; see Sambrook, supra.
  • the present invention furthermore relates to host cells transformed with a polynucleotide or vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces , in particular those of the species S. cerevisiae .
  • prokaryotic is meant to include all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of an antibody of the invention or the corresponding immunoglobulin chains.
  • Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis .
  • eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells, most preferably HEK 293, NSO and CHO cells.
  • the antibodies or immunoglobulin chains encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated.
  • Antibodies of the invention or the corresponding immunoglobulin chains may also include an initial methionine amino acid residue.
  • a polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (“Catalogue of Cell Lines and Hybridomas,” Fifth edition (1985) Rockville, Md., U.S.A., which is incorporated herein by reference).
  • transgenic animals, preferably mammals, comprising cells of the invention may be used for the large scale production of the antibody of the invention.
  • the present invention relates to a method for the production of an antigen of the present invention or of a tumor antigen and taa-specific binding molecule, respectively, an antibody or a binding fragment or immunoglobulin chain(s) thereof, said method comprising
  • the transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth.
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, “Protein Purification”, Springer Verlag, N.Y. (1982).
  • the antibody or its corresponding immunoglobulin chain(s) of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions.
  • the isolation and purification of the, e.g., recombinantly expressed antibodies or immunoglobulin chains of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody of the invention.
  • the antibodies of the invention can be further coupled to other moieties for, e.g., drug targeting and imaging applications. Such coupling may be conducted chemically after expression of the antibody or antigen to site of attachment or the coupling product may be engineered into the antibody or antigen of the invention at the DNA level.
  • the DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.
  • Substantially pure immunoglobulins of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity most preferred, for pharmaceutical uses.
  • the antibodies Once purified, partially or to homogeneity as desired, the antibodies may then be used therapeutically (including extracorporally) or in developing and performing assay procedures.
  • the present invention also involves a method for producing cells capable of expressing an antibody of the invention or its corresponding immunoglobulin chain(s) comprising genetically engineering cells with the polynucleotide or with the vector of the invention.
  • the cells obtainable by the method of the invention can be used, for example, to test the interaction of the antibody of the invention with its antigen.
  • the immunoglobulin or its encoding cDNAs may be further modified.
  • the method of the present invention comprises any one of the step(s) of producing a chimeric antibody, humanized antibody, single-chain antibody, Fab-fragment, bi-specific antibody, fusion antibody, labeled antibody or an analog of any one of those.
  • Corresponding methods are known to the person skilled in the art and are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988.
  • xenogeneic antibodies Further sources of antibodies to be utilized in accordance with the present invention are so-called xenogeneic antibodies.
  • the general principle for the production of xenogeneic antibodies such as human antibodies in mice is described in, e.g., international applications WO91/10741, WO94/02602, WO96/34096 and WO 96/33735.
  • the antibody of the invention may exist in a variety of forms besides complete antibodies; including, for example, Fv, Fab and F(ab) 2 , as well as in single chains; see e.g. international application WO88/09344.
  • the antibodies of the present invention or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination.
  • Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994).
  • Modifications of the antibody of the invention include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C-terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
  • the present invention encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus; see, e.g., international application WO00/30680 for corresponding technical details.
  • the present invention encompasses small peptides including those containing a binding molecule as described above, for example containing the CDR3 region of the variable region of any one of the mentioned antibodies, in particular CDR3 of the heavy chain since it has frequently been observed that heavy chain CDR3 (HCDR3) is the region having a greater degree of variability and a predominant participation in antigen-antibody interaction.
  • Such peptides may easily be synthesized or produced by recombinant means to produce a binding agent useful according to the invention. Such methods are well known to those of ordinary skill in the art.
  • Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available.
  • the peptides can be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.
  • the present invention relates to any binding molecule, antibody or binding fragment which are obtainable in accordance with above described means and display the mentioned properties, i.e. which specifically recognize a tumor antigen or taa, and which for therapeutic use preferably maintain a substantially human framework so as to be devoid of immunogenicity in a patient.
  • Such antibodies and binding molecules can be tested for their binding specificity and affinity by for example by using the method of isolating human tumor (associated) antigen specific binding molecules described hereinbefore.
  • the binding molecule, antibody, immunoglobulin chain or a binding fragment thereof or the antigen is detectably labeled.
  • Labeling agents can be coupled either directly or indirectly to the antibodies or antigens of the invention.
  • One example of indirect coupling is by use of a spacer moiety.
  • the antibodies of the present invention can comprise a further domain, said domain being linked by covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art and described above or can be performed by, e.g., chemical cross-linking as described in, e.g., international application WO94/04686.
  • the additional domain present in the fusion protein comprising the antibody of the invention may preferably be linked by a flexible linker, advantageously a polypeptide linker, wherein said polypeptide linker comprises plural, hydrophilic, peptide-bonded amino acids of a length sufficient to span the distance between the C-terminal end of said further domain and the N-terminal end of the antibody of the invention or vice versa.
  • the therapeutically or diagnostically active agent can be coupled to the antibody of the invention or an antigen-binding fragment thereof by various means. This includes, for example, single-chain fusion proteins comprising the variable regions of the antibody of the invention coupled by covalent methods, such as peptide linkages, to the therapeutically or diagnostically active agent.
  • molecules which comprise at least an antigen-binding fragment coupled to additional molecules covalently or non-covalently include those in the following non-limiting illustrative list.
  • Traunecker, Int. J. Cancer Surp. SuDP 7 (1992), 51-52 describe the bispecific reagent janusin in which the Fv region directed to CD3 is coupled to soluble CD4 or to other ligands such as OVCA and IL-7.
  • the variable regions of the antibody of the invention can be constructed into Fv molecules and coupled to alternative ligands such as those illustrated in the cited article. Higgins, J.
  • Infect Disease 166 (1992), 198-202 described a hetero-conjugate antibody composed of OKT3 cross-linked to an antibody directed to a specific sequence in the V3 region of GP120.
  • Such hetero-conjugate antibodies can also be constructed using at least the variable regions contained in the antibody of the invention methods: Additional examples of specific antibodies include those described by Fanger, Cancer Treat. Res. 68 (1993), 181-194 and by Fanger, Crit. Rev. Immunol. 12 (1992), 101-124.
  • Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins.
  • the antibodies of the present invention can be used in a corresponding way to obtain such immunotoxins.
  • Illustrative of such immunotoxins are those described by Byers, Seminars Cell. Biol. 2 (1991), 59-70 and by Fanger, Immunol. Today 12 (1991), 51-54.
  • the above described fusion protein may further comprise a cleavable linker or cleavage site for proteinases.
  • These spacer moieties can be either insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to enable drug release from the antigen at the target site.
  • therapeutic agents which can be coupled to the antibodies and antigens of the present invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • the drugs with which can be conjugated to the antibodies and antigens of the present invention include compounds which are classically referred to as drugs such as mitomycin C, daunorubicin, and vinblastine.
  • the present invention relates to the use of a binding molecule or an antibody of the present invention or binding fragment thereof for the preparation of a composition for in vivo detection of or targeting a therapeutic and/or diagnostic agent to a tumor.
  • the present invention relates to compositions comprising the aforementioned binding molecule, antibody or binding fragment or antigen of the present invention or chemical derivatives thereof, or the polynucleotide, vector or cell of the invention.
  • the composition of the present invention may further comprise a pharmaceutically acceptable carrier.
  • the term “chemical derivative” describes a molecule that contains additional chemical moieties that are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule.
  • the pharmaceutical composition of the present invention may comprise further anti-tumor agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.
  • the present invention relates to the use of the binding molecule, antibody or binding fragment of the present invention or of a binding molecule having substantially the same binding specificities of any one thereof, the antigen, the polynucleotide, the vector or the cell of the present invention for the preparation of a pharmaceutical or diagnostic composition for the preparation of a pharmaceutical or diagnostic composition for treating or preventing the progression of a tumor; for the amelioration of symptoms associated with a tumor; for diagnosing or screening a subject for the presence of a tumor or for determining a subject's risk for developing a tumor.
  • Said pharmaceutical composition can be designed to be administered intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, parenterally or as an aerosol; see also infra.
  • the present invention relates to a method of treating or preventing the progression of a tumor in a subject; for ameliorating the symptoms associated with a tumor; for diagnosing or screening a subject for the presence of a tumor or for determining a subject's risk for developing a tumor, which method comprises administering to said subject an effective amount of any one of the afore-described binding molecules, antibodies, antigens, polynucleotides, vectors or cells of the instant invention.
  • the therapeutic and diagnostic applications in accordance with the present invention include melanoma and breast cancer, and are most suitable for use in targeting a tumor comprising primary breast carcinoma and/or metastases. Unless stated otherwise, the terms “tumor”, “cancer”, “carcinoma” and the like are used interchangeably herein.
  • the present invention encompasses any use of a tumor antigen binding molecule comprising at least one CDR of the above described human antibody, in particular for diagnosing and/or treating a disorder related to a tumor.
  • said binding molecule is an antibody of the present invention or an immunoglobulin chain thereof.
  • the present invention relates to anti-idiotypic antibodies of any one of the mentioned antibodies described hereinbefore. These are antibodies or other binding molecules which bind to the unique antigenic peptide sequence located on an antibody's variable region near the antigen binding site.
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising any one of the above described binding molecules, antibodies, antigen-binding fragments, polynucleotides, vectors or cells of the invention and optionally suitable means for detection such as reagents conventionally used in immuno or nucleic acid based diagnostic methods.
  • the antibodies of the invention are, for example, suited for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize the antibody of the invention are competitive and non-competitive immunoassays in either a direct or indirect format.
  • examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western blot assay.
  • RIA radioimmunoassay
  • sandwich immunometric assay
  • flow cytometry flow cytometry
  • Western blot assay Western blot assay.
  • the antigens and antibodies of the invention can be bound to many different carriers and used to isolate cells specifically bound thereto.
  • Examples of well known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble or insoluble for the purposes of the invention.
  • the present invention relates to a method for determining cancer cells, e.g., breast cancer cells in a sample comprising assaying said sample for expression of at least one tumor associated antigen like NY-ESO-1 by assaying for presence of the antigen protein, with an antibody of the present invention which specifically binds to said antigen, wherein expression of at least one of said antigens is indicative of the presence of cancer cells in said sample.
  • a similar method which may be adapted in accordance with the present invention is described in U.S. Pat. No. 6,338,947 for SCP-1, NY-ESO-1 and SSX-2.
  • the method of the present invention may further comprise assaying said sample for at least one of SCP-1, NY-ESO-1, SSX-1, SSX-2, SSX-4, MAGE-1, GAGE, MAGE-3, and LAGE-1.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.
  • subject or “patient” refers to a mammal, preferably a human, in need of treatment for a condition, disorder or disease.
  • compositions of the present invention can be formulated according to methods well known in the art; see for example Remington: The Science and Practice of Pharmacy (2000) by the University of Sciences in Philadelphia, ISBN 0-683-306472.
  • suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc.
  • Compositions comprising such carriers can be formulated by well known conventional methods.
  • These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intra-muscular, topical or intradermal administration.
  • the regimen is a continuous infusion, it should also be in the range of 1 ⁇ g to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the pharmaceutical composition of the invention may comprise further agents such as anti-tumor agents and cytotoxic drugs, depending on the intended use of the pharmaceutical composition.
  • a therapeutically effective dose or amount refers to that amount of the active ingredient sufficient to ameliorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the therapeutic agent in the composition is present in an amount sufficient to prevent metastasis and neoplastic growth of cells.
  • compositions in accordance with the present invention can be used for the treatment of tumors and cancer including but not limited to melanoma, primary breast cancer, hepatocellular carcinoma and metastases as well as other human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, carcinoma of the head/neck, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, pa
  • Tumor material as well as normal tissue not necessary for the routine histopathological analysis was frozen in liquid nitrogen. Serum and blood for the isolation of memory B cells was collected from patient C1 in accordance with the informed consent that was approved by the local Ethical committee and signed by the patient.
  • Memory B cells were isolated from human peripheral blood lymphocytes by a two step selection procedure.
  • the pan B cell marker CD22 was used for the positive selection of B cells using the MACS technology (Miltenyi, Bergisch Gladbach, Germany).
  • PBL were labeled using MACS-conjugated anti human CD22 mAbs, phycoerythrin-conjugated mAbs anti human IgD and APC-conjugated antibodies anti human IgM, IgA, CD3, CD8, CD56 (Becton Dickinson, Basel, Switzerland).
  • Pan B cells were isolated by positive selecting CD22-positive cell using a midi MACS device and LS columns (Miltenyi) followed by selection of phycoerythrin- and APC-negative cells using a MoFlo cell sorter (DakoCytomation, Fort Collins, USA).
  • CD22-positive IgM-, IgD-, IgA-negative B cells were then incubated with EBV containing supernatant obtained from B95-8 cells in the presence of CpG 2006 (6, 15) in B cell medium containing RPMI 1640 supplemented with 10% fetal calf serum. Cells were seeded in at 50 cells per well in B cell medium on 30.000 irradiated feeder PBL prepared from voluntary donors.
  • 96 well strip well microplates (Corning, N.Y., USA) were coated with 25 ⁇ l/well of a 1 ⁇ g/ml recombinant NY-ESO-1 protein in PBS overnight at 4° C. Plates were washed with PBS-T and blocked overnight at 4° C. with PBS containing 5% milk powder (Rapilait, Migros, Switzerland). B cell conditioned medium, patient serum and recombinant antibody preparations were incubated for 2 h at room temperature.
  • Binding of human antibodies to NY-ESO-1 was determined using a donkey anti-human IgG-HRP secondary antibody (Jackson ImmunoResearch Europe Ltd., Cambridgeshire, UK) followed by measurement of the HRP activity using a TMB substrate solution (TMB, Sigma, Buchs, Switzerland).
  • Cylinders of tumor tissues measuring 0.6 mm in diameter were punched out of paraffin embedded NY-ESO-1-positive tumor tissue and healthy control tissue. Pairs formed of a cylinder of tumor tissue and of healthy control tissue were placed at each position of a 2 ⁇ 4 grid whose dimensions were compatible with the microtiter format of the B cell culture plates and conventional multi channel pipettes.
  • Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue. Heat-based antigen retrieval was applied to all slides. Non-specific fluorescence was blocked using polyclonal rabbit anti-human IgG (Dako, Baar, Switzerland) for 30 min at room temperature followed by a second block in 1% of low fat milk (Rapilait, Migros, Switzerland) for 10 min. Primary antibody or B cell conditioned medium was incubated overnight at 4° C. Binding of human antibodies to NY-ESO-1 was revealed using Cy 3-conjugated secondary antibodies to human IgG (Jackson ImmunoResearch Europe Ltd., Soham, UK).
  • Single cells obtained from a memory B cell culture were deposited into PCR tubes.
  • cDNA was prepared using primers specific for the constant regions of immunoglobulin G heavy, ⁇ -light and ⁇ -light chains.
  • PCR amplification of immunoglobulin heavy and light chain variable regions was performed according to standard protocols (7, 16). Immunoglobulin heavy and light chain variable regions were amplified using a semi nested PCR approach. 1st round PCR was performed with primers specific for the IgG constant region and pools of primers specific for conserved framework 1 regions of heavy and light chain Ig variable region families (7).
  • 293-T human embryonic kidney cells were cultured in DMEM supplemented with 10% ultra-low IgG FCS, 1% penicillin-streptomycin and 1% L-glutamin (Invitrogen, Basel, Switzerland). Co-transfection with immunoglobulin heavy and light chain encoding plasmid DNA was performed by the standard calcium phosphate precipitation method. Thereafter the cells were cultured in serum free D-MEM supplemented with 1% Nutridoma SP (Roche, Rotnch, Switzerland). Supernatants were collected after 8 days of culture and IgG was purified on a protein G column (Amersham Biosciences, Upsala, Sweden) using fast protein liquid chromatography (FPLC) (Amersham Biosciences, Upsala, Sweden). Purified Manhattan antibody was biotinylated following the manufacturers instructions (SIGMA, Buchs, Switzerland).
  • SK-MEL-37 cells were grown onto microscope slide, fixed with formaldehyde and permeabilized with 1% Triton X-100 for 10 min at room temperature. After blocking with 10% goat serum for 1 h RT cells were incubated with Manhattan at a concentration of 1 ug/ml or negative control antibody (hu8-18c5 (17) expressed in recombinant fashion with a human Fc region) in PBS/1% goat serum/0.2% Triton X-100 overnight at 4°. Bound antibodies were visualized by staining with goat anti-human IgG Alexa Fluor® 546 (1:300, Molecular Probes, Leiden, Netherland) for 1 h RT. Microscopy was performed using a Leica SP 5 microscope.
  • a melanoma patient was selected with a serum titer to the taa NY-ESO-1 in ELISA and on autologous lymph node sections obtained at biopsy. Post vaccination with recombinant vaccinia virus expressing full length NY-ESO-1 a partial clinical response demonstrated by the regression of two NY-ESO-1-positive metastases in the liver was observed. 50 ml Peripheral blood was collected from the patient and surface IgM/IgD double-negative B cells representing the Ig-switched memory B cells were isolated and cultured after immortalization using a modified Epstein Barr virus transformation protocol (6). 100.000 memory B cells were obtained and were seeded into 96 well microtiter templates at 50 cells per well.
  • the setup of the tissue screen consisted of 8 pairs of tissue rods of NY-ESO-1-positive mamma tumors and healthy mamma tissue as controls mounted on to glass slides. Due to the miniaturization of this assay 15 ⁇ l of B cell conditioned medium were sufficient to perform the assay. The ability to compare the conditioned medium of several memory B cell cultures and of negative controls on a single slide facilitated the evaluation of the fluorescence staining.
  • tissue-reactive culture 12D7 The evaluation of the 9 ELISA-positive B cell cultures in this tissue assay identified one culture that yielded a higher staining intensity as compared to that of the other 8. This is illustrated in FIG. 1B , where immunofluorescence obtained with tissue-reactive culture 12D7 is compared to immunofluorescence obtained with well 9D1 which was rated as being not tissue-reactive.
  • NY-ESO-1 Specific Human Monoclonal Antibody Manhattan Binds to peptide NY-ESO-1 11-30 with a KD of 10 ⁇ 10
  • the avidity of Manhattan was determined by competition ELISA using increasing concentrations of soluble NY-ESO-1 11-30 peptide to compete for the plate bound peptide. As depicted in FIG. 3C , the antigen-binding equilibrium dissociation constant (KD) of the interaction of Manhattan with its cognate peptide was in the lower nanomolar range.
  • KD antigen-binding equilibrium dissociation constant
  • the above experiments provide a general method for the identification and molecular cloning of antibodies directly from peripheral blood lymphocytes (PBLs) of human subjects.
  • the method of the present invention could be proven by isolating a human monoclonal antibody to the tumor-associated antigen NY-ESO-1 from a melanoma patient.
  • This primary screen was followed by a molecular cloning step the purpose of which was to identify and isolate the single clone of B cells that secreted the antibody detected in the primary screening.
  • a second object of the present invention was the isolation of an antibody to the tumor-associated antigen NY-ESO-1 with tissue-reactivity. This was motivated by the observation, that serum of the patient contained antibodies that reacted with NY-ESO-1-positive autologous tissue taken at biopsy. To that end the micro-array technology was adapted for the screening of memory B cell cultures. This had several advantages as compared to classical methods of immunohistochemistry. First, the availability on one single slide of several replica positions allowing to assay and to compare several samples. Second, the possibility to place positive tissue adjacent to negative tissue greatly improves assay sensitivity, a feature which was crucial since incubation with conditioned medium of memory B cell cultures often resulted in very weak staining. Third, this miniaturization of the assay format needs much less of conditioned medium which also is a decisive factor since the culture volume of memory B cell cultures was generally less than 200 ⁇ l.
  • NY-ESO-1 has been described as an intracellular antigen (11) and was shown in this study to be localized in the nucleus, at least in the cell line SK-Me-37. In this context, surface staining on live SK-Me-37 using biotinylated Manhattan had been negative.
  • NY-ESO-1 frequently induces humoral responses which correlate with a bad prognosis for the patient (13). While this could be a mere correlation due to increased abundance of antigen as the tumor grows, a tolerogenic role of this B cell response could also be hypothesized. According to this scenario, free antigen released by necrotic or apoptotic tumor cells would induce a strong B cell response, the B cells then would present antigen as a result of Fc-receptor-mediated uptake of immune complexes (14). As B cells may be poor APC, this presentation could result in the induction of tolerance of NY-ESO-1-reactive T cells and thus prevent tumor rejection.

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