Classifications

• • 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
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • 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
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • 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

• TECHNICAL FIELD This invention relates generally to human immune response to antigens expressed on the surface of tumor cells, and more specifically, to human monoclonal antibodies which recognize major tumor- associated gangliosides, particularly GD3, GM3 and GD2.
• the invention relates also to methods of using the antibodies and derivatives thereof in diagnosing and therapeutically treating human cancers, especially melanoma and other cancers of neuroectodermal origin.
• Cancer has remained one of the most deadly threats to human health. In spite of extensive efforts on cancer research, existing methods of cancer diagnosis and therapy are of limited effectiveness. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult. Furthermore, cancers can arise from almost any tissue in the body through malignant transformation of one or a few normal cells within the tissue, and each type of cancer with particular tissue origin differs from the others. Accordingly, a desirable method of diagnosis or treatment should be able to not only distinguish malignant cells from surrounding normal cells, but also selectively diminish the particular type of cancer. Current methods of cancer treatment are relatively non-selective. Surgery removes the diseased tissue, radiotherapy shrinks solid tumors and chemotherapy kills rapidly dividing cells. Chemotherapy, in particular, results in numerous side effects, in some cases so severe as to preclude the use of potentially effective drugs. Moreover, cancers often develop resistance to chemotherapeutic drugs.
• Gangliosides are glycolipids containing sialic acids. They are molecules composed of a carbohydrate chain with sialic acid at the cell surface and a hydrophobic ceramide in the lipid bilayers. Svennerholm (1963) J. Neurochem. 10:863-871.
• Gangliosides act as regulators of cell growth by altering growth factor signals and are also involved in cell adhesion and cell matrix interactions. Cheresh et al. (1986) J. Biol. Chem. 102:688-696. Ganglioside expression is heterogeneous in terms of cell type, organ of origin and species. Although present in normal and cancer tissues, gangliosides are most abundant in tissues originating in the neural crest.
• Neoplastic transformation of normal cells results in both quantitative and qualitative changes in cell surface gangliosides. Hokomori (1985) Cancer Res.
• the monosialoganglioside GM3 makes up approximately 90% of the gangliosides found on the cell surface, with the disialoganglioside GD3 constituting only 5% of the ganglioside population.
• Human tumors of neuroectodermal origin such as glioma and neuroblastoma express large amounts of GM2, GD2 or GD3, whereas in melanoma the most common and abundant gangliosides are GM3, GM2, GD2 and GD3. Tsuchida et al. (1987).
• GM3:GD3 ratio varies with the advent of the neoplastic transformations that take place within a tumor and serves as a useful index of prognosis.
• Tumor tissue can arbitrarily be categorized into three major groups based on GM3:GD3 ratios. Group I, in which the ratio ranges from 13:1 to 1:5; Group II, with a ratio from 1.4:1 to 1 : 1.4 and Group III with a ratio from 1 : 1.5 to 1:5. Groups II and III were found to have the least survival time of 60 to 90 months.
• ganglioside antigens desirable targets for immunotherapy because these antigens undergo specific changes during malignant transformation.
• Human monoclonal antibodies that would interact more efficiently with the human effector system without eliciting HAMA responses.
• the number of human MAb cell lines that stably secrete human antibodies to gangliosides are notably few. Yamaguchi et al. (1987) Proc. Natl. Acad. Sci. USA, 84:2416-2420; and Wu et al. (1986) Cancer Res. 46:440-443.
• small-scale trials of these HuMAbs have demonstrated clinical efficacy, further studies have been hindered, largely due to the lack of sufficient antibody production.
• EBV-imortalization techniques where B lymphocytes from melanoma patients were infected with Epstein-Barr virus (EBV) in vitro and cloned for the selection of antibody producing B-lymphoblasts.
• EBV-imortalization technique One major disadvantage of the EBV-imortalization technique is that the EBV-infected B-cell lines frequently lose their ability to secret sufficient amount of antibody in culture. Yamaguchi et al. (1987). The lack of production of EB V-transformed cells is thought to be due to the instability of antibody secreting genes or outgrowth of non-producing cell populations within the culture.
• IgG class antibodies are useful in primarily complement-dependent immunoreactivities against tumors
• IgG class antibodies activate the more effective, longer sustained antibody- dependent, cell-mediated killing system.
• production of IgM antibody in large quantity is difficult and costly due to the antibody's complex pentamer structure and large molecular weight. Therefore, there is a need to produce IgG class MAbs in facilitating large scale and more effective clinical immunotherapy.
• One disadvantage of using ganglioside antigens as targets for HuMAbs is their relatively low density on tumor cells.
• the present invention encompasses compositions of antigen binding fragments of a newly identified human monoclonal antibody (HuMAb), which is immunoreactive to specific types of cancers, especially melanoma and other cancer cells of neuroectodermal origin.
• Human monoclonal antibody (HuMAb)
• the antibody is characterized as having specificity for the major tumor associated gangliosides, particularly GD3, GM3 and GD2.
• the antibody is GMA1, which is a human IgGl.K antibody comprising H chains having the amino acid sequence of SEQ ID NO:2 and L chains having the amino acid sequence of SEQ ID NO:4.
• the invention further encompasses an isolated polynucleotide encoding the antigen binding fragments with the properties described above.
• the polynucleotide encodes at least a portion of the antibody that is distinguishable from other antibodies.
• the polynucleotide encodes a portion of the H or L chain V region of the antibody.
• the polynucleotide encodes a portion of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.
• the invention also encompasses isolated polynucleotides having at least a portion of the sequence of SEQ ID NO: 1 or SEQ ID NO:3.
• the polynucleotides encode at least about 5 amino acid residues.
• Another embodiment of the present invention is a polypeptide comprising at least a portion of the V region of the antibody with the properties described above.
• the invention provides a polypeptide fragment of the antibody V region, comprising at least 5 consecutive amino acid residues of SEQ ID NO:2 or
• the 5 consecutive amino acid residues are from a CDR of the L or H chain V region of the antibody.
• Also provided in the present invention are methods for detecting major tumor associated gangliosides in a sample, preferably a biological sample, wherein the antigen binding fragments are used to contact the subject samples and recognize the specific ganglioside antigens.
• the major tumor associated gangliosides to be detected are GD3, GM3 and GD2.
• the antigen binding fragments can be detectably labeled in various ways to facilitate the detection of the antibody- antigen complex so formed.
• the present invention further encompasses methods for cancer diagnosis by detecting the tumor associated gangliosides in a sample as described above, wherein a diagnostically effective amount of detectably labeled antigen binding fragment is used to image the subject in need of cancer diagnosis or as an ex vivo tissue or cell labeling procedure.
• the present invention encompasses methods of treating cancers expressing the major tumor associated gangliosides.
• the methods comprise administering an amount of a physiologically acceptable composition containing the antigen binding fragment effective to achieve the desired effect, be it palliation of an existing tumor mass or prevention of recurrence.
• GMA1 reactivity with live unfixed human tumor cells.
• Column 1 Human IgG; column 2, hybridoma GMA1 (GMA1); column 3, recombinant GMA1 (rGMAl).
• Rows 1, 2, 3 and 4 show reactivity of the antibodies against SK-Mel-28, PANC-1,
• Lanes 2-6 show GMA1 immunostaining with 5, 2.5, 1.25, 0.6, and 0.3 nmoles of ganglioside mixture, respectively.
• Figure 5 Nucleotide and deduced amino acid sequences of GMAl heavy
• A GMAl V H sequence from amino acids -19 to 118. The first 19 amino acids encode the signal sequence. Italicized amino acids represent CDR regions, CDRl (31-35), CDR2 (50-66) and CDR3 (99-107).
• B GMAl V L sequence from amino acids -20 to 1 12. The first 20 amino acids encode the signal sequence. The italicized amino acids represent CDR regions CDRl (24- 39), CDR2 (55-61) and CDR3 (94-102).
• FIG. 1 Diagram of the pMEX-8 GMAl antibody expression vector.
• the vector is shown with the sites of cleavage by selected restriction endonucleases. Regions of the vector encoding different transcriptional regions are indicated by the heavy black bars. The insertion points for the GMAl heavy (V H C H ) and light (V L C L ) chains are shown.
• Ap R represents the B-lactamase gene and ORI is the origin of replication from plasmid pUC 19.
• SS signal sequence
• CMV cytomegalovirus promoter
• BGH bovine growth hormone polyadenylation region
• SV40 PE enhancerless SV40 promoter
• DHFR dihydrofolate reductase gene
• NEO neomycin resistance gene
• SV40 PA SV40 polyadenylation site.
• the present invention encompasses compositions of antigen binding fragments of a newly identified human monoclonal antibody (HuMAb), which is immunoreactive to specific types of cancers, especially melanoma and other cancer cells of neuroectodermal origin.
• the antibody is characterized as having specificity for the major tumor associated ganglioside GD3, GM3 and GD2.
• the antibody is GMAl, which is a human IgGl.K antibody comprising H chains having the amino acid sequence of SEQ ID NO:2 and L chains having the amino acid sequence of SEQ ID NO:4.
• the invention further encompasses an isolated polynucleotide encoding the antigen binding fragments with properties described above.
• the polynucleotide encodes at least a portion of the antibody that is distinguishable from other antibodies.
• the polynucleotide encodes a portion of the H or L chain V region of the antibody. More preferably, the polynucleotide encodes a portion of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 encoding at least about five amino acid residues.
• the invention also encompasses isolated polynucleotides having at least a portion of the sequence of SEQ ID NO: 1 or SEQ ID NO:3 of at least about fifteen nucleotides.
• Another embodiment of the present invention is a polypeptide comprising at least a portion of the V region of the antibody having the properties described above.
• the invention provides a polypeptide fragment of the antibody V region, comprising at least 5 consecutive amino acid residues of SEQ ID NO:2 or SEQ ID NO:4.
• the 5 consecutive amino acid residues are from a CDR of the L or H chain V region of the antibody.
• Also provided in the present invention are methods for detecting major tumor associated gangliosides in a sample, wherein the antigen binding fragments are used to contact the subject samples and recognize the specific ganglioside antigens.
• the major tumor associated gangliosides to be detected are GD3, GM3 and GD2.
• the antigen binding fragments can be detectably labeled in various ways to facilitate the detection of the antibody-antigen complex so formed.
• the present invention further encompasses methods for cancer diagnosis by detecting the tumor associated gangliosides in a sample as described above, wherein a diagnostically effective amount of detectably labeled antigen binding fragments are used to image the subject in need of cancer diagnosis or as an ex vivo tissue or cell labeling procedure.
• the present invention further encompasses methods for in vivo detection of cancer cells.
• a diagnostically effective amount of detectably labeled antigen binding fragment is given to the subject in need of tumor imaging.
• diagnosisically effective means that the amount of detectably labeled antigen binding fragment is administered in sufficient quantity to enable detection of the neoplasia.
• the concentration of detectably labeled antigen binding fragment which is administered should be sufficient such that the binding to those cells having elevated ganglioside levels is detectable compared to the background. Further, it is desirable that the detectably labeled antigen binding fragment be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
• the dosage of detectably labeled antigen binding fragment for in vivo diagnosis is somewhat patient-specific and depends on such factors as age, sex, and extent of disease.
• the dosage of antigen binding fragment can vary from about 0.01 mg/m 2 to about 500 mg/m 2 , preferably 0.1 mg/m 2 to about 200 mg/m 2 , most preferably about 0.1 mg/m 2 to about 10 mg/m 2 .
• Such dosages may vary, for example, depending on number of injections given, tumor burden, and other factors known to those of skill in the art. For instance, tumors have been labeled in vivo using cyanine- conjugated Mabs. Ballou et al. (1995) Cancer Immunol. Immunother. 41:257-263.
• the type of detection instrument available is a major factor in selecting a given radioisotope.
• the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
• Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the individual is minimized.
• a radioisotope used for in vivo imaging lacks a particle emission, but produces a large number of photons in the 140-250 keV range, to be readily detected by conventional gamma cameras.
• ⁇ ⁇ In- scFv for instance, 2 mg of scFv labeled with 5 mCi of '"Indium
• the range administered is about 0.01 mg to 20 mg, more preferably about 0.1-10 mg and even more preferably about 1 -5 mg per patient.
• radioisotopes can be bound to antigen binding fragments either directly or indirectly by using an intermediate functional group.
• Intermediate functional groups which are often used to bind metallic ion radioisotopes to immunoglobulins are the bifunctional chelating agents such as diethylene triaminepentacetic acid (DTP A) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
• DTP A diethylene triaminepentacetic acid
• EDTA ethylenediaminetetraacetic acid
• Typical examples of metallic ions which can be bound to ⁇ C are '"In, 97 Ru, 67 Ga, 68 Ga, 72 As, 89 Zr, 90 Y, and 20I T1.
• Antigen binding fragments can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR).
• MRI magnetic resonance imaging
• ESR electron spin resonance
• any conventional method for visualizing diagnostic imaging can be utilized.
• gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
• Elements which are particularly useful in such techniques include 157 Gd, "Mn, 162 DY, 52 Cr, and 56 Fe.
• ⁇ C can also be labeled with a fluorescent dye for the purpose of in vivo diagnosis.
• Antigen binding fragments can also be used to detect neoplasias using in vitro assays. Samples are taken from the patient and subject to any suitable immunoassay with the antigen binding fragments to detect the presence of elevated ganglioside levels. This is particularly useful in detecting lymphomas and leukemias where the tumor cells are circulating in the patient's bloodstream.
• Antigen binding fragments can also be used to monitor the course of amelioration of malignancy in an individual. Thus, by measuring the increase or decrease in the number of cells expressing gangliosides or changes in the level of gangliosides, it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the malignancy is effective.
• the present invention encompasses methods of treating cancers expressing the major tumor associated gangliosides. The methods comprise administering an amount of a physiologically acceptable composition containing the antigen binding fragments effective to achieve the desired effect, be it palliation of an existing tumor mass or prevention of recurrence.
• carcinomas can be any known in the field of oncology that express on its surface the tumor-associated gangliosides.
• carcinomas include, but are not limited to, melanoma, glioblastoma, meningioma, astrocytoma, neurofibrosarcoma, leukemia, thyroid cancer, lung carcinoma, glioma, colon carcinoma, ovary carcinoma, breast carcinoma, kidney carcinoma, and prostate carcinoma.
• cancers of neuroectodermal origin such as malignant melanoma express a large amount of tumor-associated gangliosides, making them the most preferred targets for immunotherapy using the antigen binding fragments.
• compositions and methods described herein relate generally to oligospecific anti-ganglioside antibody derivatives which are routinely generated by classical techniques of immunochemistry .
• This includes antigen binding fragments that have been coupled to another compound by chemical conjugation, or by mixing with an excipient or an adjuvant.
• antigen binding fragment includes any peptide that binds oligospecifically to gangliosides in essentially the same manner as GMAl .
• these derivatives include immunoglobulin fragments such as Fab, F(ab') 2 , Fab 1 , scFv (both monomeric and polymeric forms) and isolated H and L chains.
• An antigen binding fragment retains the specificity of GMAl, although avidity and/or affinity may be altered.
• polypeptide polypeptide
• peptide protein
• polymers of amino acid residues of any length may be linear or non-linear (e.g., branched), it may comprise modified amino acids or amino acid analogs, and it may be interrupted by chemical moieties other than amino acids.
• the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling or bioactive component.
• polynucleotide or “nucleic acid” is meant a single-, double- or triple- stranded DNA, genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and pyrimidine bases, or other natural, chemically or biochemically modified or containing non-natural or derivatized nucleotide bases.
• any embodiment described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double stranded form of either the DNA, RNA or hybrid molecules.
• the backbone of the polynucleotide can comprise sugars and phosphate groups (as may typically be found in RNA or DNA), or modified or substituted sugar or phosphate groups.
• the backbone of the polynucleotide can comprise a polymer of synthetic subunits such as phosphoramidates and thus can be a oligodeoxynucleoside phosphoramidate (P-NH 2 ) or a mixed phosphoramidate- phosphodiester oligomer.
• P-NH 2 oligodeoxynucleoside phosphoramidate
• Peyrortes et al. (1996) Nucleic Acids Res. 24: 1841-8; Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; and Schultz et al.
• a phosphorothiate linkage can be used in place of a phosphodiester linkage.
• a double-stranded polynucleotides can be obtained from the single stranded polynucleotide product of chemical synthesis either by synthesizing the complementary strand and annealing the strands under appropriate conditions, or by synthesizing the complementary strand de novo using a DNA polymerase with an appropriate primer.
• polynucleotides genes or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, r ⁇ combinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
• a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, uracyl, other sugars and linking groups such as fluororibose and thioate, and nucleotide branches.
• sequence of nucleotides can be interrupted by non-nucleotide components.
• a polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
• Other types of modifications included in this definition are caps, substitution of one or more of the naturally occurring nucleotides with an analog, and introduction of means for attaching the polynucleotide to proteins, metal ions, labeling components, other polynucleotides, or a solid support.
• recombinant polynucleotide means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which either does not occur in nature or is linked to another polynucleotide in a nonnatural arrangement.
• a "vector” refers to a recombinant DNA or RNA plasmid or virus that comprises a heterologous polynucleotide to be delivered into a target cell, either in vitro or in vivo.
• the heterologous polynucleotide can comprise a sequence of interest for purposes of therapy, and can optionally be in the form of an expression cassette.
• a vector need not be capable of replication in the ultimate target cell or subject.
• the term includes cloning vectors for the replication of a polynucleotide, and expression vectors for translation of a polynucleotide encoding sequence. Also included are viral vectors, which comprise a polynucleotide encapsidated or enveloped in a viral particle.
• a "cell line” or “cell culture” denotes bacterial, piant, insect or higher eukaryotic cells grown or maintained in vitro.
• the descendants of a cell may not be completely identical (either morphologically, genotypically, or phenotypically) to the parent cell but are included in the present invention.
• a "host cell” denotes a prokaryotic or eukaryotic cell that has been genetically altered, or is capable of being genetically altered by administration of an exogenous polynucleotide, such as a recombinant plasmid or vector.
• an exogenous polynucleotide such as a recombinant plasmid or vector.
• genetically altered cells the term refers both to the originally altered cell, and to the progeny thereof.
• Heterologous means derived from a genotypically distinct entity from the rest of the entity to which it is being compared.
• a polynucleotide can be placed by genetic engineering techniques into a plasmid or vector derived from a different source, and is a heterologous polynucleotide.
• a promoter removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous promoter.
• a “signal peptide” or “leader sequence” is a short amino acid sequence that directs a newly synthesized protein through a cellular membrane, usually the endoplasmic reticulum in eukaryotic cells, and either the inner membrane or both inner and outer membranes of bacteria.
• Signal peptides are typically at the N- terminal portion of a polypeptide and are typically removed enzymatically between biosynthesis and secretion of the polypeptide from the cell. The signal peptide is not present in the secreted protein, only during protein production.
• An “isolated" polynucleotide or polypeptide is one that is substantially free of the materials with which it is associated in its native environment. By substantially free is meant at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% free of these materials.
• treatment refers to clinical intervention in an attempt to alter the natural course of the individual or cell being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of the treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Treatments include vaccines, described below.
• an "immunogen” refers to composition for human or animal use, which is administered with the intention of conferring to the recipient a degree of specific immunologic reactivity against a particular antigen.
• the immunologic reactivity can be carried out by antibodies or cells (particularly B cells, plasma cells, T helper cells, and cytotoxic T lymphocytes, and their precursors) that are immunologically reactive against the target, or any combination thereof.
• the target is major tumor-associated gangliosides.
• the immunologic reactivity may be desired for experimental purposes, for treatment of a particular condition, for the elimination of a particular substance, or for prophylaxis.
• An active immunogen is intended to elicit an immune response that persists in the absence of the vaccine components.
• an "effective amount” is an amount sufficient to effect a beneficial or desired clinical result.
• An effective amount can be administered in one or more doses.
• an effective amount is amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease.
• an adjuvant an effective amount is one sufficient to enhance the immune response to the immunogen.
• the effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition and the form of the antibody being administered.
• mammals include, but are not limited to, humans, farm animals, sport animals, and pets.
• the basic immunoglobulin (Ig) structural unit is composed of two identical light (“L”) polypeptide chains (approximately 23 kDa), and two identical heavy (“H”) chains (approximately 53 to 70 kDa).
• L light
• H heavy
• the four chains are joined by disulfide bonds in a "Y" configuration.
• the two H chains are bound by covalent disulfide linkages.
• the L and H chains are each composed of a variable (V) region at the N- terminus, and a constant (C) region at the C-terminus.
• V variable
• C constant
• V L J L is composed of a V (VJ region connected through the joining (JJ region to the C region ( J.
• the V region (V H D H J H ) 1S composed of a variable (V ⁇ ) region linked through a combination of the diversity (D H ) region and the joining (J H ) region to the C region (C H ).
• the V L J L and V H D H J H regions of the L and H chains, respectively, are associated at the tips of the Y to form the antigen binding portion and determine antigen binding specificity.
• the (C H ) region defines the isotype, i.e., the class or subclass of antibody.
• Antibodies of different isotypes differ significantly in their effector functions, such as the ability to activate complement, bind to specific receptors (e.g., Fc receptors) present on a wide variety of cell types, cross mucosal and placental barriers, and form polymers of the basic four-chain IgG molecule.
• specific receptors e.g., Fc receptors
• Antibodies are categorized into "classes" according to the C H type utilized in the immunoglobulin molecule (IgM, IgG, IgD, IgE, or IgA).
• C H type utilized in the immunoglobulin molecule
• C ⁇ , C ⁇ , C ⁇ 2 , C ⁇ 3 , and C ⁇ 4 C H subtypes
• Each H chain class can be associated with either of the L chain classes
• a C H ⁇ region can be present in the same antibody as either a K or ⁇ L chain
• the C regions of the H and L chains within a particular class do not vary with antigen specificity (e.g., an IgG antibody always has a C ⁇ H chain C region regardless of the antigen specificity).
• Each of the V, D, J, and C regions of the H and L chains are encoded by distinct genomic sequences.
• Antibody diversity is generated by recombination between the different V H , D H , and J ⁇ gene segments in the H chain, and V L and J L gene segments in the L chain.
• the recombination of the different V H , D H , and J H genes is accomplished by DNA recombination during B cell differentiation. Briefly, the H chain sequence recombines first to generate a D H J H complex, and then a second recombmatorial event produces a V H D H J H complex.
• a functional H chain is produced upon transcription followed by splicing of the RNA transcript. Production of a functional H chain triggers recombination in the L chain sequences to produce a rearranged V L J L region which in turn forms a functional V L J L C L region, i.e., the functional L chain.
• the HuMAb of the present invention can be produced in several ways.
• the antibody can be generated using human-human hybridoma methods for monoclonal antibody production, wherein isolated human B-lymphocytes are fused with human lymphoblastoid partners, and the resulting hybridomas are used as antibody source.
• the growth of antibody producing cells can be enriched by pre-selection of the targeted B-lymphocytes using matrix-coated channels or magnetic beads.
• subpopulations of human B-lymphocytes that have certain predetermined characteristics, such as producing a particular isotype antibody can be enriched by using magnetic beads coated with antigens against the interesting isotype.
• McKnight et al. (1996) Hybridoma 15:255-261.
• the method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse.
• the mammal can be primed for ascites production by prior administration of a suitable composition; e.g., Pristane.
• antigen binding fragments can be chemically synthesized using sequence data and other information provided in this disclosure, in conjunction with standard methods of protein synthesis.
• a suitable method is the solid-phase Merrifield technique.
• Automated peptide synthesizers are commercially available, such as those manufactured by Applied Biosystems, Inc. (Foster City, CA).
• the antigen binding fragments can be recombinantly produced by employing routine molecular cloning methods such as described in Sambrook et al. (1989).
• a polynucleotide encoding either the H or L chain of the antibody can be inserted into a suitable expression vector, such as pMEX-8 (Example 2).
• the resulting expression plasmid is in turn introduced into a host cell.
• the host cell is grown under suitable conditions such that the incorporated polynucleotide is transcribed and translated into polypeptide.
• H and L chains of the antibody can be produced separately, and then combined by disulfide bond rearrangement.
• vectors with separate polynucleotides encoding each chain of the antibody can be transfected into a single host cell which can then produce and assemble the entire molecule.
• a wide variety of mammalian cells can be used as host cells, including, for example, Chinese hamster ovary (CHO) cells.
• the antibody thus produced can be purified using standard techniques in the art.
• HuMAbs are desired to overcome the usual problems of genetic instability and low production rate attached with some of the conventional methods for producing antibody.
• the antibody specificity against particular types of cancer cells can be determined by screening the antibody against panels of cell lines. Routine enzyme immunoassay (EIA) can be used for the initial screening assay. All HuMAbs that bind with greater than 2 times the absorbance of a control antibody to a relevant tumor cell line are assumed positive in preliminary tests. Glassy (1993) Hum. Antibod. Hybrid 4:154-165; Glassy et al. (1983) J. Immunol. Methods 58:1 19-126; and Glassy et al. (1985) J. Immunol. Methods 81 : 1 15-122. Following the initial assessment of the specificity of the MAb, flow cytometry analysis is carried out with the antibody to further define the specificity of the antibody. McKnight et al. (1996).
• Antigen binding fragments according to the present invention are characterized as capable of binding oligospecifically to the major tumor associated gangliosides GD3, GM3, and GD2.
• the binding activity with selected gangliosides can be determined by both ELISA and immunostaining of thin layer chromatography
• the invention also encompasses antigen binding fragments conjugated to a chemically functional moiety.
• the moiety is a label capable of producing a detectable signal.
• conjugated antibody are useful, for example, in detection systems such as quantitation of tumor burden, and imaging of metastatic foci and tumor imaging.
• labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, bioluminescent compounds substrate cofactors and inhibitors. See, for examples of patents teaching the use of such labels, U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
• the moieties can be covalently linked to the antibody, recombinantly linked, or conjugated to the antibody through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.
• Other functional moieties include signal peptides, agents that enhance immunologic reactivity, agents that facilitate coupling to a solid support, vaccine carriers, bioresponse modifiers, paramagnetic labels and drugs.
• Signal peptides are described above and include prokaryotic and eukaryotic forms.
• Agents that enhance immunologic reactivity include, but are not limited to, bacterial superantigens.
• Agents that facilitate coupling to a solid support include, but are not limited to, biotin or avidin.
• Immunogen carriers include, but are not limited to, any physiologically acceptable buffers.
• Bioresponse modifiers include cytokines, particularly tumor necrosis factor (TNF), interleukin-2, interleukin-4, granulocyte macrophage colony stimulating factor and ⁇ interferons.
• Suitable drug moieties include antineoplastic agents. These include, but are not limited to, radioisotopes, vinca alkaloids such as the vinblastine, vincristine and vindesine sulfates, adriamycin, bleomycin sulfate, carboplatin, cisplatin, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, duanorubicin hydrochloride, doxorubicin hydrochloride, etoposide, fluorouracil, lomustine, mechlororethamine hydrochloride, melphalan, mercaptopurine, methotrexate, mitomycin, mitotane, pentostatin, pipobroman, procarbaze hydrochloride, streptozotocin, taxol, thioguanine, and uracil mustard.
• vinca alkaloids such as the vinblastine, vincris
• Immunotoxins including single chain molecules, can be produced by recombinant means. Production of various immunotoxins is well-known in the art, and methods can be found, for example, in "Monoclonal Antibody-toxin Conjugates: Aiming the Magic Bullet," Thorpe et al. (1982) Monoclonal Antibodies in Clinical Medicine, Academic Press, pp. 168-190; Vitatta (1987) Science 238:1098-1104; and
• Suitable toxins include, but are not limited to, ricin, radionuclides, pokeweed antiviral protein, Pseudomonas exotoxin A, diphtheria toxin, ricin A chain, fungal toxins such as restrictocin and phospholipase enzymes. See, generally, “Chimeric Toxins,” Olsnes and Pihl, Pharmac. Ther. 15:355-381 (1981); and “Monoclonal Antibodies for Cancer Detection and Therapy," eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985).
• the chemically functional moieties can be made recombinantly for instance by creating a fusion gene encoding the antigen binding fragment and functional regions from other genes (e.g. enzymes). In the case of gene fusions, the two components are present within the same polypeptide gene.
• the antigen binding fragments can be chemically bonded to the moiety by any of a variety of well known chemical procedures. For example, when the moiety is a protein, the linkage can be by way of heterobifunctional cross linkers, e.g., SPDP, carbodiimide glutaraldehyde, or the like.
• the moieties can be covalently linked, or conjugated, through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.
• a secondary reagent such as a second antibody, protein A, or a biotin-avidin complex.
• Paramagnetic moieties and the conjugation thereof to antibodies are well- known in the art. See, e.g., Miltenyi et al. (1990) Cytometry 11 :231-238.
• the antigen binding fragment is to be administered to an individual, it is preferably at least 80% pure, more preferably it is at least 90% pure, even more preferably it is at least 95% pure and free of pyrogens and other contaminants.
• the percent purity is calculated as a weight percent of the total protein content of the preparation, and does not include constituents which are deliberately added to the composition after the fragment is purified.
• the invention also encompasses an isolated polynucleotides encoding the antibody with properties as described above or the fragments thereof.
• the polynucleotide encodes at least a portion of the antigen binding fragments that is distinguishable from other antibodies. Preferably, this portion is related in some way to the immunologic reactivity of the antigen binding fragments.
• the invention encompasses a polynucleotide encoding a portion of the GMAl L chain V region, comprising at least about 70 consecutive nucleotides, preferably at least about 80 consecutive nucleotides, more preferably at least about 100 consecutive nucleotides, even more preferably at least about 150 nucleotides of SEQ ID NO:3.
• the invention also encompasses a polynucleotide encoding a portion of the GMAl L chain V region, comprising at least about 25 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, and even more preferably at least about 35 consecutive nucleotides of the CDRl encoding sequence thereof.
• the invention also encompasses a polynucleotide encoding a portion of the GMAl L chain V region, comprising at least about 20 consecutive nucleotides, preferably at least about 25 consecutive nucleotides, and even more preferably at least about 35 consecutive nucleotides of the CDR2 or CDR3 encoding sequence thereof.
• the invention also encompasses a polynucleotide encoding a portion of the GMAl H chain V region, comprising at least about 70 consecutive nucleotides, preferably at least about 80 consecutive nucleotides, more preferably at least about
• the invention also encompasses a polynucleotide encoding a portion of the GMAl L chain V region, comprising at least about 25 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, and even more preferably at least about 35 consecutive nucleotides of the CDRl encoding sequence thereof.
• the invention also encompasses a polynucleotide encoding a portion of the GMAl L chain V region, comprising at least about 20 consecutive nucleotides, preferably at least about 25 consecutive nucleotides, and even more preferably at least about 35 consecutive nucleotides of the CDR2 or CDR3 coding sequence thereof.
• the invention includes an isolated polynucleotide encoding a polypeptide having immunologic activity of GMAl.
• the polypeptide encodes at least 5 amino acids of a V L chain of GMAl as depicted in SEQ. ID NO:4.
• the invention also includes an isolated polynucleotide encoding a polypeptide having immunologic activity of GMAl.
• the polynucleotide encodes at least 5 amino acids of a V H chain of GMAl as depicted in SEQ. ID NO:2.
• the polynucleotide sequence can be similar to those depicted in SEQ. ID NO:l or SEQ ID NO:3 with changes designed to optimize codon usage, stability, facilitate cloning, or any other purpose.
• polynucleotides encode at least five amino acids of a GMAl CDR.
• the invention also encompasses polynucleotides encoding for functionally equivalent variants and derivatives of GMAl and functionally equivalent fragments thereof which can enhance, decrease or not significantly affect properties of the polypeptides encoded thereby.
• These functionally equivalent variants, derivatives, and fragments display the ability to specifically recognize major tumor-associated gangliosides. For instance, changes in a DNA sequence that do not change the encoded amino acid sequence, as well as those that result in conservative substitutions of amino acid residues, one or a few amino acid deletions or additions, and substitution of amino acid residues by amino acid analogs are those which will not significantly affect properties of the encoded polypeptide.
• Conservative amino acid substitutions are glycine/alanine; valine/isoleucine/leucine; asparagine glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tyrosine/tryptophan.
• the polynucleotides can comprise additional sequences, such as additional encoding sequences within the same transcription unit, controlling elements such as promoters, ribosome binding sites, and polyadenylation sites, additional transcription units under control of the same or a different promoter, sequences that permit cloning, expression, and transformation of a host cell, and any such construct as may be desirable to provide embodiments of this invention.
• the invention also provides polynucleotides covalently linked with a detectable label. Such polynucleotides are useful, for example, as probes for detection of related nucleotide sequences.
• the polynucleotides of this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof.
• the polynucleotides can be isolated by RT-PCR as described in Example 2, wherein degenerate nucleotides based on human H or L chain region amino acid sequences were used as primers to amplify cDNAs encoding the specific antibody designated as GMAl.
• the sequences of the polynucleotides can be determined by standard sequencing techniques.
• the polynucleotides can be inserted into a suitable vector, and the vector in turn is introduced into a suitable host cell for amplification or expression.
• the construction of the vector and the introduction of the vector into a host cell can be accomplished using well known recombinant cloning techniques.
• a variety of vectors can be used for cloning and expression of the polynucleotides.
• Cloning vectors can be used to obtain replicate copies of the polynucleotides they contain, or as a means of storing the polynucleotides in a depository for future recovery.
• Expression vectors (and host cells containing these expression vectors) can be used to obtain polypeptides produced from the polynucleotides they contain.
• Suitable cloning and expression vectors include any known in the art, for example, those for use in bacterial, mammalian, yeast and insect expression systems.
• pMEX-8 is a mammalian expression vector designed for high level expression of human antibodies. Nasoff et al. (1991) Hybridoma 16:427-439. This vector is constructed by inserting eight control elements into plasmid pUC19. pMEX-8 is assembled using an "interchangeable cassette" strategy. Thus, any control element can easily be replaced with a different sequence to achieve more effective synthesis or secretion of a particular antibody. pMEX-8 contains four transcription units.
• the nucleotides encoding the H and L chains of the antigen binding fragments can be incorporated onto the same pMEX-8 vector, resulting an expression plasmid capable of producing the desired antigen binding fragments.
• Transcription of the H- and L-chain genes is initiated using the promoter-enhancer region of the major immediate early gene of human cytomegalovirus and terminated using a bovine growth hormone polyadenylation/termination region.
• the third transcription unit encodes dihydrofolate reductase (DHFR).
• DHFR dihydrofolate reductase
• the key to expression in host cells such as CHO cells is amplification of the DHFR gene and adjacent tightly linked antibody sequences by the addition of methotrexate (MTX).
• MTX is a competitive inhibitor of DHFR.
• Stepwise selection for cell growth in progressively increasing concentrations of MTX results in cells with increased levels of DHFR as a consequence of a proportional increase in gene copy number.
• cells secreting higher levels of the HuMAb are selected following increases in the level of MTX.
• pMEX-8 is designed to express both H and L chains from the same plasmid. Nearly equimolar amounts of H and L chains produced from identical promoters maximize the effective assembly and secretion of the antibody.
• a significant feature of pMEX-8 is the use of a very weak promoter to drive the expression of DHFR. This allows for effective amplification using low levels of MTX, which is necessary due to the cytotoxic effects of high level MTX.
• the vectors containing the polynucleotides can also contain polynucleotide sequences encoding other polypeptides that enhance, facilitate, or modulate the desired result, such as lymphokines, including, but not limited to, IL-2, IL-4, GM- CSF, TNF- ⁇ , and IFN- ⁇ .
• lymphokines including, but not limited to, IL-2, IL-4, GM- CSF, TNF- ⁇ , and IFN- ⁇ .
• a preferred lymphokine is GM-CSF.
• Preferred GM-CSF constructs are those which have been deleted for the AU-rich elements from the 3 ' untranslated regions and sequences in the 5' untranslated region that are capable of forming a hairpin loop.
• vaccinia vectors encoding for recombinant HuMAb variants, such as scFvs, chimeras, and polymers.
• the vectors containing the polynucleotides can be introduced into the host cells by any of a number of appropriated means, including but not limited to, electroporation, transfection, microinjection, calcium phosphate precipitated DNA, lipofection, and protoplast fusion.
• Host cells can be either prokaryotic or eukaryotic.
• Prokaryotic hosts include bacterial cells, for example E. coli and Mycobacteria.
• eukaryotic hosts are yeast, insect, avian, plant and mammalian cells. Host systems are known in the art and need not be described in detail herein.
• a mammalian host cell is CHO, as described in Example 2.
• the polynucleotides of this invention have several uses. They are useful, for example, in expression systems for the production of the HuMAb. They are also useful as hybridization probes to assay for the presence of the HuMAb polynucleotide or related sequences in a sample using methods well known to those in the art. Further, the polynucleotides are also useful as primers to effect amplification of desired polynucleotides. The polynucleotides of this invention are also useful in pharmaceutical compositions including vaccines and for gene therapy.
• the polynucleotides can also be used as hybridization probes for detection of sequences encoding the HuMAb.
• Suitable samples include cells transformed ex vivo for use in gene therapy.
• DNA or RNA is extracted from a sample, and optionally run on a gel and/or digested with restriction endonucleases.
• the processed sample polynucleotide is typically transferred to a medium suitable for washing.
• the sample polynucleotide is then contacted with a GMAl polynucleotide probe under conditions that permit a stable duplex to form if the sample contains a matching GMAl sequence. Any stable duplexes formed are detected by any suitable means.
• the GMAl polynucleotide probe can be supplied in labeled form, and label remaining with the sample after washing will directly reflect the amount of stable duplex formed.
• hybridization is performed in situ. A suitably prepared tissue sample is overlaid with a labeled probe to indicate the location of the GMAl encoding sequences.
• a short polynucleotide can also be used as a primer for a PCR reaction, particularly to amplify a longer sequence comprising a region hybridizing with the primer. This can be conducted preparatively, in order to produce polynucleotide for further genetic manipulation. It can also be conducted analytically, to determine whether a HuMAb encoding polynucleotide is present, for example, in a sample of diagnostic interest.
• polynucleotides are in vaccines and gene therapy.
• the general principle is to administer the polynucleotide so that it either promotes or attenuates the expression of the polypeptide encoded therein.
• the present invention includes methods of inducing an immune response and methods of treatment comprising administration of an effective amount of the polynucleotides to an individual.
• a polynucleotide encoding HuMAb such as GMAl is administered to an individual, either directly or via cells transfected with the polynucleotide.
• the polynucleotide is in the form of a circular plasmid, preferably in a supercoiled configuration.
• the polynucleotide is replicated inside a cell.
• the polynucleotide is operatively linked to a suitable promoter, such as a heterologous promoter that is intrinsically active in cells of the target tissue type.
• plasmids persist as circular non-replicating episomal molecules.
• In vitro mutation can be carried out with plasmid constructs to encode, for example, molecules with greater affinity and/or avidity.
• cells such as COS-7, CHO, or HeLa can be transfected with the plasmids.
• Expression of the antibody is then determined by immunoassay; for example, by Western blot. Smaller antibody polypeptides can be detected, for example, by constructing the plasmid so that the resultant polypeptide is fused with a tag, such as a target epitope or enzyme label. Further characterization of the expressed polypeptide can be achieved by purifying the peptide and then conducting one of the functional assays described herein.
• the polynucleotides of this invention are used for genetically altering cells ex vivo.
• cells removed from a donor or obtained from a cell line are transfected or transduced with vectors encoding a HuMAb polypeptide, and then administered to a recipient.
• Suitable cells for transfection include peripheral blood mononuclear cells.
• the polynucleotides of this invention are used for genetically altering cells in vivo.
• the purpose includes, but is not limited to, treating various types of cancer.
• the invention also encompasses polypeptides comprising at least a portion of the V region of the antigen binding fragments with properties described above.
• Preferred polypeptides are those with the immunologic activity of GMAl .
• polypeptides that comprise amino acid sequences substantially different from other immunoglobulins are also preferred.
• the invention includes a polypeptide fragment of the HuMAb H chain V region, comprising at least 25 consecutive amino acids, more preferably 30 consecutive amino acids of SEQ ID NO:2, or 5 consecutive amino acids of the CDRl thereof, or at least 7 consecutive amino acids, preferably at least 9 consecutive amino acids of the CDR2 or CDR3 thereof.
• the invention also includes a polypeptide fragment of the HuMAb L chain V region, comprising at least 25 consecutive amino acids, more preferably 30 consecutive amino acids of SEQ ID NO:4, or 7 consecutive amino acids of the CDR2 thereof, or at least 8 consecutive amino acids, preferably 10 consecutive amino acids of the CDRl or CDR3 thereof.
• the size of the polypeptides can be only the minimum size required to provide a desired function.
• the polypeptides can optionally comprise additional sequence. Polypeptides comprising 7 amino acids, more preferably about 10 amino acids, more preferably about 15 amino acids, more preferably about 25 amino acids, more preferably about 50 amino acids, more preferably about 75 amino acids from the L or H chain V region of GMAl are also included. Even more preferred are polypeptides comprising the entire GMAl L or H chain V region.
• the invention includes modified polypeptides that are functionally equivalent to the antigen binding fragments, or have altered but measurable immunologic activity of GMAl.
• modified polypeptides include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously alter the immunologic activity.
• GMAl polypeptide comprising one or more amino acid substitution in comparison with the prototype GMAl sequence.
• Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region, such as the V region.
• Amino acid substitutions if present, are preferably conservative substitutions that do not deleteriously affect folding or functional properties of the peptide.
• Groups of functionally related amino acids within which conservative substitutions can be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serme/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan.
• Polypeptides of this invention can be in glycosylated or unglycosylated form, can be modified p ⁇ st-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group).
• polypeptides of this invention can be made by any suitable procedure, including proteolysis of the antibody, by recombinant methods or by chemical synthesis. These methods are known in the art and need not be described in detail herein.
• proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin.
• Intact antibody can be incubated with one or more proteinases simultaneously or sequentially. Alternatively, or in addition, intact antibody can be treated with disulfide reducing agents. Peptides can then be separated from each other by techniques known in the art including, but not limited to, gel filtration chromatography, gel electrophoresis, and reverse-phase HPLC.
• polypeptides can also be made by expression from a polynucleotide encoding the peptide according to the information provided elsewhere in this application, in a suitable expression system.
• polynucleotides encoding the polypeptide are Iigated into an expression vector under control of a suitable promoter and used to genetically alter the intended host cell. Both eukaryotic and prokaryotic host systems can be used.
• the polypeptide is then isolated from lysed cells or from the culture medium and purified to the extent needed for its intended use.
• compositions encompasses pharmaceutical compositions and immunogenic compositions containing the antigen binding fragments either alone or in combination.
• Such pharmaceutical compositions and vaccines are useful for eliciting an immune response and treating neoplastic diseases, either alone or in conjunction with other forms of therapy, such as chemotherapy or radiotherapy.
• compositions that contain HuMAb, or a polynucleotide or a polypeptide derivative thereof as an active ingredient is conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E.W. Martin ed., Mack Publishing Co., PA. Depending on the intended use and mode of administration, it may be desirable to process the active ingredient further in the preparation of pharmaceutical compositions. Appropriate processing can include sterilizing, mixing with appropriate non-toxic and non-interfering components, dividing into dose units, and enclosing in a delivery device.
• Liquid physiologically acceptable compositions can, for example, be prepared by dissolving or dispersing a polypeptide embodied herein in a liquid excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol.
• the composition can also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.
• Pharmaceutical compositions of the present invention are administered by a mode appropriate for the form of composition. Typical routes include subcutaneous, intramuscular, intraperitoneal, intradermal, oral, intranasal, and intrapulmonary (i.e., by aerosol).
• Pharmaceutical compositions of this invention for human use are typically administered by a parenteral route, most typically intracutaneous, subcutaneous, or intramuscular.
• compositions for oral, intranasal, or topical administration can be supplied in solid, semi-solid or liquid forms, including tablets, capsules, powders, liquids, and suspensions.
• Compositions for injection can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolution or suspension in liquid prior to injection.
• a preferred composition is one that provides a solid, powder, or liquid aerosol when used with an appropriate aerosolizer device.
• pharmaceutical compositions are preferably supplied in unit dosage form suitable for administration of a precise amount. Also contemplated by this invention are slow release or sustained release forms, whereby a relatively consistent level of the active compound are provided over an extended period.
• test compounds are prepared as a suitable pharmaceutical composition and administered to test subjects. Initial studies are preferably done in small animals such as mice or rabbits, optionally next in non- human primates and then ultimately in humans. Immunogenicity is preferably tested in individuals without a previous antibody response. A test composition in an appropriate dose is administered on an appropriate treatment schedule. It may be appropriate to compare different doses and schedules within the predicted range.
• compositions of this invention are particularly suitable for administration to humans with a neoplastic disease. Especially relevant are melanoma, neuroblastoma, glioma, sarcoma, lymphoma, and small cell lung cancer. Diagnostic Methods
• the major tumor associated gangliosides to be detected are GD3, GM3 and GD2.
• Any sample containing a detectable amount of the targeted ganglioside antigen can be used.
• a sample can be a solid such as tissue, or a liquid such as urine, cerebrospinal fluid, serum, amniotic fluid, saliva, and those commonly used in histological diagnosis.
• the antigen binding fragments can be detectably labeled in various ways to facilitate the detection of the antigen binding fragments- antigen complex so formed.
• Detection of the ganglioside antigens using the antigen binding fragments can be done utilizing various immunoassays known in the art.
• the present invention further encompasses methods for cancer diagnosis by way of detecting the tumor associated gangliosides in a sample as described above.
• Certain types of cancer cells, especially melanomas and other cancer cells of neuroectodermal origin, are known to express characteristically high levels of the major tumor associated gangliosides.
• a diagnostically effective amount of detectably labeled antibody is given to the subject in need of cancer diagnosis.
• diagnosis means that the amount of detectably labeled antibody is administered in sufficient quantity to enable detection of the neoplasia.
• Methods of Treatment encompasses methods of treating cancers expressing the major tumor associated gangliosides.
• the methods comprise administering an amount of a pharmaceutical composition containing the antibody effective to achieve the desired effect, be it palliation of an existing tumor mass or prevention of recurrence.
• the amount of a pharmaceutical composition administered is an amount effective in producing the desired effect.
• An effective amount can be provided in one or a series of administrations.
• the effective amount of the antigen binding fragments to be administered will depend upon several factors, such as the route of administration, the condition of the individual, and the desired objective.
• the term "therapeutically effective” means that the amount of antigen binding fragment used is of sufficient quantity to ameliorate the cancer.
• “Ameliorate” denotes a lessening of the detrimental effect of the cancer on the individual.
• the amount per administration is about 10 ⁇ g to 20 mg, preferably 250 ⁇ g to 10 mg, more preferably 300 ⁇ g to 5 mg, even more preferably 500 ⁇ g to 2.5 mg.
• Administrations are typically conducted on a weekly or biweekly basis until a desired, measurable parameter is detected, such as diminution of disease symptoms.
• compositions of this invention can be used alone, or in conjunction with other active agents that promote the desired objective, or provide a desirable adjunct therapy.
• suitable active agents include the anti-neoplastic drugs and bioresponse modifiers described above and effector cells such as those described by Douillard et al. (1986) Hybridomas (Supp. 1:5139).
• the compositions of this invention can be unlabeled or labeled with a therapeutic agent as described above. These agents can be coupled either directly or indirectly to the polypeptides.
• indirect coupling is by use of a spacer moiety.
• spacer moieties can be either insoluble or soluble (Diener et al. (1986) Science 231 : 148) and can be selected to enable drug release at the target site.
• a HuMAb and a therapeutic agent can be translated, synthesized, Iigated or otherwise produced as a single molecule which has both the antibody and therapeutic agent functions.
• therapeutic agents which can be coupled to the HuMAb for immunotherapy include, but are not limited to, bioresponse modifiers, drugs, radioisotopes, lectins, and toxins.
• Bioresponse modifiers include lymphokines which include, but are not limited to, tumor necrosis factor, interleukins 1, 2, and 3, lympho toxin, macrophage activating factor, migration inhibition factor, colony stimulating factor, and interferon.
• Interferons with which the antibody can be labeled include ⁇ -interferon, ⁇ -interferon, and ⁇ -interferon (IFN- ⁇ ) and their subtypes.
• IFN- ⁇ ⁇ -interferon
• certain isotypes may be more preferable than others depending on such factors as leukocyte distribution as well as isotype stability and emission. If desired, the malignant cell distribution can be evaluated by the in vivo diagnostic techniques described below.
• alpha and beta particle-emitting radioisotopes are preferred in immunotherapy.
• a high energy beta emitter capable of penetrating several millimeters of tissue such as " ⁇ ”
• a short range, high energy alpha emitter such as 2I2 Bi, may be preferable.
• Radioisotopes which can be bound to the antigen binding fragments for therapeutic purposes include, but are not limited to, 125 I, l3l I, 90 Y, 67 Cu, 212 Bi, 211 At, 212 Pb, 47 Sc, ,09 Pd, and ,88 Re.
• Lectins are proteins, usually isolated from plant material, which bind to specific sugar moieties. Many lectins are also able to agglutinate cells and stimulate lymphocytes. However, ricin is a toxic lectin which has been used immunotherapeutically. This is preferably accomplished by binding the alpha- peptide chain of ricin, which is responsible for toxicity, to the antibody molecule to enable site specific delivery of the toxic effect.
• Toxins are poisonous substances produced by plants, animals, or microorganisms that, in sufficient dose, are often lethal.
• Diphtheria toxin is a substance produced by Corynebacterium diphtheria which can be used therapeutically. This toxin consists of an alpha and beta subunit which under proper conditions can be separated.
• the toxic A chain component can be bound to an antibody and used for site specific delivery to a neoplastic cell.
• the administration of both usually occurs substantially contemporaneously.
• substantially contemporaneously means that they are administered reasonably close together with respect to time.
• the therapeutic agent can be administered 1 to 6 days before.
• the administration of the therapeutic agent can be daily, or at any other suitable interval, depending upon such factors, for example, as the nature of the malignancy, the condition of the patient and half-life of the agent.
• the present invention also encompasses the use of uposomes with membrane bound antibodies to specifically deliver the liposome to the area of the tumor or neoplastic cells expressing major tumor-associated gangliosides.
• uposomes can be produced such that they contain, in addition to the antibody, such immunotherapeutic agents as those described above which would then be released at the site of malignancy.
• Another such delivery system described by Brown et al. (1994) Virology 198:477-488; and Miyamura et al. (1994) Proc. Natl. Acad. Sci. USA 91 :8507-851 1 utilizes chimeric parvovirus B19 capsids for presentation of the antigen binding fragments. Such chimeric systems are encompassed for use in the claimed methods.
• the dosage ranges for the administration of the HuMAb are those large enough to produce the desired effect in which the symptoms of the malignant disease are ameliorated without causing undue side effects such as unwanted cross-reactions, anaphylactic reactions, and the like.
• the dosage will vary with the patient's age, condition, sex and extent of the disease and can be determined by one of skill in the art.
• the dosage can be adjusted by the individual physician in the event of any complication. Dosage can vary from about 0.1 mg/kg to about 2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one or more dose administrations daily, for one or several days.
• lower dosages comparable to those used for in vivo immunodiagnostic imaging, can be used.
• compositions can be administered by injection or by gradual perfusion.
• the antigen binding fragments can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, intrathecally or transdermally, alone or in combination with effector cells.
• Another method of administration is intralesionally, for instance by direct injection directly into the tumor.
• Intralesional administration of various forms of immunotherapy to cancer patients does not cause the toxicity seen with systemic administration of immunologic agents.
• compositions may be desirable to administer the compositions locally to the area in need of treatment; this can be achieved by, for example, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as silastic membranes, or fibers.
• a suitable such membrane is Gliadel® provided by Guilford Pharmaceuticals Inc.
• Suitable subjects include those who are suspected of being at risk of a pathological effect of any neoplasia, particularly carcinoma, are suitable for treatment with the pharmaceutical compositions of this invention. Those with a history of cancer are especially suitable.
• Suitable human subjects for therapy comprise two groups, which can be distinguished by clinical criteria.
• Patients with "advanced disease” or "high tumor burden” are those who bear a clinically measurable tumor.
• a clinically measurable tumor is one that can be detected on the basis of tumor mass (e.g., by palpation, CAT scan, or X-Ray; positive biochemical or histopathological markers on their own can be insufficient to identify this population).
• a pharmaceutical composition embodied in this invention is administered to these patients to elicit an anti-tumor response, with the objective of palliating their condition. Ideally, reduction in tumor mass occurs as a result, but any clinical improvement constitutes a benefit. Clinical improvement includes decreased risk or rate of progression or reduction in pathological consequences of the tumor.
• a second group of suitable subjects is known in the art as the "adjuvant group". These are individuals who have had a history of cancer, but have been responsive to another mode of therapy.
• the prior therapy may have included, but is not restricted to, surgical resection, radiotherapy, and traditional chemotherapy.
• these individuals have no clinically measurable tumor.
• they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastases.
• This group can be further subdivided into high-risk and low-risk individuals.
• a pharmaceutical composition embodied in this invention is administered to patients in the adjuvant group, or in either of these subgroups, in order to elicit an anti-cancer response.
• the composition delays recurrence of the cancer, or even better, reduces the risk of recurrence (i.e., improves the cure rate).
• Such parameters may be determined in comparison with other patient populations and other modes of therapy.
• compositions of this invention can be administered at any time that is appropriate.
• immunotherapy can be conducted before or during traditional therapy of a patient with high tumor burden, and continued after the tumor becomes clinically undetectable. Immunotherapy can be continued in a patient who initially fell in the adjuvant group, but is showing signs of recurrence.
• the attending physician has the discretion to determine how or when the compositions of this invention are to be used.
• EXAMPLE 1 Generation of the original GMAl human hybridoma Lymphocytes from three separate patients, two melanoma and one lung carcinoma were used for the generation of the original GMAl human hybridoma. These lymphocytes were aseptically processed and prepared as previously described and cryopreserved. Glassy (1987) Cancer Res. 47:5181-5188; and Koda et al. (1990) Arch. Surg. 125:1591-1597. When needed, ampules were thawed and resuspended at 5 x lOVml in RPMI-1640 medium supplemented with 10% FCS and glutamine and incubated overnight at 37°C, 5% CO 2 /95% air prior to fusion.
• a human B lymphoblastoid cell line derived from parental WIL-2 cells, SHFP-1 was the human fusion partner cell line used in the fusion. Details of the fusion and cloning of human-human hybridomas generated from SHFP-1 and pooled human lymphocytes have been previously described. Glassy (1987); and Glassy
• 5'oligonucleotide primer 5 -GTCGAATTCATGGAG(TC)TTGGGCTGA(GC)CTGG(GC)TTT(TC)T-3' (SEQ ID NO: 1
• the 3'PCR primer is complementary to the end of the IgGl constant H chain region.
• Amplification of H chain sequence was carried out for 30 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 2 min.
• the 1.4 kilobase PCR product which resulted was cut with restriction endonuclease Eco RI and Iigated into the cloning vector pUC18 for further analysis.
• 5'-GTCGAATTCATGAGGCTCCCTGCTCAG-3' (SEQ ID NO:7) was utilized as a PCR primer along with a 3' oligonucleotide primer
• PCR conditions were similar to those used for H chain cloning except that extension time at 72°C was reduced from 2 min to 1 min.
• the -750 b.p. fragment which resulted was cut with restriction endonuciease Eco RI and inserted into pUCl 8 for further characterization.
• Nucleic acid sequencing of GMAl H and L chain genes was carried out on a 373 automated DNA sequences (ABI, Foster City, CA) using a Taq fluorescent dideoxy terminator cycle sequencing kit (ABI).
• the resulting nucleotide sequence and deduced amino acid sequence of V H and V L , and their corresponding signal sequences are shown in Figure 5.
• Analysis of the sequence data shows that the GMAl H chain variable region belongs to the V H 3 family.
• the V H CDR regions are associated with the cannonical class 1-3.
• GMAl is similar to the germline sequence V H 26/DP-47, demonstrating 91% homology at the amino acid level.
• GMAl V L is a member of the subgroup II family of kappa L chains, derived from the germline gene V K II A17/DPK18. Cox et al. (1994) Eur. J. Immunol. 24:827-836.
• EXAMPLE 3 Expression of the recombinant GMAl antibody (rGMAl) using the mammalian expression vector pMEX-8
• the complete GMAl H and L chain DNA sequences were inserted into the plasmid expression vector pMEX-8.
• pMEX-8 is a mammalian expression vector designed for high level expression of human antibodies ( Figure 6). Detailed procedure describing its expression has been discussed in Nasoff et al. (1997). pMEX-8 DNA sequencing was carried out at each intermediate stage of construction as described above.
• CHO/DHFR ' cells were obtained from the ATCC and grown in IMDM medium supplemented with 10% dialyzed FCS and gentamicin (50 ⁇ g/ml). 20 mg of plasmid DNA was electroporated into CHO/DHFR " cells (1 x 10 7 cells ml) at 174 volts, 400 mF of capacitance, and 13 ohms of resistance using an Electro Cell Manipulator 600 (BTX, San Diego, CA). After 24 hours of growth at 37°C in IMDM media (Gibco BRL, Gaithersberg, MD), G418 was added to a final concentration of 500 mg/ml.
• IMDM media Gibco BRL, Gaithersberg, MD
• G418 resistant colonies were pooled and reseeded into IMDM medium containing 10% dialyzed fetal calf serum (Summit Biotechnology, Fort Collins, CO), i.e., medium depleted of nucleotides. Selection for both G418 resistance and DHFR expression produced cells containing stable chromosomal integration of the plasmid vector. Individual colonies were isolated using 10 mm cloning cylinders and transferred to 24 well plates. Supernatants were assayed for IgG levels by immunoblotting or ELISA using a goat anti-human IgG conjugated with alkaline phosphatase.
• EXAMPLE 4 Binding specificity of GMAl against selected human tumor cell lines The binding specificity of GMAl was analyzed against a panel of human cell lines by cell binding assay.
• the following human cell lines were used: melanoma cell lines SK-MEL-5, SK-MEL-24, SK-MEL-28, SK-MEL-31, M14, M21 and A375; pancreatic cancer cell line, PANC-1; neuroblastoma cell lines Lanl, Lan5, U87MG; gastric carcinoma, KATO-III; breast carcinoma cell fine, SK-Br-3; and colon carcinoma HT-29, CACO2 and COLO205. All cell lines were obtained from the American Type Culture Collection (ATCC).
• ATCC American Type Culture Collection
• Immunoreactive HuMAbs were identified by an indirect enzyme immunoassay (EIA) to panels of cell lines immobilized on Immulon I (Dynatech; Winooski, VT) micro- EIA plates. Glassy et al. (1983). All HuMAbs that bind with greater than 2 times the absorbance of a control antibody to a relevant tumor cell line are assumed positive in preliminary tests. The strategies used in screening for tumor-reactive HuMAbs have been previously described. Glassy et al. (1986) Methods Enzymol. 121 :525-541; and Gaffar et al. (1986) Bioessays 4:119-123.
• GMAl showed binding activity mainly against melanoma (SK-MEL-28), stomach (KATO III), pancreas (PANC-1) and neuroblastoma (U87MG) cell lines
• GMAl was not reactive against cell lines from colon (HT29), ovary (SK-OV-3) and breast (SK-Br- 3) which are known to be ganglioside negative.
• Flow cytometric analysis was carried out with both recombinant and hybridoma antibodies to further define the specificity of GMAl against antigens expressed on the cell surface or in the cytoplasm of targeted cells.
• Fresh or 70% alcohol cold acetone fixed whole cells were harvested by 1 mM ethyl enediamine tetraacetic acid (EDTA) in Ca 2+ , phosphate-buffered saline (PBS), washed and incubated with 1 ⁇ g of GMAl for 4 hr at 4°C.
• EDTA ethyl enediamine tetraacetic acid
• PBS phosphate-buffered saline
• GMAl was reactive against both live as well as methanol/acetone fixed SK- MEL-28 and PANC-1 cell lines. Binding to unfixed SK-MEL-28 and PANC-1 was used to determine the cell surface activity of the antibody ( Figure 1). Reactivity to fixed tumor cells was used to determine intracellular binding. Both recombinant and hybridoma antibodies reacted to cell surface antigen present on malignant melanoma (SK-MEL-28) and pancreatic carcinoma (PANC-1), whereas no reactivity could be detected with breast (SK-Br-3) and colon carcinoma (HT-29) cells.
• SK-MEL-28 malignant melanoma
• PANC-1 pancreatic carcinoma
• EXAMPLE 5 Specificity of GMAl against major tumor-associated ganglioside antigens The specificity of GMAl was examined by TLC immunostaining and ELISA using a variety of purified gangliosides and neutral glycolipids. Commercially available human and bovine gangliosides and neutral glycolipids used in ELISA and immuno-TLC were purchased from Sigma (St. Louis, MO) and Calbiochem (San Diego, CA). Murine monoclonal antibodies R24 (Pukel et al. (1982) J. Exp. Med. 155:1133-1147) and DMAb-7 (He et al. (1989) Acta Neuropathol.
• ELISA was performed by a previously reported method. Ravindranath et al. (1994) J Immunol. Methods 169:257-272. Briefly, gangliosides were dissolved in ethanol, added to each well of a 96-well maxisorb plate (Nunc) at concentrations varying between 0.1 nmoie to 10 nmole, and dried in a desiccator for 48 hours. After blocking with PBS-4% BSA at 37°C for 90 min, GMAl (100 ⁇ l) was reacted for 2 hrs at 37°C. The intensity of the reaction was measured using peroxidase-labeled goat anti-human antibody (American Qualex; La Mirada, CA).
• FIG. 1 The color development of the o-phenylenediamine dihydrochloride substrate (Sigma) was measured at 490 nm in a spectrophotometer.
• Figure 2 shows specificity analysis of GMAl by ELISA using varying concentrations of purified gangliosides and neutral glycolipids, ranging from 100 picomoles (pmoles) to 10 nmoles.
• GD3 showed the best activity profile followed by GM3 and GD2.
• GMAl also showed minimal cross reactivity with GDlb, GTlb and GQlb in decreasing order.
• the plates were sprayed with, then submerged in, 10% FBS containing RPMI-1640 medium for 20 minutes, incubated with rGMAl (or R24 or DMAb-7) at room temperature for 2 hours followed by biotinylated goat anti-human IgG (Pierce; Rockford, IL) or biotinylated goat anti- mouse IgG (Zymed; San Francisco, CA) for one hour followed by peroxidase conjugated streptavidin (Pierce) for 45 minutes and finally developed with diaminobenzidine (Sigma) in the presence of H 2 O 2 for 2-3 minutes.
• rGMAl or R24 or DMAb-7
• rGMAl reacted with the gangliosides in a manner similar to GMAl, demonstrating that the specificity of the antibody was not altered during molecular cloning.

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