The present invention relates to monoclonal antibody 1A7. This is an anti-idiotype produced by immunizing with an antibody specific for ganglioside GD2, and identifying a hybridoma secreting antibody with immunogenic potential in a multi-step screening process. Also disclosed are polynucleotide and polypeptide derivatives based on 1A7, including single chain variable region molecules and fusion proteins, and various pharmaceutical compositions. When administered to an individual, the 1A7 antibody overcomes immune tolerance and induces an immune response against GD2, which comprises a combination of anti-GD2 antibody and GD2-specific T cells. The invention further provides methods for treating a disease associated with altered GD2 expression, particularly melanoma, neuroblastoma, glioma, soft tissue sarcoma, and small cell carcinoma. Patients who are in remission as a result of traditional modes of cancer therapy may be treat with a composition of this invention in hopes of reducing the risk of recurrence.

Description of the Invention

This invention relates to the discovery of an anti-idiotype antibody that is capable of recruiting a tumor-specific response against GD2. The antibody is designated 1A7. The immune response elicited by 1A7 typically comprises both humoral and cellular components, and is therefore expected to be useful in palliating the clinical conditions related to GD2-associated tumors. The invention comprises the 1A7 antibody molecule, along with polynucleotide and polypeptide derivatives thereof, and methods for using these compounds in diagnosis and treatment.

Cancer patients are typically tolerized to various tumor associated antigens (TAA), including GD2. 1A7 successfully circumvents immune tolerance, and elicits an immune response against GD2. According to the network theory Ab1 represents anti-tumor monoclonal antibody; Ab2 represents anti-idiotypic monoclonal antibody; and Ab3 represents anti-anti-idiotypic monoclonal antibody. 1A7 is an Ab2 that stimulates Ab3 with the same specificity as Ab1, and can therefore recruit effector functions against tumors bearing the tumor antigen. An Ab3 response reactive against the original TAA is referred to herein as Ab1'.

While not wishing to be bound by theory, one explanation is that the 1A7 combining site may present a region that at least partly resembles an epitope in GD2 in the context of one or more other epitopes which render it more immunogenic. The epitope of GD2 which may resemble that of 1A7 is identified by the Ab1 (14G2a) used to generate 1A7. As a result, 1A7 escapes the normal immune tolerance against GD2, and is able to elicit an anti-GD2 response.

FIGS. 1 and 2 (see Original Patent) show the nucleotide encoding region of the 1A7 light and heavy chain variable regions, respectively, along with the corresponding amino acid translation. These sequences were compared with those of other known immunoglobulin molecules (Example 2). Both the polynucleotide and polypeptide variable region sequences for 1A7 are unique.

Amongst the 50 database sequences matched most closely to that of the 1A7 light chain variable region, none was identical 1A7 differed from the five closest sequences by 2 substitution differences at residues 50 and 55, which are contained in the second CDR. The two differences at these positions were non-conservative substitutions, and persisted in comparisons with other light chain sequences.

Amongst the 50 database sequences matched most closely to that of the 1A7 heavy chain variable region, none was identical FIG. 3, Panels A and B (see Original Patent)). The following summarizes the main points deduced from the comparison. The closest match was with a heavy chain fragment. There were 2 deletions and 12 substitution differences. The closest match with a full length heavy chain variable region had the following features: There were 3 deletions and 17 substitution differences 1A7 differed in length from all sequences but one, due to insertions or deletions of 1 to 8 residues about the VDJ junction. For the sequence of equal length, there were 26 substitution differences. All other comparisons showed a total of at least 22 insertions, deletions and substitution differences. Differences appeared throughout the variable region.

FIG. 3 Panel C (see Original Patent) provides a comparison of the 1A7 amino acid light and heavy chain sequences with consensus sequences derived from the database sequences. Other than splicing differences about the heavy chain VDJ junction, there appear to be about 16 differences between 1A7' and the consensus sequences that have likely arisen from somatic mutation during antibody maturation.

Particularly of interest in developing 1A7 derivatives with 1A7 immunologic activity are regions of the polynucleotide or polypeptide sequence comprising part of the VDJ junction. Also of interest are regions spanning at least one, preferably-2, and more preferably 3 or more of the point differences between the 1A7 amino acid sequences and the consensus sequences.

The full sequences of the 1A7 light and heavy chain constant regions have not been determined, but are expected to be identical or nearly identical to those of other mouse immunoglobulin molecules.

For the mouse kappa light chain constant region, four genetic allotypes encoding two protein allotypes have been published by Solin et al. (1993) Immunogenetics 37:401-407, which is hereby incorporated herein by reference. FIG. 1 of Solin et al. depicts mouse and rat immunoglobulin kappa chain gene sequences, comparing the sequences within the kappa chain constant region for different strains and highlighting allotypic differences. Included are kappa chain constant region sequences for BALB/c, PL, SJL, and M spretus. Other naturally occurring allotypes are possible.

The mouse .gamma..sub.1 heavy chain constant region DNA sequence from newborn mice has been published by Honjo et al. (1979) Cell 18:559-568, which is hereby incorporated herein by reference. FIG. 5 of Honjo et al. shows the germ-line DNA sequence, along with the encoded protein sequence. Shown in the line above is another protein sequence obtained from the mouse myeloma MOPC 21. Other naturally occurring allotypes are possible.

The 1A7 antibody and derivatives thereof are useful, for example, for eliciting an anti-GD2 immune response, for treating a GD2-associated disease, and as reagents for detecting the presence of anti GD2.

Certain compounds, compositions and methods described in this application relate generally to 1A7 and derivatives thereof which are routinely generated by classical techniques of immunochemistry. This includes 1A7 which has been coupled to another compound by chemical conjugation, or by mixing with an excipient or an adjuvant It includes such immunoglobulin fragments as Fab, F(ab').sub.2, Fab', and isolated heavy and light chains. First generation therapies are those based on such compounds and compositions.

Other compounds, compositions and method described in this application relate to polynucleotide and polypeptide derivatives of 1A7 that fall outside the preceding classification. The compositions are typically generated by genetic engineering, although they may alternatively be obtained by other methods and combinations of methods. This classification includes (but is not limited to) isolated polynucleotide fragments, recombinant polynucleotides, engineered peptide fragments and fusion peptides, and polynucleotides or polypeptides prepared by chemical synthesis based on the sequence data. Preferred compounds include polypeptide fragments of the 1A7 CDRs, antibody fusion proteins comprising cytokine effector components, single-chain variable region proteins and the polynucleotides encoding them, therapeutic plasmids comprising 1A7 polynucleotides, and vectors such as vaccinia vectors.

Unless explicitly stated otherwise or required by their nate, methods of making or using certain compounds embodied in this invention may be applied to other compounds of this invention, as appropriate.

Pharmaceutical compositions and treatment modalities of this invention may be brought to bear whenever it is desirable to elicit a response against GD2, especially in humans. Human patients with GD2-associated tumors, including melanoma, neuroblastoma, glioma, soft tissue sarcoma, and small cell carcinoma (including small cell lung cancer) are especially appropriate subjects. The subjects may have an advanced form of disease, in which case the objective may include mitigation or reversal of disease progression, and amelioration of side effects. The subjects may have had a history of the condition, for which they have already been treated, in which case the objective will typically include a decrease or delay in the risk of recurrence of clinical pathology.

General Techniques

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including-recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C. Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology" (J. E. Coligan et al., eds., 1991).

All patents, patent applications, articles and publications mentioned herein, both supra and infra, are hereby incorporated herein by reference.

These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

How Monoclonal Antibody 1A7 was Generated and Selected

1A7 was obtained by immunizing naive mice with 14G2a anti-GD2 antibody to obtain an anti-idiotype response. 14G2a binds to a unique epitope of GD2. Syngeneic BALB/c mice-were immunized four times with 14G2a (Ab1) and their spleen cells were fused with the non-secretory mouse myeloma P3-653 cells.

To obtain an anti-idiotype with all the features desired, an extensive screening process was employed, comprising the following four important steps: (1) Positive selection for antibody binding to 14G2a; (2) Negative selection against antibody recognizing isotypic or allotypic determinants; (3) Positive selection for an ability to inhibit the binding of 14G2a to GD2; and (4) Positive selection for an ability to induce a humoral immune response against the original tumor-associated antigen (GD2) in both mice and rabbits.

Several anti-idiotype (Ab2) hybridomas were obtained that were specific for the idiotype components of the 14G2a immunogen, and did not react with its isotypic or allotypic determinants. To determine whether the anti-14G2a were directed against the paratope of 14G2a, the binding of radiolabeled 14G2a to the GD2-positive cell line M21/P6 was studied in the presence of varying amounts of Ab2 hybridoma culture supernatants. With as little as 5 .mu.l of culture supernatant, three of the Ab2 tested inhibited the binding by at least 70%. 1A7 showed the highest level of inhibition. Accordingly, 1A7 was grown and purified from ascites fluid for further studies, while the others were kept in reserve.

The purified Ab2 was prepared as a vaccine and injected into naive mice and rabbits. After 4 injections, serum samples were titered for the presence of Ab3 that bound not only to the immunizing Ab2, but also to GD2. The Ab2 passing all of these screening stages was designated 1A7. Further details of the method used to obtain 1A7 are provided in Example 1.

Ab3 produced in animals immunized with 1A7 has been further characterized. The immune sera from both mice and rabbits competed with 14G2a for binding to the GD2-associated cell line M21/P6 and inhibited the binding of radioiodinated 14G2a to 1A7. This indicated that anti-anti-Id (Ab3) in mice and rabbits may share idiotopes with 14G2a and they probably bind to the same epitope as Ab1. Administration of 1A7 to non-human primates (cynomolgus monkeys) also generated a specific immune response, comprising activity against GD2 (Example 3).

The nucleic acid sequence encoding the light and heavy chain variable regions of 1A7 have also been deduced, along with the translated protein sequences (Example 2).

The invention also encompasses 1A7 conjugated to a label capable of producing a detectable signal These conjugated antibodies are useful, for example, in detection systems such as quantitation of anti-GD2 or tumor imaging. Such labels are known in the art and include, but are not limited to, radioisotopes, enzymes, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. The labels may be covalently linked to 1A7, or conjugated to the 1A7 through a secondary reagent, such as a second antibody, protein A, or a biotin-avidin complex.

Preparation of 1A7 Antibody

The 1A7 antibody of this invention can be prepared in several ways.

It is most conveniently obtained from the hybridoma deposited with the ATCC under Accession No. HB-11786, or the progeny thereof. For example, the cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Optionally, matrix-coated channels or beads and cell co-cultures may be included to enhance growth of antibody-producing cells. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal is optionally primed for ascites production by prior administration of a suitable composition; for example, Pristane.

Alternatively, 1A7 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, Calif.).

1A7 may also be obtained by employing routine recombinant methods such as described in Sambrook et al. (1989). For instance, using the sequences and information provided herein, a polynucleotide encoding either the 1A7 heavy or light chain can be cloned into a suitable expression vector (which contains control sequences for transcription, such as a promoter). The expression vector is in turn introduced into a host cell. The host cell is grown under suitable conditions such that the polynucleotide is transcribed and translated into a protein. Heavy and light chains of 1A7 may be produced separately, and then combined by disulfide bond rearrangement. Alternatively, vectors with separate polynucleotides encoding each chain of 1A7, or a vector with a single polynucleotide encoding both chains as separate transcripts, may be transfected into a single host cell which may then produce and assemble the entire molecule. Preferably, the host cell is a higher eukaryotic cell that can provide the normal carbohydrate complement of the molecule. The 1A7 thus produced in the host cell can be purified using standard techniques in the art. A polynucleotide encoding 1A7 for use in the production of 1A7 by any of these methods can in turn be obtained from the hybridoma producing 1A7, or be produced synthetically or recombinantly from the DNA sequence provided herein.

Methods of antibody isolation are well known in the art See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. The 1A7 antibody is a mouse immunoglobulin of the IgG1 subclass, and may be isolated by any technique suitable for immunoglobulins of this isotype. Purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin. 1A7 may also be purified on affinity columns comprising the 14G2a paratope; for example, in the form of a purified Ab1 or Ab3. Preferably, 1A7 is purified from BALB/c ascites using Protein-A-CL-SEPHAROSE.TM. 4B chromatography followed by chromatography on a DEAE-SEPHAROSE.TM. 4B ion exchange column.

If 1A7 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. In this context, 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 1A7 is purified.

Uses of 1A7 Antibody

The 1A7 antibody may be used for a number of purposes. These include eliciting an antibody response to 1A7 or GD2, eliciting a T cell response to 1A7 or GD2, and treating various types of cancer. These uses are elaborated more fully in a later section.

1A7 may also be used to purify anti-1A7 (Ab3), anti-GD2 (Ab1'), or 14G2a (Ab1). The method comprises contacting a biological sample containing the antibody with a 1A7 polypeptide, producing a complex, and recovering the Ab3 or Ab1 from the complex. Typically, the 1A7 polypeptide is coupled to an affinity matrix for affinity column purification. Such methods are routine in the art and need not be described in detail herein.

The invention also encompasses methods of detecting anti-1A7 or anti-GD2 in a biological sample. Anti-OD2 is detectable whenever (like 14G2a) it cross-reacts with 1A7. Anti-GD2 with this activity may spontaneously arise during the course of a GD2-associated disease. Anti-GD2 with this activity is especially likely in individuals who have received a course of therapy with 1A7, or a derivative thereof. These methods are applicable in a clinical setting, for example, for monitoring antibody levels in an individual, as well as an industrial setting, in which commercial production of anti-1A7 or anti-GD2 is desired.

The assay methods entail coning any anti-1A7 or anti-D2 target antibody in the sample with a 1A7 antibody or polypeptide under conditions sitable to allow the formation of a stable complex between the target and the A7, and detecting any stable complex formed. The sample is suitably prepared before conducting the assay, optionally by enriching for antibody activity. When using intact 1A7, it is generally preferable to deplete the sample of any anti-mouse immunoglobulin activity that may be present. Anti-mouse immunoglobulin antibody can be removed from a sample, for example, by precipitation with normal mouse IgG or adsorption with a mouse Ig adsorbant. Binding of anti-mouse immunoglobulin antibody, particularly that specific for the Fc region, can be minimized by judicious choice of the reagents of the assay. F(ab').sub.2 or Fab fragments of 1A7 and other reagents with fewer mouse determinants are appropriate.

After the sample is suitably prepared, it is mixed with a excess functional equivalent of 1A7 under conditions that permit formation of a complex between 1A7 and any target antibody that may be present. The amount of complex is then determined, and compared with complexes formed with standard samples containing known amounts of target antibody in the range expected. Complex formation may be observed by immunoprecipitation or nephelometry, but it is generally more sensitive to employ a reagent labeled with such labels as radioisotopes like .sup.125I, enzymes like peroxidase and .beta.-galactosidase, or fluorochromes like fluorescein.

Antibody assays may be conducted entirely in fluid phase. For example, anti-GD2 may be mixed with labeled 1A7. Alternatively, the anti-GD2 in the sample may be used to compete with a labeled anti-GD2 for binding sites on 1A7. Generally, bound and unbound label is separated to quantitate the percent bound. Suitable separation methods include gel filtration chromatography, and precipitation with antibody against immunoglobulin of the species from which the sample is obtained, optionally in the presence of polyethylene glycol. Alternatively, the proportion of bound and unbound label may be determined in situ, for example, using fluorescene/quench labeling pairs or enzyme/inhibitor labeling pairs. See, e.g., U.S. Pat. No. 3,996,345 (Ullman et al.).

It is generally more convenient to conduct a capture assay using a reagent linked to a solid phase, such as a polyethylene test tube, microtiter plate well, or magnetic bead. In a competition-type capture assay, unlabeled target antibody in the sample competes with a labeled analog reagent for binding to 1A7. The 1A7 may be attached directly to the solid support, or captured later, for example, using an anti-immunoglobulin. In this assay, the amount of label associated with the solid phase is inversely related to the amount of anti-GD2 in the sample.

In the sandwich-type capture assay, target antibody is captured by 1A7 attached directly or through a secondary reagent to a solid phase. After washing, the anti-GD2 is detected using anti-immunoglobulin of the appropriate species, or a second 1A7 antibody, to which a label is directly or indirectly attached. Alternatively, the anti-immunoglobulin may be attached to the solid phase and labeled 1A7 is used to complete the sandwich. If the anti-immunoglobulin used is isotype-specific, then the class of the antibody may also be determined. In this type of assay, the amount of label associated with the solid phase correlates positively with the amount of anti-GD2 in the sample. Other methods of measuring specific antibody are known in the art, and may be adapted to measure anti-1A7 or anti-GD2 by using 1A7 as the specific reagent

1A7 may also be used to measure the level of cellular anti-1A7 or anti-GD2 activity. In one example, 1A7 is used to identify anti-GD2 expressing cells in a cell suspension, perhaps B or T lymphocytes expressing a receptor that binds 1A7. 1A7 may be labeled and mixed into the cell suspension. Alternatively, unlabeled 1A7 may be mixed with the cells, and followed with a labeled secondary reagent such as labeled anti-mouse immunoglobulin or protein A. Suitable labels for this purpose include radiolabels and fluorescent labels. The use of fluorescent labels also allows anti-GD2 cells to be separated from non-specific cells in a fluorescence-activated cell sorter. In a second example, anti-GD2 expressing cells are detected in a tissue section. Typically, the tissue is fixed and embedded in a suitable medium, overlaid with 1A7, and then developed with a secondary anti-immunoglobulin coupled with a fluorescent or enzyme marker.

Polynucleotide Derivatives of 1A7

The invention provides various polynucleotides encoding the anti-idiotype antibody 1A7 or fragments of 1A7, based on the polynucleotide sequences provided herein. Various embodiments are described in this section, comprising a number of different combinations of the 1A7 heavy or light chain variable region sequences. In general, a 1A7 polynucleotide of this invention encodes at least one feature that is unique to the 1A7 molecule (in comparison with other immunoglobulins). Preferably, this feature is related in some way to an immunological reactivity of 1A7.

The invention encompasses a polynucleotide encoding a portion of the 1A7 light chain variable 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:1. The invention also encompasses a polynucleotide encoding a portion of the 1A7 light chain variable region, comprising at least about 25 consecutive nucleotides, preferably at least about 30 consecutive nucleotides, even more preferably at least about 35 consecutive nucleotides of the CDR1 encoding sequence thereof. The invention also encompasses a polynucleotide encoding a portion of the 1A7 light chain variable region, comprising at least about 20 consecutive nucleotides, preferably at least about 25 consecutive nucleotides, 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 1A7 heavy chain variable 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 1A7 light chain variable region, comprising 15 consecutive nucleotides of the CDR1 encoding sequence thereof. The invention also encompasses a polynucleotide encoding a portion of the 1A7 light chain variable region, comprising at least about 20 consecutive nucleotides, preferably at least about 25 consecutive nucleotides, even more preferably at least about 35 consecutive nucleotides of the CDR2 or CDR3 encoding sequence thereof.

The invention includes isolated 1A7 polynucleotides encoding a polypeptide having immunological activity of 1A7, wherein the polypeptide encodes at least 5 amino acids of a variable light chain of 1A7 as depicted in SEQ. ID. NO:2. In another embodiment, the invention includes isolated 1A7 polynucleotides encoding a polypeptide having immunological activity of 1A7, wherein the polynucleotide encodes at least 5 amino acids of a variable heavy chain of 1A7 as depicted in SEQ. ID. NO:4. The polynucleotide sequence may be similar to those depicted in SEQ ID NO:1 (FIG. 1 (see Original Patent)) or SEQ ID NO:3 (FIG. 2 (see Original Patent)) with changes designed to optimize codon usage, stability, or to facilitate cloning, or for some other purpose. It is within the skill of one in the art, given the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:4, to design such polynucleotides. Preferred are polynucleotides encoding at least five amino acids of a 1A7 complementarity determining region (CDR).

The invention also encompasses polynucleotides encoding for functionally equivalent variants and derivatives of 1A7 and functionally equivalent figments thereof which may enhance, decrease or not significantly affect properties of the polypeptides encoded thereby. These functionally equivalent variants, derivatives, and fragments display the ability to induce an immune response, preferably an anti-GD2 immune response. 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.

The 1A7 polynucleotides of the invention may 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 includes a polynucleotide of at least about 15 consecutive nucleotides, preferably at least about 20 nucleotides, more preferably at least about 25 consecutive nucleotides, more preferably at least about 35 consecutive nucleotides, more preferably at least about 50 consecutive nucleotides, even more preferably at least about 75 nucleotides, still more preferably at least about 100 nucleotides, still more preferably at least about 200 nucleotides, and even more preferably at least about 300 nucleotides that forms a stable hybrid with a polynucleotide encoding the light chain or heavy chain variable region of 1A7, but not with other immunoglobulin encoding regions known at the time of filing of this application. Any set of conditions may be used for this test, as long as at least one set of conditions exist wherein the test polynucleotide demonstrates the required specificity. Preferably, the 1A7 encoding sequences to which the test polynucleotide binds are those shown in SEQ. ID NOS:1 and 3. Since the known immunoglobulin sequences fill into a hierarchy of similarity with that of 1A7, the test may be performed by comparing the hybridization of the test polynucleotide with the 1A7 sequence with the hybridization with the most closely related sequences. Preferred is a panel of about 10 of the most closely related sequences to SEQ. ID NO:1 or 3, as appropriate. Sequences to which the test polynucleotide should not form a duplex are the light chain variable region encoding sequences listed in SEQ. ID NOS: 17-26 and the heavy chain variable region encoding sequences listed in SEQ. ID NOS:27-44.

Hybridization reactions can be performed under conditions of different "stringency". Conditions that increase stringency of a hybridization reaction are published. See, for example, Sambrook and Maniatis. Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25.degree. C., 37.degree. C., 50.degree. C. and 68.degree. C.; buffer concentrations of 10.times.SSC, 6.times.SSC, 1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and heir equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2, or 15 minutes; and wash solutions of 6.times.SSC, 1.times.SSC, 0.1.times.SSC, or deionized water.

"T.sub.m" is the temperature in degrees Centigrade at which 50% of a polynucleotide duplex made of complementary strands hydrogen bonded in antiparallel direction by Watson-Crick base pairing dissociates into single strands under conditions of the experiment. T.sub.m may be predicted according to a standard formula, such as: T.sub.m=81.5+16.6 log[Na.sup.+]+0.41 (% G/C)-0.61 (%F)-600/L where [Na.sup.+] is the cation concentration (usually sodium ion) in mol/L; (% G/C) is the number of G and C residues as a percentage of total residues in the duplex; (% F) is the percent formamide in solution (wt/vol); and L is the number of nucleotides in each strand of the duplex.

Useful 1A7 polynucleotides encoding fragments of 1A7 may be identified by generating polynucleotide fragments (based on SEQ ID NO:1 or SEQ ID NO:3, for example) and testing the polypeptides encoded thereby for a function of interest. Alternatively, the polypeptide fragment encoded by a particular polynucleotide may be prepared and tested for a function of interest. Alternatively, given a 1A7 polypeptide with desirable properties, a polynucleotide could be designed that encodes it.

Included in all these embodiments are polynucleotides with encoding regions for 1A7 polymers, fusion proteins, humanized immunoglobulins, single-chain variable regions, and other particular polypeptides of interest. These polypeptides are described in a later section.

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.

Preparation of 1A 7 Polynucleotides:

The polynucleotides of this invention can be obtained using chemical synthesis, recombinant cloning methods, PCR, or any combination thereof.

Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequence data provided herein to obtain a desired polynucleotide by employing a DNA synthesizer or ordering from a commercial service.

Alternatively, 1A7 polynucleotide sequences can be obtained from a 1A7 antibody producing cell line, 1A7 cloning vector, or 1A7 expression vector. RNA or DNA encoding the desired sequence may be isolated, amplified, and processed by standard recombinant techniques. Such techniques include digestion with restriction nucleases, and amplification by polymerase chain reaction (PCR), or a suitable combination thereof. PCR technology is described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR. The Polymerase Chain Reaction, Mullis et al. eds, Birkauswer Press, Boston (1994). A polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification. Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome. Amplified DNA can be isolated from the host cell by standard methods: see, e.g., Sambrook et al. (1989). RNA may also be obtained from transformed host cell, it may be obtained by using an DNA-dependent RNA polymerase.

If used as a vaccine, plasmids containing 1A7 polynucleotides are preferably prepared by the method of Horn et al. ((1995) Human Gene Therapy 6:565-573), which produces a pharmaceutical grade plasmid DNA suitable for administration.

Cloning and Expression vectors Comprising a 1A7 Polynucleotide

The present invention further includes a variety of vectors comprising a 1A7 polynucleotide. These vectors can be used for expression of recombinant polypeptides as well as a source of 1A7 polynucleotides. Cloning vectors can be used to obtain replicate copies of the 1A7 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. They may also be used where it is desirable to express 1A7 polypeptides in an individual and thus have intact cells capable of synthesizing the polypeptide, sum as in gene therapy. Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and need not be described in detail herein. For example, see Gacesa and Ramji Vectors, John Wiley & Sons (1994).

Cloning and expression vectors typically contain a selectable marker (for example, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector), although such a marker gene can be carried on another polynucleotide sequence co-introduced into the host cell. Only those host cells into which a selectable gene has been introduced will grow under selective conditions. Typical selection genes either: (a) confer resistance to antibiotics or other toxins substances, e.g., ampicillin, neomycyin, methotrexate; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper marker gene will depend on the host cell, and appropriate genes for different hosts are known in the art. Cloning and expression vectors also typically contain a replication system recognized by the host.

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28. These and many other cloning vectors are available from commercial vendors such as BioRad, Stratagene, and Invitrogen.

Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide encoding a 1A7 polypeptide of interest The polynucleotide encoding the 1A7 polypeptide is operatively linked to suitable transcriptional controlling elements, such as promoters, enhancers and terminators. For expression (i.e., translation), one or more translational controlling elements are also usually red such as ribosome binding sites, translation initiation sites, and stop codons. These controlling elements (transcriptional and translational) may be derived from the 1A7 gene, or they may be heterologous (i.e., derived from other genes or other organisms). A polynucleotide sequence encoding a signal peptide can also be included to allow a 1A7 polypeptide to cross or lodge in cell membranes or be secreted from the cell. A number of expression vectors suitable for expression in eukaryotic cells including yeast, avian, and mammalian cells are known in the art One example of an expression vector is pcDNA3 (Invitrogen, San Diego, Calif., in which transcription is driven by the cytomegalovirus (CMV) early promoter/enhancer. This vector also contains recognition sites for multiple restriction enzymes for insertion of the 1A7 polynucleotide of interest. Another example of an expression vector (system) is the baculovirus/insect system.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (where the vector is an infectious agent, such as vaccinia virus, which is discussed below). The choice of means of introducing vectors or 1A7 polynucleotides will often depend features of the on the host cell.

Once introduced into a suitable host cell, for example, E coli or COS-7, expression of a 1A7 polypeptide can be determined using any of the assays described herein. For example, presence of 1A7 polypeptide can be detected by RIA or ELISA of the culture supernatant (if the 1A7 polypeptide is secreted) or cell lysates.

A particularly useful expression vector for 1A7 polynucleotides is a vaccinia virus comprised of a 1A7 polynucleotide sequence, which can also be used in vaccine preparations (Moss (1991) Science 252:1662-1667). To introduce polynucleotide sequences encoding 1A7 polypeptide, including 1A7 polypeptide fragments, into vaccinia, the polynucleotide sequence of interest is first inserted into a plasmid containing a vaccinia virus promoter with flanking sequences homologous to vaccinia DNA inessential for replication. Plasmid-containing cells are then infected with vaccinia, which leads to a low level of homologous recombination between plasmid and virus, with resultant transfer of the vaccinia promoter and 1A7 polypeptide-encoding polynucleotide sequence into the vaccinia virus genome. Typically, the 1A7 polynucleotide is inserted into the viral tk (thymidine kinase) gene. Insertion into the tk site attenuates the virus more than 10,000 fold compared to wild type (Flexner et al. (1980) Vaccine 88 (Cold Spring Harbor Laboratory), 179-184). Recombinant virus is identified by the tk.sup.- phenotype. Preferably, expression of the 1A7 polynucleotide is under the control of the vaccinia early/late promoter (7.5 K), whereby the resultant 1A7 polypeptides can be expressed in infected cells throughout the life cycle of the virus. However, other promoters known in the art can be used, such as pH6, or synthetic promoters. Expression of the 1A7 polypeptide occurs in cells infected with the recombinant vaccinia or individuals which are immunized with the live recombinant vaccinia virus. Any one of several strains of vaccinia can be used, including but not limited to WR, ALVAC, and NYVAC.

A vaccinia vector of this invention can contain one or more polynucleotides encoding a 1A7 polypeptide. It 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 and GM-CSF. 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. Also embodied in this invention are vaccinia vectors encoding for recombinant 1A7 variants containing 1A7 polypeptides, such as scFvs, chimeras, and polymers, as described below.

Host Cells Transformed with LA 7 Polynucleotides

Other embodiments of this invention are host cells transformed with 1A7 polynucleotides and vectors comprising 1A7 polynucleotide sequences, as described above. Both prokaryotic and eukaryotic host cells may be used. Prokaryotic hosts include bacterial cells, for example E. coli and mycobacteria. Among 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. One example of a mammalian host cell is NS0, obtainable from the European Collection of Cell Cultures (England). Transfection of NS0 cells with a plasmid, for example, which is driven by a CMV promoter, followed by amplification of this plasmid in using glutamine synthetase provides a useful system for protein production. (Cockett et al. (1990) Bio/Technology 8:662-667).

The host cells of this invention can be used, inter alia, as repositories of 1A7 polynucleotides, or as vehicles for production of 1A7 polynucleotides and polypeptides. They may also be used as vehicles for in vivo expression of 1A7 polypeptides.

Uses for 1A 7 Polynucleotides

The polynucleotides of this invention have several uses. 1A7 polynucleotides are useful, for example, in expression systems for the production of 1A7 or 1A7 fragments. They are also useful as hybridization probes to assay for the presence of 1A7 polynucleotide or related sequences in a sample using methods well known to those in the art Further, 1A7 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.

1A7 polynucleotides can also be used as hybridization probes for detection of 1A7 encoding sequences. Suitable samples include cells transformed ex vivo for use in gene therapy. In one illustration, DNA or RNA is extracted from a sample, and optionally run on a gel and/or digested with restriction nucleases. The processed sample polynucleotide is typically transferred to a medium suitable for washing. The sample polynucleotide is then contacted with the 1A7 polynucleotide probe under conditions that permit a stable duplex to form if the sample contains a matching 1A7 sequence. Any stable duplexes formed are detected by any suitable means. For example, the 1A7 polynucleotide probe may be supplied in labeled form, and label remaining with the sample after washing will directly reflect the amount of stable duplex formed. In a second illustration, hybridization is performed in situ. A suitably prepared tissue sample is overlaid with a labeled 1A7 probe to indicate the location of 1A7 encoding sequences comprised therein.

A short 1A7 polynucleotide may also be used as a primer for a PCR reaction, particularly to amplify a longer sequence comprising a region hybridizing with the primer. This may be conducted preparatively, in order to produce polynucleotide for further genetic manipulation. It may also be conducted analytically, to determine whether a 1A7 encoding polynucleotide is present, for example, in a sample of diagnostic interest.

The 1A7 polynucleotides of this invention can be used in expression systems to produce 1A7 polypeptides, intact 1A7, or recombinant forms of 1A7, such as are described below.

Another use of 1A7 polynucleotides is in vaccines and gene therapy. The general principle is to administer the polynucleotide so that it either promoters or attenuates the expression of the polypeptide encoded therein. Thus, the present invention includes methods of inducing an immune response and methods of treatment comprising administration of an effective amount 1A7 polynucleotides to an individual. In these methods, a 1A7 polynucleotide encoding a 1A7 polypeptide is administered to an individual, either directly or via cells transfected with the 1A7 polynucleotide. Preferably, the 1A7 polynucleotide is in the form of a circular plasmid, preferably in a supercoiled configuration. Preferably, the 1A7 polynucleotide is replicated inside a cell. Thus, the 1A7 polynucleotide is operatively linked to a suitable promoter, such as a heterologous promoter that is intrinsically active in cells of the target tissue type. Preferably, once in cell nuclei, plasmids persist as circular non-replicating episomal molecules. In vitro mutation may be carried out with plasmid constructs to provide, for example, molecules that are more immunogenic or that comprise a T cell epitope with a desirable HLA motif.

To determine whether plasmids containing 1A7 polynucleotides are capable of expression in eukaryotic cells, eukaryotic cells such as COS-7, CHO, or HeLa cells can be transfected with the plasmids. Expression of 1A7 polypeptide is then determined by immunoassay; for example, by Western blot. Smaller 1A7 polypeptides can be detected, for example, by constructing the plasmid so that the resultant 1A7 polypeptide is fused with a tag, such as a target epitope or enzyme label. Further characterization of the expressed 1A7 polypeptide can be achieved by purifying the peptide and then conducting one of the functional assays described herein.

In one mode of gene therapy, the polynucleotides of this invention are used for genetically altering cells ex vivo. In this strategy, cells removed from a donor or obtained from a cell line are transfected or transduced with vectors encoding a 7 polypeptide, and then administered to a recipient Suitable cells for transfection include peripheral blood mononuclear cells.

In another mode of gene therapy, the polynucleotides of this invention are used for genetically altering cells in vivo. The purpose may include (but is not limited to) eliciting an antibody response to 1A7 or GD2, eliciting a T cell response to 1A7 or GD2, and treating various types of cancer. These uses are elaborated more fully in a later section.

Polypeptide Derivatives of 1A7

The present invention encompasses polypeptide fragments of 1A7 containing at least a portion of a variable region of 1A7. Preferred fragments are those with immunological activity of 1A7.

Also preferred are fragments which comprise amino acid sequences substantially different from other immunoglobulins, and fragments comprising a CDR. In one embodiment, the invention includes a polypeptide fragment of the 1A7 light chain variable region, comprising at least 5 consecutive amino acids, more preferably 15 consecutive amino acids, still more preferably 30 consecutive amino acids of SEQ ID NO:2, wherein the fragmet contains one or the other (preferably both) of the lysine residues at positions 50 and 55 of the mature light chain variable region sequence. In other embodiments, the invention includes a polypeptide fragment of the 1A7 heavy chain variable region, comprising the 5 amino acids from the CDR1 or comprising at least 8, preferably 10, more preferably 12, and still more preferably 16 of the amino acids from the CDR2; or comprising at least 4, preferably 6, more preferably 8, and still more preferably 9 of the amino acids from the CDR3. In yet other embodiments, the peptide comprises a combination of two or more sequences from positions 50-55 of the 1A7 light chain and the three CDRs of the heavy chain as outlined in this paragraph, optionally in combination with other 1A7 variable region sequences.

The size of the 1A7 polypeptide fragments may be only the minimum size required to provide a desired function. It may optionally comprise additional sequence, either native to 1A7, or from a heterologous source, as desired. 1A7 fragments may contain only 5 consecutive amino acids from a 1A7 variable region 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 1A7 light or heavy chain variable region are also included. Even more preferred are polypeptides comprising the entire 1A7 light or heavy chain variable region.

The invention includes modified 1A7 polypeptides which are functionally equivalent to 1A7, or have altered but measurable 1A7 immunologic activity. Fragments with improved 1A7 immunologic activity are preferred. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously change the functional activity.

One example of this is 1A7 polypeptides comprising one or more amino acid substitution in comparison with the prototype 1A7 sequence. Substitutions can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable 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 may be made are glycine/alanine; valine/isoleucine/leucine; asparagine/glutamine; aspartic acid/glutamic acid; serine/threonine/methionine; lysine/arginine; and phenylalanine/tryosine/tryptophan. Polypeptides of this invention may be in glycosylated or unglycosylated form, may be modified post-translationally (e.g., acetylation, and phosphorylation) or may be modified synthetically (e.g., the attachment of a labeling group).

The invention also encompasses fusion proteins comprising one or more 1A7 polypeptide. A 1A7 fusion polypeptide can be prepared, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship. Alternatively, fusion proteins may be provided in expression systems constructed by co-transfection with plasmids comprising encoding regions for different functional regions of the protein.

Useful heterologous sequences for inclusion in a fusion polypeptide include sequences that provide for secretion from a host cell, enhance immunological reactivity, or facilitate the coupling of the polypeptide to an immunoassay support or a vaccine carrier. One example is a bacteria "super antigens", such as staphylococcal enterotoxin A (SEA) (Dohlsten et al. (1994) Proc. Nad. Acad. Sci. USA 91:8945-8949). In a preferred example, a 1A7 polypeptide is fused with a bioresponse modifier, particularly a cytokine, which may enhance immunogenicity. Examples of bioresponse modifiers include cytokines and lymphokines such as GM-CSF, interleukin-2 (IL 2), interleukin 4 (IL-4), and y-interferon. GM-CSF and IL-2 are especially preferred The preferred arrangement is for the cytokine effector unit to be fused to the C-terminal of the immunoglobulin heavy chain

1A7 polypeptide derivatives comprising both a 1A7 light chain and a 1A7 heavy chain may be formed as separate light and heavy chains and then assembled, or assembled in situ by an expression system for both chains. Such expression systems may be created by tranfecting with a plasmid comprising separate transcribable regions for the light and heavy chain, or by co-transfecting the same cell with plasmids for each chain. In a third method, a suitable plasmid with a heavy chain encoding region is transfected into a heavy chain loss mutant. For example, heavy chain loss mutants can be obtained by treating 2.times.10.sup.7 1A7 cells with fluorescein-labeled rabbit anti-mouse IgG (H chain specific, DAKO Corporation, Carpinteria, Calif.) according to the supplier's instruction. The stained and unstained cell populations are analyzed in a fluorescence-activated cell sorter. The unstained cells are collected in a sterilized tube and placed in 96-well plates with 1 cell/well by limiting dilution. The culture supernatants are then assayed by ELISA using goat anti-mouse IgG (heavy chain specific) and goat anti-mouse kappa The clones with kappa-positive and IgG-negative phenotype are subcloned at least 3 times to obtain stable 1A7.sup.(-H) mutants. mRNA from putative heavy chain loss mutant 1A7.sup.(-H) cones can be isolated and the sequence of the light chain variable region cDNA determined. Reverse PCR of the mRNA for the 1A7 V.sub.H is performed with 2 sets of 5'- and 3'-primers, used for cloning of 1A7.sup.(-H) cDNA (Example 2). A heavy chain loss mutant should yield no detectable DNA band. Transfection of these cells may then proceed using a suitable heavy chain plasmid construct.

The invention also encompasses a hybrid antibody, in which one pair of heavy and light chains is obtained from a first antibody, which the other pair of heavy and light chains is obtained from a different second antibody. For purposes of this invention, one pair of light and heavy chains is from 1A7. In one example, each light-heavy chain pair binds different epitopes of GD2. Such hybrids may also be formed using chimeric heavy or light chains.

Another embodiment is a humanized version of 1A7. "Humanized" antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. In one version, the heavy chain and light chain constant regions are replaced with human sequence. This is a fusion polypeptide comprising a 1A7 variable region and a heterologous immunoglobulin constant region. In another version, the CDR regions comprise 1A7 amino acid sequences, while the variable framework regions have also been converted human sequences. See, for example, EP 0329400. In a third version, variable regions are humanized by designing consensus sequences of human and mouse variable regions, and converting residues outside the CDRs that are different between the consensus sequences.

Another 1A7 derivative contemplated by this invention is an antibody in which the 1A7 heavy or light chain has been modified to provide additional properties. For instance, a change in amino acid sequence can result in greater immunogenicity of the resultant 1A7 polypeptide. The changes range from changing of one or more amino acids to the complete redesign of a region such as a constant region domain. Changes contemplated affect complement fixation, interaction with membrane receptors, and other effector functions. A recombinant 1A7 antibody may also be designed to aid the specific delivery of a substance (such as a lymphokine) to an effector cell. Also contemplated are proteins in which various immunoglobulin domains have been placed in an order other than occurs in nature.

The invention also encompasses single chain variable region fragments ("scFv") of 1A7. Single chain variable region fragments are made by linking light and/or heavy chain variable regions by using a short linking peptide (Bird et al (1988) Science 242: 423-426). Any peptide having sufficient flexibility and length can be used as a linker in a scFv. Usually the linker is selected to have little to no immunogenicity. An example of a linking peptide is (GGGGS).sub.3 (SEQ. ID NO:45), which bridges approximately 3.5 nm between the carboxy terminus of one variable region and the amino terminus of another variable region. Other linker sequences may also be used, and may provide additional functions, such as a means for attaching a drug or to a solid support.

All or any portion of the heavy or light chain can be used in any combination. Typically, the entire variable regions are included in the scFv. For instance, the light chain variable region can be linked to the heavy chain variable region. Alternatively, a portion of the light chain variable region can be linked to the heavy chain variable region, or portion thereof. Also contemplated are scFvs in which the heavy chain variable region is from 1A7, and the light chain variable region is from another immunoglobulin. It is also possible to construct a biphasic, scFv in which one component is a 1A7 polypeptide and another component is a different polypeptide, such as a T cell epitope.

The scFvs can be assembled in any order, for example, V.sub.H-(linker)-V.sub.L or V.sub.L-(linker)-V.sub.H. There may be a difference in the level of expression of these two configurations in particular expression systems, in which case one of these forms may be preferred. Tandem scFvs can also be made, such as (X)(linker)-(X)-(linker)-(X), in which X are 1A7 polypeptides, or combinations of 1A7 polypeptides with other polypeptides. In another embodiment, single chain 1A7 antibody polypeptides have no linker polypeptide, or just a short, inflexible linker. Possible configurations are V.sub.L-V.sub.H and V.sub.H-V.sub.L. The linkage is too short to permit interaction between V.sub.L and V.sub.H within the chain, and the chains form homodimers with a V.sub.L/V.sub.H antigen binding site at each end. Such molecules are referred to in the art as "diabodies".

Single chain variable regions may be produced either recombinantly or synthetically. For synthetic production of scFv, an automated synthesize can be used. For recombinant production of scFv, a suitable plasmid containing polynucleotide that encodes the scFv can be introduced into a suitable host cell, either eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli, and the expressed protein may be isolated using standard protein purification techniques.

A particularly useful system for the production of 1A7 scFv's is plasmid vector pET-22b(+) (Novagen, Madison, Wis.) in E. coli. pET-22b(+) contains a nickel ion binding domain consisting of 6 sequential histidine residues, which allows the expressed protein to be purified on a suitable affinity resin. Another example of a vector that can be used is pcDNA3 (Invitrogen, San Diego, Calif.), described above.

Conditions of expression should be such that the scFv polypeptide can assume optimal tertiary structure. Depending on the plasmid used (especially the activity of the promoter) and the host cell, it may be necessary to modulate the rate of production. For instance, use of a weaker promoter, or expression at lower temperatures, may be necessary to optimize production of properly folded scFv in prokaryotic systems; or it may be preferably to express scFv in eukaryotic cells.

Preferred single chain variable regions comprise at least 10 consecutive amino acids of SEQ. ID NO:2 and at least 10 consecutive amino acids of SEQ. ID NO:4, especially wherein the amino acids of SEQ. ID NO:2 and the amino acids of SEQ. ID NO:4 are joined by a linker polypeptide of 5 to 20 amino acids, or comprising the light chain variable region and the heavy chain variable region of monoclonal antibody 1A7.

The invention also encompasses polymeric forms of 1A7 polypeptides, containing a plurality of 1A7 polypeptides. One embodiment is a linear polymer of 1A7 polypeptides, optionally conjugated to carrier. These linear polymers can comprise multiple copies of a single 1A7 polypeptide, or combinations of different 1A7 polypeptides, and can have tandem 1A7 polypeptides, or 1A7 polypeptides separated by other amino acid sequences. Another embodiment is 1A7 multiple antigen peptides (MAPs) MAPs have a small immunologically inert core having radially branching lysine dendrites, onto which a number of 1A7 polypeptides are covalently attached. See Posnett et al. (1988). Biol. Chem. 263:1719-1725; Tam (1989) Methods Enzymol. 168:7-15. The result is a large macromolecule having a high molar ratio of 1A7 polypeptides to core. MAPs are efficient immunogens and useful antigens for immunoassays. The core for creating an 1A7 MAP can be made by standard peptide synthesis techniques, or obtained commercially (Quality Controlled Biochemicals, Inc., Hopkinton, Mass.). A typical core matrix is made up of three levels of lysine and eight amino acids.

In another embodiment of the invention, the immunogenicity of the 1A7 polypeptides is enhanced by preparing them in expression systems in which they are assembled with particle-forming proteins such as, for example, those associated with hepatitis B virus. See, e.g., U.S. Pat. No. 4,722,840. Constructs wherein the 1A7 polypeptide is linked directly to particle-forming protein coding sequences produce hybrids which are immunogenic for an anti-GD2 response. The vectors also comprise immunogenic HBV epitopes; for example, the pre-S peptide and stimulate a response against HBV. Such expression systems may be provided in eukaryotic cells, including yeast or mammalian cells.

In another embodiment, 1A7 polypeptides are conjugated to a carrier molecule. This is desirable for a 1A7 peptide that comprises a suitable epitope for eliciting anti-GD2, but is too small to be immunogenic. Any conjugation method known in the art may be used. Any carrier can be used which is not harmful to the host. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins; polysaccharides (such as latex functionaliaed SEPHAROSE.TM., agarose, cellulose, cellulose beads and the like); polymeric amino acids (such as polyglutamic acid, polylysine, and the like); amino acid copolymers; and inactive virus particles or attenuated bacteria, such as Salmonella. Especially useful carrier proteins are serum albumins, keyhole limpet hemocyanin (KLH), certain immunoglobulin molecules, thyroglobulin, ovalbumin, and tetanus toxoid. KLH is especially preferred.

1A7 polypeptides of the invention can be identified in a number of ways. For example, the variable regions of the light and heavy-chains can be screened by preparing a series of short polypeptides that together span the entire variable region amino acid sequence. Using a series of polypeptides of 20 or 50 amino acids in length, each 1A7 variable region may be surveyed for useful functional properties. It is also possible to carry out a computer analysis of a protein sequence to identify potentially interesting polypeptides, such as those that bear the shape of D2, or those involved in idiotype-anti-idiotype contact.

Those skilled in the art will readily appreciate that the various adaptations of 1A7 described in this section may be combined in various fashions to yield other 1A7 polypeptides with desirable properties. For instance, 1A7 polypeptides with modified residues may be comprised in a MAP. In another example, a 1A7 scFv is fused to a cytokine, such as IL-2. AU such combinations are contemplated in this invention.

Preparation of 1A 7 Polypeptides

The polypeptides of this invention can be made by any suitable procedure, including proteolysis of the 1A7 antibody, by recombinant methods or by chemical synthesis.

1A7 polypeptides, especially shorter polypeptides up to about 50 amino acids, are conveniently made by chemical synthesis, based on the sequence data and other information provided herein.

Certain 1A7 polypeptides which are fragments of the whole molecule may alternatively be prepared from enzymatic cleavage of intact 1A7. Examples of proteolytic enzymes include, but are not limited to, trypsin, chymotrypsin, pepsin, papain, V8 protease, subtilisin, plasmin, and thrombin. Intact 1A7 can be incubated with one or more proteinases simultaneously or sequentially. Alternatively, or in addition, 1A7 can be treated with disulfide reducing agents. Peptides may 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.

A 1A7 polypeptide can also be made by obtaining a polynucleotide encoding it according to the information provided elsewhere in this application, and introducing it into a suitable expression system. Typically, polynucleotides encoding a 1A7 polypeptide are ligated 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. Examples of prokaryotic host cells appropriate for use with this invention include E. coli. Examples of eukaryotic host cells include avian, insect, plant, and animal cells such as COS7, HeLa, and CHO cells.

In certain applications, such as when a 1A7 polypeptide is expressed in a suitable storage medium such as a plant seed, the 1A7 polypeptide can be used without purification (Fiedler et al. (1995) Biotechnology 13:1090-1093). For most applications, it is generally preferable that the polypeptide is at least partially purified from other cellular constituents. Preferably, the polypeptide is at least about 50% pure as a weight percent of total protein. More preferably, the protein is at least about 50-75% pure. For clinical use, the polypeptide is preferably at least about 80% pure, according to the criteria given in another section.

Characterization of 1A 7 Polypeptides

The 1A7 polypeptides of this invention can be char d in several ways. For instance, a 1A7 polypeptide may be tested for its ability to bind specifically to 14G2a, for its ability to specifically inhibit the binding between 14G2a and intact 1A7, or for its ability to specifically inhibit the binding between 14G2a and GD2. Alternatively, a 1A7 polypeptide can be tested for its ability to elicit an immune response, preferably an anti-GD2 response. 1A7 polypeptides can also be tested for their ability to palliate or ameliorate GD2-associated disease, such as GD2-associated tumors. It is understood that only one of these properties need be present in order for a polypeptide to come within this invention, although preferably more than one of these properties is present.

The ability of a 1A7 polypeptide to bind 14G2a may be tested by immunoassay. Any form of direct binding assay is suitable. In one such assay, the 14G2a or alternatively the 1A7 polypeptide is labeled. Suitable labels include radioisotopes such as .sup.125I, enzymes such as peroxidase, fluorescent labels such as fluorescein, and chemiluminescent labels. Typically, the other binding partner is insolubilized (for example, by coating onto a microtiter plate) to facilitate washing. After combining the labeled component with the insolubilized component, the solid phase is washed and the amount of bound label is determined. Another such assay is a sandwich assay, in which the putative 1A7 polypeptide is captured by a first anti-immunoglobulin on a solid phase and developed with a second anti-immunoglobulin. Either the insolubilized or labeled anti-immunoglobulin or both is 14G2a. In either of these examples, the extent of binding of 1A7 is directly related to the amount of label bound to the solid phase.

To conduct the inhibition assays, the putative 1A7 polypeptide is titered for its ability to decease the binding of 1A7 to 14G2a, or 14G2a to GD2. Either of the binding pairs in the reaction to be inhibited is labeled, while the other is typically insolubilized in order to facilitate washing. GD2, if it is used, may be provided as the purified ganglioside, or as a GD2-expressing cell line, like M21/P6. The 1A7 polypeptide is typically mixed with the labeled component, and then the name is combined with the solid phase. Polypeptides with the characteristics of 1A7 will proportionately decrease the amount of label attached to the solid phase, compared with control polypeptides. This test may be more sensitive than measuring direct binding, because lower affinity interaction between 1A7 and Ab1 may be too weak to form a stable bond, but adequate to interfere with the binding of another ligand-receptor pair when present at sufficient concentration.

A 1A7 polypeptide or a molecule comprising such a peptide that has an Fc-like effector component may be tested for their ability to mediate immune effector reactions, particularly complement-mediated cytolysis (CMC) and antibody-dependent cellular cytotoxicity (ADCC). Suitable assays are described elsewhere in this disclosure.

Another way of characterizing 1A7 polypeptides, particularly those intended for use in therapy, is to test their ability to generate an immune response. Suitable techniques for characterizing the immune response are given in a later section.

Uses of Polypeptides

Polypeptide fragments, fusion proteins, and other derivatives of 1A7 have many uses The uses generally parallel those of the intact 1A7 antibody as outlined earlier in this disclosure. Certain derivatives may have desirable effects for particular uses. For example, humanized or single-chain variable region molecules may be desirable for human administration, because they are less likely to stimulate an anti-mouse isotype response. Fusion proteins comprising a cytokine may be more immunogenic.

Preferred uses of these compounds include eliciting an antibody response to 1A7 or more preferably GD2, eliciting a T cell response to 1A7 or more preferably GD2, and treating various types of GD2-associated cancer. These uses are elaborated more fully in a later section.

Pharmaceutical Compositions and Vaccines Comprising 1A7 Antibody and Polynucleotide and Polypeptide Derivatives

The present invention encompasses phaamceuacal compositions and vacines containing 1A7 antibody, or polynucleotide or polypeptide derivatives of 1A7 either alone or in combination. Such pharmaceutical compositions and vaccines are useful for eliciting an immune response and treating GD2-associated diseases, either alone or in conjunction with other forms of therapy, such as chemotherapy or radiotherapy.

Pharmaceutical compositions include vaccines for direct administration to an individual. Vaccines may comprise 1A7 antibodies, polynucleotides, or polypeptide derivatives, or a combination thereof.

Vaccines containing naked 1A7 polynucleotides can be used for genetic immunization (see generally Tang et al. (1992) Nature 356: 152-154). Once in the cell nuclei, plasmids comprising 1A7 encoding regions may persist as circular non-replicating episomes leading to dose-dependent and long-lived expression. (Spooner et al. (1995) Gene Therapy 2:173-180). Immunization using polynucleotides has been shown to generate cellular as well as humoral responses (Spooner et al.; Wang et al. (1995) Human Gene Therapy 6:407-418). Genetic immunization has many of the advantages of live or attenuated microorganisms as vehicles for eliciting an immune response, without the risk of infection.

Preferably, 1A7 polynucleotides are in the form of plasmid vectors containing appropriate control sequences for transcription and translation, such as promoters, enhancers, and signal sequences. One or more 1A7 polynucleotides can be used within a single cloning vector, or multiple vectors can be used. A preferred 1A7 encoding region for use in a polynucleotide vaccine encodes a 1A7 scFv. The polynucleotide vaccine may also comprise encoding regions for other substances which will enhance the immune response. A preferred example is GM-CSF.

Another preferred embodiment of an 1A7 polynucleotide suitable for use as a vaccine is a viral vector. Examples include adenovirus, adeno-associated retroviruses (AAV), and SV40. Especially preferred is a vaccinia vector, whereby the polypeptide encoded by the 1A7 polynucleotide is expressed along with the immunogenic viral particle. Recombinant vaccinia comprising polynucleotides encoding 1A7 polypeptides such as scFv may be used for direct vaccination at about 10.sup.7 to 10.sup.8 plaque forming units per dose. Vaccinia can be administed parenterally, by subcutaneous or intramuscular injection, for example, as well as through mucosal membranes, such as nasally, orally or by inhalation. Alternatively, vaccinia can be administered via vaccinia-infected cells. In this technique, suitable cells, such as tumor cells, are infected with vaccinia in culture. The infected cells are then reintroduced to the individual. Methods for infecting cells with vaccinia and reintroducing these infected cells, have been described (see, e.g., Moss (1991)).

Other 1A7 polynucleotide vaccines may be designed using other delivery vehicles known in the art. Another such delivery vehicle is cationic liposomes, to which DNA may be readily attached by virtue of its charge.

Vaccines can also be prepared from the 1A7 antibody, polypeptide derivatives thereof, or an combination thereof. A protein can optionally be treated chemically to enhance its immunogenicity, especially if 100 amino acids or less. This may include cross-linking, for example, with glutaraldehyde; or linking to a protein carrier, such as keyhole limpet hemocyanin (KLH) or tetanus toxoid.

The preparation of pharmaceutical compositions that contain 1A7 antibody, or polynucleotide or polypeptide derivative 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 may include sterilizing, mixing with appropriate non-toxic and non-interfering components, dividing into dose units, and enclosing in a delivery device.

Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving or dispersing a vector embodied herein in a liquid excipient, such as water, saline, aqueous dextrose, glycerol, or ethanol. The composition may optionally also contain other medicinal agents, pharmaceutical agents, adjuvants, carriers, and auxiliary substances such as wetting or emulsifying agents, and pH buffering agents.

Protein vaccines of this invention typically comprise an adjuvant, which may be the same as or in addition to the excipient or carrier. Examples of adjuvants include but are not limited to aluminum hydroxide, alum, QS-21 (U.S. Pat. No. 5,057,540), DHEA (U.S. Pat. Nos. 5,407,684 and 5,077,284) including its precursors and modified forms (e.g., DHEA-S, the sulfonated form of DHEA), .beta.2 microglobulin (WO 91/16924), muramyl dipeptides, muramyl tripeptides (U.S. Pat. No. 5,171,568), monophosphoryl lipid A (U.S. Pat. No. 4,436,728; WO 92/16231) and its derivatives, such as various forms and generations of DETOX.TM. and BCG (U.S. Pat. No. 4,726,947). Other suitable adjuvants are aluminum salts, squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875. For veterinary use and for production of antibodies in animals, complete and incomplete Freund's adjuvant can be used. The choice of an adjuvant will depend, in part, on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the regulatory acceptability of the adjuvant, particularly when intended for human use. For instance, alum is approved by the United States Food and Drug Administration (FDA) for use as an adjuvant in humans.

A preferred vaccine composition comprising 1A7 antibody or peptide derivative is prepared by mixing with aluminum hydroxide and incubated to about 48.degree. C. for about 30 min. Even more preferred are vaccine compositions comprising 1A7 and QS-21 or RIBI.TM.PC. QS-21 and RIBI.TM.PC are equally preferred; and the selection between them is made on the basis of availability.

Pharmaceutical compositions of the present invention are administered by a mode appropriate for the form of composition. Possible routes include intracutaneous, subcutaneous, intramuscular, intraperitoneal, intradermal, oral, intranasal, intradermal, and intrapulmonary (i.e., by aerosol). Protein vaccines of this invention for human use are typically administered by a parenteral route, most preferably subcutaneous. A series of injections is preferably given at different subcutaneous sites.

Pharmaceutical 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. For administration via the respiratory tract, a preferred composition is one that provides either a solid or liquid aerosol when used with an appropriate aerosolizer device. Although not required, 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.

It is recognized that a number of alternative vaccine compositions, not limited to those described herein, may be efficacious in inducing an immune response. All such compositions are embodied within the present invention, providing they include a 1A7 polynucleotide or polypeptide as an active ingredient. The pharmaceutical compositions of this invention can be used in conjunction with other modes of therapy, whether established or experimental, whenever his is clinically desirable.

Testing 1A7 Compounds and compositions for the Ability to Elicit a Specific Immune Response

Compounds embodied in this invention may be assessed for their ability to elicit a specific immune response. Accordingly, 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 anti-1A7 response.

A test composition in an appropriate test dose is administered on an appropriate immunization schedule. It may be appropriate to compare different doses and schedules within the predicted range.

Samples (usually blood samples) are collected regularly during treatment, and assayed for a specific immune response. The response may include antibody, helper-inducer T cells, or cytotoxic T cells, or a combination thereof. As a screening test, the samples may be measured for an anti-1A7 response. Since the objective is typically to identify compositions useful in cancer therapy, the samples are preferably measured for an anti-GD2 response, as manifest in direct or inhibition type experiments.

This section outlines some non-limiting examples of assays that are suitable to monitor the immune response.

Presence of anti-1A7 (Ab3) and anti-GD2 (Ab1') activity in a humoral response is preferably determined after first pre-incubating sera with autologous immunoglobulin or adsorbing on a suitable affinity resin to remove antibody activity against isotypic and allotypic determinants. The adsorbed serum is then tested for Ab3 or Ab1' activity, for example, using ELISA or RIA. For instance, different dilutions of pre-reacted sera are reacted with 1A7 (or 1A7 polypeptide) coated on microtiter plates. An unrelated Ab2 serves as a control. After washing, the Ab3-1A7 complex is labeled using, for example, .sup.125I-labeled 1A7 in a homogeneous sandwich assay. Results from this assay are compared to those obtained before administration of the 1A7 polypeptide (Example 1). Alternatively, binding to GD2 positive cells, such as M21/P6 cells, can be tested using immune flow cytometry. In a third example, the specificity of Ab3 is determined by Western blot GD2 is separated by SDS-PAGE and blotted to a nitrocellulose filter. The filter is then incubated with sera containing Ab3, and the reaction developed by a suitably labeled anti-immunoglobulin. If the Ab3 binds to GD2, a band at the appropriate molecular weight should appear.

If desired, the specificity of the Ab3 can be further characterized. For example, competition assays can be performed to determine whether Ab3 share Ab1 idiotopes. Accordingly, competition experiments are conducted in which Ab3 is tested for inhibition of binding between 1A7 and 14G2a Inhibition indicates that Ab3 and 14G2a contain at least some similar binding determinants. Competition of Ab3 with the binding of 14G2a to GD2 may also be measured.

Another way of characterizing a composition of this invention is testing its ability to elicit an antibody that is cytotoxic. For determination of complement mediated cytotoxicity (CMC), M21/P6 target cells expressing GD2 are labeled with .sup.51Cr. Labeling may be accomplished by incubating about 10.sup.6 cells with approximately 200 .mu.Ci Na.sub.2SO.sub.4 for 60 minutes at 37.degree. C., followed by washing. The assay is conducted by adding and incubating serum suspected of containing antibody. Guinea pig serum pre-adsorbed with M21/P6 cells (or other source of complement) is then added. After a suitable incubation period at 37.degree. C., extent of .sup.51Cr release is then measured and compared with that of unopsonized control cells. Release of .sup.51Cr correlates with CMC activity (Herlyn et al. (1981) Int J. Cancer 27:769).

Another way of characterizing a composition of this invention is by testing its ability to elicit an anti-GD2 antibody that participates in an ADCC response (Cheresh et al. (1986) Cancer Research 46:5112-5118). In this assay, cultured human M21/P6 cells (which express GD2 in their surface) are labeled with .sup.51Cr and are used as target cells. Normal human peripheral blood mononuclear cells (PBMC) are used as effector cells. The Ab3 containing serum from immunized subjects is supplied to mediate the ADCC reaction. Preferably, the ADCC assay is conducted in the presence of heat-inactivated serum with an effector to target cell ratio of 100:1 for 4 hours, although other suitable conditions may be used. The amount of .sup.51Cr released is then measured.

The 1A7 antibodies, polynucleotides and polypeptides of this invention can also be characterized by their ability to elicit a cellular immune response. As used herein, a "cellular immune response" is a response that involves T cells, and can be observed in vitro or in vivo. Subjects are immunized with the 1A7 composition, and cells are recovered for assaying the response. Where the subject is a small animal, cells are typically obtained from the spleen. For larger animals including humans, cells are typically obtained from peripheral blood. A suitable cell population is recovered from the sample by standard separation techniques, typically involving centrifugation over an appropriate medium such as FICOLL-HYPAQUE.TM..

One way of detecting a cellular immune response is by assaying for T cell proliferative activity. In this test, cellular immune response is measured by proliferation of peripheral blood mononuclear cells (PBMCs) incubated with 1A7 polypeptide. PBMCs are isolated after a requisite number of administrations of 1A7 polypeptide, and are incubated with varying concentrations of 1A7 polypeptide. A non-specific mitogen such as PHA serves as a positive control; incubation with an unrelated anti-idiotype antibody serves as a negative control. Preferably, the stimulator cells are autologous with the responder cells, particularly in terms of histocompatibility Class II antigens. After incubation of the PBMCs for an appropriate period (typically 5 days), [.sup.3H]thymidine incorporation is measured. If desired, determination of which subset of T cells are proliferating can be performed using flow cytometry. Optionally, splenic T cells can be pre-depleted of either CD4.sup.+ or CD8.sup.+ cells before the proliferation assay by incubation with monoclonal antibody RL.172 (anti-CD4.sup.+) or monoclonal antibody.168 (anti-CD8.sup.+) and complement.

Another way of detecting a cellular immune response is to test for T cell cytotoxicity (CTL) activity. In this test, an enriched T cell population are used as effectors in a standard .sup.51 Cr release assay (Kantor et al. (1992) J. Natl. Cancer Inst. 84:1084-1091). An example of a .sup.15Cr release assay is the following. GD2-positive tumor cells (typically 1-2.times.10.sup.6 cells) are radiolabeled as target cells with about 200 .mu.Ci of Na.sub.2 .sup.51CrO.sub.4 (Amersham Corp., Arlington Heights, Ill.) for 60 minutes at 37.degree. C., followed by thorough washing to remove unincorporated isotopes. T cells and targets (1.times.10.sup.4/well), both resuspended in culture medium, are then be combined at various effector-to-target ratios in 96-well, U-bottom plates (Costar Corp.). The plates are centrifuged at 100.times.g for 5 minutes to initiate cell contact, and are incubated for 4 or 16 hours at 37.degree. C. with 5% CO.sub.2. After incubation, supernatants are collected using a Supernatant Collection System (Skatron, Inc., Sterling, Va.) and radioactivity will be quantitated in a gamma counter (Beckman Instruments). Spontaneous release of .sup.51Cr is determined by incubation of targets in the absence of effectors, while maximum or total release of .sup.51Cr will be determined by incubation of targets in 0.1% TRITON X-100. Percentage of specific release of .sub.51Cr is calculated in relation to spontaneous and maximal release.

Another way of chat a 1A7 polypeptide is testing its ability to ameliorate, delay the progression or reduce the extent of GD2-associated disease, as outlined in the following section.

Use of Pharmaceutical Compositions for Eliciting an Immune Response and Treating Disease

Compositions embodied in this invention such as those outlined in the previous section may be used for administration to individuals. They may be administered for experimental purposes, or to obtain a source of anti-GD2.

Compositions of this invention are particularly suitable for administration to human individuals with a GD2-associated disease. A GD2 associated disease is one in which expression of the GD2 ganglioside is altered at the affected tissue site, usually an elevation in cell-surface expression Relevant diseases are those in which an active immune response against GD2 would confer a clinical benefit Especially relevant are GD2-associated cancers; particularly melanoma, neuroblastoma, glioma, sarcoma, and small cell lung cancer.

The compositions of this invention may be administered to an individual with one of several objectives in mind. For example, the various compositions of this invention may be used to elicit an immune response. This includes an anti-1A7 specific response, and more preferably an anti-GD2 response. The desired response may be a specific antibody response; a specific T helper-inducer repines, or a specific cytotoxic T cell response. An ADCC response or a cytotoxic T cell response is especially preferred in the context of cancer therapy, since these arms of the immune system are believed to be important effector elements in immune surveillance. The presence of an antibody response may provide a convenient means for routine clinical monitoring. Thus, a response that involves several components of the immune response in combination is especially preferred.

Assays for measuring and characterizing antibody response, ADCC, antibody-mediated cytolytic activity, T cell proliferative activity, and cytotoxic T cell activity are all described elsewhere in this disclosure.

Also included in this invention are methods for tinting GD2-associated disease, such as a tumor expressing GD2. The method comprises administering an amount of a pharmaceutical composition effective to achieve the desired effect be it palliation of an existing tumor mass or prevention of recurrence.

Dose

For treatment of a GD2-associated disease in vivo, the amount of a pharmaceutical composition administered is an amount effective in producing the desired effect. An effective amount may be provided in one or a series of administrations.

For intact 1A7, a mouse requires approximately 100 .mu.g of KLH-coupled 1A7 emulsified in CFA and IFA in each of at least about three and typically at least four administrations. Monkeys require approximately 2 mg. The range of intact 1A7 that can be appropriately administered to humans is from about 10 .mu.g to 20 mg, preferably 200 .mu.g to 15 mg, more preferably 500 .mu.g to 10 mg, still more preferably 1 mg to 4 mg, and even more preferably 2 mg. Smaller peptides and fusion proteins may be more potent on a per-weight basis, and the preferred dose may be lower than with the intact molecule. Appropriate doses can easily be determined by comparing various doses of intact 1A7 and derivatives thereof in animal models, and scaling appropriately for human use.

The amount of 1A7 polynucleotide to be administered will depend upon several factors, such as the route of administration, the condition of the individual, and the desired objective. Typically, if administered directly, the amount per administration is about 10 .mu.g to 1 mg, preferably 25 .mu.g to 500 .mu.g, more preferably 30 .mu.g to 250 .mu.g, even more preferably 50 to 100 .mu.g.

Administrations are typically conducted on a weekly or biweekly basis until a desired, measurable parameter is detected, such as elicitation of an immune response. Administration can then be continued on a less frequent basis, such as biweekly or monthly, as appropriate.

The various compounds 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.

The exact dose and timing for the administration of any composition of this invention depends on the individual to be treated, the capacity of the individual's immune system to synthesize antibodies, the route of administration, the degree of protection desired, and the immunological and clinical response to previous doses. The immunological response may be assessed by assays given in the previous section. Choosing an appropriate amount to administer accordance with the guidelines suggested are the responsibility of the administering physician.

Appropriate Subjects for Therapy, and Desirable Effects

Suitable subjects include those who are suspected of being at risk of a pathological effect of any GD2-associated condition are suitable for treatment with the pharmaceutical compositions of this invention. Those with a history of a GD2-associated cancer are especially suitable.

The clinical studies described in the Example section are designed to exclude subjects who have not been treated previously with mouse immunoglobulin. The concept is that a proportion of subjects who have been so treated may have circulating anti-mouse immunoglobulin (HAMA). This selection criteria has been implemented to facilitate initial testing. However, the presence of HAMA is not believed to be an impediment to therapy, since the purpose of the 1A7 is to elicit a response, not remain in the circulation. Patients are more typically chosen for therapy irrespective of their history of previous treatment with mouse immunoglobulin. Of course, for certain engineered compounds like humanized 1A7 and scFv, most mouse isotype determinants have been deleted, and the presence of HAMA in a potential recipient is even less of a consideration.

Suitable human subjects for therapy comprise two groups, which may 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 are insufficient to identify this population). A pharmaceutical composition embodied in this invention is administered to these patients to elicit an anti-GD2 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 a GD2-associated 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. As a result, these individuals have no clinically measurable tumor. However, they are suspected of being at risk for progression of the disease, either near the original tumor site, or by metastasis.

This group may be further subdivided into high-risk and low-risk individuals. The subdivision is made on the basis of features observed before or after the initial treatment These features are known in the clinical arts, and are suitably defined for each different GD2-associated cancer. Features typical of high risk subgroups are those in which the tumor has invaded neighboring tissues, or who show involvement of lymph nodes.

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-GD2 response. Ideally, 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.

Of course, cross-overs between these two patient groups are possible, and the pharmaceutical compositions of this invention may be administed at any time that is appropriate. For example, 1A7 therapy may be conducted before or during traditional therapy of a patient with high tumor burden, and continued after the tumor becomes clinically undetectable. 1A7 therapy may 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.

It is recognized in the art that the immunological status of each of the aforementioned category differs one from another by several criteria. For example, patients with active disease are generally immunosuppressed, either due to tumor-related pathology or to recent radiotherapy or chemotherapy. Their immune system may be under a barrage of tumor-associated antigen from the tumor site. On the other hand, patients who are in remission may have stronger active suppression against autoantigens. Accordingly, the ability of an anti-idiotype based vaccine to elicit an anti-tumor response, or improve the clinical condition, must be determined separately for each patient category.

Other Clinical Indications

Various compounds and compositions of this invention have other clinical indications, of which this section provides only a survey.

One indication is the treatment of cells ex vivo. This may be desirable for experimental purposes, or for treatment of an individual with a GD2-associated disease. In one example, the 1A7 antibody, or a polynucleotide or polypeptide derivative are administered to a culture of cells, such as peripheral blood cells obtained from a donor, or a suitable cell line. This may be done, for example, with the objective of stimulating T cell activity. About 0.5 to 2 .mu.g/mL of 1A7 is an effective dose for this purpose. If desired, the stimulated cells may then be administered to a recipient, in an effort to convey passive immunity. In a second example, donor cells are genetically altered with an expression vector of this invention, to provide for ongoing secretion of 1A7 antibody after administration of the cells to the recipient.

The invention also encompasses compositions and methods using 1A7 antibodies and polypeptide derivatives to remove a label (particularly a radiolabel) from an individual who has received a labeled anti-GD2 antibody (such as 14G2a) in the course of radioscintigraphy or radiotherapy. Effective imaging using radiolabeled antibodies is hampered due to excess circulating radiolabeled antibody, which often takes several days to clear. Accordingly, 1A7 antibody or a polypeptide derivative is administered to the individual at a specified time after administration of the labeled anti-GD2. The intention is for the 1A7 polypeptide to complex with anti-GD2 at sites other than the tumor, such as in the circulation and interstitial spaces, and thereby promote its clearance. As a result, the level of label in unaffected tissues is reduced, and the image of the tumor (in comparison to neighboring tissues) is enhanced. Similarly, when radionuclides are given to subjects for irradiation of a tumor site, it is desirable to reduce collateral exposure of unaffected tissue. This invention thus includes methods of treatment in which a radiolabeled anti-GD2 antibody is administered in a therapeutic dose, and followed by a molar excess of 1A7.

In either of these applications, an amount of 1A7 polypeptide is chosen that is in sufficient molar excess over the labeled anti-GD2 to locate and bind any anti-GD2 that is not localized at the tumor site. The timing of administration and amount of 1A7 polypeptide will depend upon the nature of the radiolabeled antibody, the type of radioisotope used and the condition of the individual. Preferably, the molar ratio of 1A7 polypeptide to the anti-GD2 antibody is at least about 5:1, more preferably about 25:1 to 200:1. Preferably, 1A7 polypeptide is administered 5 to 24 hours after the individual has received the anti-GD2 antibody.

The invention also includes methods of detecting the presence of an anti-GD2 antibody bound to a tumor cell comprising the steps of treating an individual with 1A7 for a sufficient time to allow binding to the anti-GD2 antibody, and detecting the presence of any complex formed. The invention is for the 1A7 to detect anti-GD2 that has pre-attached to the tumor cell; or alternatively, to promote the binding of anti-GD2 to the tumor cell by forming a polyvalent anti-GD2/1A7 immune complex. In the former instance, the anti-GD2 is provided with a detectable label or a means by which a label can be attached. In the latter instance, either the anti-GD2 or the 1A7 is provided with a label. Suitable labels include radiolabels such as .sup.111In, .sup.131I and .sup.99mTc. The anti-GD2 and 1A7 are administered (usually sequentially) into the subject and allowed to accumulate at the tumor site. The tumor is then detected or visualized using standard techniques of radioscintigraphy.

Diagnostic Kits

The present invention encompasses kits containing 1A7 antibodies, polynucleotides, or polypeptides. Diagnostic procedures using the 1A7 polynucleotides or polypeptides of this invention can be performed by diagnostic laboratories, experimental laboratories, practitioners, or private individuals. Kits embodied by this invention include those that allow someone to conduct an assay for anti-GD2 or anti-1A7 activity, or for an 1A7 encoding sequence. An alteration in one of these components resulting, for example, from the presence of a GD2-associated disease or treatment directed towards it is typically compared with that in a sample from a healthy individual. The clinical sample is optionally pre-treated for enrichment of the target being tested for. The user then applies a reagent contained in the kit in order to detect the changed level or alteration in the diagnostic component.

Each kit necessarily comprises the reagent which renders the procedure specific: a reagent 1A7 antibody or polypeptide, used for detecting anti-1A7 or anti-GD2 in the sample; or a reagent 1A7 encoding polynucleotide, used for detecting a 1A7 encoding polynucleotide in the sample. Optionally, the reagent may be conjugated with a label to permit detection of any complex formed with the target in the sample. In another option, a second reagent is provided that is capable of combining with the first reagent after it has found its target. For example, labeled anti-mouse IgG may be provided as a secondary reagent for use with intact 1A7. Labeled avidin may be provided as a secondary reagent when the primary reagent has been conjugated with biotin.

The kits may be employed on a variety of biological samples, including both liquid samples, cell suspensions and tissue samples. Suitable assays using 1A7 antibodies, polypeptides, and polynucleotides that can be supplied in kit form include those described elsewhere in this disclosure.

Each reagent is supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for exchange or addition into the reaction medium when the test is performed. Suitable packaging is provided. The kit may optionally provide additional components that are useful in the procedure. These optional components include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.

Deposit

The foregoing description provides, inter alia, detailed methods for preparing monoclonal antibody 1A7, along with 1A7 encoding polynucleotides, 1A7 polypeptide fragments, and other derivatives.

A practitioner of ordinary skill in the art may practice embodiments of this invention by referring to the sequence data for 1A7, which is provided herein. Alternatively, a practitioner may practice the invention by first purifying the 1A7 antibody, or a 1A7 encoding polynucleotide from a 1A7 antibody producing cell. A hybridoma cell line producing 1A7 antibody has been deposited with the American Type Culture Collection (ATCC) under terms of the Budapest Treaty, and has been given Accession No. HB-11786.

Claim 1 of 5 Claims

1. A monoclonal antibody 1A7 produced from hybridoma cell line deposited under ATCC Accession No. HB-11786.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.