WO1998012227A1

WO1998012227A1 - Recombinant single chain antibodies directed against the gp54 cancer marker, composition comprising same and use thereof

Info

Publication number: WO1998012227A1
Application number: PCT/CA1997/000690
Authority: WO
Inventors: Yves Fradet; André DARVEAU
Original assignee: Diagnocure Inc
Current assignee: Diagnocure Inc
Priority date: 1996-09-19
Filing date: 1997-09-19
Publication date: 1998-03-26
Anticipated expiration: 1999-03-19
Also published as: AU4373197A

Abstract

This invention relates to a novel single chain antibody molecules. The starting antibodies from which are derived these molecules are T16 and 48-127. Theses antibodies recognize different epitopes of the gp54 antigen which is expressed at the surface of a plurality of carcinoma cells. Are disclosed: the sequence of the light and heavy chain and of a single chain antibody molecule (scFv) for each antibody. Both scFvs bind gp54-expressing cells, and the immune complexes are internalised by cells. Therapeutic and diagnostic compositions/methods are also disclosed.

Description

TITLE OF THE INVENTION

RECOMBINANT SINGLE CHAIN ANTIBODIES DIRECTED AGAINST THE GP54 CANCER MARKER, COMPOSITION COMPRISING SAME AND USE THEREOF

FIELD OF THE INVENTION

The present invention relates to cancer diagnosis and therapy, more particularly, to cancer expressing the gp54 or trop-2 marker, by the use of antibodies thereto, parts, variants and derivatives thereof.

BACKGROUND OF THE INVENTION Antigen gp54 or trop-2 is present in a majority of carcinoma such as bladder, breast, uterine and prostate cancers. The gp54 antigen is expressed by normal urothelial cells and by all human bladder tumors. Immunofluorescence studies, however, suggested that it was not expressed on the luminal surface of normal urothelial umbrella cells and could therefore represent a valuable candidate for specific targeting of tumor cells by the intravesical route.

Selective expression of gp54 has been confirmed by de Harven et al. (1992). It has been showed that a gp54-antibody complex was internalized by bladder cancer cells in vitro (de Harven et al. op. cit. and Battelli 1996). This particular property of the immune complex confers thereto a promising utility for the targeting of various diagnostic and cytotoxic agents to cells from superficial tumors (TCC and CIS) of the human urinary bladder.

Since there is a phenotypic heterogeneity of many human carcinomas as well as antigenic modulations, no monoclonal antibody to gp54 antigen is up to now capable of recognizing 100% of carcinoma cells. Many antibodies have been developed against this antigen. Some compete with one another, others do not. A combination of non-competing antibodies may increase the number of detected cells particularly when different epitopes of a molecule are exposed at different stages of cancerization in an heterogenous cell population.

Amongst the antibodies already developed against gp54 antigen, monoclonal antibody T16 has been described by Fradet et al (1986). Another monoclonal antibody, called 48-127, has been recently developed by the present inventors. This antibody recognizes a different epitope from that recognized by T16. Antibody 48-127 has been developed by using a different source of cancer cells and a different immunization protocol and has a unique binding epitope.

In our knowledge, there is no known combination of antibodies or parts or variants or derivatives thereof that have been used to increase the recognition of carcinoma cells which expose different epitopes at different times. There thus remains a need to increase recognition of carcinoma cells to improve the diagnostic accuracy or the therapeutic success of labelled or cytotoxic-conjugated antibodies.

Furthermore it is known that the use of single chain recombinant antibodies (scFvs) is preferred to entire antibodies because they do not provoke an immune response against them and take much less time to distributed and captured by target tissues (Chester; 1995). Yet, the main inconvenients of scFvs are that they do not remain bounded to the target for a long time and they are rapidly cleared because of their small size (Verharr; 1996 and Begent;1996). However, taking advantage of the internalisation of gp54-antibody immune complexes, it is now possible to administer to a patient a 48-127 scFv or a T16 scFv, or a mixture of both which is capable to increase cell targeting over and above each scFv alone.

SUMMARY OF THE INVENTION

It is now an object of the present invention to meet needs for acceptable diagnostic or therapeutic tools against gp54-expressing cells. The present invention features recombinant single chain antibody molecules

(scFvs).

By "recombinant single chain antibody molecule" as used herein is meant a peptide having a heavy and a light antibody chain component with a flexible linker positioned therebetween, which peptide is encoded by a DNA generated by recombinant DNA technology.

This invention features a method of generating an isolated DNA sequence encoding a recombinant single chain 48-127 or T16 antibody molecules, which method comprises amplifying a first nucleic acid sequence encoding a heavy chain and a second nucleic acid sequence encoding a light chain of 48-127 or T16 antibody molecules to generate a heavy and a light chain specific DNA sequences, amplifying by PCR the heavy and light chain specific DNA sequences using a first set of primers A and B, A being a heavy chain specific 5' primer, B being a light chain specific 3' primer and a second set of primers C and D, C being a heavy chain specific 3' primer and D being a light chain specific 5' primer, wherein at least 5 contiguous nucleotides of each of the C and D primers are complementary to each other, and recovering the formed scFvs.

The invention features a method of generating a recombinant single chain T16 or 48-127 antibody molecule, the method comprises the same above-steps and further comprises a step of expressing the recombinant single chain T16 or 48-127 antibody so amplified in expression vectors. Of course an inverse construction may be easily contemplated e.g. a VL-linker-VH construct in lieu of a VH-linker-VL one.

Also featured in the invention is an isolated DNA encoding the single chain antibody 48-127 and T16 molecules of the invention.

By "isolated DNA" as used herein is meant a DNA sequence which has been purified from the sequences which flank it in a naturally occurring state, e.g. a DNA sequence which has been removed from the sequences which are normally adjacent to the DNA sequence, i.e., those sequences which are adjacent to the DNA sequence in the genome in which it naturally occurs. The term also applies to a DNA sequence which as been substantially purified from other components which naturally accompany it, such as RNA, protein and lipid, i.e., those components which naturally accompany it in a cell. Isolated DNA sequence also denotes a synthetically prepared DNA sequence corresponding to the cloned sequence. It is an object of the present invention to provide the sequences of 48-127 and

T16 heavy and light chains isolated DNAs and variants thereof.

It is another object to provide the sequences of 48-127 and T16, recombinant single chain molecules directed against the gp54.

It is further an object aspect of the invention to provide expression vectors comprising the above scFv antibody encoding DNA molecules.

It is yet another object of the invention to provide 48-127 and T16 scFvs.

It is another object of the invention to provide 48-127 and T16 scFvs derivatives.

By "derivatives" as used herein is meant scFv molecules which are coupled to a cytotoxic agent, or which are labelled.

It is still another object of the invention to provide 48-127 and T16 scFvs variants.

By "variants" as used herein is meant light and heavy chain and scFv polypeptides, isolated heavy and light chain DNA molecules, scFv DNA molecules and which are mutated to change or increase their avidity towards gp54 expressing cells, or that are proteins or nucleic acids which are substantially similar in structure and biological activity to the proteins and nucleic acids of the present invention. Variants may therefore be obtained by directed mutagenesis or by searching cross reacting proteins with the aid of a ligand specific to the protein of the present invention, or by searching nucleic acids hybridizing with the nucleic acids of the present invention.

"Nucleic acid hybridization" refers generally to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences, which under appropriate conditions will form a thermodynamically favored double-stranded structure. Examples of hybridization conditions can be found in the two laboratory manuals referred above (Sambrook et al., 1989, and Ausubel et al., 1989) and are commonly known in the art. In the case of a hybridization to a nitrocellulose filter, as for example in the well known Southern blotting procedure, a nitrocellulose filter can be incubated overnight at 65°C with a labeled probe in a solution containing 50% formamide, high salt ( 5 x SSC or 5 x SSPE), 5 x Denhardt's solution, 1% SDS, and 100 μg/ml denatured carried DNA ( i.e. salmon sperm DNA). The npn-specifically binding probe can then be washed off the filter by several washes in 0.2 x SSC/0.1% SDS at a temperature which is selected in view of the desired stringency: room temperature (low stringency), 42°C (moderate stringency) or 65°C (high stringency). The selected temperature is based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions of hybridization and washing can be adapted according to well known methods by the person of ordinary skill. Stringent conditions will be preferably used (Sambrook et al.,1989, supra).

It is also an object of the invention to provide diagnostic and therapeutic compositions which comprise the 48-127 and/or T16 scFv derivatives or variants.

It is further another object of the present invention to provide methods for diagnosing in a patient a cancer which cancer cells express gp54 antigen, which comprises the step of administering a T16 scFv labelled derivative or a 48-127 scFv labelled derivative or a mixture of both.

It is further another object of the present invention to provide methods for treating a patient affected by a cancer which cancer cells express gp54 antigen, which comprises the step of administering a therapeutic amount of a T16 scFv cytotoxic derivative or a therapeutic amount of 48-127 scFv cytotoxic derivative or a mixture of both.

DESCRIPTION OF THE INVENTION Immunoscintiαraphv using Recombinant Single Chain mAbs 48-127 and T16 In the majority of cases involving invasive forms of cancer, the main problem consists in evaluating the stage (or spread) of cancer. In about 50% of cases, micrometastases are present which cannot be detected using current methods, thus condemning radical surgical excision of the primary tumor to failure. Despite modern imaging, the risk of error involved in gauging the spread of these tumors can approach 40%. The gp54 antigen's characteristics make it an ideal target for immunolocalization of metastases since it is expressed by most tumoral cells and because the antibody which attaches to the antigen forms a complex which is rapidly internalized within the cell. Targeting the gp54 antigen with mAbs T16 and/or 48-127 labelled with radioactive isotopes has rendered potentially possible the detection of micrometastases from cancers of the bladder, prostate, breasts and uterine cervix. At the present time, to the best of applicants' knowledge, there is no test available to detect with enough sensitivity the small sites of metastases from bladder, breast and cervical cancers. To optimize a test in nuclear medicine, it is necessary to have antibody or peptide fragments that are much shorter than a whole antibody. Instead of the test being performed several days after being injected into the patient, which results in longer hospitalization or reduced patient compliance, the use of the technetium isotope, for example, coupled to mAb fragments (scFvs or chimerea) would make it possible to test the patient within a few hours of being injected. For the purpose of showing scFvs' binding effectiveness, an immunofluorescent label has been coupled thereto. Intravesical and Systemic Therapy using Genetically Engineered R48-127 and

I1& Therapy will be based on the targeting of the gp54 tumor antigen with a polyvalent vector specifically directed against this antigen as a general carrier. This antigen will be targeted by recombinant scFv fragments of mAbs 48-127 and/or T16.

MAbs 48-127 and T16 have been cloned and molecularly engineered into polyvalent fragments that still react with gp54 antigen. One advantage of such recombinant antibody molecules is that a majority of the mouse mAbs has been removed thus minimizing the potential for this reagent to be rejected by the patients after a few injections, due to the immune reactivity to the mouse immunoglobulin. Another advantage of these recombinant single chain antibody molecules is the possibility to genetically engineer the reagent to attach either one of three different types of agents as cancer treatments. One such family of agents is recombinant toxins which, when internalized into the cancer cells, will kill them. Another family of molecules, is the biological response modifiers, such as G-CSF, Interleukin 2 and Interleukin 12. Bringing these reagents in close contact with tumor cells will result in a mobilization of the immune system to destroy these cancer cells. The third type of reagent could be nanoerythrosomes or liposomes, that can be used as a 48-127/T16- targeted delivery system to carry drugs, photodynamic agents or other therapeutic gene vectors to cancer cells.

Intravesical therapy with these various agents is a natural first step for investigation.

Preliminary studies have shown that mAbs T16 and 48-127 aimed at the gp54 antigen preferably adhere to cancerous and pre-cancerous cells in the bladder. Furthermore, several studies using antibodies coupled with radioactive or toxic substances have demonstrated that mAbs 48-127 and T16 linked to the antigen is internalized in the cell. Intravesical therapy offers a relatively safe means of testing the anti-tumor effectiveness of coupled antibodies in human beings. Due to the frequently recurrent nature of superficial bladder cancer, this effectiveness can be monitored on a short term basis. Since the antibodies have been proven to be effective as well as their scFvs in binding to target cells, treatment of bladder, breast, cervical and prostate cancer is contemplated.

Combination of T16/48-127 and chimaera:

A chimeric construction of single chain variable fragment (scFv) of T16/48-127 is contemplated as an alternative to the use of a mixture of T16 and 48-127 scFvs. Each member of the chimaera is capable of recognizing its corresponding epitope on gp54 - expressing cells. This chimaera can be coupled to a large variety of diagnostic and cytotoxic reagents, and provide compositions for diagnosing and eliminating carcinoma cells expressing the gp54 antigen, respectively. Chimaera can bind a large number of cancer cells by recognizing different parts of the same antigen, which parts are exposed differentially during the cancerization process, as each of T16 and 48-127 scFvs does.

Immunocapturing agent comprising 48-127 antibody:

Enrichment of cells expressing the gp54 antigen is particularly desirable for detecting the presence of a few carcinoma cells in a diluted environment. Such is particularly the case when a staging procedure is undertaken to detect the presence of metastatic cells in the blood stream. Therefore, if the combination 48-127/T16 or the chimaera is useful in detecting a larger population of heterogenous carcinoma cells, it is another object of the invention to provide a composition for immunocapturing these cells comprising these two scFv antibody molecules. ScFvs would be fixed on a solid support, and a diluted sample, such as a blood or urine sample, would be contacted therewith. Detection of captured cells may be performed directly or preceded by PCR amplification of gp54-encoding nucleic acids to increase test sensitivity.

Staging:

Immunocapture is one of the first steps of a staging procedure. This step is followed by an amplification of the cDNA of the captured cells, which makes use of primers ¹⁾ specific to the trop-2-expressing cells and ²⁾tissue-specific (origin of the metastatic cells). Primers specific to trop-2 have been designed from a unique region found by aligning trop-1 and trop-2 nucleic acid sequences. These primers have been successfully used to amplify gp54-expressing cDNAs obtained from carcinoma cell lines. It is therefore another object of the invention to provide primers specific to the trop-2 antigen coding sequence. It is further another object of the invention to provide a staging method comprising the steps of: ^a> immunocapturing carcinoma cells with the aid of a composition comprising T16 and/or 48-127 antibody molecules, ^b) detecting the presence of carcinoma cells by amplifying the cDNA of the captured cells by the action of a polymerase which activity is primed with the above-primers specific to trop-2.

Detection of the gp54 antigen in prostate metastatic cells, for example, is unlikely to be affected by hormone therapy, contrarily to the well known prostatic marker PSA. Detection of gp54 thus provides a more reliable measure of remaining cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the invention, reference will now be made to the accompanying drawings, showing by way of illustration a preferred embodiment thereof, and in which: Figure 1 represents the sequences of the nucleotides used as primers in PCR reactions (nucleotides in brackets are understood to be used in the alternative).

Figure 2 represents the sequence of the 48-127 light chain variable region.

Figure 3 represents the sequence of the 48-127 heavy chain variable region.

Figure 4 represents the sequence of the T16 light chain variable region. Figure 5 represents the sequence of the T16 heavy chain variable region.

Figure 6 represents the use of various oligonucleotides for the construction of scFv molecules.

Figure 7 represents the sequence of the 48-127 scFv.

Figure 8 represents the sequence of the T16 scFv. Figure 9 shows the recognition of gplg by 48-127 and T16 scFvs. Plates were coated with gplg. 48-127 and T16 whole antibodies, 48-127 (GPFv48) and

T16(GPFv16) scFvs, and a control monoclonal antibody (NUH82 were added to the plate and allowed to bind to gplg. IN the case of scFvs, anti-FLAGM2

(Kodak) was added. Complexes were revealed using an anti-murine Ig coupled to horseradish peroxidase (GAM-HRP). As a control, anti-FLAGM2 and GAM-

HRP were used in the absence of antibodies or scFvs.

Figures 10 a, b, c and d show the reaction between T24, a gp54 expressing cell line and 48-127 antibody and its scFv. The immunofluorescence pattern is similar whether using the antibody (Figure 10a) or scFv (Figure 10c). EFFRON, a human cell line negative for the expression of gp54, was not recognized by either antibody (Figure 10b) or scFv (Figure 10d).

Figures 11 a, b, c and d show the reaction between T24, a gp54 expressing cell line and T16 antibody and its scFv. The immunofluorescence pattern is similar whether using the antibody (Figure 11a) or scFv (Figure 11c). EFFRON, a human cell line negative for the expression of gp54, was not recognized by either antibody (Figure 11 b) or scFv (Figure 11d).

Other objects, advantages and features of the present invention will become more apparent upon reading of the following non-restrictive description of preferred embodiments with reference to the accompanying drawing which is exemplary and should not be interpreted as limiting the scope of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT Obtention of the antibody 48-127 The monoclonal antibody 48-127 has been obtained by immunizing a female

BALB/c mice with a sequential administration spaced by two to four week intervals of, membranes of SW-780 cells (a low grade bladder tumor cells), superficial bladder tumor cells, and membranes of MGH-U3 cells (an other low grade tumor bladder cell line). The mice were previously submitted to an injection of normal kidney cells followed three days later by an injection of cyclophosphamide. One mouse was killed three days after the last injection and the cloned cells were fused to mice myeloma cells SP2/0-Ag14. The obtained hybridomas have been selectioned upon the reactivity with MGH-U3, SW-780 and normal kidney cells (indirect immunofluorescence). The selected clones have been then characterized upon the reactivity with a variety of cultured cells (immunofluorescence) and by their coloration on normal tissues (immunoperoxidase). Monoclonal antibody 48-127 is an immunoglobulin lgG1 in an Ouchterlony diffusion test. In immunofluorescence tests, it reacts with a majority of human bladder cell lines and with breast carcinoma cell lines, these cells being fixed or not. In normal tissues as well as in normal urothelium, 48-127 reacts with epidermial and with distal and collecting tubules of kidney. It also reacts with a normal human kidney cells in culture (3 primary cultures/5), but it does not react with cultured fibroblasts. In a capturing test with T24 cells (kidney tumor cells), we have demonstrated the co-existence of epitopes T16 and 48-127 on the same antigen. However, these two antibodies are not competing in RIA on T24 line ceils. Analysis in flux cytometry shows an increased expression of T16 and 48-127 on confluent cells, when compared to exponentially growing cells. 48-127 does not react in Western blots and therefore does not seem to recognize a denatured antigen. Preliminary results suggest that the epitope 48-127 resists to neuraminidase and would therefore not be sialated. Monoclonal antibody 48-127 has been produced in large quantities by culturing the hybridoma in a mini flow-path model FP400-30 Amicon bioreactor. The quality of the antibody has been controlled by evaluating the proportion of active antibody, by HPLC and immunofluorescence. In order to evaluate the active proportion of the antibody, increasing quantities of T24 cells and Effron cells (melanoma cell line negative for 48-127) were intubated 30 minutes at room temperature in a 1/10000 dilution of antibody produced in the bioreactor. After centrifuging, the quantity of residual immunoglobulins in the supernatant was measured by Elisa. The produced immunoglobulins seem totally precipitable with T24 but does not react significantly with Effron cells. The chromatography has indicated the presence of contaminants of a molecular weight inferior to 10000. For removing them, the antibody was dialyzed against PBS or a distilled water through a dialysis membrane with an exclusion limit of 12-14000 Daltons. After dialysis, the protein concentration was measured by absorbance at 280 nm. This concentration was equal to the concentration of immunoglobulin measured by Elisa while, before dialysis, it was two to three times superior, which indicates an important enrichment of antibody. After dialysis, the activity of the antibody was measured in immunofluorescence test on T24 cells fixed and non fixed: serial dilutions of non dialysed 48-127, of dialysed 48-127 against PBS or water, or of 48-127 purified from ascites (caprylic acid) demonstrated similar levels of activity. However, about half the antibody precipitates during dialysis against water. The antibody 48-127 produced by a bioreactor is practically 100% active and can be rid of contaminants of low molecular weight by dialysis against PBS. Reactivity against αp54 48-127 antibody reacts with the gp54 antigen purified by affinity chromatography column made of T16 monoclonal antibody.

In competition tests, both T16 and 48-127 antibodies are capable of capturing the same molecule, but they react on different epitopes thereof. It has been shown that antibodies against gp54 antigen are internalized upon reaction with the antigen born at the surface of a tumorous cells (T24 cells). Internalization occurs with monoclonal antibodies coupled to a fluorescein, colloidal gold as well as a toxin. 48-127 and T16 monoclonal antibodies: sequencing of their variable regions

RNA from 48-127 and T16 monoclonal antibody-producing cells lines grown in cell culture was prepared using standard molecular biology protocols (Ausubel et al.). Variable regions were directly amplified from total RNA by RT-PCR both for the heavy (VH) and light (VL) chain immunoglobulin genes using VIN61 (5') and VIN62 (3') for VH and Roy68 (5') and VIN60 (3') for the light chain (Figure 1). To confirm the validity of the sequence and to discard any variations introduced during PCR, at least two amplification reactions were performed for each chain. Amplified fragments were purified and cloned into pBluescript (Stratagene). Their sequence was determined using the Prism Dye Deoxy with Taq FS kit (ABI PRISM) on an Applied Biosystems 373 DNA Sequencer. Sequences are presented in Figures 2 to 5. Construction of 48-127 and T16 scFvs

Single chain Fvs (scFvs) are polypeptide combining in a single molecule the variable regions of the heavy and light chains of a given antibody, linked together by a short linker. These recombinant molecules retain from the parental antibody the ability of recognizing a specific antigen. 48-127 and T16 scFvs were constructed by PCR-mediated ligation as described previously. The Vhsense, Jhser, OC-3 and OC-4 oligos (Figure 1) were used for the scFvs construction (Figure 6). Amplified fragments were cloned into the pFLAG-CMV1 vector (Kodak). This vector allows the addition of a short sequence at the 5' end of the molecule. This sequence (FLAG) codes for a peptide that can be specifically recognized by a commercially available antibody. In addition, the vector contains all the sequences essential for the expression and secretion of scFvs in eukaryotic cells. Recombinant vectors were transfected into COS-7 cells using lipofectin (Gibco/BRL) and production of scFvs were analyzed by Western blot analysis of culture supernatants using anti-flag M1 and M2 antibodies. Recombinant genes, capable of directing the secretion of scFvs, were sequenced as described above. Not every construct will direct the expression of scFvs. Degenerate primers used for PCR will lead to a variation in the 5' end of the heavy and light chain variable regions. Some amino acids will prevent the correct folding of the scFvs and will block their secretion. So some scFvs will be secreted and others not. This explains why those genes that have been sequenced come from secreting clones. Sequences are presented in Figures 7 and 8. Other constructs

The reverse forms VL-linker-VH have also been obtained. Specificity of 48-127 and T16 scFvs

Specificity of scFvs were determined using two different approaches. 1. Recognition of the GP-lg recombinant molecule

48-127 and T16 recognize two different epitopes on gp54. Gp54 is identical to Trop2 (also called GA733-1 , M1S1, EGP-1). A soluble molecule (GP-lg) consisting of the extracellular portion of gp54 was linked to the constant region of a human immunoglobulin gene. The recombinant molecule was then expressed in SP2/0, a murine cell line. GP-lg was then used to coat test plates. Both 48-127 and T16 recognize GP-lg in an ELISA based assay (Figure 9). When 48-127 and T16 scFvs were used in the same assay, both of them were shown to recognize GP-lg (Figure 9). ?. Recognition of T24. a gp54-expressing cell line Both 48-127 and T16 antibodies can specifically recognize T24, a gp54 expressing cell line (Figures 10 and 11, respectively).48-127 and T16 scFvs (Figures 10c and 11c, respectively) were shown to recognize T24 by immunofluorescence, similarly to their parental antibody (Figure 10a and 11a, respectively). In addition, EFFRON, a human cell line negative for the expression of gp54, was not recognized neither by 48-127 and T16 antibodies (Figures 10b and 11 b, respectively, nor by their respective scFvs (Figures 10d and 11d). Diagnostic and therapeutic methods and compositions

Having now shown that the scFvs recognize a gp54-expressing cell line, diagnostic and therapeutic methods and compositions are readily contemplated. They would make use or comprise T16 scFv or 48-127scFv or a mixture thereof. After binding to gp54-expressing cells, the so formed immune complexes should be internalized. Affinity of 48-127 and T16 scFvs

Affinity of 48-127 and T16 scFvs towards gp54 is currently being analyzed by plasmon surface resonance (BIAcore) and compared to the parental antibodies using GP-lg as a ligand. Modification of 48-127 and T16 scFvs

DNA of the heavy and light chain variable regions of 48-127 and T16 antibodies being isolated, genetic engineering of these molecules can be performed using various molecular biology approaches. This can lead to the creation of new variants with increased affinities and altered avidities. Mutations can be inserted into scFvs and molecules with increased affinity can be further selected through the phage display technology or the equivalent. As an example, hybrid molecules composed of 48-127 and T16 can also be made by linking DNA of both scFvs, generating a new product capable of recognizing different parts of gp54. Production of 48-127 and T16 scFvs

ScFvs can be easily produced in large amount in bacterial expression systems. This greatly reduces the cost of production. Attachment of 48-127 and T16 scFvs

Modification of the sequence of 48-127 and T16 scFvs through genetic engineering can facilitate the binding to various matrices, including drug delivery systems such as nanoerythrosomes. This will target specific attachment of the matrices to certain cells. In addition, it will help capturing cells expressing gp54 for improved diagnostic of bladder cancer directly from urine samples. Immunoscintigraphy

ScFvs possess many advantages over whole antibody for immunoscintigraphy. These include rapid uptake, improved penetration, faster clearance and high tumor to tissue ratio. In addition, modification of their sequences trough genetic engineering allows attachment of alternative isotopes for an improved detection of cancer cells (Verhaar et al., 1996). Immunotherapy

Through genetic engineering, scFvs can be combined to immune modulators molecules such as 11-12 or GM-CSF to mobilize the action of the immune system to specific cell types. Alternatively, scFvs can be linked to toxins, to induce specific killing of cancer cells.

Although the present invention has been described hereinabove by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A recombinant antibody molecule having the amino acid sequence defined in Figure 2 or a functional variant thereof.

2. A recombinant antibody molecule having the amino acid sequence defined in Figure 3 or a functional variant thereof.

3. A recombinant antibody molecule having the amino acid sequence defined in Figure 4 or a functional variant thereof.

4. A recombinant antibody molecule having the amino acid sequence defined in Figure 5 or a functional variant thereof.

5. A recombinant antibody molecule having the amino acid sequence defined in Figure 7 or a functional variant thereof.

6. A recombinant antibody molecule having the amino acid sequence defined in Figure 8 or a functional variant thereof.

7. An isolated nucleic acid encoding the molecule of claim 1.

8. An isolated nucleic acid encoding the molecule of claim 2.

9. An isolated nucleic acid encoding the molecule of claim 3.

10. An isolated nucleic acid encoding the molecule of claim 4.

11 An isolated nucleic acid encoding the molecule of claim 5.

12. An isolated nucleic acid encoding the molecule of claim 6.

13. An isolated nucleic acid as defined in claim 7, having the nucleic acid sequence shown in Figure 2 or hybridizing to said nucleic acid sequence.

14. An isolated nucleic acid as defined in claim 8, having the nucleic acid sequence shown in Figure 3 or hybridizing to said nucleic acid sequence.

15. An isolated nucleic acid as defined in claim 9, having the nucleic acid sequence shown in Figure 4 or hybridizing to said nucleic acid sequence.

16. An isolated nucleic acid as defined in claim 10, having the nucleic acid sequence shown in Figure 5 or hybridizing to said nucleic acid sequence.

17. An isolated nucleic acid as defined in claim 11 , having the nucleic acid sequence shown in Figure 7 or hybridizing to said nucleic acid sequence.

18. An isolated nucleic acid as defined in claim 12, having the nucleic acid sequence shown in Figure 8 or hybridizing to said nucleic acid sequence.

19. A recombinant expression vector comprising the isolated nucleic acid of any one of claims 7 to 18.

20. A recombinant host transformed with the recombinant vector of claim 19.

21. A method for producing the molecule of any one of claim 1 to 6 which comprises the step of culturing the recombinant host of claim 20 in a culture medium capable of supporting growth of said recombinant host and the expression of said nucleic acid, and recovering said molecule from the culture medium or from said recombinant host.

22. A derivative of the molecule of claim 5 or 6, said derivative being a labelled derivative.

23. A derivative of the molecule of claim 5 or 6, said derivative being a cytotoxic conjugated derivative.

24. A diagnostic composition comprising labelled derivatives as defined in claim 22, or a combination of both labelled derivatives.

25. A therapeutic composition comprising cytotoxic conjugated derivatives as defined in claim 23, or a combination of both cytotoxic conjugated derivatives.