MXPA97009526A - Chemical antibodies for the administration of antigens to cells selected from the inm system - Google Patents

Abstract

The present invention relates to antibody molecules specific for the surface structures of cells that contain antigens and that have been modified to include an antigenic entity at a specific site thereof, in order to produce novel molecules of conjugated antibodies. These conjugated molecules are produced by the genetic modification of the genes encoding heavy and light chains of the antibody specific for the structure of the surface, and by the expression of mammalian cells to produce the conjugated antibody. The conjugated antibody retained the specificity for the antigen presenting cells and contained the antigenic entity. The conjugated antibody molecules deliver the antigen to the cells presenting the antigen in order to produce an improved immune response in a host immunized therewith. The conjugated antibody molecules and the nucleic acid molecules that encode them are useful as antigens and as immunogens in diagnostic and prophylactic applications

Description

CHEMICAL ANTIBODIES FOR THE ADMINISTRATION OF ANTIGENS TO SELECTED CELLS OF THE IMMUNE SYSTEM FIELD DS IA INVENTION The present invention relates to novel recombinant antibody molecules, genetically modified to contain an antigen portion for the purpose of administering the antigen portion to the cells presenting the antigen of the immune system.

BACKGROUND OF THE INVENTION Current theories of immunology suggest that, in order to provide a potent antibody response, an antigen must be seen both by B cells, which subsequently develop in the cells that produce antibodies, and also by T cells. auxiliaries, which provide the growth and differentiation signals to the antigen-specific B cells. Auxiliary T cells recognize the antigen on the surface of antigen-presenting cells (APC) in association with the gene products of the Histocopatent Class II (MHC) complex. There are significant advantages in the use of proteins and peptides derived from proteins of infectious organisms as part of the subunit vaccines. The search for these adequate subunits constitutes a very active area of both present and past research. Advances in recombinant DNA manipulation techniques, protein purification, peptide synthesis and cellular immunology have greatly assisted in this attempt. However, a major stumbling block in the use of these materials as vaccines has been the relatively poor in vivo immunogenicity of most protein and peptide subunits. In general, immune response to vaccine preparations is improved by the use of adjuvants. However, the only adjuvants currently licensed for use in humans are aluminum hydroxide and aluminum phosphate, collectively called alum, which are limited in their effectiveness as a potent adjuvant. In this way, there is a need for new adjuvants, with the desired efficiency and safety profiles. Various adjuvants, such as Freund's complete adjuvant (FCA), syntex and QS21, have been widely used in animals (ref 1 - Throughout this application, reference is made to several references in parentheses to more fully describe the state of the art to which this invention corresponds The complete bibliographic information for this situation is found at the end of the specification immediately preceding the claims The descriptions of these references are hereby incorporated by reference in the present description). In animals, the administration of peptides and protein antigens with these adjuvants has been shown to result in neutralizing antibodies against a variety of infectious organisms (references 3 to 8). A new way has been described for coupling both the B and T cell components of an immune response, which uses anti-class II (mab) monoclonal antibodies coupled to the antigens to the cells presenting the target class II carrier antigen (APC) (references from 9 to 11, also Patents North American Nos. 5,194,254 and 4,950,480, assigned to the assignee hereof). The experiments carried out in vivo in rodents and rabbits using this technology, (references 9 to 12), have demonstrated convincing evidence of improvement in the immunogenicity of antigens, in the absence of conventional adjuvants.

Several research groups have used other cell surface markers such as surface immunoglobulin (slg) (reference 13), Fc? Receptors, CD45 and MHC class I (references 14 to 17), to achieve the selection of the target to the APC; however, most of these latter studies involve in vitro experiments and the lack of data in animals. Other groups of studies report the use of antibodies of non-pertinent specificity to carry antigen epitopes (references 18 to 24). In vivo studies that use these "antigen antibodies"; however, they comprise the use of conventional adjuvants and some of them require multiple injections for the desired effect (reference 24). In previous studies using anti-class II mab as a targeting molecule (references 9 to 11), the biotin-streptavidin-based interaction was used to bind the antibody and the antigen. There are some inherent disadvantages with these chemical coupling techniques, such as productions (approximately 20%) and also the factor of variability between the different preparations. There is also inadequate control over the amounts of the coupled peptide as well as the exact location of the reaction. Additionally, purification is usually required and therefore, there can be significant losses in the material. Recently, a study reporting in vitro data using Fab-peptide class II, anti-human fusions generated by the recombinant DNA methodology has been published (reference 27). There are several differences between these fusions and the present invention since the above is a fragment of expressed monovalent protein of E. Coli of a complete immunoglobulin molecule, divalent and is also an in vitro study. Common problems encountered in bacterial expression systems include expression as inclusion bodies that require solubilization and refolding with costly product losses. Expression of the complete antibody is currently not possible in E. Coli, and therefore, the monovalent Fab may not have the affinity necessary for targeting in vivo. Thus, there are several advantages in the use of a recombinant whole IgG system as described herein. Therefore, the need remains, to produce conjugates to select antibodies and antigens of reproducible, specific structure, in high yields. These conjugated antibody molecules and the nucleic acid molecules encoding them are useful in immunogenic preparations including vaccines, for protection against disease caused by a selected pathogen and for use as reagents and diagnostic equipment and for the generation of them.

SUMMARY OF THE INVENTION The present invention includes new, conjugated, recombinant antibody molecules that have been genetically modified to contain a portion of antigen for the administration of the antigen portion to the cells that present the antigen of the immune systems. Accordingly, in one aspect of the present invention, there is provided an antibody molecule, conjugated, comprising a portion of monoclonal antibody specific for a surface structure of the cells presenting the antigen, genetically modified to contain at least a portion of antigen exclusively in at least one site selected in the monoclonal antibody. The conjugated antibody molecule is capable of distributing the antigen portion to the cells presenting the antigen of a host and is capable of producing an immune response to the antigen portion in the host. The genetic modification of the antibody portion, to contain the antigen portion only at preselected sites, ensures that a product with consistent composition and structure is obtained. The cells presenting the antigen can be any of the cells that present the antigen, suitable, of the immune system, including the cells that express the main histopathology, class I, class II (MHC), B cells, T cells or cells that present the antigen, professionals that include dendritic cells, and CD4 + cells. The at least one portion of antigen is preferably located on at least one end of at least one of the heavy and light chain of the monoclonal antibody portion, particularly the C-terminal end of both the heavy and light chain. The at least one portion of antigen binds directly preferentially to the C-terminal end of both the heavy and light chain of the monoclonal antibody portion. One characteristic of the present invention is the ability to obtain an improved immune response to an antigen without the use of an adjuvant. Accordingly, in one embodiment of the invention, the at least one antigenic portion may comprise a weakly immunogenic antigen portion, inherently. The at least one portion of antigen may comprise a plurality of antigen portions, which may be the same or different. In addition, the at least one portion of antigen can be a peptide having from 6 to 100 amino acids and containing at least one epitope. The new antibody molecules, conjugated, provided herein are produced by recombinant methods that include the provision of new nucleic acid molecules and vectors containing them. In accordance with another aspect of the present invention, there is provided a nucleic acid molecule containing a first nucleotide sequence encoding a monoclonal antibody chain specific for a surface structure of the antigen-presenting cells, selected from of the group consisting of the heavy chain and light chain of the monoclonal antibody, a second nucleotide sequence coding for at least one antigen and a third nucleotide sequence comprising a promoter for the expression of eukaryotic cells of a fusion protein comprising the monoclonal antibody chain and at least one antigen. The cells that present the antigen can be any of those described above. The first nucleotide sequence and the second nucleotide sequence are preferably linked directly in an individual transcriptional unit under the control of the promoter. The third nucleotide sequence is preferably placed at the 5 'end of the first nucleotide sequence. The present invention further includes vectors comprising the nucleic acid molecules provided herein. In a specific embodiment of this aspect of the invention, this vector can contain a first nucleic acid molecule comprising a first nucleotide sequence coding for the heavy chain of a monoclonal antibody specific for a surface structure of the cells presenting the antigen, a second nucleotide sequence coding for at least one antigen and a third nucleotide sequence comprising a promoter for the expression of eukaryotic cells of a fusion protein comprising the heavy chain of the monoclonal antibody and at least one antigen as the first transcriptional unit, and a second molecule of nucleic acid comprising a first nucleotide sequence coding for the light chain of a mo? oconal antibody specific for a surface structure of the cells presenting the antigen, a second nucleotide sequence coding for at least one antigen and a third nucleotide sequence comprising u A promoter for the expression of eukaryotic cells of the fusion protein comprising the light chain of the monoclonal antibody and at least one antigen as a second transcriptional unit. A particular vector has the characteristics of the plasmid pCMVdhfr. chLCHC (ATCC Access No. 97.202).

The production of the conjugated antibody molecule comprising a portion of monoclonal antibody specific for a surface structure of the cells presenting the antigen and at least a portion of antigen in mammalian cells constitute a further aspect of the invention. This method comprises: constructing a first nucleic acid molecule containing a first nucleotide sequence encoding a heavy chain of the monoclonal antibody and a second nucleotide sequence encoding at least one antigen, constructing a second nucleotide acid molecule which has a first nucleotide sequence encoding a light chain of the monoclonal antibody and a second nucleotide sequence encoding at least one antigen, and coexpressing the first and second nucleic acid molecules in mammalian cells to form the antibody molecule, conjugated The expression of the first and second nucleic acid molecules includes the construction of an expression vector containing the first and second nucleic acid molecules as the independent transcriptional units, which also preferably contain a promoter that can be operated on mammalian cells. to direct coexpression. Coexpression includes the secretion of the conjugated molecule and the conjugated molecules can be separated from the culture medium and purified, preferably by binding to protein A and selectively eluting the conjugated molecules. A further aspect of the invention provides an immunogenic composition comprising a conjugated antibody molecule as provided herein or a nucleic acid molecule as provided herein. The immunogenic composition is preferably formulated as a vaccine for administration in vivo to a host to produce an immune response against the disease (s) caused by a pathogen that causes at least one antigen. According to a further aspect of the invention, there is provided a method for generating an immunoresponse in a host, comprising administering thereto an effective amount of an immunogenic composition as provided herein. The new antibody molecules, conjugates provided herein are also useful in diagnostic applications. Accordingly, in yet a further aspect of the invention, there is provided a method for determining the presence of a selected antigen in a sample, comprising: (a) immunizing a host with an antibody molecule, conjugate, as provided in present, wherein the at least one portion of antigen is the antigen selected to produce the antibodies specific to the selected antigen; (b) isolating the antibodies; (c) contacting the sample with the isolated antibodies to produce complexes comprising any selected antigen in the sample and the antigen-specific antibodies, selected; and (d) determining the production of complexes. The invention further comprises diagnostic equipment for determining the presence of a selected antigen in a sample, comprising: (a) a conjugated antibody molecule as provided herein, wherein the at least one portion of antigen is the selected antigen; (b) a means for detecting the production of complexes comprising any selected antigen in the sample and the antigen-specific antibodies, selected for the selected antigen; and (c) a means for determining the production of the complexes. The invention further includes methods for producing antibodies specific for a selected antigen. This method comprises: (a) immunizing a host with an effective amount of an immunogenic composition as provided herein, wherein at least one antigen is a selected antigen to produce antibodies specific for the selected antigen; and (b) isolating host antibodies; Another method comprises: (a) administering an immunogenic composition as provided herein, wherein the at least one antigen is a selected antigen, to at least one mouse to produce at least one immunized mouse; (b) removing the B-lymphocytes from at least one immunized mouse; (c) fusing the B-lymphocytes from at least one mouse immunized with myeloma cells, thereby producing hybridomas; (d) cloning the hybridomas; (e) selecting the clones that produce the selected anti-antigen antibody, (f) culturing the clones that produce the selected anti-antigen antibody; and then (g) isolating the anti-antigen antibodies selected from the cultures.

BRIEF DESCRIPTION OF > S DRAWINGS The invention is further described (detail herein with reference to the accompanying drawings, in which: Figure IA shows the DNA sequence (SEQ ID NO: 1) and the derived amino acid sequence (SEQ ID NO: 2) of the variable region of the murine 44H104 mab light chain. The sequence of the peptide that mediates the secretion is shown in cursive writing. Figure IB shows the DNA sequence (SEQ ID NO: 3) and the derived amino acid sequence (SEQ ID NO: 4) of the variable region of the murine 44H104 mab heavy chain. The sequence of the secretory peptide that mediates the secretion is shown in cursive writing. Figure 2A shows the amino acid sequence (SEQ ID NO: 5), in an individual letter code of the peptide CTLB36, and the nucleotide sequence and coding therefor (SEQ ID NO: 6), which includes the two codons of termination. Figure 2B shows a schematic for the construction and assembly of a gene encoding CTLB36 using extension PCR by overlap. Figure 2C shows the synthetic polynucleotides CTLB 63.1, CTLB 36.2 and CTLB 36.3 and their sequences (SEQ ID NOS: 7, 8 and 9) used in the scheme of Figure 2B and the primers LC.F, HC.F and their sequences (SEQ ID NOS: 10, 11 and 12) used in the PCR reaction. Figure 3A shows a scheme for the construction of the light chain gene of 44H104 using the VL and CL DNA cartridges generated by PCR. Figure 3B shows the oligonucleotide primers Pr. 1, Pr. 2, Pr. 3 and Pr. 4 (SEQ ID NOS: 13, 14, 15 and 16) synthesized for the PCR reactions to obtain the Vtj gene cartridges and CL. Figure 4A shows a schematic for the construction of 44H104 heavy chain gene, chimeric using the CH DNA cartridges generated by PCR. Figure 4B shows the oligonucleotide primers Pr. 5, Pr. 6, Pr. 7 and Pr. 8 (SEQ ID NOS: 17, 18, 19 and 20) synthesized for PCR reactions to obtain the VH and CH gene cartridges . Figure 5 contains the structures and schemes for the construction of expression vectors based on pRc / CMV for the genes encoding chimeric light or heavy chain fusions with CLTB36. The pCMV.chLCHC plasmid is a co-linear construct, in tandem with both genes in the same sector. The plasmid pCMVdhfr.chLCHC is a co-linear plasmid with the dhfr cartridge that codes for the gene cartridge. Figure 6 shows the flow cytometry data demonstrating the binding of chimeric antibody conjugates to HUT78 cells. The conjugate is stained with the anti-human Fc specific antibody in panel A and anti-CLTB36 guinea pig serum in panel B. Figure 7 illustrates anti-CLTB36 IgG titers in macaque sera as measured by ELISA, after immunization and reinforcement with the ch-44H104-CLTB36 conjugates. Figure 8 illustrates the titers of anti-rP24 IgG in bleeding 1 and 4 of macaques immunized with the ch.44H104-CLTB36 conjugates. Figure 9 depicts SDS / PAGE gels stained with 7.5% Coomassie blue (A) and 10% (B). Gel A was run with samples in non-reducing buffer, and gel B in reducing buffer. The bands corresponding to the intact antibody (A) and the heavy and light chains (B) are marked with arrows. Figure 10 depicts Western blots that correspond to the gels stained with Coomassie blue of Figure 9. Bands corresponding to the conjugate (A) of intact antibody and light and heavy chain conjugates (B) are indicated by arrows. The primary antibody used was anti-sera from guinea pigs anti-CLTB36.

GENERAL DESCRIPTION OF THE INVENTION In the present invention, an antigen, against which it is desired to promote antibodies in a host, is generally conjugated to the C-terminus of both light and heavy chains of a monoclonal antibody, which is specific to a structure of particular surface of the cells that present the antigen. This arrangement allows the distribution of the antigen to the relevant cells in the immune system in the injection of the conjugate to a host. The monoclonal antibody, therefore, acts as a "vector" or "distribution vehicle" to select the target of antigenic determinants to cells presenting the antigen, thereby facilitating its recognition by the helper T cells. The cells that present the antigen possess a variety of cell surface structures, specific or markers that are selected as the target by any particular monoclonal antibody. In that way, the antigens can be conjugated to a monoclonal antibody specific for any of the surface structures in the cells that present the antigen, including the gene products of the major histocompatibility complex (MHC) class I and class II. The surface structures in the cells presenting the antigen of the immune system that can be recognized and targeted by the monoclonal antibody portion of the monoconjugates are numerous and the surface-specific antigen structure selected by the monoclonal antibody depends on the antibody specific monoclonal The monoclonal antibody may be specific for an MHC product, and in particular, may be specific for MHC class I molecules or for MHC class II molecules. However, the invention is not limited to these specific surface structures and conjugates having the corresponding monoclonal antibodies, but rather, as will be apparent to those skilled in the art, the invention is applicable to any other suitable surface substructure. of the cells that present the antigen that can be recognized and selected by a specific monoclonal antibody to which an immunogenic molecule is conjugated. For example, strong serological responses, independent of the adjuvant, can be obtained to an antigen administered, with conjugates formed with the specific monoclonal antibody of the dendritic cells and the monoclonal antibody specific for CD4 + cells.

In the present invention, the raonoclonal antibody specific for the target structure is provided in the form of a conjugate with an antigen against which it is desired to elicit an immune response, conveniently attached to the C-terminus of the heavy and / or light chains of the monoclonal antibody. . While the conjugated antibody molecules are illustrated by this C-terminal connection, the antigen portion can alternatively be inserted into the light and heavy chains of the antibody and these inserts can establish a repressed, particular conformation of the antigen, and in particular epitopes , within the known structural framework of an antibody molecule. These conjugated antibody molecules can conveniently be produced by genetic modification of a gene encoding the heavy and light chains of the antibody to contain a gene encoding one or more antigen (s) and co-expressing the nucleic acid molecules, resulting The invention is particularly useful for antigen molecules that normally have a weakly immunogenic response, since the response is potentiated by the present invention. The antigen molecule may be in the form of a peptide or protein, as discussed above, but is not limited to these materials. The present invention can be applied to any antigen that is desired, to select the cells that present the antigen using the monoclonal antibody. The antigen can be a protein or a peptide of 6 to 100 amino acids comprising an amino acid sequence of an epitope. Representative organisms from which antigen can be derived include influenza viruses, parainfluenza viruses, respiratory viruses, measles viruses, mumps viruses, human immunodeficiency viruses, polioviruses, rubella viruses, herpes simplex viruses, type 1 and 2, hepatitis viruses types A, B and C, yellow fever virus, smallpox virus, rabies virus, vaccinia virus, reovirus, rhinovirus, Coxsackie virus, Ecovirus, rotavirus, virus of papilloma, the paravoviruses and adenoviruses, E. coli, V. cholera, BCG, M. tuberculosis, C. diphtheria, Y. pestis, S. typhi, B. pertussis, S. aureus, S. pneumoniae, S. pyogenes , S. mutans, myoplasmas, yeasts, C. ni, meningococci (for example, N. meningitidis), Plasmodium spp. Mycobacteria spp, Shigella spp, Campylobacter spp, Proteus spp, Neisseria gonorrhea, and Haemophilus influenzae. The antigen portion can also be derived from hormones, such as human HCG hormone, and antigens associated with tumor. The present invention attempts to address some of the problems of the prior art, referred to above, by incorporating a peptide antigen at the C-terminus of the heavy and light chains * of the selection antibody by a recombinant DNA medium. The model peptide used herein is CLTB36, which is a TB tandem TB peptide found to elicit neutralizing responses in several animals (as described in copending USSN 08 / 257,528 filed June 9, 1994, assigned to the transferee). of the present and the description of which is incorporated herein by reference and which corresponds to International Publication No. WO 94/29339), although the principles of the invention are applicable to any antigen. The DNA sequence encoding this peptide is incorporated at the 3 'ends of the genes encoding an anti-human, chimeric, mouse / human class II (44H104) mab. When these genes are included in a suitable expression vector and are expressed, there is a class II / antigen, antihuman, chimeric, recombinant fusion. This can be easily purified in a single step by affinity purification with protein A or other suitable method. The present description reports the in vivo responses of macaques at a priming and reinforcement dose of the antibody / CLTB36, chimeric, anti-class II fusion generated by a recombinant medium. The genes for the fusion protein were generated by the polymerase chain reaction (PCR) using the cloned cDNA and the synthetic oligonucleotides. The antigen gene (CLTB36) was constructed using PCR by extension overlap. The genes were cloned into an expression vector, transfected into YB2 / 0 cells and gene amplification was carried out using a murine dhfr cartridge cloned in the same expression plasmid. Several clones were identified that segregate appropriate levels of the product properly folded and assembled. C-term antigen fusions of the heavy and light chain do not affect the proper assembly of the antibody (see Figure 9) that also maintains its binding specificity (see, Figure 6). As described in U.S. Patent Nos. 4,950,480 and 5,194,254, the coupling of a weak antigen to the specific monoclonal antibody results in an improvement in the immunogenicity of this antigen, while avoiding the use of adjuvants and therefore represents a more secure immunization that can use materials from which only a weak immune response is achieved. Examples of these materials are small peptides that are epitopes of larger proteins or are protein subunits of a pathogen. For use in humans, it is desirable that the antibody be modified to produce a chimeric mouse / human antibody, since. Generous, anti-human monoclonal antibody responses would be generated by the administration of a murine antibody to humans. Since the invention can be broadly applied to any of the species, it is desirable that, when administering an antibody molecule conjugated to a specific species, the sequences of the murine antibody are replaced by the corresponding sequences of the specific species in a manner analogous to that described herein for the mouse chimeric antibodies. human. The experimental data presented herein and detailed in the following examples demonstrate the ability of a mouse / human chimeric antibody, which targets the antigen-presenting cells (APCs) of the immune system via the MHC class II surface receptors. , to improve the immune response to a peptide antigen conjugated to the C-terminus of both heavy and light chains. This conjugate can conveniently be produced, as detailed in the examples, using recombinant DNA methodology, specifically by assembling the clones encoding both heavy and light chains with CLTB36 or another antigen of interest into a suitable expression vector. The pRC / CMV vector was selected as the basic expression plasmid in the experimental work performed herein, since it uses the immediately preceding CMV promoter with a powerful and broad host range to drive transcription. The final construct was designed to contain the genes of the heavy and light chains in the same vector as independent transcriptional units. The murine dhfr gene coding cartridge was also incorporated into this specific vector to provide a suitable means of gene amplification. This expression vector was subjected to electrophoresis in YB2 / 0 cells of rat myeloma. The cell lines expressing the recombinant antibody were established. Using the amplification procedure summarized in the later examples and reported in the literature (reference 32) stable cell lines that secrete viable amounts of the recombinant antibody conjugate (approximately 30 μg / ml) were established relatively quickly (in approximately 4 months). The recombinant chimeric conjugate was assembled correctly and had the same specificity as the mab 44H104 parent.

The recombinant conjugate, when administered to the macaques without an extrinsic adjuvant (eg, alum or syntex), produces good priming immunoresponses, as measured by the IgG titers to the peptide antigen in the conjugate. This response is also directed towards the native antigen as measured by the reactivity of recombinant P24. The priming response then disappears as it is reinforced in two of the three animals by another dose of the chimeric mab conjugate in PBS. The experimental data presented herein and detailed below, demonstrate the improvement of the immune response to a peptide antigen in the absence of conventional adjuvants, by coupling a chimeric anti-class II antibody conjugate that is generated by the recombinant medium. The conjugate can be obtained in large amounts by expression in cells, such as YB2 / 0 cells. It is clearly apparent to one skilled in the art that the various embodiments of the present invention have many applications in the fields of vaccination, diagnosis and treatment of diseases caused by selected pathogens. A further non-limiting discussion of these uses is further presented below: 1. Vaccine preparation and use Immunogenic compositions, suitable for use as vaccines, can be prepared from the conjugated antibody molecules as described herein. The vaccine produces an immune response in a subject that produces antibodies that includes antibodies to the anti-antigen portion. The vaccinated subject must be stimulated by a pathogen that produces the antigen portion, the antibodies bind and activate the pathogen. The immunogenic compositions that include the vaccines can be prepared as injectable products, such as liquid solutions or emulsions. The conjugated antibody molecules can be mixed with pharmaceutically acceptable excipients which are compatible therewith. These excipients may include water, saline, dextrose, glycerol, ethanol, and combinations thereof. Immunogenic compositions and vaccines should additionally contain auxiliary substances, such as wetting or emulsifying agents, or pH buffering agents. Immunogenic compositions and vaccines can be administered parenterally, by subcutaneous or intramuscular injection. Alternatively, the immunogenic compositions formed according to the present invention can be formulated and administered or distributed in a manner to cause an immune response on the mucosal surfaces. In this way, the immunogenic composition can be administered to the mucosal surface, for example, by nasal or oral (intragastric) routes. Alternatively, other modes of administration including suppositories and oral formulations may be desirable. For suppositories, binders and carriers can include, for example, polyalkylene glycols or triglycerides. Other formulations may include incipients normally employed such as, for example, pharmaceutical grades of saccharin, cellulose and magnesium carbonate. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powder and may contain about 1 to 95% of the conjugated antibody molecules. Immunogenic preparations and vaccines are administered in a compatible manner by the dose formulation, and in such amount as to be effective, protective and immunogenically therapeutically. The amount to be administered depends on the subject to be treated, including, for example, the ability of the individual's immune system to synthesize antibodies, if desired, to produce a cell-mediated immune response. The precise amounts of the active ingredient required to be administered depend on the practitioner's judgment. However, suitable dose ranges can be easily determined by one skilled in the art and can be in the order of micrograms to milligrams of the conjugated antibody molecules. Suitable regimens for initial administration and booster doses are also variable, but may include an initial administration followed by subsequent administrations. The dose may also depend on the route of administration and will vary according to the size of the host. The concentration of the antigen in an immunogenic composition according to the present invention is generally from about 1 to 95%. A vaccine containing antigenic material from only one pathogen is a monovalent vaccine. Vaccines containing antigenic material of various pathogens are combined vaccines and also correspond to the present invention. These combined vaccines contain, for example, material from various pathogens or from several strains of the same pathogen, or combinations of the various pathogens. The nucleic acid molecules encoding the conjugated antibody molecules of the present invention can also be used directly for immunization by the administration of the DNA directly, for example, by injection for genetic immunization. Processes for direct injection of DNA in test subjects for genetic immunization are described in, for example, Ulmer et al., 1993 (reference 33). 2. Immunoassays The conjugated antibody molecules of the present invention are useful as immunogens for the generation of anti-antigen portion antibodies (including monoclonal antibodies for use in immunoassays, including enzyme-linked immunosorbent assays (ELISA), RIA and other assays. binding to antibodies not linked to enzymes or methods known in the art In ELISA assays, anti-antigen portion antibodies are immobilized on a selected surface, for example, a surface capable of binding proteins such as wells on a plate polystyrene microtitre After washing to remove completely absorbed antibodies, a non-specific protein such as a solution of bovine serum albumin (BSA) which is known to be antigenically neutral with respect to the sample of The test can be attached to the selected surface.This allows you to block the nonspecific absorption rates on the mobilization surface and in this way reduces the background caused by the nonspecific bonds of the test samples on the surface. The immobilization surface is then contacted with a sample, such as clinical or biological materials, which is to be tested in a manner conducive to immunocomplex formation (antigen / antibody). This may include dilution of the sample with. diluents, such as solutions of BSA, bovine gamma-globulin (BGG) and / or phosphate buffered saline (PBS) / Tween. The sample is then allowed to incubate for 2 to 4 hours, at temperatures such as on the order of about 25 ° to 37 ° C. After incubation, the surface put in contact with the sample is washed to remove the unformed material in immunocomplex. The washing process may include washing with a solution, such as PBS / Tween or a borate buffer. After the formation of the specific immunocomplexes between the test sample and the antigen-bound, bound antibodies, and the subsequent washing, and the occurrence, and even the amount, of the immunocomplex formation can be determined.

Biological Deposits Plasmid pCMVdhfr.chLCHC containing portions coding for the conjugated antibody molecules described and referenced herein has been deposited with the American Species Crop Collection (ATCC) located at 12301 Parklawn Drive, Rockville, Maryland, USA, 20852, in accordance with the Budapest Treaty and before the filing of this application, under Accession No. 97,202 on June 23, 1995. Samples of the deposited plasmid will become available to the public in the concession of a patent based on this North American Patent Application and all restrictions on the availability of the deposit will be removed at that time. The invention described and claimed herein is not to be limited in scope by the deposited plasmid, since the deposited mode is proposed only as an illustration of the invention. Any equivalent or similar plasmids encoding similar antigens or equivalents as described in this application are within the scope of this invention.

EXAMPLES The foregoing description generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described only for purposes of illustration and are not intended to limit the scope of the invention. Changes in the form or substitution of equivalents that are considered as circumstances may be suggested or made convenient. Although specific terms have been employed herein, these terms are proposed in a descriptive sense and not for purposes of limitations. Enzymes and reagents commonly used in normal recombinant DNA technology manipulations were purchased from Boehringer Mannheim, New England Biolabs, Gibco / BRL and Pharmacia. Many specific reactions were performed using the reagent kits that were purchased from various sources indicated in the subsequent specific examples. Antibody reagents for ELISAs were purchased from Caltg unless otherwise indicated. Plasmid vectors were purchased from Gibco / BRL or Invitrogen. The polymerase chain reaction (PCR) was performed using the protocols and equipment (Gene Amp PCR System) supplied by Perkin Elmer Cetus. The thermal cycle apparatus used in the PCR reactions was purchased from Perkin Elmer Cetus. The synthesis of the oligonucleotides was carried out using an Applied Biosystems 380B DNA synthesizer. The synthesized oligonucleotides were purified in OPC cartridges supplied by Applied Biosystems following the protocols of the manufacturers. DNA sequencing was performed in an automated DNA sequencer (370A; Applied Biosystems), using the chemistry of the dideoxy terminator and the reagents supplied by the manufacturer.

Example 1 This example illustrates the synthesis of the cDNA and the determination of the sequence. The hybridoma cell line 44H104 secreting the anti-human, murine class II mab (IgG2aK) was cultured in the RPMI medium (Gibco-BRL) supplemented with glutamine (2 mM), penicillin (50 μg / ml) and streptomycin ( 50 U / ml) and containing 10% FBS. Cells were harvested (106) and the mRNA was isolated using a Fast Track mRNA isolation kit (Invitrogen). The first and second strand cDNA was prepared using the "cDNA synthesis Plus" (Amersham) kit and the protocols supplied by the manufacturer. The cDNA generated in this step was cloned into μgOlO using the "cDNA Cloning System-? GtlO" kit (Amersham) using to generate a cDNA library of lamda phage. A cDNA library from the mRNA of the cell line secreting mab 44H104 was made in the lamda phage. Phage clones containing the genes encoding the heavy and light chains were identified. The PCR reactions were also performed on the cDNA (50 ng) using the primers and the conditions used by Winter and colleagues (Reference 28). The amplified products corresponding to VL and VH, of 44H104 were labeled with P32 using the 'Random priming system I1' (New England Biolabs) and used as probes to isolate the phage clones containing the inserts encoding the genes of heavy and light chain. The inserts were excised and cloned in the multi-linker region of pUC18. These were sequenced and the nucleotide sequence of both VL and VH are shown in Figure 1 and IB respectively (SEQ ID NOS: 1 and 2). The sequences in italics in this figure are the sequences of the signal peptide that precedes the mature sequences of the heavy and light chains. The majority of normal manipulations were performed using well-described protocols (reference 29).

Example 2: This example illustrates the construction of a gene encoding the peptide antigen CTLB36. The CLTB36 antigen peptide (Figure 2A, SEQ ID NO: 5), which consists of an epitope of T and B cells, linked in tandem, derived from the sequence of the MN strain of HIV, was constructed by PCR using the overlap extension method (illustrated in Figure 2B ).

The sequence of nucleic acids encoding CLTB36 was deduced from the amino acid sequence of the peptide antigen (Figure 2A, SEQ ID NO: 6). The procedure consists of the synthesis of three oligonucleotides (CLTB36.1, CLTB36.2 and CLTB36.3, Figure 2C, SEQ ID NOS: 7, 8 and 9) that encompasses the complete gene. Oligonucleotide CLTB36.1 was designated as having 16 bases at the 3 'end, complementary (overlap) to the 5' end of CLTB32.2, which in turn has an overlap of 16 bases at its 3 'end with nucleotides of 5. corresponding to the oligonucleotide CLTB36.3. The polynucleotide primers designated as PrLC.F and PrHC.F were also synthesized; these were designated to overlap with the 5 'end of the gene encoding CLTB36 and to provide a BamHI site for incorporation into the light chain gene or a Kpn I site for fusion with the heavy chain gene (Figure 2C; SEQ ID NOS: 10 and 11). This last primer (Pr.R) is the "back" primer and has homology to the 3 'end of the CLTB36 gene and was designed to provide a Hind III site to be cloned into the expression plasmid (Figure 2C, SEQ ID NO: 12 ). Oligonucleotides CLTB36.1, CLTB36.2 and CLTB36.3 were mixed together (30 μm each) in PCR reaction buffer heated to 90 ° C and quenched slowly at about 45 ° C.

Subsequently, the volume was increased to 100 μl by suitable additions of the buffer, the dNTP primers . { PrLC.F and PrR for the light chain antigen; PrHC.F and Pr.R for the heavy chain antigen; 100 pmol each) using the material and protocols from a computer Gene Amp PCR and a PCR reaction was performed. The aqueous phase of the reaction mixture was removed to another tube and an aliquot (5 μl) was ligated into the pCRII vector and cloned using a "TA cloning" kit (Invitrogen). The insert was sequenced and the clones containing the correct sequence that can be cleaved by the correct combination of the restriction sites were established.

Example 3: This example illustrates the assembly of the gene encoding the chimeric light chain of 44H104 conjugated to mab CTLB36. The VL of 44H104 and its natural signal sequence was obtained for PCR amplification using pUCld.LC (vector pCU18 containing a light chain encoding the cDNA insert) as a template. The two primers used in the reaction (Pr 1 and 2; Figure 3B, SEQ ID NOS: 13, 14) were designed to (a) incorporate a Hind III restriction site followed by the Kozak consensus sequence (CCGCC, reference 3) at the 5 'end of the amplified product and (b) incorporate a restriction site Xho I at the junction of V and C by increasing an imperceptible mutation. PCR reactions were carried out using 50 ng of the template, 1.00 pmol of each of the primers in a volume of 100 μl using. buffers, dNTP and the enzyme supplied in the GeneAmp equipment. The parameters of the cycle were: 95 ° C for 1 minute, 55 ° C for 1 minute followed by 72 ° C for 2 minutes, for a total of 25 cycles. An aqueous aliquot of the final reaction mixture was analyzed on a 10% agarose gel and another aliquot (5 μl) was ligated into the pCR II vector supplied in a "TA cloning" kit (Invitrogen). The ligation reaction was used to transform the competent E. coli cells placed on X-Gal agar plates containing ampicillin. The plasmid was isolated from several colonies that have a white phenotype and was sequenced. Approximately one in three clones that have the correct sequence were found. The human light chain constant gene (Kappa) required for the chimeric 44H104 light chain coding construct was also obtained by PCR amplification. The template was a plasmid pCU19-k containing an insert that codes for the human kappa gene. The primers used in the PCR reaction (items 3 and 4, Figure 3B, SEQ ID NOS: 15 and 16) were designed to incorporate an Xho I restriction site at the 5 'end of the cartridge suitable for ligation with the gene VL obtained previously. These primers also incorporate a Ba HI site at the 3 'end to allow ligation to the antigen-CLTB36 gene. The PCR reaction was carried out in the same manner as described above for the V gene of 44H104, cloned into pCRII vector and the clones having identified and sequenced inserts. The two clones that have the correct sequence were placed separately for additional work. The PCRII vector containing the V gene insert was digested with a combination of the restriction endonucleases Hind III and Xho I and the 400 bp insert was isolated. Similarly, fragments of polynucleotides encoding the human Kappa and CLTB36 gene were excised from the cloning vectors of pCRII using digestion with combinations of Xho I / BamH I and BamH I / Hind III, respectively. All three of these fragments were mixed (10-20 ng each) were ligated into an aliquot of the pRC / CMC expression plasmid digested with Hind III (Invitrogen) using standard protocols. The ligation reaction was used to transform the competent and recombinant E. coli TG1 cells analyzed for inserts. The orientation of the insert was determined by the digestion patterns with restriction enzymes and confirmed by DNA sequencing. This plasmid was designated pCMV.chLC (Figure 5).

Example 4: This example illustrates the assembly of a gene encoding the chimeric heavy chain of mab 44H104 conjugated to CTLB36. The gene for the chimeric heavy chain conjugated to CTLB36 was constructed from gene cartridges, generated in a manner similar to that described for the light chain of Example 3. The scheme and detailed sequences of the oligonucleotide primers are shown in FIG. Figure 4. Synthetic oligonucleotide primers 5 and 6 (SEQ ID NOS: 17, 18) were used in generating the VH gene from a plasmid template (pUClβ) containing a cDNA insert encoding the heavy chain of MAB 44H104. The primers were designed to incorporate a 5 'Hind III restriction site, a kozak sequence and an imperceptible mutation at the 3 'end (V-VH junction) resulting in a Spe I site for binding to the constant domain gene. The PCR product was cloned into the pCRII vector and the integrity of the nucleotide sequence of the insert was confirmed. The human constant domain gene (C? L) was obtained by amplifying the insert encoding it in the plasmid pUC19-Gl using the PCR primers 7 and 8 (SEQ ID NOS: 19, 20). As with primers Pr. 5 and Pr. 6, the primers were designed to direct a Spe I site at 5 'for ligation to the VH gene and a fusion of the Kpn I recognition site to the antigen gene. The PCR products were cloned into pCRII as before, and the identified clones were corrected by sequencing the DNA. Gene cartridges encoding human VH, C? L and CLTB36 were obtained from the sequences inserted into the pRCII plasmid by digestion with combinations of the restriction enzymes Hind III / Spe I, Spe I / Kpn I and Kpn I / Hind III, respectively. the correct DNA fragments were isolated on agarose gels, mixed and ligated into the plasmid pRC / CMV digested with Hind III. These were used to transform the competent E. coli cells and the plasmid was isolated from the selected colonies. The plasmid was verified for the inserts that verify for the chimeric CLTB36 heavy chain conjugate. The orientation of the gene with respect to the rest of the expression plasmid was established using the restriction enzyme digestion patterns. The insert was also sequenced, the expression plasmid was designated pCMV.chHC (Figure 5).

Example 5: This example illustrates the construction of the expression plasmids. The DNA sequences encoding the CLTB36 fusions with the chimeric light and heavy chains were mounted in pRC / CMV (Invitrogen) to give the plasmids pCMV.chLC and pCMV.chHC respectively (Figure 5) as described in Examples 3 and 4. An individual expression vector containing the genes for both the heavy and light chains and different transcription units each under its own CMV promoter was constructed (the scheme is shown in Figure 5). Plasmid pCM.chHC was digested with Nru I and Dra III and a DNA fragment of 2.8 kb was isolated on a 0.8% agarose gel. The DNA fragment was turned blunt-ended following a normal protocol (reference 30) and using dNTP and DNA polymerase (Klenow). The resulting DNA fragment was then ligated into the plasmid pCMV.chLC linearized by digestion with the restriction enzyme Nru I and the resulting co-linear vector was designated pCMV.chLCHC. the orientation and general structure of the plasmid is as shown in Figure 5 and is confirmed by the digestive analysis with digestion enzymes, extensive. The expression plasmid pCMVdhfr.chLCHC was constructed by inserting a 1.9 kb Pvu II / BamH I fragment from blunt ends from the pSV2.dhfr plasmid (reference 31), in the Bgl II restriction site of the pCMV.chLCHC vector. This DNA fragment codes for a dihydrofolate reductase, murine under the control of an SV40 promoter and ending in a SV40 poly. The orientation of the insert was confirmed by restriction digestion analysis and is as shown for pCMVdhfr.chLCHC in Figure 5. The plasmid was isolated from transformed TGl cells by cleaving in cesium chloride (reference 30) and used in transfection experiments.

Example 6: This example illustrates an expression of the chimeric conjugates 44H104-CLTB36. Initial expression was attempted by co-transfecting the plasmids pCMV.chLC and pCMV.chHC prepared as described in Examples 3 and 4, into murine SP2 / 0 myeloma cells that do not secrete Ig by electroporation. The SP2 / 0 cells were cultured to the middle phase and then harvested; 1 x 10 7 cells were washed with cold PBS, centrifuged (4-5 x g, for 5 minutes) and redispersed in 0.5 ml of PBS. The plasmid DNA linearized with the Bgl II enzyme (10 μg of each plasmid) was added to the cell suspension and the mixture was incubated on ice for 10 minutes. The suspension was transferred to a 0.4 cm electroporation probe, cold and subjected to an electrical pulse at a setting of 700 V and capacitance of 25 μF in a 'Gene Pulsar * electroporation apparatus (Biorad). The mixture was further incubated on ice (5 min.) And then left in RPMI supplemented (with 10% FBS) for 48 hours. Subsequently, the cells were plated in the selective medium consisting of RPMI medium supplemented with 10% FBS and 600 μg / ml of g418. { Sigma) in 96-well plates (1 x 104 cells per well). The medium was replaced every three days and after two weeks, the wells that exhibited cell growth were verified for the secretion of recombinant antibodies in the supernatants by ELISA. Several mixtures of the wells were selected and cloned by the cloning method by dilution (Reference 30) and again verified for the secretion of CH.mad. A few selected clones were excised and stored as sources with DMSO in liquid nitrogen. The CMV.chHC expression plasmid was then used to transfect SP2 / 0 cells. The methodology of the electroporation and the establishment of the cloned cell lines secreted by the chimeric mab-CLTB36 conjugates are as described above. However, the total yield was completely low. The expression plasmid pCMVdhfr.chLCHC, prepared as described in Example 5, was transfected with rat myeloma cells YB2 / 0 (ATCC CRL 1662) following the protocols detailed by Shitara et al. (Reference 32). Essentially, YB2 / 0 cells were cultured in supplemented RPMI (containing 2 mM glutamine).; penicillin 50 U / ml and streptomycin 50 U / ml) containing 10 FBS. Aliquots of 1x101 were collected, washed in PBS and taken in 250 μl of PBS. These were mixed with the non-linearized pCMVdhfr.chLCHC plasmid (10 μg) and electroporated at 200 V, 250 μF capacitance in the Gene Pulsar electroporation apparatus (Bio Rad). Then the cells were treated exactly the same as the electroporated SP2 / 0 cells described above and after 48 hours in the non-selective medium they were plated in 10 96-well plates in RPMI supplemented having 600 μg / ml of G418. The well media exhibiting cell growth were analyzed for the recombinant antibody and the mixtures secreting the desired product were identified. Selected mixtures were transferred to 6-well plates and media was replaced with supplemented RPMI containing 10% FBS, 600 μg / ml G418 and 50 nM methotrexate (Sigma). The mixtures were adapted to methotrexate (MTX) concentration and then the level was increased to 100 nM. Subsequently, the concentration of MTX was increased to 200 nM, then 500 nM, 1000 nM and finally 1500 nM. The cells were adapted to each of these levels through the various passages and finally cloned by limiting the dilution. The various clones that secrete the recombinant products from 3 to 30 μg / ml of the spent culture medium (after purification of protein A purification) were obtained and used to obtain the chimeric mab in amounts large enough to allow the experimentation on animals. 96-well microtiter plates were coated (Maxisorp Immuno; Nuc) with a goat anti-human kappa light chain antibody fragment. Plates were washed in PBST, (PBS containing 0.05% Tween 20), blocked with 0.1% casein in PBST, incubated with aliquots (100 μl) of the culture supernatants. A human myeloma IgGlK (Pharmingen) was used as a positive control). After washing, the plates were washed with F (ab ') 2 (specific for goat anti-human IgG Fc, conjugated to alkaline phosphatase.) The unbound conjugate was washed and the substrate pNPP (Gibco / BRL) was added to the Wells in phosphatase buffer After approximately 15 minutes and color development was measured in a Dynatech MR5000 ELISA plate reader at a setting of 405-410 nm.

Example 7: This example describes the isolation and purification of the ch 44H104-CLTB36 conjugates. The clones identified as high conjugate producers of Example 6, exclusively from transfection with pCMVdhfr.chLCHC of YB2 / 0 cells and the subsequent gene amplification experiments, were scaled in supplemented RPMI containing G418 (600 μg / ml) , methotrexate (1 μM) and FBS ultra-low 10% IgG concentration (from Gibco / BRL). The cells were grown in T-shaped flasks until approximately half of them died (approximately 1 week). The culture was centrifuged and the supernatant was collected. The spent medium was stored at 4 ° C with 0.1% sodium azide to prevent microbial growth. The ch 44H104-CLTB36 conjugates in the supernatant were isolated by purification with protein A. The supernatant was passed through a Protein A-HiperD column (Sepracor). The column was washed and the bound material was eluted with 0.2 M glycine (pH 2.8); the fractions containing the bound material were neutralized in 1.0 M Tris (pH 8.0) and mixed. The fractions were dialyzed against PBS and finally concentrated in Amicon microconcentrators. The protein content of the mixed, dialyzed and concentrated material was determined using a standard protein assay kit (Biorad Laboratories). The conjugate was stored at 4 ° C in PBS. To remove any of the high molecular weight aggregates, the material purified by Protein A was further fractionated on a Sephacryl S-300 hplc column (HR, 9.5 x 90 cm). The column was equilibrated with PBS and the sample was applied in 2 ml aliquots. The column was run at a flow rate of 1 ml / min in PBS and the effluent was inspected at 280 nm. The maximum value of the empty volume (consisting of any of the aggregates) was collected separately from the maximum value corresponding to the non-aggregated material. The last fractions were mixed and concentrated using a YM-10 ultrafiltration membrane (Amicon).

Example 8: This example describes the characterization of the conjugate ch mab 44H104-CLTB36. The conjugate produced after the procedure of Example 7 was assembled as a covalently bonded dimer of the heterodimers comprised of light and heavy chains. This was demonstrated by electrophoresis in SDS / PAGE in 7.5 and 10% gels, running samples in non-reducing buffer and redoubts, respectively (see Figure 9). The presence of the CLTB36 peptide in the conjugates was determined by Western blot using the anti-CLTB36 guinea pig serum generated at home. The second antibody used in these experiments was an anti-igG guinea pig alkaline phosphatase conjugate (Jackson Laboratories) (see Figure 10). The conjugate was also analyzed for binding to class II molecules in HUT78 cells by flow cytometry using the binding of the recombinant conjugate to HUT78 cells. HUT78 cells (T cell lymphoma cells expressing human MHC class II) were cultured in supplemented RPMI containing 10% FBS. An aliquot of cells (1 x 106 cells / tube) was distributed in 15 ml conical centrifuge tubes and washed with 2 ml of binding buffer (PBS containing 0.1% BSA and 0.1% NaN3). The cells were collected after centrifugation (400 x g for 5 minutes at 4 ° C) and the pellet was redispersed in the linear buffer containing different concentrations of the recombinant antibody conjugate.

(Figure 8). The tubes were incubated on ice for 60 minutes with occasional shaking and then washed twice with a cooled wash buffer (4 ° C) (2 ml). The cells were dispersed in 100 μl of a dilution 220 of goat anti-human IgG conjugated with fluorocein isodiocyanate (Fc-specific, Sigma Chemical Co.) and further incubated on ice for 30 minutes with occasional shaking. The cells were washed in binding buffers (2 X) and subsequently once in PBS containing 0.1% sodium azide (NaN3). The cells were finally dispersed an aliquot of 1% paraformaldehyde in PBS (0.5 ml) and analyzed in an EPICA V flow cytometer (Coulter, Harpendon UK). The recombinant conjugate was also analyzed for the presence of the CLTB36 peptide by the same technique. For this analysis, the anti-human conjugate in the previous protocol was substituted with home-generated anti-CLTB guinea pig serum. This step was followed by 100 μl of the 1:50 dilution of the mouse anti-guinea pig mab of biotin-conjugated IgG2b (sigma) for 30 minutes and finally with 100 μl of a 1: 5 dilution of a streptavidin-phycoerythrin conjugate ( Becton Dickinson, 30 minutes). The cells were washed as before and fixed with 1% paraformaldehyde in PBS and analyzed on a flow cytometer. Negative controls, consisting of cells treated as described above, but without the step of incubation with the recombinant mab conjugate, were used in both assays. The results obtained are shown in Figure 6. This analysis demonstrates the surface availability of the peptide cells for antibody binding.

Example 9: This example describes the immunization of macaques with the ch 44H104-CLTB36 conjugates. The immunogen (conjugate of mab), prepared as in Example 7, was concentrated and filtered through a 0.22 μM filter. The protein concentration of this was estimated to be about 0.58 mg / ml in PBS. Three cynomologous macaques were selected and serum samples from them were screened or examined for spontaneous viral agents, such as SA8, HSV-1, HSV-2, V. Zoster, Chimp CMV, EBV, SRV-1, SRV-2, SRV-5, SIV, STLV-1 and B virus. The selected macaques (# 197, 198 and 200) were bled and injected intramuscularly with 1.5 ml of PBS (containing 800 μg of protein, equivalent to 80 μg of peptide). The program set forth in the following Table 1 was established.

TABLE 1 Serum samples from pre-bleeding and bleeding from 1 to 5 were examined for anti-CLTB36 reactivity. 96-well microtitre plates (Polystyrene, Dynatech Labs) were coated with 10 μg / ml of CLTB36 in carbonate-bicarbonate buffer (0.05 M, pH 9.6). The cells were blocked with 5% skim milk in PBS and subsequently washed in PBS-Tween 20 (0.05%). The serum samples were serially diluted (in 1% skim milk with 0.5% Tween 20) in wells and incubated at 37 ° C for 2 hours. Plates were washed and incubated in F (ab ') 2 goat anti-monkey IgG conjugated to horseradish peroxidase (Cappel Laboratories). The excess conjugate was thoroughly washed and the TMB / H202 colorimetric substrate (ADI) was added. The reaction was stopped after 5 minutes and the absorbance at 450 and 540 nm was measured in an ELISA plate reader (EL 310, Biotech Instruments). The protocol and reagents for an ELISA for the reactivity of P24 were as described for CLTB36 above; the difference being that the 96-well microtitre plates were coated with recombinant P24 (Dupont) at a concentration of 1 μg / ml and on a carbonate-bicarbonate buffer. The titers of IgG in different bleeds reactive against CLTB36 and measured by ELISA, are shown in Figure 7. As can be seen, good priming responses are produced by the recombinant conjugate targeting targets in PBS, in all three animals (up to approximately 1 in 25, 000 in an animal). The titers observed in ELISA decrease after 4 and 6 weeks and then increase again after a booster dose of the immunogen. The reinforcement in the IgG titers was especially prominent in two animals from among the three, the third for unexplained reasons was not reinforced after this promising primary response. Sera from pre-bled monkey and bleeding 1 and 4 (2 weeks after priming and 2 weeks after reinforcement, respectively) were also evaluated for IgG responses against recombinant P24 (CLTB36 has an epitope derived from this portion of the HIV protein). The titers of detectable P24 were measured in all three animals and are presented in Figure 8. A conjugate deficient in binding to class II was constructed by mutation of the CDRH3 region of the heavy chain. This conjugate was injected into three cynomolgus monkeys as described above. The sera of the animals did not have any of the CLTB36 specific antibodies, demonstrating that the binding of class II is responsible for the immunogenicity of ch 44hl04-CLTB36 by the selective distribution to the cells presenting the antigen.

SUMMARY OF THE DESCRIPTION In summary of this description, the present invention provides novel recombinantly produced molecules containing a portion of antigen and a portion of monoclonal antibody, wherein the portion of monoclonal antibody specific for a determinant expressed in cells presenting the antigen of a host, methods for assembling these molecules, nucleic acid molecules that code for these molecules, and immunization methods using these molecules, whereby an improved immunoresponse to the antigen portion is achieved in the absence of adjuvants. Modifications are possible within the scope of this invention.

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Claims (26)

1. CLAIMS: 1. An antibody molecule, conjugated, recombinant, characterized by a portion of monoclonal antibody specific for a surface structure of the cells presenting the antigen, which has a heavy and a light chain and which is genetically modified to contain the minus one portion of antigen exclusively in at least one preselected site in the heavy chain and / or light chain of the monoclonal antibody portion, whereby the conjugated antibody molecule is capable of distributing the antigen portion to the cells presenting the antigen of a host and is capable of eliciting an immune response to the antigen portion in the host. The molecule according to claim 1, characterized in that the cells presenting the antigen are selected from the group consisting of the cells expressing the main histocompatibility class I, the cells expressing the main histocompatibility class II, the dendritic cells and CD4 + cells. The molecule according to claim 1 or 2, characterized in that at least a portion of antigen is located on at least one end of at least one of the heavy and light chains of the monoclonal antibody portion. 4. The molecule according to claim 3, characterized in that at least a portion of antigen is located at the C-terminal end of at least one of the heavy and light chains of the monoclonal antibody portion. The molecule according to claim 4, characterized in that at least a portion of antigens is located at the C-terminal end of both the heavy and light chains of the monoclonal body portion. 6. The molecule according to claim 5, characterized in that at least a portion of the antigen is linked directly to the C-terminal end of both the heavy and light chains of the monoclonal antibody portion. 7. The molecule according to any of claims 1 to 6, characterized in that at least a portion of antigen is a weakly immunogenic antigen portion inherently. The molecule according to any of claims 1 to 7, characterized in that at least one portion of antigen comprises a plurality of individual or different antigen portions. 9. The molecule according to any of claims 1 to 8, characterized in that at least a portion of antigen is a peptide having from 6 to 100 amino acids and containing at least one epitope. 10. A nucleic acid molecule, characterized by a first nucleotide sequence coding for a monoclonal antibody chain specific for a surface structure of the antigen-presenting cells selected from the group consisting of heavy chain and the light chain of the monoclonal antibody, a second nucleotide sequence coding for at least one antigen and a third nucleotide sequence comprising a promoter for the expression of eukaryotic cells of a fusion protein comprising the monoclonal antibody chain and at least one antigen. 11. The nucleic acid molecule according to claim 10, characterized in that the encoded chain is the heavy chain of the monoclonal body 12. The nucleic acid molecule according to claim 10, characterized in that the encoded strand is the light chain of the monoclonal antibody. . The nucleic acid molecule according to any of claims 10 to 12, characterized in that the cells presenting the antigen are selected from the group consisting of the cells expressing major histocompatibility class I, the cells expressing major histocompatibility class II dendritic cells and CD4 + cells. 14. The nucleic acid molecule according to any of claims 10 or 13, characterized in that the first nucleotide sequence and the second nucleotide sequence are directly linked in an individual transcriptional unit under the control of a promoter. 15. The nucleic acid molecule according to claim 14, characterized in that the third nucleotide sequence is placed at the 5 * end of the first nucleotide sequence. 16. A vector characterized by the nucleic acid molecule according to any of claims 10 to 15. 17. The vector according to claim 16, which is a single co-expression vector containing a first nucleic acid molecule comprising a first sequence of nucleotides coding for the heavy chain of a monoclonal antibody specific for a surface structure of the cells presenting the antigen, a second nucleotide sequence encoding at least one antigen and a third nucleotide sequence comprising a promoter for the expression of eukaryotic cells of a fusion protein comprising the heavy chain of the monoclonal antibody and at least one antigen as a first transcriptional unit, and a second molecule and nucleic acid comprising a first sequence of nucleotides that code for the light chain of a monoclonal antibody specific for a surface structure e of the cells presenting the antigen, a second sequence of nucleotides coding for at least one antigen and a third nucleotide sequence comprising a promoter for the expression of eukaryotic cells of a fusion protein comprising the light chain of the monoclonal antibody and at least one antigen as a second monoclonal unit and at least one antigen as a second transcriptional unit. 18. The vector according to claim 17 characterized in that the characteristic properties of plasmid pCMVdhfr.chLCHC as shown in Figure 5 and having the deposit of ATCC No. 97.202. 19. A method for making a conjugated antibody molecule, comprising a portion of monoclonal antibody specific for a surface structure of cells presenting the antigen and at least a portion of antigen, characterized by: constructing a first nucleic acid molecule which contains a first. nucleotide sequence encoding a heavy chain of the monoclonal antibody and a second nucleotide sequence encoding at least one antigen, constructing a second nucleotide acid molecule containing a first nucleotide sequence encoding the light chain of the monoclonal antibody and a second nucleotide sequence encoding at least one antigen, and co-expressing the first and second nucleic acid molecules in mammalian cells to form the conjugated antibody molecule. The method according to claim 19, characterized in that the coexpression of the first and second nucleic acid molecules includes the construction of an individual expression vector containing the first and second nucleic acid molecules as independent transcriptional units. The method according to claim 20, characterized in that each independent transcriptional unit includes a promoter operable in mammalian cells to direct co-expression. 22. The method according to any of claims 19 to 21, characterized in that the expression vector has the. characteristic properties of the plasmid pCMVdhfr.chLCHC as shown in Figure 5 and having the ATCC deposit No. 97,202. 23. The method according to any of claims 19 to 22, characterized in that the coexpression includes the secretion of the conjugated antibody molecule and further separated and purifying the conjugated antibody molecule. The method according to claim 23, characterized in that the purification comprises the binding of the antibody molecule conjugated to the protein A and the selective elution of the protein conjugated antibody molecule. 25. An immunogenic composition, comprising a conjugated antibody molecule according to any one of claims 1 to 9 or a nucleic acid molecule according to any of claims 10 to 15. 26. The immunogenic composition according to claim 25 formulated as a vaccine for administration in vivo to a host to confer protection against disease caused by a pathogen that produces at least one antigen.