WO1994014847A1

WO1994014847A1 - Genetically engineered immunoglobulins

Info

Publication number: WO1994014847A1
Application number: PCT/US1993/012339
Authority: WO
Inventors: Maurizio Zanetti; Rosario Billetta
Original assignee: The Regents Of The University Of California
Priority date: 1992-12-24
Filing date: 1993-12-17
Publication date: 1994-07-07

Abstract

This invention relates to the introduction of oligopeptide epitopes of biological receptor, preferably herein the CD4 receptor, for expressing within the three dimensional fold of an immunoglobulin (Ig) molecule, thus creating molecules useful to induce specific, biologically active anti-receptor immunity.

Description

GENETICALLY ENGINEERED IMMUNOGLOBULINS

Cross Reference to Related Applications

This is a continuing application of applications U.S. Serial No. 07/947415 and U.S. Serial No. 07/947521, both filed 18 September 1992, each being a continuation application of application U.S. Serial No. 07/316144, filed 24 February 1989.

Field of the Invention

The present invention may utilize in its preferred embodiments, the use of recombinant DNA technology to genetically engineer natural or synthetically-derived immunoglobulin molecules, imparting therein novel epitopes, so as to create novel entities that can be employed in vitro and in vivo in a variety of means, such as to immunize against pathogens, and for example, build tolerance to antigens.

In preferred embodiments, the epitopes are inserted into the so-called heavy or light chain variable domain of a given immunoglobulin molecule. Thus, known recombinant DNA technologies come to bear in the present invention, helping create novel immunoglobulin entities that retain functionality by localizing to particular cell types mechanistically via the so-called constant domains but otherwise functionally exploited to provide a novel localization of a particular antigenic determinant or epitope.

Background of the Invention

Recombinant DNA technology has reached the point currently of being capable, in principle, of providing the methodology sufficient to identify, isolate and characterize DNA sequences, configure them for insertion into operative expression vectors and transfect those vectors variously into recombinant hosts such that those hosts are harnessed in their ability to produce the polypeptide encoded by the DNA sequence. Obviously, many variations attend the methodology associated with recombinant DNA technology, and particular means are not without inventive faculty. Nonetheless, methods are generally known in the published literature enabling requisite mental equipment for the art skilled to practice recombinant DNA technology in the production of polypeptides from a given recombinant host system.

Immunoglobulins (Igs) are the main effectors of humoral immunity, a property linked with their ability to bind antigens of various types. In view of the myriad numbers of antigens to a particular host organism, it can be appreciated that there are a like number or more of immunoglobulins that contain antigenic determinants or epitopes against particular such antigens.

Immunoglobulin molecules are unique in their functionality of being capable of localizing to certain cell types, probably by means of mutual recognition of certain receptors that are located on the cell membrane. Immunoglobulins demonstrate a second general property whereby they act as endogenous modulators of the immune response. Igs and their idiotypic determinants have been used to immunize at the B- and/or T-cell level against a variety of exogenous antigens. In many cases, the immunity they evoke is comparable with that induced by the antigen itself. Although the principle underlying this phenomenon is understood, little is known about the molecular basis and the minimal structural requirements for the immunogenicity of Igs molecules and the interaction between those regions which may be responsible for such immunogenicity and the regions that are thought to provide the localization of a given immunoglobulin molecule with a particular cell/receptor type.

In the last many years, much progress has been made in endeavors to understand the immunogenic properties, structure and genetics of immunoglobulins. See Jeske, et al . , Fundamental Immunology. Paul, ed. , Raven Press, New York (1984) , p 131 and Rabat, Journal Immunology 141. 525 (1988).

Initially, the antigenicity of the so-called variable (V) domain of antibodies was demonstrated. Oudin, et al . , Academy of Sciences D 257. 805 (1963) and Kunkel, et al . , Science 140. 1218 (1963) . Subsequently, further research pointed out the existence of discrete areas of variability within V regions and introduced the notion of hypervariable (HV) or complementarity-determining regions (CDR) . u, et al . , J. Exp. Med. 132. 211 (1970) . Many studies since have indicated that the immunogenic property of Ig molecules is determined presumably primarily by amino acid sequence contained in the CDRs. Davie, et al . , Ann. Rev. Immuno1. 4_., 147 (1986) .

The basic immunoglobulin or antibody structural unit is well understood. The molecule consists of heavy and light chains held together covalently through disulfide bonds. The heavy chains are also covalently linked in a base portion via disulfide bonds and this portion is often referred to as the so-called constant region which is thought responsible for a given immunoglobulin molecule being mutually recognizable with certain sequences found at the surface of particular cells. There are five known major classes of constant regions which determine the class of the immunoglobulin molecule and are referred to as IgG, IgM, IgA, IgD and IgE. The N-terminal regions of the so-called heavy chains branch outwardly in a pictorial sense so as to give an overall Y-shaped structure. The light chains covalently bind to the Y branches of the two heavy chains. In the regions of the Y branches of the heavy chains lies a domain of approximately 100 amino acids in length which is variable, and therefore, specific for particular antigenic epitopes incidental to that particular immunoglobulin molecule.

It is to the Y branches containing the variable domains harboring the antigenic epitopes to which the particular attention is directed as a predicate of the present invention.

Prior researchers have studied and manipulated entire CDRs of immunoglobulins, producing chimeric molecules that have reported functionality. Exemplary attention is directed to Jones, et al . , Nature 321. 522 (1986) reporting on a V- region mouse-human chimeric immunoglobulin molecule. This research thus amounted to a substantially entire CDR replacement as apparently does the research reported by Verhoeyen, et al . , Science 239. 1534 (1988); Riechmann, et al . , Nature 332. 323 (1988); and by Morrison, Science 229. 1202 (1985) . See also European Patent Application Publication No. 125023A, published 14 November 1984.

Bolstered by the successful research summarized above that resulted presumably in functional chimeric molecules, the goal of the present research was to explore further the variable region contained in the N-terminus Y branches. It was a goal of the present research to manipulate these variable regions by introduction or substitution of novel determinants or epitopes so as to create novel immunoglobulin molecules that would possibly retain the localization functionality and yet contain functional heterologous epitopes. In this manner, the novel immunoglobulin molecules hereof could be employed for use within the organism at foreign sites, thereby imparting immunity characteristics in a novel site-directed manner.

A problem facing the present researchers at that time lay in the fact that epitopes are found in a region of the Y branch. Therefore, it was difficult to envision whether any manipulation of the variable region would be possible without disrupting the interaction of heavy chain with the corresponding light chain, and if that proved inconsequential, whether the resultant molecule would retain its functionality, with respect to the novel epitope, in combination with the constant region of the basic immunoglobulin molecule. Thus, even hurdling the problem of where to experiment, it was not possible to predict whether one could successfully produce such novel, bifunctional immunoglobulin molecules.

The present research and invention are based upon the successful threshold experiment, producing model, novel immunoglobulin molecules found to be fully functional by virtue of their ability to localize on certain cell/receptor sites and elicit reactivity to the antigens specific for the introduced novel antigenic determinant or epitope.

Summary of the Invention

The present invention is based upon the successful production of novel immunoglobulin molecules having introduced into the N-terminus variable region thereof a novel epitope not ordinarily found in the immunoglobulin molecule used as a starting molecule.

Expression of oligopeptide epitopes in the hypervariable loops of an antibody molecule, antigenization of antibody, is an efficient procedure for stabilizing oligopeptides within a limited spectrum of tertiary structures. As a result, peptides acquire an ordered conformation, and antigenized antibodies (^Ab) can serve as useful mimics of antigens and ligands.

More particularly, this invention relates to the introduction of oligopeptide epitopes of biological receptor, preferably herein the CD4 receptor, for expression within the three-dimensional fold of an immunoglobulin (Ig) molecule, thus creating molecules useful to induce specific, biologically active anti- receptor immunity.

The present invention is thus directed to novel immunoglobulin molecules having at least one novel heterologous epitope contained within the N-terminus variable domain thereof, said novel immunoglobulin molecule having retained functionality with respect to its C-terminus constant domain of the heavy chain specific for a particular cell/receptor type, and having novel, specific epitope in vitro and in vivo reactivity.

The present invention is further directed to pharmaceutical compositions containing, as essential pharmaceutical principle, a novel immunoglobulin hereof, particularly those in the form of an administrable pharmaceutical vaccine.

The present invention is further directed to methods useful for building tolerance to certain antigens, including those associated with autoimmune diseases, or for down-regulating hypersensitivity to allergens, or for providing active or passive immunity against certain pathogenic antigens, by administering to an individual in perceived need of such, a novel immunoglobulin molecule as defined above. The present invention is further directed to novel recombinant means and methods useful for preparing, identifying and using the novel immunoglobulin molecules hereof including DNA isolates encoding them, vectors operatively harboring such DNA, hosts transfected with such vectors, cultures containing such growing hosts and the methods useful for preparing all of the above recombinant aspects.

The new approach to generate protective immunity, antigenized antibodies, aims at interfering with the mechanisms of virus binding and cell adhesion. ^Abs is a new way to express im unologically-relevant oligopeptides based on the exploitation of hypervariable (HV) loops as a privileged site for the maintenance of conformation and three-dimensionality. HV loops are convex sites with the ability to provide contact bonds of the ionic, hydrogen, and van der aals types for receptors, ligands and antibodies.

The use of ^Abs is an excellent vehicle for immunization, possibly due to its intrinsic ability to conserve and/or confer tertiary structure to oligopeptides, an important feature to antigenicity and immunogenicity.

Among the advantages over conventional approaches, ^Abs combines molecular specificity (amino acid sequence) with three-dimensionality (the Ig-fold)-characteristics of key importance to antigenicity and immunogenicity - and the ability to target the target antigen presenting cells directly (APCs) via the Fc portion of the molecule. In our hands, ^Abs elicits immunity against the native antigen even across the boundaries of major histoco patibility complex (MHC) restriction.

It should be pointed out that although the emphasis of this application will be on active immunization, monoclonal antibodies of novel specificity and exquisite biological function that may be generated in the course of these studies may be considered for passive therapy as well to complement active immunization.

Detailed Description of the Invention

The present invention is described herein with particular detail for the preparation of model, novel immunoglobulin entities. This description is provided, as it was conducted, using recombinant DNA technology. Further detail herein defines methods by which one can test a given immunoglobulin to assure that it exhibits requisite functionality common to its starting material immunoglobulin and specially as to its novel epitopic antigenic activity. Given this information with respect to the particular novel immunoglobulin molecules described herein, coupled with general procedures and techniques known in the art, the art skilled will well enough know how to configure recombinant expression vectors for the preparation of other novel immunoglobulin molecules falling within the general scope hereof for use as herein described. Thus, having described the threshold experiment of the successful preparation of a novel immunoglobulin molecule, one skilled in the art need not follow the exact details used for reproducing the invention. Instead, the art skilled may borrow from the extant, relevant art, known techniques for the preparation of still other novel immunoglobulin molecules falling within the general scope hereof.

1. Figure Legends

Figure 1 is a diagram illustrating the construction of the pNylNANP expression vector.

Figure 2 is an SDS-PAGE of the ylNANP and T recombinant Ig. Figure 3 shows the binding of ¹²⁵I-labelled monoclonal antibody Sp-3-B4 to engineered antibody ylNANP.

Figure 4 is a Western blot binding of ¹²⁵I-labelled antibody Sp3-B4 to engineered antibody ylNANP and localization of the engineered (NANP)₃ epitope in the H chain.

Figure 5 shows results of cross-inhibition of ^12SI-labelled antibody Sp3-B4 binding to synthetic peptide (NANP)₃ (panel A) or engineered antibody ylNANP (panel B) by ylNANP Ig or peptide (NANP)₃.

Figure 6 is a stereo drawing of the a carbon backbone of the first (VI) extracellular, domain of human CD4 (residues 1-98) from the x-ray crystal analysis.

Figure 7 depicts structure and epitope expression of ^Abs expressing oligopeptides of human CD4 and reactivity with an anti-CD4 antibody.

Figure 8 shows C57BL/6 mice immunized with lCD4^B (lOOμg/injection) intraperitoneally at monthly intervals first in complete Freund's adjuvant and subsequently in incomplete adjuvant. Hybridomas were prepared from a mouse with the highest titer of inhibitory antibodies by fusing with Sp2/0 myeloma cells. Syncytia assay conditions and percent inhibition were described in the text.

Figure 9 provides an analysis of AbPLl. Upper panel: Dose-response inhibition of syncytia formation. Lower panel: FACS analysis and surface staining of CD4 on CEM cells.

Figure 10 is a Western Blot on recombinant (7)CD4. 2 μg of soluble rCD4 were loaded onto a 10% SDS/PAGE gel, electrophoresed and transferred to PVDF membrane (Millipore) . After blotting, the membrane was blocked by soaking in 5% dry milk in PBS and incubated with lOμg/ml of each mAbPL overnight at 4 °C. Binding was revealed with a HRP-conjugated goat antibody to mouse Ig (γ-chain specific) in PBSA. The 0KT4D and a noncorrelate Ab have been used as a positive and negative control, respectively.

2. General Methods and Definitions

"Expression vector" includes vectors which are capable of expressing DNA sequences contained therein, where such sequences are operatively linked to other sequences capable of effecting their expression. It is implied, although not always explicitly stated, that these expression vectors may be replicable in the host organisms either as episomes or as an integral part of the chromosomal DNA. "Operative," or grammatical equivalents, means that the respective DNA sequences are operational, that is, work for their intended purposes. In sum, "expression vector" is given a functional definition, and any DNA sequence which is capable of effecting expression of a specified DNA sequence disposed therein is included in this term as it is applied to the specified sequence. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" referred to as circular double stranded DNA loops which, in their vector form, are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and which become known in the art subsequently hereto.

Apart from the novelty of the present invention involving the introduction of novel epitopes by means of repositioning or augmentation of a parent immunoglobulin, it will be understood that the novel immunoglobulins of the present invention may otherwise permissively differ from the parent in respect of a difference in one or more amino acids from the parent entity, insofar as such differences do not lead to a destruction in kind of the basic activity or bio-functionality of the novel entity.

"Recombinant host cells" refers to cells which have been transfected with vectors defined above.

Extrinsic, support medium is used to support the host cells and includes those known or devised media that can support the cells in a growth phase or maintain them in a viable state such that they can perform their recombinantly harnessed function. See, for example, ATCC Media Handbook. Ed. Cote et al . , American Type Culture Collection, Rockville, MD (1984) . A growth supporting medium for mammalian cells, for example, preferably contains a serum supplement such as fetal calf serum or other supplementing component commonly used to facilitate cell growth and division such as hydrolysates of animal meat or milk, tissue or organ extracts, macerated clots or their extracts, and so forth. Other suitable medium components include, for example, transferrin, insulin and various metals.

The vectors and methods disclosed herein are suitable for use in host cells over a wide range of prokaryotic and eukaryotic organisms.

"Heterologous" with reference herein to the novel epitope for a given immunoglobulin molecule refers to the presence of (at least one) such epitope in the N-terminus domain of an immunoglobulin that does not ordinarily bear that epitope(s) in its native state. Hence, that chain contains heterologous epitope sequence(s). Such heterologous epitope sequences shall include the classic antigenic epitopes as well as receptor binding domains or binding regions that function as receptor sites, such as the human CD4 binding domain for HIV, hormonal receptor binding ligandε, retinoid receptor binding ligands and ligands or receptors that mediate cell adhesion.

"Chimeric" refers to immunoglobulins hereof, bearing the heterologous epitope(s), that otherwise may be composed of parts taken from immunoglobulins of more than one species. Hence, a chimeric starting immunoglobulin hereof may have a hybrid heavy chain made up of parts taken from corresponding human and non-human immunoglobulins.

In addition to the above discussion and the various references to existing literature teachings, reference is made to standard textbooks of molecular biology that contain definitions and methods and means for carrying out basic techniques encompassed by the present invention.

See, for example, Maniatis, et al . , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1982 and the various references cited therein, and in particular, Colowick et al . , Methods in Enzvmology Vol 152. Academic Press, Inc. (1987) . All of the herein cited publications are by this reference hereby expressly incorporated herein.

The foregoing description and following experimental details set forth the methodology employed initially by the present researchers in identifying and characterizing and preparing particular immunoglobulins. The art skilled will recognize that by supplying the present information including the wherewithal of the location and makeup of the epitope containing domain of a given immunoglobulin, and how it can be manipulated to produce the novel immunoglobulins hereof. Therefore, it may not be necessary to repeat these details in all respects in their endeavors to reproduce this work. Instead, they may choose to employ alternative, reliable and known methods, for example, they may synthesize the underlying DNA sequences encoding a particular novel immunoglobulin hereof for deployment within similar or other suitable, operative expression vectors and culture systems. Thus, in addition to supplying details actually employed, the present disclosure serves to enable reproduction of the specific immunoglobulins disclosed and others, and fragments thereof, such as the individual chains for in vitro assembly, using means within the skill of the art having benefit of the present disclosure. All of such means are included within the enablement and scope of the present invention.

3. Description of Particularly Preferred Embodiments

Research focused on the engineering, expression and testing of antibodies containing discrete sequences of human CD4. The four regions used for "antigenization" are listed below (Table I) and are also depicted in Figure 6 in the context of three-dimensional folding of CD4. The first two sequences used correspond to the putative HIV- binding site were 42-49 and 41-55. The two new sequences correspond to amino acid residues 38-49. Among the latter, one is the wild type (WT) sequence, the other contains a mutation glutamine (Q) to alanine (A) in position 40 that according to published data (24) possesses significantly increased affinity for gpl20. In each instance, these oligopeptide sequences were inserted in the CDR3 loop of a murine heavy chain V region gene expressed along with a human γl constant region gene to yield a chimeric (mouse/human) H chain. The insertion of the CD4 peptides was confirmed by nucleotide sequence analysis. TABLE I

CD4

The engineering steps were as follows, with reference made to Figure 7. a) General structure of PN ICD4 expression vector modified to encode peptides 42-49^<Λ) or 41-55^(li) of human CD4. The CDR3 region of the host V_H (KAYSHG; residues 93-98) was utagenized to introduce a single Kpnl/Asp718 site and yield the intermediate sequence KVPYSHG; residues 93-99. Here the amino acid 94A was deleted and substituted with the VP doublet which is encoded in the Asp718 cloning site. Subsequently, complementary oligonucleotides coding for peptides 42-49 or 41-55 of human CD4 sequence were introduced between 94V and 95P of the mutagenized V_H region. Only the coding oligonucleotide strands are shown here. The engineered V_HCD4^Λ or V_HCD4^IJ coded by the 2.3-kilobase (kb) EcoRI fragment was cloned upstream from a human 71 constant (C) region gene contained in the 12.8 kb vector PN7I. [Solazzo et al. , Focus 10. 64 (1988) . This is a PSV vector harboring a human 7, gene, encoded downstream from the EcoRI site. It also carries a neomycin resistance gene under the control of the SV40 promoter for the selection of stable transformant cells. See also Solazzo et al. , Eur. J. Immunol. 19. 453 (1989).]

About 30 μg of the final DNA constructs pN7lCD4^Λ or PN7ICD4¹¹ were electroporated in the murine J558L cell line (20 x 10⁶ cells) using a field strength of 750 V/cm.

Transfected cells were incubated without selection for 24 h and then selected in presence of 1.2 mg/ml G418 (GIBCO) . G418-resistant clones secreting high level of the ^Abs were identified by enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) -conjugated goat antibody to human immunoglobulin (Ig) (SIGMA) .

^Abs were first concentrated by (NH₄)₂S0₄ precipitation and then purified by affinity chro atography on a Protein A (Pharmacia-LKB, Alameda, CA) column equilibrated with 3M NaCl/lM glycine, pH 8.9. Elution was performed using Glycine 0.1M- HC1/0.5M NaCl pH 2.8. The eluted fractions were neutralized using 1M Tris-HCl pH 8.0 and dialysed against phosphate-buffered saline (PBS) . B, BamHI; RI, EcoRI; Neo, neomycin (G418) resistance; Amp, ampicillin resistance.

b) Schematic view of ^AεAbs expressing CD4 peptides. The heavy chain is the fusion product of a human 7IC region with a murine V_H region engineered to express the CD4 sequences 42-49 and 41-55. The CDR3 region of the H chain was modified by inserting between 94V and 95P the residues SFLTKGPS(upper)^(A) or GSFLTKGPSKLNDRA (lower)^(B sequences. The inserted sequences are flanked at each side by a VP doubet. The λ, light (L) chain is provided by the murine myeloma J558L used for transfection.

c) Recognition of the two ^AgAbs by OKT4D a monoclonal antibody to CD4. ^AεAbs were electrophored on a 10% Sodium dodecyl Sulfate (SDS) /Polyacrylamide Gel (PAGE) and 1.2 μg per lane were run under reducing conditions (5% β- mercaptoethanol) . Protein were blotted onto 0.45-μm Polyvinylidene difluoride paper (PVDF) (Millipore) . after blotting, the membrane was blocked by soaking in 5% dry milk in PBS and incubated for 1 hour at room temperature with OKT4D (2.5μg/ml) , Pharmaceutical Research Institute- Raritan-NJ) a murine monoclonal antibody (IgGl,k) that binds residues 44-52 of human CD4. The bound antibody was revealed by ^{l 5}I-labeled goat antibody to mouse k . PVDF paper was exposed to Kodak X-OMAT AR film for 3 days at - 70 °C. A band in correspondence of the heavy chain is visible on the ^AgAb 7lCD4^Λ and ^AgAb 7ICD4" but not in the 7IWT control.

The antigenized antibodies produced were expressed as transfectoma products obtained by introducing the appropriate plasmid vector (containing the engineered V domain) into J558L mouse myeloma cell line by electroporation. This cell line is a H chain-defective variant of myeloma J558, and carries the rearrangement for a λl light (L) chain. The overall structure of the antibodies that are obtained by this procedure is depicted in Figure 7. The supernatants of neomycin resistant colonies (stable transformants have been tested by enzy e- linked immunosorbent assay (ELISA) for immunoglobulin (Ig) production using goat antibodies to human Ig in a sandwich assay.

By this method, we selected cultures secreting >20 μg/ml. ^^sAbs have been purified from culture supernatants by affinity chromatography on Protein A-Sepharose. The purified proteins have been concentrated and analyzed by SDS-PAGE for purity using Coomassie blue staining. Verification of the expression of the CD4 inserts has been done by solid phase radioimmunoassay (RIA) and Western blot using OKT4D, a monoclonal antibody to CD4 whose recognition site is around amino acid residue 47. Table II indicates that the four ^AgAbs^cw all bound in a dose dependent manner glycosylated, recombinant gpl20 in a solid-phase assay, hence suggesting that the residues grafted into the host antibody molecule are sufficient to mediate binding. A reference HIV⁺ serum was used as a positive control. TABLE II

Antibodies Antigenized with Peptide Structures of Human CD4 Bind Recombinant gpl20 in ELISA

The first two ^Abs^CD4 were tested for their ability to induce site-specific and biologically-active antibodies to CD4. In particular, we intended to generate antibodies against the HIV interactive site of CD4 that could block syncytia formation. Precisely, we used a rapid method for the formation and analysis of syncytia in vitro . This test utilizes CD4^" 8E5 T cells (transfected with a HIV defective for reverse transcriptase) and CD4⁺ human T cells MOLT3. When these two cells are incubated for 3 hours at 37°C, syncytia form in large number and these can be quantified by visual inspection.

8E5 (1.2 10⁰/ml) and M0LT3 (2xl0⁶/ml) cells were mixed in equal volumes in a final volume of 300 μl, plated in 96- well plate and incubated at 37°C for 30 minutes. The plate was then spun for 5 minutes at 1200 rpm and incubated at 37°C for an additional 3 hours. At the end of the incubation the cells were transferred to a flat- bottom 96-well plate and the formation of syncytia recorded.

Rabbits were immunized at regular intervals for a total of 5 injections. Sera were taken 10 days after the immunizing injection. Only rabbits immunized with ^AbCD4^B (residues 41-55) produced syncytia-inhibiting antibodies (Figure 7) . By FACS, 2 out of 5 bound, to some extent, to CD4. CD4 spontaneously binds Ig of unrelated specificity, thereby making it difficult to discriminate between paratope-dependent and paratope-independent binding. See Table III.

TABLE III

NZW Rabbits

100 μg/injection x5 subcutaneously

* sera were used at the dilution of 1:500 ** Inhibition was evaluated as follows:

% of inhibition postimmune serum - % of inhibition preimmune serum

Similarly, mice were immunized using only ^AbCD4^B and obtained syncytia inhibiting antibodies in every instance. From a mouse with the highest titer (1:500) several hybridomas were generated and screened using the microsyncytia assay. Out of many positive clones, we retained four (Figure 8) . All were of the IgGl, k isotype and inhibited when diluted 1:2 with values ranging from 45 to 82 %. The 4 clones were further subcloned, purified and characterized. As an example, we have displayed the results obtained with one of them (mAbPLl) (Figure 9) . The upper panel shows a dose-dependent effect of this antibody on syncytia formation. In the lower panel, a FACS profile on CEM cells is shown using mAbPLl and by comparison Leu3a. Antibody PL1 bound with a unimodal peak, but with intensity lower than Leu3a. All antibodies also bound with CD4 in Western blot and the immunizing ^Ab (Figure 9) .

Taken together, these results indicate that ^Ab^CD4B efficiently elicited antibodies against the site of CD4 engineered into the Ig V_H domain, further indicating that the conformation retained by the CD4 I4^mcr integrated within the Ig loop is adequate for immunogenicity. Thus, this method appears to. provide an ideal solution to the limitation of immunogenicity of synthetic peptides.

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The foregoing description details specific methods that can be employed to practice the present invention. Having detailed specific methods initially used to identify, isolate, characterize, prepare and use the immunoglobulins hereof, and a further disclosure as to specific model entities, the art skilled will well enough know how to devise alternative reliable methods for arriving at the same information and for extending this information to other intraspecies and interspecies related immunoglobulins. Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope hereof; rather, the ambit of the present invention is to be governed only by the lawful construction of the appended claims.

Claims

Claims :

1. An immunoglobulin molecule containing at least one CD4 HIV binding domain within the third complementarity- determining region (CDR) in the N-terminus variable domain thereof, said immunoglobulin molecule having the effector function conferred by the constant region of the immunoglobulin, and having specific CD4 epitope reactivity.

2. An immunoglobulin according to Claim 1 wherein said binding domain is the region of a ino acid residues selected from the group consisting of 42 to 49, 41 to 55, and 38 to 49.

3. As a product of recombinant DNA technology, an immunoglobulin according to Claim 1.

4. A heavy chain of an immunoglobulin containing within the third complementarity-determining region (CDR) in the N-terminus variable domain thereof at least one CD4 HIV binding domain.

5. As a product of recombinant DNA technology, the heavy chain according to Claim 3.

6. The heavy chain according to Claim 4 in a form unassembled with its counterpart heavy chain.

7. The heavy chain according to Claim 6 in a form unassembled with its associated light chain.

8. A chimeric immunoglobulin molecule according to Claim 1.

9. The chimeric immunoglobulin molecule according to Claim 7 comprising hybrid heavy chain comprising both human and non-human sequences.

10. A pharmaceutical composition containing as an essential principle an immunoglobulin molecule according to Claim 1.

11. The composition according to Claim 10 suitable for administration to a human subject.

12. The composition according to Claim 10 in the form of an administrable vaccine.

13. A DNA molecule that is a recombinant DNA molecule or a cDNA molecule encoding an immunoglobulin molecule according to Claim 1.

14. An expression vector operatively harboring DNA encoding an immunoglobulin, defined according to Claim 13.

15. A recombinant host cell transfected with an expression vector according to Claim 14.

16. A process of preparing an immunoglobulin molecule according to Claim 1 which comprises expressing in a recombinant host cell transfecting DNA encoding said immunoglobulin molecule.