CA2163125A1 - Genetically engineered immunoglobulins - Google Patents

Abstract

This invention relates to the introduction of oligopeptide epitopes of influenza virus nucleoprotein for expressing within the three dimensional fold of an immunoglobulin (Ig) molecule, thus creating molecules useful to induce specific, biologically active anti-viral immunity.

Description

. .,, ~- ` t -' 21 631 25 W094t28026 ~ PCT~S94/06090 GENETICALLY ENGINEERED IMMUNOGLOBULINS

Field of the Invention We have developed a new method for the presentation of immunogenic epitopes to cytotoxic T lymphocytes (CTL). The new method is based on antibody antigenization, a process whereby one or several loops of an immunoglobulin molecule are re-engineered to encompass the sequence of selected portions of pathogens (virus and parasites), self antigens and tumor antigens.

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 ln 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.

W094/28026 ~ 2 1 ~ 3 1 2 5 ~CT~594/06090 ~

Back~round 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 lmml~nlze 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 ~ W094/28~6 ~ 2163125 PC1~594/06090 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 Immunoloqy, Paul, ed., Raven Press, New York (1984), p. 131 and Kabat, Journal Immunoloqy 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 complementarily-determining regions (CDR). Wu, 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. Immunol. 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-

2 1 6 3 1 2 5 W094/28026 PCT~S94/06090 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 o~ 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, 5cience 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 ~or use c ~ 2 1 6 3 1 2 5 W094/~8026 PCT~S94/06090 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.

Recognition of antigen peptides by T lymphocytes is restricted by the major histocompatibility complex (MHC) gene products and is mediated by the T-cell receptor (TCR) recognition structure.

CD4+ T helper lymphocytes recognize antigen peptides presented in the context o~ class I I MHC molecules (unanue, Curr. OPin. Immunol. 4:63 (1992)) while CD8+ cytotoxic T
lymphocytes (CTL) require that antigen peptides be presented by class I MHC molecules (Braciale, Curr. Opin.
Immunol. 4 :59 (1992) ) . In both instances, antigens need to be processed into small peptides, 9 residues for peptides that bind class I molecules ~Rotzschke et al., Nature 348:252 (1990)) and 13-17 residues for peptides that bind class II molecules (Chicz et al., Nature 358:764 (1992);
Rudensky et al., Nature 353:622 (1991)). Antigens presented to CD4' T cells derive primarily ~rom exogenous proteins that are processed into peptides that bind class II MHC molecules in the endocytic compartment (Unanue, Curr. Opin. Immunol. 4 :63 (1992)), albeit proteins from the cytosol can also bind class II MHC molecules (Malnati et xr~ ~ ~ 6 3 ~ 2 5 W094l28026 PCT~S9~/06090 al., Nature 357:702 (1992)). Antigens presented to CD8+ T
cells derive mostly from proteins processed in the cytosol and bind to class I MHC molecules in the endoplasmic reticulum (ER) (Braciale, Curr. Opin. Immunol. 4:59 (1992)). Prototype antigens for this type of presentation are viral proteins generated by intracellular replication of an infectious virus (Long et al., Immunol. Today 10:45 (1989)).

While cytosolic proteins are fragmented into peptides and translocated across the ER membrane by proteasomes, intracellular polypeptides that map to the class II MHC
complex (Glynne et al., Nature 353:357 (1991)), endogenous proteins destined for secretion are synthesized by ribosomes attached to the rough ER. In both instances peptides from endogenous proteins complex with class I MHC
molecules in a pre-Golgi compartment.

B lymphocytes are specialized antigen presenting cells (APC) (Lanzavecchia, Immunol. Today 10:157 (1989)) that express both class II and class I MHC molecules. As such, B cells constitutively present immunoglobulin peptides within the MHC molecules expressed at their surface in the context of class II molecules (Rudensky et al., Nature 359:429 (1992). However, there exists only indirect evidence that endogenous Ig peptides are presented in class I MHC molecules (Shinohara et al., Nature 336:481 (1988);
Weiss et al., Cell 64:767 (1991); Yamamoto et al., Eur. J.
Immunol. 17:719 (1987)) and no endogenous Ig peptide has beer isolated from B cells that mediates CTL function.

It was predicted that the translated polypeptide encoded by antigenized antibody gene would follow the secretory pathway. There is large indirect evidence in favor of this assumption. First, is the demonstration that unassembled immunoglobulin ~ chains accumulate in the ER where they undergo processing (Sitia et al., 1990). In the absence of W094/2~026 ~ .3l ~ 2 1 6 3 1 25 PCT1'594/06090 any L chain, the H chain is retained in the ER bound to the H chain-binding protein (BiP) (Bole et al., 1986). By analogy, processing of the ~2 315 L chain and generation of an idiotype peptide presented in class II molecules was shown to occur in the ER (Weiss et al., 1989). Recently, it was demonstrated that the ER possess an enzyme necessary to cleave the leader sequence from a nascent class I
molecule.

One important issue in current immunology is to be able to program the immune system towards preselected T-cell epitopes, whether these be restricted by class I or class II MHC molecules. This is significant in defensive immune responses against pathogens, and, in particular, intracellular pathogens for eliciting CTL specific for protective epitopes. A CTL response can be achieved through a number of strategies like immunization with synthetic peptides (Aichele et al., 1990), recombinant proteins (Kleid et al., 1981), vaccinia virus constructs (Mackett et al., 1985), soluble proteins osmotically vehicled to the cytosol (Carbone et al., 1990) and cells pulsed in vi tro with synthetic peptides (external loading) (De Bruijn et al., 1991). While synthetic peptides are scarcely immunogenic and vaccinia vectors have drawbacks in previously vaccinated individuals (Lane et al., 1968), external loading of peptide provides a limited availability of empty class I MHC molecules at the cell surface.

The present research and invention is 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 spec fic for the introduced novel antigenic determinant or epitope. This invention demonstrates a new method for the engineering of cellular vaccines that can be used for the in vivo or in vi tro induction of CTL.

W094/~026 ' ~ 2 1 6 3 1 2 5 PCT~S9~/06090 ~

A plasma cell can secrete about 103 molecules of Ig cell/sec, Ig can be an extraordinary source of endogenous peptides and B cells efficient APCs for presentation of peptide epitopes in the context of class I MHC molecules.
In this invention, it is demonstrated that one can use B
cells as APC to process and present a peptide from an endogenous Ig heavy (H) chain to a class I MHC restricted CTL clone specific for Ig peptide.
Sl ~y 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.

Successful model systems of the foreign molecules include the hydrophilic tetrapeptide Asn-Ala-Asn-Pro (NANP) of Plasmodium falciparum circumsporozite protein, the tripeptide Arg-Gly-Asp (RGD) involved in the interaction of a variety of adhesive proteins, and oligopeptide epitopes of the human CD4 HIV binding domain.

More particularly, this invention relates to introduction of oligopeptide epitopes of a nucleoprotein (NP) peptide of influenza virus for expression within the third fold of an immunoglobulin molecule. This virus peptide is recognized by CTL in the context of the H-2 Db allele. It is shown that the NP peptide engineered in the H chain: 1) mediates killing of B cell lymphomas by a CTL clone specific for that peptide restricted by the Db molecule, and 2) could be purified from the H-2 Db molecules at the cell surface.
This study formally demonstrates that peptides from the hypervariable loops of Ig are presented by class I MHC
molecules and validates a role for the processing and presentation of self immunoglobulin V regions to CD8+ T
cells in the regulation of the immune response.

~ W094/23026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 This invention demonstrates the possibility to program class I-restricted presentation of intracellular pathogens peptides using antigenized antibody genes as non-infectious "replicating" material .

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 aparticular 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 principal, a novel immunoglobulin hereof, particularly those in the form of an administrable pharmaceutical vaccine.

The present invention is further directed to methods useful ~or 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.

W094/28026 w . ~ 2 1 ~ 3 1 2 5 PCT~S94/06090 ~

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. Fiqure Leqends Figure 1 is a diagram illustrating the construction of the pN~lNANP expression vector.

Figure 2 is an SDS-PAGE of the ~lNANP and WT recombinant Ig.

Figure 3 shows the binding of 12;I-labelled monoclonal antibody Sp3-B4 to engineered antibody ~lNANP.

~ W094/28026 ~ y~,~s~ ~; 2 1 63 1 25 PCT~'S94/06090 1~ .
Figure 4 is a Western blot binding of 12~-labelled antibody Sp3-B4 to engineered antibody rlNANP and localization of the engineered (NANP) 3 epitope in the H chain.
.

Figure 5 shows results of cross-inhibition of l25I-labelled antibody Sp3-84 binding to synthetic peptide (NANP) (panel A) or engineered antibody ~lNANP (panel 8) by ~lNANP Ig or peptide (NANP) 3 .

Figure 6 is a diagram of pN~lNP expression vector and general strategy of transfection.

Figure 7 depicts specificity of target cell recognition by cold target competition. The inset shows the dose response of killing of B6-2 HNP transfectants. 5lCr-labeled B6-2.503 cells (2.5 x 105 cells/ml) were mixed with CTL clone 34 cells at an E:T ratio of 10:1, 1:1, 0.1:1 or 0:1. Percent cytotoxicity was calculated 4 hours later from triplicate cultures as described.

Figure 8 shows lack of interference of soluble ~lNP with external loading of peptide and lysis of target cells by a CTL clone.

Figure 9 shows inhibition of lysis of B6-2 HNPtransfectants by a monoclonal antibody to Db.

Figure 10 shows presentation of processed NP peptide by engineered cells is restricted by H-2b.

Figure 11 shows elution of influenza virus NP peptide from B6-2 HNP tranfectants. (a) HPLC profile of the synthetic peptide ASNENMETM of influenza virus (100 ~g). (b) Cytotoxic assay using single HPLC fractions from the - experiment shown in a. (c) HPLC profile of the peptide mixture eluted from the Db molecules purified from B6-2 HNP
transfectants. (d) Cytotoxic assay using single HPLC
-W094/28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 ~

fractions from the experiment shown in c.

Figure 12 shows that addition of exogenous peptide does not increase lysis of cells engineered with the HNP gene.

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 ~ W094l28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 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 m~mm~l ian 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 like binding domains or binding regions that function as receptor sites, such as the human CD4 binding domain for HIV, hormonal receptor binding ligands, retinoid receptor binding ligands and W094l28026 ~ '` ~ 2 i 6 3 i 2 5 PCT~S94/06090 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 Cloninq: A
LaboratorY Manual, Cold Spring Harbor Laboratory, New York, 1982 and the various references cited therein, and in particular, Colowick et al., Methods in Enzvmoloqy 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 methodoloqy 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 ~ W094/28026 ~ ~ 2 1 63 1 25 PCT~S94/06090 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 o~ the present disclosure. All of such means are included within the enablement and scope of the present invention.

3. DescriPtion of ParticularlY Preferred Embodiments Protein engineering was used to introduce a foreign epitope into the CDR3 of the H chain of a mouse/human chimeric antibody (C~162). This epitope consists of three copies of the tetrapeptide Asn-Ala-Asn-Pro (NANP). The tetrapeptide occurs naturally as a 37 tandem repeat in the Plasmodium falciparum circumsporozoite (CS) protein, interspersed with four repeats of the variant sequence Asn-Val-Asp-Pro [Dame et al., Science 229:593 (1984)]. In the construct described here, the epitope is flanked by Val and Pro residues at each end [VP (NANP) 3 VP] . The experiment verified that the (NANP) 3 epitope could be inserted in the NV region of a host H chain (VH) without altering the framework folding of the Ig molecule, i.e., its molecular assembly with the light (L) chain and it determined that the antigenic and immunogenic properties of the recombinant Ig molecule were expressed. It is known that the CDR3 of VH regions of antibody is often the structural correlate of an immunodominant idiotope [Davie, et al., Ann. Rev.
Immunol. 4:147 (1986)], which indicates that the CDR3 is at the surface of the molecule. Moreover, it is well established that because of recombination of the variable-diversity-joining (VDJ) regions, as well as N-addition mechanisms [Tonegawa, Nature 302:575 (1983); Miller et al., Immunol. TodaY 7:36 (1986)], the CDR3 may vary considerably in length (from 3 to 19 amino acids) [Kabat, et al., W094/28026 ~ .~ t ~ 2 ~ 63 1 25 PCT~TSg~06090 ~

Proteins of Immunoloqical Interest, U.S. Dept. of Health and Human Service NIH (1987)], implying a high degree of plasticity at the structural level. Second, the (NANP) 3 epitope selected for this study is relatively short, repetitive and of proven immunogenicity in mice and humans [Good et al., Ann. Rev. Immunol. 6:663 (1988)l.

Particularly developed is a new method for the presentation of immunogenic epitopes to cytotoxic T lymphocytes (CTL).
The new method is based on antibody antigenization, a process whereby one or several loops of an immunoglobulin molecule are re-engineered to encompass the sequence of selected portion of pathogens (virus and parasites), self antigens and tumor antigens.

Antibodies may be antigenized by inserting immunogenic epitopes in any of the three CDR regions of each heavy chain and any of the three CDR regions of the light chain.
A preferred site of engineering an immunogenic epitope is the third CDR region of the heavy chain.

An immunogenic epitope may be inserted into one or more of the 9iX CDRs, thus generating an antibody antigenized with between one and six epitopes. In a preferred embodiment, one immunogenic oligopeptide sequence is engineered within the third complementarity-determining region (CDR3) of the heavy chain of the immunoglobulin.

Immunogenic epitopes may be engineered within any or all of the CDRs by inserting a nucleic acid sequence encoding the epitope at a site unique to the CDR and absent from the nucleic acid sequence of the immunoglobulin chain wherein the epitope sequence is to be inserted. Insertion may be accomplished, for example, using a restriction enzyme capable of recognizing the unique sequence in the CDR.

- 094/2~026 ~; `~i~i`` 2 1 6 3 1 2 5 PCT~S94/06090 Mature lymphocytes of the CD8 phenotype recognize antigen in conjunction with class I MHC molecules. The best studied systems relate to CTL that recognize virally-infected cells (Long et al., Immunol. Today 10:45, 1989).
CTL's function requires active replication of the virion within the cell. Few examples do however, exist to indicate that inactivated (non-replicating) virus (Wraith et al., J. Gen. Virol. 66:1327, 1985) or soluble proteins (Moore et al., Cell 54:777 (1988); Staerz et al., Nature 329:449 (1987)) can also be presented to class I-restricted T cells provided that they reach the inside of the cell.

To further demonstrate the invention, an antibody was engineered to encompass the oligopeptide sequence ASNENMETMESSTL representing a CTL epitope of influenza virus nucleoprotein (NP) (Bastin et al., J. Exp. Med.
165:1508, 1987; Townsend et al., Cell 44:959 (1986)). This epitope has been characterized as a short nonglycosylated protein sequence which is recognized as a target by NP-specific CTL clones in a MHC-restricted way. More recently, it has been directly proven that this peptide is indeed responsible for targeting CTL on in~luenza virus-infected cells as it could be eluted from the MHC class I
molecule (Rotzschke et al., Nature 348:2 (199O)).

4. Examples Exam~le I
A. Construction of the pN~lNANP expression vector The production of hybridoma 62 and BlOH2, and the purification of mAb 62 and 109.3 (anti-2,4-dinitrophenol) have been described previously [Zanetti et al., J. Immunol.
131:2452 (1983) and Glotz et al., J. Immunol. 137:223 (1986)].

A DNA library was constructed from size-selected 2-2.5-kb Eco RI fragments from hybridoma 62 genomic DNA. Fragmen~s were eluted from low melting point agarose and ligated into W094/28026 ~ 2 1 ~ 3 1 2~ PCT~S9~/06090 the AgtlO vector [Huynh et al., DNA Cloninq Techniques 1:49 (1985)]. After ligation and packaging, 5 x 104 plaque-forming units were screened by replicate hybridization with the JH [Sakano et al., Nature 286:676 (1980)] and pSAPC15 [Brodeur et al., Eur. J. Immunol. 14:922 (1984)] probes.
Four clones were isolated and plaque purified; the 2.3-kb EcoRI insert form one of them was subcloned into pEMBL18 vector [Dente et al., DNA Clonina Techniques l:101 (1985)].
The VHBlOH2 coding se~uence was determined by cloning the cDNA from the parental hybridoma by primer extension of the poly(A)+ RNA with a synthetic oligonucleotide (5'-GGGGCCAGTGGATAGAC-3') that anneals at the 5' end of the CHl region. The same oligonucleotide was used as a probe for screening the library after 5' end-labeling by kinase with 32P-ATP. The nucleotide sequence of both clones was determined by dideoxy method on both strands after subcloning suitable restriction fragments into the pEMBL18 vector.

Plasmid pNr162 containing DNA encoding Cl, 62 antibody was constructed by subcloning in the proper orientation the 2.3-kb EcoRI DNA fragment carrying the VH62 rearrangement into the unique EcoRI site of the PN~1 vector [Sollazo et al., Focus 10: 64 (1988)] (a PSV derived vector harboring an human ~1, gene). This vector encodes a human r1 gene 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. Transfectoma cells were constructed by introducing the plasmids pN~162 and pN~lCHA, a chimeric construct encoding an antibody lacking Id62 and Ig binding into J558L mouse by electroporation. This cell line is an H chain-defective variant of myeloma J558 [Morrison et al., Science 229:229 (1985)] and carries the rearrangement for a A1 light (L) chain. Briefly 3 x lC6 cells in 1 ml of Dulbecco's modified minimum essential medium (DMEM) containing 10 ~g of supercoiled plasmid DNA were pulsed for 17 ms at 650 094/28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 V/cm in a Cell Porator apparatus (Bethesda Research Laboratories, Bethesda, MD). After pulsing, the cells were resuspended in 10 ml of DMEM supplemented with 10 mM Hepes buffer, 2 mM L-glutamine, penicillin (50 ~g/ml), streptomycin (50 ~g/ml) and 10~ fetal calf serum (cDMEM), and incubated for 48 h at 37OC in a 10~ CO2 atmosphere.
The cells were then resuspended in 20 ml of cDMEM and an aliquot (2 ml) was diluted into 20 ml of cDMEM containing 1.2 mg/ml of G418 (Gibco, Grand Island, NY), plated on a 96-well microtiter plate and cultured for 14 days. The supernatants of neomycin-resistant colonies (stable transformants) were tested by solid-phase radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

The presence of Id62 in the supernatant of J558L cells transfected with pN~l62 vector was tested by competitive inhibition in ELISA. This measures the inhibition (percent) of the binding of horseradish peroxidase (HP)-conjugated mAb 62 (ligand) to anti-Id62 antibody coated on 96-well polyvinyl microtiter plate (Dynatech, Alexandria, VA) [Zanetti et al., J. Immunol. 131:2452 (1983)]. The supernatant of J558L cells transfected with pN~lCHA plasmid and purified mAb 62 and 109.3 (an IgGl, x anti-2,4-dinitrophenol) served as controls [Zanetti et al., J.
Immunol. 131:2452 (1983)]. A second method to test for Id62 expression was by Western blot [Towbin et al., Proc.
Natl. Acad. Sci. USA 71:4350 (1979)]. Briefly, approximately 5 ~g of antibody C~l62 purified by affinity chromatography on an anti-human Ig Sepharose 4B column (Pharmacia, Uppsala, Sweden) was electrophoresed on a 10~
sodium dodecyl sulfate polyacrylamide gel electrophoresed on a 10~ sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions. The gel was then blotted onto 0.45 M nitrocellulose paper (Millipore, - Bedford, MA) and probed withl2~I-labelled affinity-purified syngeneic anti-Id62 antibody [Zanetti et al. J. Immunol.
135:1245 (1985)]. Antibodies 62 and 109.3 served as W094/28026 i~ T f~ 2 ~ 63 1 2~ PCT~S91/06090 positive and negative control, respectively. The filter was exposed a first time for 24 h at -70C with intensifier screen. To demonstrate the co-expression of the human C
region on the H chain of the chimeric C~162 antibody, the nitrocellulose paper was re-probed with l25I-labelled goat anti-human Ig antibody and exposed for 2 h at 70C.

Sequence data is publicly available from EMBL/Gene Bank Data Library under Accession No. Y00744.

The rlNANP antibody carrying the malarial CS immunodominant B-cell epitope NANP in the CDR3 of its H chain was engineered as follows:

Figure 1 is a diagram illustrating the construction of the pN~lNANP expression vector. In panel A: (a) The productively rearranged VH gene of the hybridoma cell line 62 isolated from a size-selected lambda gtlO library and subcloned into pBluescript (publicly available from Stratagene, San Diego, CA) is described infra.; (b) The restriction site KpnI/Asp718 of the polylinker region was deleted by Kpn I digestion, filled in with T4 polymerase and ligated, yielding the plasmid pH62~k; (c) pH62~k was used as a template for site-directed mutagenesis to introduce a unique Asp718 restriction site in CDR3 of the VH gene. The synthetic oligonucleotide (5-'CAAGAAAGGTACCCTACTCTC-3'), which encodes a 3 bp insertion (TAC), was annealed to the uracylated single-stranded complementary template and elongated; (d) Complementary synthetic oligonucleotides (5'-GTACCCAATGCAAACCCAAATGCAAACCCA~ATGCAAACCCA-3' 3'- GGTTACGTTTGGGTTTACGTTTGGGTTTACGTTTGGGTCATG-5') were annealed and subcloned into the unique Asp718 site of pH62k. The construction was verified by sequence analysis by using a 15m'r primer corresponding to the 5' end of VH62 gene (5'-GACGTGAAGCTGGTG-3'); (e) The 2.3-~ 094/~8026 2 1 6 3 1 2 5 PCT~S94/06090 kb Eco RI fragment carrying the engineered VHNANP genewas subcloned upstream from the human yl C region into the 15-kb pNyl vector. The pN~lNANP construct was electroporated into J558L cells subsequently cultured in the presence of G418. Resistant clones were - screened for Ig production by a sandwich enzyme-linked immunosorbent assay (ELISA) using goat anti-human antibodies immobilized on microtiter wells as the capturing antibodies and horseradish peroxidase (HP) conjugated goat anti-human Ig (Sigma) as the revealing antibodies. Clones producing >2-5 ~g Ig/ml of protein 106 cells were expanded and the antibody purified from culture supernatants. Sequence modifications illustrated in panel A are shown in detail in panel B.
Abbreviations used: Asp - Asp 718; B - BamHI; RI -EcoRI; FR - framework region; CDR - complementarily-determining region; neo - neomycin (G418) resistance;
amp - ampicillin resistance.

The restriction fragment encoding the VH gene of a murine monoclonal antibody to thyroglobulin (mAb 62) was modified as shown in Figure l. A double-stranded synthetic DNA fragment encoding three copies of the NANP tetramer (NANP) 3 and carrying Asp718 protruding ends was inserted in frame between Pro 95 and Tyr 96 of VH62k coding region. The pH62NANP construct was verified by dideoxy sequencing. The Eco RI restriction fragment encoding the engineered Vg was subcloned into the pNYl expression vector upstream from the human yl constant (C) region to obtain the pNr1NANP construct.
This plasmid was electroporated into the murine J558L
cell line, a H chain-defective variant of myeloma J558L
that carries the rearrangement for a lambda-1 L chain [Morrison et al., Science 229:1202 (1985)].

Transfectoma cells were cultured, subcloned and screened for secretion of the engineered Ig molecule ~ A~ 2 ~ ~ 3 1 2 5 W094/~8026 PCT~S94/06090 using a sandwich enzyme-linked immunosorbent assay (ELISA) with goat anti-human Ig antibodies. Clones producing 2-5 ~g/ml of protein 106 cells were selected and expanded, and the chimeric protein was purified by means of affinity chromatography on a Sepharose 48-Protein-A column. The purified Ig molecule was analyzed by SDS-PAGE under reducing and nonreducing conditions.

Figure 2 is an SDS-PAGE of the ~lNANP and WT
recombinant Ig. Five ~g of Protein A-purified antibody were loaded on a 7.5~ polyacrylamide gel under nonreducing conditions. The gel was stained with Comassie blue. The inset shows the resolution into heavy (H) and light (L) chains of engineered antibody ~lNANP electrophoresed on a 10~ polyacrylamide gel under reducing (5~ ~-mercaptoethanol) conditions.

Figure 2 shows that the nonreduced ~lNANP chimeric antibody has an apparent molecular weight of 160 kD, suggesting a proper H2L2 assembly to form a tetrameric Ig protein. When the ~lNANP antibody was compared with the wild-type (WT) Ig, a chimeric antibody lacking the (NANP) 3 insert, purified from culture supernatant fluid of J558L cells transfected with pNyl62, a slight difference in size was observed due to the presence of the inserted epitope. However, the molecular weight of the ~lNANP antibody is well in the range of a tetrameric complex. Both preparations also showed a smear in the region below the 160 kD band, suggesting some degradation and/or noncorrectly assembled protein products. Under reducing conditions, the engineered ~lNANP antibody was appropriately resolved into an H
and na L chain (Figure 2, inset). As determined by ELISA of NP-40 lysates, transfectoma cells secreting the ~lNANP antibody had approximately the same cytoplasmic levels of H chains as cells producing the ~V~94/28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 WT Ig. Collectively, these results indicate that the insertion of 15 amino acids into the CDR3 of VH62 did not appreciably alter the interaction between VH and VL
polypeptide chains nor the assembly and secretion of the tetrameric (H2L2) Ig molecule.

B. Bindinq of monoclonal antibody Sp-3-B4 to enqineered antibodY ~lNANP
To determine if the engineered ~lNANP antibody indeed expresses the (NANP) 3 epitope in an immunological accessible form, solid-phase radioimmunoassay (RIA) and Western blot techniques were used and a murine monoclonal antibody (Sp3-B4) generated against P.
falciparum and specific the NANP epitope.

Figure 3 shows the binding of 125I-labelled monoclonal antibody Sp-3-B4 to engineered antibody ~lNANP. Murine monoclonal antibody (mAb) Sp3-B4, an IgG2a,k antibody produced by immunization with the P. falci~arum parasite and reacting with the repetitive epitope NANP.
Specific for the NANP epitope, any antimalainal 2 0 antibody could be so used as a tool and generated via analogous techniques. Polyvinyl microtiter wells were coated by drying at 37C with 5 ~g/ml solution in 0.9~
NaCI of purified ~lNANP Ig (solid diamonds), WT (solid triangles), (NANP) 3 synthetic peptide (solid squares), a 16mersynthetic peptide (YYCARKAYSHGMDYW) encompassing the CDR3 of the VH region of prototype antibody 62 (open squares), and the 15mer synthetic peptide YPQVTRGDVFTMPED of the cell-adhesive molecule vitronectin (open diamonds). The 125I-labelled antibody Sp3-B4 (20 X 104 cpm/50~1) was incubated overnight at +4C. After extensive washing, the bound radioactivity was counted in a gamma counter. The test was done in triplicate.

W094/28026 ~ tl~ 2 ~ 6 3 ~ 25 PCT~S91/06090 The results of the direct RIA binding (Figure 3) showed that 125I-labelled mAb Sp3-B4 bound both the synthetic peptide (NANP) 3 and the recombinant ~lNANP antibody immobilized on microtiter wells. However, the binding to antibody ~lNANP can be considered more efficient; in molar terms, the estimated ratio of peptide to antibody was about 50 to 1, assuming that the antibody expresses two copies of the (NANP~ 3 epitope per Ig molecule. No binding occurred to either the WT Ig or two irrelevant synthetic peptides, one corresponding to the CDR3 sequence of prototype V~62 and the other to residues YPQVTRGDVFTMPED of vitronectin.

Figure 4 is a Western blot binding of 25I-labelled antibody Sp3-B4 to engineered (NANP) 3 epitope in the H
chain. Ten ~g of purified ~lNANP Ig, recombinant WT
Ig, native monoclonal antibody 62, and polyclonal human gamma globulins (HGG) (Cohn fraction II, Miles) were loaded onto a 10~ SDS-PA~E and electrophoresed at 150 V under nonreducing (left panel) and reducing (right panel) conditions. Resolved proteins or polypeptide chains were transferred from the gel to 0.45-~m nitrocellulose paper. After blotting, the filter was blocked with 10~ solution of dry milk in 0.9~ NaCI for two hours at room temperature. The sheet was then incubated overnight at +4C by rocking with 125I-labelled antibody Sp3-B4 (40 x 104 cpm/ml) in phosphate-buffered saline, pH 7.3, containing 1~ bovine serum albumin and 1~ Tween 20. After incubation, the filter was washed extensively, dried and exposed to Kodak XAR-5 film at -700C for 18 hours. Binding to ~lNANP Ig, recombinant WT Ig, antibody 62 and HGG in RIA by the same 12sI-labelled probe (105 cpm/50~1) was 10,560; 420; 360; and 330 cpm, respectively.

Western blot analysis (Figure 4) showed that l2'I-labelled mAb Sp3-B4 specifically bound antibody rlNANP

~ 094/28026 -~ T ,~ 2 t 63 1 25 PCT~S91/06090 in both the nonreduced (left panel) and reduced (right panel) forms. In the latter, as expected, binding occurred on the H- but not the L-chain, confirming that the engineered ~lNANP antibody bears the (NANP) 3 epitope on the H chain. No binding occurred to controls for the H and L chain and the human C region.

C. Efficiency of enqineered ~lNANP antibody in ex~ressinq the (NANP) 3 epitope A cross-inhibition assay was employed to assess the engineered ~lNANP antibody's relative efficiency in expressing the (NANP) 3 epitope. The synthetic peptide (NANP) 3 and antibody ~lNANP were used to inhibit the binding of 125I-labelled mAb Sp3-B4 to either the (NANP) 3 peptide or the ~lNANP antibody immobilized on microtiter plates.

Figure 5 shows results of cross-inhibition of l25I-labelled antibody Sp3-B4 binding to synthetic peptide (NANP) 3 (panel A) or engineered antibody ~lNANP (panel 8) by ~lNANP Ig or peptide (NANP) A fixed amount of l25I-labelled antibody Sp3-B4 (probe) was mixed vol/vol with decreasing amounts o~ the various inhibitors diluted in phosphate-buffered saline, pH 7.3, containing l~ bovine serum albumin and l~ Tween 20.
The mixture was incubated at +4C overnight by rocking.
Fifty ~l of each mixture were incubated on individual polyvinyl microtiter wells coated with either synthetic peptide (NANP) 3 (panel A) or purified engineered ~lNANP
Ig (panel B). The conditions of coating are as detailed in the legend to Figure 4. The following inhibitors were used: purified ~lNANP Ig, WT Ig, and synthetic peptides (NANP) 3, CDR3 and vitronectin. The percentage of inhibition was calculated as ~ollows:
[(average binding of the probe alone) - (average binding of the probe incubated in the presence of W094l28026 ~ S ~l ~ 3 ~ 2 5 PCT~S94/06090 inhibitor)]/(average binding of the probe alone) x 100.
Tests were done in duplicate.

Figure 5 shows that both the peptide and the engineered antibody efficiently inhibited the binding to both physical forms of the ~NANP) 3 epitope, i.e., synthetic peptide and antibody borne. However, whereas the ~lNANP antibody was about four times more effective than the peptide itself (panel A) in inhibiting binding to the synthetic peptide, it was approximately 150 times more effective than the peptide in inhibiting binding to the engineered Ig (panel B). The WT Ig and control peptides (CDR3 and vitronectin) caused no inhibition. Thus, when compared with the synthetic peptide it appears that the (NANP) 3 epitope borne on the ~lNANP antibody assumes a three-dimensional configuration that in immunological terms more closely mimics that of the active CS protein.

D. Induction in vivo of anti-NANP antibodies by recombinant ~lNANP antibody To determine whether the recombinant ~lNANP antibody could be used to induce anti-NANP antibodies, in vivo experiments were performed in rabbits. Two rabbits were immunized with the engineered ~lNANP antibody, and two controls receive the WT Ig. As indicated in Table I, infra., as early as 30 days after the first immunization, both rabbits immunized with the rlNANP
antibody produced anti-NANP antibodies detectable by ELISA and RIA. After booster immunizations, the titer rose in both rabbits; the maximal titer was 1/3200 on day 70. Importantly, this antiserum was positive when tested by indirect immunofluorescence on P. falci~arum sporozoite showing that the epitope expressed by the ~lNANP Ig is indeed mimicking the native antigen. Sera from control rabbits immunized with the WT Ig did not ~ 094/28026 ~ t ~ 2 1 6 3 1 2 5 PCT~S94/06090 react with the (NANP) 3 peptide immobilized on microtiter wells nor with the parasite. Rabbits of both groups produced an anti-human response as determined by agglutination of red cells coated with human gamma globulin. Rabbits' antisera were tested by direct immunofluorescence on P. falciparum (strain Indochina III) dried onto glass slides in the presence of 10~ fetal bovine serum.

The observation that the VH region of an antibody molecule can be engineered to express 15 amino acid residues containing an epitope of an unrelated molecule shows that the VH/CH polypeptide chain containing the foreign epitope is properly assembled with the endogenous L chain to form a (H2L2) tetramer, so it appears that the insertion of this epitope in the CDR3 was tolerated and did not affect the overall Ig framework folding. Based upon the present research, as long as the recombinant epitope is stereochemically compatible with contiguous CDR residues, it can be inserted or substituted for a CDR and can be expected to be exposed at the sur~ace o~ the molecule, although it cannot be ruled out that the results reported here may be due to the nature of the epitope itself. In the construct described here, the (NANP) 3 sequence is flanked on both sides by the amino acids Val and Pro.
Possibly, this helps stabilize the inserted epitope by anchoring it at each end. The large ramification at the C~ atom and the C~-methyl group of the Val residue may hinder the main chain by decreasing its flexibility; the side chain of Pro by curling back to the main chain seizes it, leading to the formation of an almost rigid side chain.

Studies in vitro using the binding site of a NANP-specific monoclonal antibody as a probe for the protein-surface interaction and in vivo demonstrating W094/28026 ~ 2 1 ~ 3 1 2 5 PCT~S94/06090 ~

that rabbits immunized with the engineered Ig molecule produce anti-NANP antibodies that react with the plasmodium antigen show that the (NANP) 3 epitope expressed by the engineered Ig is both antiyenic and immunogenic. In other terms, neither the molecular environment nor the globular folding of Ig modified the immunologic structure of the tNANP) 3 epitope. From a biological standpoint, the (NANP) 3 epitope engineered into an Ig molecule can be viewed as an idiotope a la carte built into the CDR3 of a host VH domain. Based on what is known of the immunogenicity of idiotypes and the predictable events that follow induction of immunity via the idiotype network [Jerne Ann. Immunol.
(Paris) 125:373 (1974); Cozenave et al., PNAS 74:5122 (1977); Urbain et al., PNAS 74:5126 (1977); Bona et al., J. Exp. Med 153: 951 (1981)], these results imply that an immune response of predetermined epitope specificity can be dictated in molecular terms and predicted in vitro. This strategy can be exploited to render a B-cell epitope T-independent, proving its utility not only for analyses of the structure and function of epitopes and Igs but also for the development of new antibody vaccines, for example, as an alternative to peptide based vaccines. Preparation of vaccines may be accomplished using extant methodology, already developed for immunoglobulins as such.

094l28026 ~ PCT~S94/06090 TABLE I
Induction of Anti-NANP Antibodies in Rabbits Immunized with the Engineered ~lNANP Antibodya Rabbit Immunogen Days After Immunization No.
0 30* 40 60* 70 49 ~lNANP 0 1/100 1/400 1/400 1/3200 ~lNANP 0 0 1/400 1/200 1/1600 aAdult white rabbits were immunized subcutaneously in several points of the back with 50 ~g of recombinant ~lNANP or the WT antibody emulsified in complete Freund's adjuvant tCFA). Booster injections of 50 ~g of the same immunogen in incomplete Freund's adjuvant were given at monthly intervals (denoted by an asterisk). Sera were collected on the days indicated and tested for reactivity with the synthetic (NANP) 3 peptide by solid-phase ELISA and RIA. Briefly, serial two-fold dilutions of individual sera in phosphate-buffered saline, pH 7.3, containing 1~ bovine serum albumin and 1~ Tween 230 were incubated overnight at +4C on microtiter plates coated with the (NANP) 3 peptide at 5 ~g/ml in 0.9~ NaCI. After the incubation, the plates were washed and incubated with either a horseradish peroxidase conjugated goat anti-rabbit Ig, or 12sI-labelled Protein A (Amersham) for one hour at room temperature. Next, the plates were washed and the bound antibodies determined by using a Bio-Rad (Richmond, CA) ELISA reader or a gamma counter. The binding of the preimmune sera was considered the reference background value. The titer was determined from the mean binding of triplicate samples after subtracting the background binding values and is expressed as the reciprocal serum dilution.

2 ~ ~ 3 ~ .2~
W094/28026 PCT~S9~/06090 ExamPle II
Materials and Methods Monocl onal an tibodi es The murine monoclonal antibody 28-14-8S (~2a, k) specific for H-2b (Db allele) was purchased from the American Tissue Type Collection (ATCC No. HB27).
Fluorescein-conjugate murine monoclonal antibody AMS-32.1 reacting with I-Ad was purchased from Pharmigen (San Diego, CA).

~y~ the ti c Pep ti des A/PR/8/34 influenza virus nucleoprotein synthetic peptide ASNENMETM (amino acid residues 366-374) was synthesized on an ABI 430-A automated synthesizer (Applied Biosystems, Inc., Foster City, CA).

Cells B6-2 is a nonsecreting murine B cell hybridoma (H-2d~) originally established by fusing C57Bl/6 (B6) (H-2b) splenic B cell with M12.4.1 lymphoma cells of BALB/c (H-2d) origin, and were kindly obtained from Dr. R. Abe (National Institute of Health, Bethesda, MD). J558L is a murine myeloma of BALB/c (H-2d) origin and is a H-chain defective variant of J558 myeloma carrying the rearrangement for a ~l light (L)-chain (Morrison, Science 229:1202 (1985)). J558L cells lack constitutive Ig secretion, but they secrete a H2L2 Ig molecule when transfected with a H-chain gene. The non-secreting Sp2/0 myeloma (H-2d) was obtained through passage from ATCC No. CRL 1581. CD8+ murine CTL clone 34 (Vitiello et al., J. Immunol. 143:1512 (1989);
Vitiello et al., J. Immunol. 131:1635 (1983)) is specific for the monopeptide ASNENMETM (residues 366-374) of the nucleoprotein (NP) antigen of A/PR8 influenza virus (Bastin et al., J. Ex~. Med. 165:1508 (1987); Rotzschke et al., Nature 348:252 (1990);

~'094/28026 PCT~S94/06090 Townsend et al., Cell 44:959 (1986)), and is restricted by the class I histocompatibility Db gene product. The clone was maintained in culture by stimulation at weekly intervals with irradiated syngeneic spleen cells pulsed with the ASNENMETM synthetic peptide.

~ngineering techniques The D region of the parental VH gene (KAYSHG; residues 93-98) was mutagenized (Sollazzo et al., Eur. J.
Immunol. 19:453 (1989)) to introduce a single KpnI/Asp718 site to yield the intermediate sequence KVPYSHG (residues 93-99). The amino acid 94A was deleted and substituted by the VP doublet encoded by the nucleotide sequence of the Asp718 cloning site.
Subsequently, complementary oligonucleotides 5' GTA CCC
GCT TCC AAT GAA AAT ATG GAG ACT ATG GAA TCA AGT ACA CTT
3', 5' GTA CAA GTG TAC TTG ATT CCA TAG TCT CCA TAT TTT
CAT TGG AAG CGG 3' coding for residues 366-379 of the influenza nucleoprotein (NP) (ASNENMETMESSTL) were introduced between 94V and 95P of the mutagenized VH
region. The engineered VHNP coded by the 2.3 kb EcoRI
~ragments was cloned upstream from a human rl constant (C) region gene contained in the 12.8 kb vector pN~1 (Sollazzo et al., Eur. J. Immunol. 19:453 (1989)).
Thirty ~g of the DNA construct pN~lNP were electroporated in B6-2, J558L and Sp2/0 cells (2 x 107) using a field strength of 625 V/cm. Transfected cells were cultured in RPMI 1640 supplemented with 10~ fetal calf serum (FCS), 4 mM glutamine, 0.1 mM non-essential amino-acids, 1 mM sodium pyruvate, 0.1 mM HEPES, 100 U/ml penicillin, 100 ~g/ml streptomycin and 0.5 ~M ~-mercaptoehanol for 24 hours, and then selected in the presence of neomycin (0.8 mg/ml) (G418; Gibco-BRL).

~ ~ r 2 1 6 3 1 25 W094/28026 PCT~S9~/06090 Esterase Release Assay B6-2 HNP transfectants cells were screened for presentation of the NP peptide by using an immunoenzymatic method that measures the release of esterase by CTL upon specific peptide antigen recognition (Kane et al., Mol. Immunol. 26:759 (1989);
Pasternack et al., Nature 322:740 (1986)). Briefly, 105 effector cells (CTL clone 34) and 104 cells from each transfectoma were coincubated in a final volume of 100 ~l of culture medium in 96-well flat-bottom plates.
Untransfected B6-2 cells and B6-2 cells pulsed with the NP peptide (5 ~g/ml) served as negative and positive controls, respectively. Spontaneous and maximum esterase release were assessed on effector cells incubated in medium alone or in the presence of 1 Triton 100-X. After 4 hours of incubation at 37 ) C, the plates were centrifuged of 400 rpm for 2 min and the supernatants collected. Twenty five microliters of each supernatant were transferred to 96-well flat-bottom plates to which were added 175 ~l of phosphate buffered saline (PBS) containing 2 x 10-4 M N-benzyloxycarbonyl-L-lysine thiobenzil ester and 2.2 x 10-4 M 5,5'-dithiobis(2-nitrobenzoic acid) (Sigma, St Louis, MO). After an incubation period of 30 min. at 25 C the absorbance at 412 nm was determined in an ELISA plate reader. The results were expressed in percent secretion as follows: [sample secretion -spontaneous secretion / maximum secretion - spontaneous secretion] x 100.
Cytotoxicity Assay Cytotoxicity was tested in a 4 hours s1Cr release assay.
Briefly, target cells were labeled with NaslCrO4 (150 ~Ci/1 x 106 cells) for 1 hour at 37 C in an atmosphere of 5~ of CO2 with or without NP peptide (10 ~g/ml or as specified), then washed and resuspended in culture medium supplemented with 10~ FCS. One hundred ml of ~ 094/28026 ~ A ~ 2 1 6 3 1 2 5 PCT~S94/06090 5lCr-labeled target cells (2.5 x 105 cells/ ml) were mixed with 100 ~l of CTL clone 34 (effector cells) at an effector:target cells (E:T) ration of 10:1, or as specified. The plates were incubated for 4 hours at 37 C in 5~ CO2, then centrifuged at 500 g for 4 minutes.
One hundred microliters of supernatant was removed and counted in a gamma counter. Spontaneous and maximal 5lCr release were determined by incubating target cells in medium alone or in the presence of 1~ Triton X-100, respectively. The cytotoxic activity was calculated from triplicated wells as follows: [experimental release - spontaneous release / maximal release -spontaneous release] x 100. Cold target competition was done by mixing 50 ~1 of 5lCr-labeled B6-2.503 cells (5 x 105 cells/ml) with 50 ~1 of EI-4 or B6-2 cells pulsed with NP peptide (10 ~g/ml) at a cold:hot cell ratio of 0:1, 5:1, 25:1, and 50:1. Then, 100 ~l of CTL
clone 34 (effector cells) were added at a E:T ratio of 10:1. Percent cytotoxicity was calculated 4 hours later as described above. ~lNP, or ~lNANP as negative control, was added at a final concentration of 100 ~g/ml either during the pulsing or the cytotoxicity phase. The murine monoclonal antibody 28-14-8S (~2a/K) specific for Dbq or a mouse monoclonal antibody of the same isotype but of unrelated specificity as control was added during the cytotoxic assay at a final concentration of 50, 5, and 0.5 ~g/ml, respectively.
HPLC fractions were tested for their capacity to pulse B6-2 cells as follows: 100 ~1 of 5lCr-labeled B6-2 cells (2.5 x 105 cells/ml) were mixed with 5 ~l of each fraction. After 1 hour incubation at 37 C, 100 ~1 of CTL clone 34 (effector cells) were added at a E:T ratio of 10:1. The NP peptide (10 ~g/ml) was used as positive control.

W094/28026 ~i ~ a ~ ~ 2 1 6 3 1 2 5 PCT~S9~/06090 Isolation of NP peptide from the class histocompatibility D surface molecule The NP peptide was isolated from B6-2 HNP transfectants by acid elution. Briefly, Db-specific monoclonal antibody 28-14-8S (~2a,k) was immobilized on Protein-A
beads at a ratio of 500 ~1 of beads:3 mg of 28-14-8S
antibody for 1 hour at 4 C. Bulk cultures of 10~
stable B6-2 HNP transfectants (clone 514 or 503) were pelleted and resuspended at 2 x 108 cells/ml in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5~ N~-40, 5mM EDTA) containing a freshly-made cocktail of proteases inhibitors [A-Protinin (5 ~g/ml), Leupeptin (10 ~g/ml), Pepstatin-A (10 ~g/ml), and PMSF 1 mM] for 30 min. at 4 C. Nuclei were pelleted by centrifugation at 3,000 g for 15 min. Lysates were then mixed with antibody 28-14-8 S/Protein-A
immunosorbent by rocking for 1 hour at 4 C. Protein-A
beads conjugated with the influenza peptide/Db complexes were then pelleted by centrifugation at 1,000 g for 5 min., washed three times with wash buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.5~ NP40, 5 mM EDTA) followed by a wash with PBS and HPLC-grade H2O. The NP
peptides were acid-extracted by washing the Protein A-class I MHC molecules beads twice with 0.2 trifluoroacetic acid (TFA). The low molecular weight material was separated by filtration through a Centricon 10 titer (Amicon) with a molecular weight cut off of 10,000 kD. The filtrate was lyophilized and kept at -20 C until used.

HPLC analysis of peptides Low molecular weight material containing peptides purified from Db+ B6-2-HNP transfectants (clones 514 cr 503) were analyzed by reverse phase HPLC using a SMART
System unit (Pharmacia) and a mRPC C2/C18 2.1 /iO
column (Pharmacia). Peptides were eluted using 0.1~
TFA in H2O (v/v) (solution A) and 0.08~ TFA in ~ 094/28026 ~ `'` 2 ~ 6 3 1 25 PCT~S94/06090 acetonitrile (solution B). The flow rate was 100 ~l/min and fractions of 100 ~1 were collected. The following gradient conditions were used: 0-61 min. a linear increase to 60~ B; 61-66 min. 6090 B; 66-71 min.
increase to lOO9o- B; 71-76 min. decrease to 0~ B. One hundred micrograms of synthetic NP peptide (residues 366-374) was purified using the same conditions and served as a reference.

A. Generation of B cells that present the influenza virus NP peptide (residues 366-379) to a CTL clone s~ecific for the same viral peptide A H chain gene was engineered to encompass in the third complementarity-determining region (CDR3) a nucleotide sequence encoding for the amino acid sequence ASNENMETMESSTL (residues 366-379) of influenza virus NP
antigen through the process of antibody antigenization (Zanetti, Nature 355:466 (1992)). The engineered H
chain gene (HNP) was used to transfect B6-2 (H-2db), SP2-0 (H-2d) and J558L (H-2d) cells, respectively.
J558L cells carry the gene for the A1 light chain and served to produce a H2L2 antibody molecule.

The H chain plasmid is the product of the fusion of a human ylC region with a murine VH engineered to express the NP sequence 366-379 in CDR3. The coding strand of the CDR3 region is shown in bold, with the NP-coding sequence underlined. The amino acid sequence of the influenza peptide 356ASNENMETMESSTL379 is shown in bold.
B, BamHI; RI, EcoRI; Neo, neomycin (G418) resistance;
Amp, ampicillin resistance. The DNA construct (pN~lNP) was electroporated in the murine B6-2 (H-2db) B cell hybridoma to generate target cells. The stable transfectants were initially screened using a serine-esterase release assay to select clones that could activate CTL clone 34 to release serine esterase used as a cellular probe (peptide presentation). The selected clones were then tested in a conventional s;Cr-W094/~80~6 ~''i ~CT~S9~/06090 release assay; clones that confirmed positive were expanded. Alternatively, the DNA construct was electroporated into murine myeloma cell line J558L (H-2d), a H chain-defective variant of myeloma J558

5 carrying the rearrangement for a A1 L chain.
Supernatants of neomycin-resistant colonies (stable tranfectants) were tested by ELISA for Ig production.
The final product is a H2L2 molecule ~lNP (antigenized antibody).

Neomycin-resistant B6-2 hybrid transfectants were screened for their ability to present the NP peptide to CTL clone 34 specific for the 9m'r ASNENMETM sequence.
To simultaneously screen a large number of transfectants, a serine esterase-release assay (Pasternack et al., Nature 322: 740 (1986) ) was used.
This assay is a readily-detectable indicator of secretory granules exocytosis triggered in the CTL
clone by specific recognition of the NP peptide/class I MHC molecule complex on B6-2 HNP transfectants. Five 20 out of 120 (4~) transfectants induced a release of esterase equivalent to that of B6-2 cells pulsed with a synthetic NP peptide (Table 2) . B6 -2 cells transfected with a wild type H chain gene (H~) -lacking the NP epitope- and B6-2 cells alone served as negative 25 controls. To confirm the results of the esterase-release assay a conventional 51Cr-release assay was used. This assay showed an absolute correlation between the two tests (Table 2). In order to select transfectants with stable integration of the H~P gene 30 primary transfectants were subcloned and retested using the same strategy. It is worth noting that transfectants negative by both esterase- and 51Cr-release assays remained negative throughout subsequent tests. On the other hand, positive transfectants 3 5 maintained their ability to present the NP epitope during a twelve month period.

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B. Specificitv of the killinq of enqineered cells by the CTL clone To ascertain the specificity of lysis of B6-2 HNP
transfectants by the CTL clone cold target competition experiments were performed using B6-2 and EL-4 cells pulsed in vitro with the synthetic NP peptide (10 ~g/ml) as the cold inhibitor.

5lCr-labeled B6-2. 503 cells t5 x 105 cells/ml) were mixed with EI-4 or B6-2 cells pulsed or not with NP
peptide (10 ~g/ml) at a cold:hot target cell ratio of 0:1, 5:1, 25:1 and 50:1, respectively. Then, CTL
(effector cells) clone 34 were added at a E:T ratio of 10:1. Percent cytotoxicity was calculated 4 hours later from triplicate wells as described. Maximum and minimum slCr release were 48, 197tl,177 and 6,137+93 cpm, respectively.

As shown in Figure 7 in both instances there was complete inhibition of cytolysis at a competitor:
target ratio of 50:1. This demonstrates that killing of B6-2 lymphoma cells engineered with the HNP gene was specific for the NP peptide/class I MHC molecule complex.

C. Soluble antiqenized antibody e.-pressing the NP
epito~e in CDR3 does not inter-ere with presentation of processed pept-de from the endoqenous HNP chain Although B6-2 is a nonsecreting B cell lymphoma, it is not unprecedented that transfection with a H chain gene reactivates a latent L chain gene. For instance, threshold amounts of antibody that may be undetectable in our assay (< 1 ng/ml) could have been endocytosed, processed and presented. To rule out this possibility, it was verified whether or not soluble antigenized antibody could mediate lysis of B6-2 cells.

~ 094l28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 s1Cr-labeled EI-4 cells (5 x 105 cells/ml) were mixed with NP peptide and/or ~lNP (or ~lNANP as control) at a final concentration of 10 or 100 ~g/ml, respectively.
Then, CTL clone 34 (effector cells) was added at an E:T
ratio of 10:1. Intact ~lNP molecule (or rlNANP as control) was added during the cytotoxic phase of the assay at a final concentration of 100 ~g/ml. Percent cytotoxicity was calculated 4 hours later from triplicate wells as described.
As shown in Figure 8 untransfected B6-2 cells pulsed with ~lNP were not lysed. Moreover, the antigenized antibody added to HNP transfectants during pulsing with peptide did not affect lysis nor did it modify the percent of lysis when added during the lytic phase.

These results demonstrate that lysis of B6-2 HNP
transfectants is the result of presentation of a processed peptide derived from the endogenously synthesized HNP chain. It should be pointed out that lack of direct influence on CTL lysis suggests that the whole antibody does not function as an anti-receptor antibody.
5 D. LYsis of B6-2 HNP transfectants is restricted by the Db allele The role of the Db allele in the presentation of the NP
peptide to the CTL clone by the engineered B cells wa analyzed using a twofold approach. First, it was ascertained that a murine monoclonal antibody (28.14.8S) specific for the Db allele could block cytotoxicity.

s1Cr-labeled B6-2.503 cells (2.5 x 10~ cells/ml) were mixed with CTL clone 34 (effector cells) at an E:T
ratio of 10:1 in the presence of various doses of the murine monoclonal antibody 28.14 ( K, ~2a) specific for W094/28026 ~ 2 1 6 3 1 2 5 PCT~S94/06090 Dbq. A mouse monoclonal antibody of the same isotype, but of unrelated specificity, was used as control.
Percent cytotoxicity was calculated 4 hours later from triplicate wells as described. Figure 9 shows a dose-dependent inhibition by antibody 28.14.8S, but not byan isotype-matched control antibody.

Second, a series of HNP transfectants carrying the H-2d haplotype (Sp2/0 and J558L cells) were analyzed. 51Cr-labeled B6-2 (H-2b~d), Sp2/0 (H-2d) or J557L (H-2d) cells pulsed with NP peptide (10 ~g/ml) or transfected with HNP or HWT (2.5 x 105 cells/ml), were mixed with CTL
clone 34 (effector cells) at an E:T ratio of 10:1.
Percent cytotoxicity was calculated 4 hours later from triplicate wells as described. As illustrated in Figure 10 lysis of these cells did not exceed that of control H~ transfectants. Thus, the NP peptide resulting from the proteolytic fragmentation of the endogenous HNP chain is presented in association with D~ allele as if it were generated from an intracellular replicating virus.

E. Purification of viral NP PePtide from B6-2 cells transfected with the HNP gene It was important to demonstrate that the NP peptide could be purified from surface class I Db molecules and that it could be used to pulse untransfected B6-2 cells and mediate lysis by CTL clone 34. A lysate of 109 B6-2 HNP transfectants was mixed with Protein-A Sepharose beads coated with monoclonal antibody 28.14.8S. MHC-bound peptides were extracted by acid elution using 0.2~ TFA, and the peptides were separated from class I
MHC molecules by centrifugation on a low molecular weight Centricon filter. The low molecular weight material was fractionated by reverse-phase HPLC. The elution profile of a representative experiment is shown ,~ ?t~ 2 1 6 3 1 2 5 ~
094/28026 : PCT~S94/06090 in Figure ll (A and C). The HPLC profile of the control gmer synthetic peptide ASNENMETM is shown as a comparison. Individual fractions were used to pulse untransfected 5lCr-labelled B6-2 cells to identify the fraction(s) containing the NP peptide. Figure ll (panel B and D) shows that in both instances the active peptide was eluted in fractions l9 and 20, suggesting that the peptide purified from the B6-2 HNP
transfectants has physicochemical and biological characteristics similar to the 9mer synthetic peptide ASNENMETM. Thus, a proteolytic fragment of the endogenously-synthesized HNP chain bound the Db allele, was transported at the cell surface and mediated lysis by the NP-specific CTL clone.
F. Ouantitat-ve analysis of ~rocessinq and presentat-on of the NP peptide in B6-2 cells enqineere~ with the HNP
That B6-2 and HNP transfectants were killed efficiently by a specific CTL clone and the NP peptide could be eluted from the cell surface of these cells prompted a quantitative analysis of this phenomenon. First, the effect of exogenous addition of synthetic peptide NP on the lysis of B6-2 HNP transfectants was verified.

Cr-labeled B6-2 or B6-2.503 cells were pulsed with O.l, l or lO ~g of NP peptide for l hour at 37 C, then mixed with CTL clone 34 (effector cells) at an E:T
ratio of lO:l. Percent cytotoxicity was calculated 4 hours later from triplicate wells. As shown in Figure 12 excess amounts (lO ~g/ml) of NP peptide added at the beginning of the cytotoxicity assay failed to induce an increase of lysis, hence implying that occupancy of MHC
class I molecules by processed peptide from the endogenous HNP chain was already maximum.
The foregoing description details specific methods that can be employed to practice the present invention.

2 1 6 3 ~ 25 W094/28026 PCT~S94/06090 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.

For example, antigen sequences can be engineered at any restriction site unique to the CDR sequence within which the antigen sequence is to be inserted, and absent from the sequence of the immunoglobulin chain wherein the CDR is located. Unique sequences in the six CDRs can be identified and located using a combination of known immunoglobulin nucleic acid sequences and cleavage sites of restriction enzymes.
Further, a desired unique restriction site may be introduced into the CDR wherein the antigenic determinant is to be inserted using molecular techniques well known to those skilled in the art. In addition, it is well within the knowledge of those skilled in the art to modify the present invention by, for example, engineering an antigen within any of the six complementarity-determining regions of an immunoglobulin.

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 (12)

Claims:

1. An immunoglobulin molecule containing at least one nucleoprotein (NP) peptide of influenza virus within a complementarity-determining region (CDR), said immunoglobulin molecule having the effector function conferred by the constant region of the immunoglobulin, and having specific influenza virus NP epitope reactivity.

2. An immunoglobulin molecule according to Claim 1, wherein said CDR is the third CDR in the N-terminus variable domain of said molecule.

3. An immunoglobulin according to Claim 1 wherein said nucleoprotein peptide comprises residues 366 to 374 of the influenza nucleoprotein.

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

5. A heavy chain of an immunoglobulin containing within the third complementarity-determining region (CDR) in the N-terminus variable domain thereof at least one influenza virus nucleoprotein peptide.

6. A pharmaceutical composition comprising as an essential principle an immunoglobulin molecule according to Claim 1.

7. The composition according to Claim 6 suitable for administration to a human subject.

8. The composition according to Claim 6 in the form of an administrable vaccine.

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

10. An expression vector operatively harboring DNA
encoding an immunoglobulin, defined according to Claim 9.

11. A recombinant host cell transfected with an expression vector according to Claim 10.

12. 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.