Cross Reference to Related Applications

This is a continuing application of applications U.S. Serial No. 07/947415 and U.S. Serial No. 07/947,521, 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.

Irαmunoglobulins (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

im unity 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 im unogenic 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. Immunol. A, 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. ¹ As a result, peptides acquire an ordered conformation, and antigenized antibodies (^Ab) can serve as useful mimics of antigens and ligands.

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.

More specifically, the present invention is directed to the introduction of the conformation of the Arg-Gly-Asp (RGD) tripeptide, a tripeptide involved in the interaction of a variety of adhesive proteins. Demonstrated is an antibody expressing up to three RGD repeats within the third complementarity region of the heavy chain efficiently blocks the adhesion of human tumor cells to fibronectin and inhibits the lysis of human erythroleukemia cells K-562 by natural killer (NK) cells.

A three-dimensional model of the engineered antibody loop reveals the structure and physicochemical characteristics likely required for the ligand activity. This study demonstrates that expression of the RGD motif in a jS-loop of an antibody imparts adhesive ligand properties to the antibody molecule.

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, otherwise bioequivalent, immunoglobulin molecules falling within the general scope hereof.

The present approach demonstrates that ^*Ab engineered to express an adhesion motif acquire ligand properties not found with the peptide in solution. Engineered were two antibodies expressing one or three RGD repeats. RGD is a tripeptide motif present on a variety of adhesive proteins, including fibronectin ² , vitronectin ³ , fibrinogen ⁴ and von Willebrand factor ⁵ . RGD functions as the binding site for a number of integrins ⁶ and participates in biological processes such as adhesion of cells to the extracellular matrix ⁷ , platelet aggregation", and migration of tumor cells ^{9 10} .

In the parent application, we demonstrated specifically that the hydrophilic tetrapeptide Aεn-Ala-Aεn-Pro (NANP) of Plaεmodium falciparum circumsporozoite protein grafted into the loop of the third complementarity-determining region (CDR3) of an antibody heavy (H) chain acquires a three-dimensional conformation that leads to i munogenecity in vivo ¹¹ .

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 ¹²⁵ I-labelled monoclonal antibody Sp-3-B4 to engineered antibody ylNANP.

Figure 4 is a Western blot binding of ^{1 S} I-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 ¹²⁵ I-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 illustrates the general configuration of the two molecules [referred to as γ,RGD and 7,(RGD) ₃ ] and the site of insertion of the hydrophilic motifs in CDR3.

Figure 7 illustrates the adhesion of human non-small cell lung carcinoma cells (TV1) to fibronectin is inhibited by γ,(RGD) ₃ . (A) The following inhibitors were used: ^RGD^ (D), γ^GD ( O ) , 7,NANP (Δ), fibronectin (■) , GdRGDSP

(A) , RGDS (O) . (B) x40 magnification of TV1 cells in complete medium (upper left, a) , with 7, (RGD), (upper right, b) , with fibronectin (lower left, c) , and with 7,NANP (lower right, d) .

Figure 8 shows the lysis of K-562 cells by NK cells of peripheral blood lymphocytes from healthy donors is inhibited by "*Ab 7 ¹ (RGD) ₃ .

Figure 9 depicts molecular models of the V regions containing RGD or (RGD) ₃ . The RGD-containing molecule is shown in the left panels, and (RGD) ₃ in the right panels. The L chain is colored in pink, and the H chain is colored in green. The loops are colored yellow except for: Arg, blue; Gly, white; and Aspartic acid, red. Panels (a) and (d) depict the molecules as solid ribbons through their backbone atoms with the (RGD) ₃ loop projecting much farther from the main body of the V region than RGD. Both loops are fairly rigid, as shown by high temperature

molecular dynamics, and are roughly planar. The side- chains of the loop residues point away from each other, accentuating the dipolar character of the loops. Panels (J )-( ) and (e) - (f) show contours of the electrostatic fields of the two molecules. Panels (b) and (e) are side views of the RGD and (RGD) ₃ loops, respectively. Panels (c) and (f) are top views. Positive contours are shown in blue, and negative contours are shown in red. Contour values are +/- 0.01, +/- 0.02, +/- 0.03, +/- 0.05, +/- 0.07, +/- 0.1, and +/- 0.15 kT/e. In the side view, RGD is largely negative, while (RGD) ₃ has alternating positive and negative lobes. However, (RGD) ₃ also is seen to be more positive at the N-terminal end of the loop, and negative at the C-terminal end. In the top views, RGD is still largely negative, presumably due to the proximity effects of the rest of the protein. On the other hand, (RGD) ₃ is markedly dipolar in this view.

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

-li¬ 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 ligands, 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 Enzymology 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

The ligand function of the two ^Ab were tested in an in vitro assay by measuring their ability to inhibit the attachment and spreading of tumor cells to a surface coated with fibronectin ^{10 12} . Human lung carcinoma cell TV1 adhere to surface-bound fibronectin and spread thereafter to form monolayers ¹³ . 7,(RGD) ₃ inhibited the adhesion and spreading of these tumor cells in a dose-dependent manner (Figure 2, A) . Inhibition by 7,RGD was apparent at the highest concentration (200 μg/ml) only. Also used as inhibitors were two synthetic peptides from the sequence of fibronectin, RGDS and GdRGDSP. Of these, GdRGDSP

caused inhibition only at a concentration of 1 mg/ml. RGDS did not inhibit. Thus, 7,(RGD) ₃ was a more efficient inhibitor than synthetic peptides known to block cell attachment to fibronectin ^{1014 15} . Figure 2 (B) depicts the inhibitory effect of 7,(RGD) ₃ on the adhesion/spreading of TV1 cells. Interestingly, 7,(RGD) ₃ but not 7,(RGD) bound tumor cells in a fluorescence-activated cell sorter (FACS) assay.

Adhesion plays an important role in the function of the immune system and apparently involves a large number of molecules, including RGD-containing molecules. Whether or not RGD is involved in specialized activities of lymphocytes remains unclear. Although adhesion is an initial step in cell lysis by, natural killer (NK) cells ¹⁶ , little is still known about the molecular requirements for a productive interaction between NK cells and target cells ¹⁷ . Notably, RGD-containing synthetic peptides are ineffective in inhibiting both NK cells-mediated lysis ¹⁸ and adhesion of NK cells to target cells ¹⁹ . Because this could simply reflect an intrinsic inability of synthetic peptides to meet the appropriate conformational requirements we used ^ b to explore the role of RGD in NK cells activity.

As shown in Figure 3, 7,(RGD) ₃ markedly inhibited lysis of K-562 target cells in a conventional ⁵¹ Cr-release assay. Inhibition by 7,RGD was somewhat weaker, a finding seemingly reflecting the lower accessibility of the adhesion motif. An isotype-matched ^Ab expressing a hydrophilic peptide of different amino acid composition in CDR3 (7,NANP) ^π the RGD-containing synthetic peptide

GdRGDSP failed to inhibit. This rules out a nonspecific effect through the Fc receptor, and implies the involvement of the RGD motif and the requirement for a stabilized tertiary structure in this phenomenon.

Conformational models for the two engineered V regions were constructed and analyzed. Each model was refined through a combination of energy minimization and molecular dynamics. The lowest energy conformations were the ones that were analyzed further. As can be seen in Figure 4, both loops project outward from the main body of the protein, ensuring that they are fully accessible for binding to a putative receptor. A comparison of panels (a) and (jb) shows that the (RGD) ₃ -containing loop extends much farther into solvent.

An examination of all low energy conformations of the models of the (RGD) ₃ loop showed that the models were quite similar to each other, indicating that each loop possessed a fairly rigid structure. This is probably due to residue Arg-102 that was highly strained in the model, being crowded by the main body of the protein, mostly the H chain. The backbone structure of the loop also constrained the charged residues to be quite far apart, so that oppositely charged side-chains did not intertwine to neutralize any long-range electrostatic effect.

The high density of charged amino acid residues in the loops generated interesting electrostatic profiles. Side views of the RGD and (RGD) ₃ loops (Figure 4, b and e) indicated the highly polar character of the loops. RGD is mostly negative whereas (RGD) ₃ has an alternating positive and negative pattern. However, the (RGD) ₃ loop is more positive at the N-terminal and more negative at the C- terminal than the RGD loop is. A remarkable difference was noted when the loops were viewed from the top (Figure 4, c and f) . Again, whereas RGD is mostly negative, (RGD) ₃ is sharply bipolar.

It is plausible to assume that this might be the electrostatic profile seen by a putative receptor approaching from a distance. Interestingly, even in the shorter loop, the side-chains of Arg-102 and Asp-104 point

away from each other, implying the existence of a dipole. Although this was not reflected in the electrostatic field shown in Figure 4 (c) , the dipole may be lost in the field for the rest of the protein, and only when the loop is large enough to project away (Figure 4, e) can the effect be seen. Thus, oligomerization of RGD in the antibody loop enhanced surface accessibility.

It also created a more pronounced electrostatic field with marked dipolar characteristics; that is, in the unbound state, the (RGD) ₃ loop contains both positively and negatively-charged residues in proximity. Both these effects could explain the greater biological activity of 7,(RGD) ₃ . It is known that the ligand function of RGD varies in different molecules depending on the molecular environment and the surrounding residues ⁷ . Interestingly, nuclear magnetic resonance analysis of two proteins that contain an integrin-binding RGD sequence, kistrin and echistatin, showed that RGD is at the tip of a β-turn ²⁰ ' ²¹ .

4. Examples

Figure 6 models for the structure of ^Ab expressing RGD. (a,jb) Diagram of pN7,RGD (a) and pN7,(RGD) ₃ (jb) expression vectors. In both cases the D region of the parental V _H gene (KAYSHG; residues 93-98) was mutagenized in the known per se manner to introduce a single Kpnl/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 coding for one or three RGD copies were introduced between 94V and 95P of the mutagenized V _H region. The coding strand of CDR3 is shown with the RGD- coding sequence in parenthesis. (c, d) The expressed ^Ab 7,RGD and 7,(RGD) ₃ are mouse/human chimeras. The H chain is the product of the fusion of a human 7,C region with the parental V _H murine engineered to express one (c) RGD

or three (d) repeats. As shown, the D region was modified by insertion of RGD or (RGD) ₃ between 94V and 95P. The inserted sequences are flanked by VP doublet [VP(RGD or RGD ₃ )VP] . The light (L) chain is the murine λj provided by the myeloma J558L host cell (H and L chains not to scale) .

The engineered V _H RGD and V _H (RGD) ₃ coded by the 2.3 kb EcoRI fragments were cloned upstream from a human 7, constant (C) region gene contained in the 12.8 kb vector pN7,. (Solazzo et _. al. , Focus 10. 64 (1988) . This is a PSV vector harboring a human 7, gene. This vector encodes a human 7, gene downstream from the EcoRI site. It also carries a neo ycin resistance gene under the control of the SV40 promoter for the selection of stable transformant cells. See also Solazzo et a_l. Eur. J. Immunol. 19. 453 (1989) .

Thirty μg of each DNA construct, pN7,RGD and pN7,(RGD) ₃ , were electroporated in the murine J558L cell line (2 x 10 ⁷ cells) using a field strength of 750 V/cm. Transfected cells were cultured in Dulbecco's modified minimum Eagle's medium (DMEM) 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 and 100 mg/ml streptomycin for 24 hours, and then selected in the presence of neomycin (1.2 mg/ml) (G418; Gibco-BRL) . G418-resistant clones secreting high level of the ^ b were identified by screening supernatants using an enzyme- linked immunosorbent assay (ELISA) and horseradish peroxidase conjugated goat antibodies to human immunoglobulin (Ig) (Sigma) as probe. ^Ab from culture supernatants were concentrated by (NH ₄ ) S0 ₄ precipitation and then purified on Protein A (Repligen) equilibrated with 3M NaCl, 1M glycine, pH 8.9. Elution was performed using 0.1 M, glycine HC1, pH 2.8, 0.5 M NaCl, and purified ^Ab were neutralized with 1 M Tris-HCl, pH 8.0, followed by dialysis against 0.15 M phosphate buffered saline, pH

7.3 (PBS) . B, BamHI; RI, EcoRI; Neo, neomycin resistance; Amp, ampicillin resistance. See Figure 6.

Cells were cultured in RPMI 1640, supplemented as described in the legend to Figure 6. Cells were harvested, washed and resuspended in RPMI 1640 containing 2 mM CaCl ₂ and MgCl ₂ . Cells (2 x 10 ⁶ /well) were incubated in a final volume of 100 μl for 18 hours at 37°C in 5% C0 ₂ atmosphere, with or without inhibitors. Non-adherent cells were removed by washing with PBS. Adherent cells were fixed with methanol and stained with 2% crystal violet (Sigma) in 2% ethanol. Cells were washed with PBS and the dye was solubilized by addition of 100 μl of 0.1 M citric acid in ethanol. Adherent cells were quantitated by reading the plates at 595 nm. Results are expressed as percent inhibition. Tests were done in triplicate.

Values refer to the mean of 5 independent experiments. The mean O.D. ₅₉₅ value of TV1 cells adhering to fibronectin was 0.78310.121. Inhibition by 7,(RGD) ₃ of the adhesion of TVl cells to fibronectin in vitro . See Figure 7.

Peripheral blood leukocytes (PBL) were isolated from heparinized peripheral blood of healthy donors by Ficoll- Hypaque (Histopaque 1077, Sigma) density gradient, washed, resuspended in RPMI 1640 and incubated for 2 hours at 37°C to deplete the adherent mononuclear cells. Lymphocytes were harvested, washed and resuspended in culture medium (RPMI 1640 supplemented as described in the legend to Figure 1) . The NK activity was tested in vitro in a standard 4 hours ⁵¹ Cr-release assay against the human erythroleukemia K-562 cells ²² . Target cells (10 ⁶ ) were labeled with 150 μCi of Na ⁵¹ Cr0 ₄ (Dupont de Nemours) for 45 minutes at 37°C. Cells were resuspended in culture medium and added to 96-well flat-bottom culture plates (Costar) (5 x 10 ³ cells/well) at various effector to target cells ratios (50:1, 25:1, 12:1) in a final volume of 200 μl alone or in the presence of 100 μg/ml of 7, (RGD) ₃ , 7,RGD, or 7,NANP as a negative control. After 4 hours incubation

at 37°C in 5% CO,, the plates were centrifuged and 100 μl of supernatant were harvested from each well. Spontaneous and maximal ⁵¹ Cr release were determined by incubating target cells alone, or in the presence of 0.5% Triton X- 100. The cytotoxicity was calculated from triplicate cultures as follows: [sample cpm - spontaneous release cpm/maximum release cpm - spontaneous release cpm] x 100. Results in Figure 8 refer to a representative experiment and are expressed as percent lysis ± SD. The inset shows the dose dependency of the inhibition by 7,(RGD) ₃ (D)

7,NANP (I) served as a control.

The coordinates of RGD and (RGD) ₃ were built using the program Homology [Biosym Technologies [Biosym Technologies, #172]]. In this regard, alignments of the sequences of the H and L chains of reference proteins with known coordinates were made using the scheme of Kabat and Wu ²³ . The coordinates for the backbone atoms of amino acid residues in the conserved regions were obtained from two reference structures. Those for the H chain came from the mouse Fab fragment J539 ²⁴ , and those for the L chain came from the anti-hemagglutinin 17/9 ²⁵ . In the conserved region, when the amino acid type of the reference protein and the model matched, the coordinates for the side-chains were copied directly. Where they differed, the side-chain was built from a standard amino acid template library. Bond lengths and angles were kept at their standard values. For the chi angles, the conformations of five other reference structures were examined. A conformation was chosen that was compatible with the majority of the other proteins. Loop coordinates also came from reference immunoglobulin structures, using the protocol of Chothia and Lesk ²⁶ .

The coordinates for the RGD and (RGD) ₃ loops were generated using the method of Jones and Thirup ²⁷ wherein an α-carbon distance matrix was constructed for peptide

segments on either side of the loop. The matrix was compared to those made from proteins of high resolution found in the Brookhaven Protein Data Bank ²¹ *. Peptide segments (loops) were selected for which the root mean square (RMS) differences of the distance matrices were lowest and the number of residues in the loop were the same. Ten such loops were obtained, and one was chosen for each model that had the least steric overlap with the rest of the protein, as judged by eye. The final conformation for the loop in each model was determined through a combination of energy minimization and molecular dynamics using the program Discover ²⁹ . All atoms of the proteins were held fixed except those of the loops. Preliminary energy minimization was done to relieve the greater part of the strain inherent in the building procedure. Then molecular dynamics was done at a system temperature of 900 degrees Kelvin. The trajectory was sampled at 1 picosecond intervals, and each snapshot was energy minimized using the steepest descents method.

For RGD, the molecular dynamics was carried out for 100 picoseconds, and for (RGD) ₃ , the run was 20 picoseconds long. For each model, the conformation studied was that with the lowest energy. Electrostatic potential energy contours were generated using the program DelPhi ²⁹ - ³⁰ . The focusing technique was used, that is, a preliminary calculation was done, and the resulting grid of potential energy values was used as input to the final calculation, thus reducing boundary edge effects. The final grid was a cube 15 A on a side centered on the engineered loop.

5. References

The following references are grouped by number referring to footnotes in the preceding text. Each of these documents is hereby expressly incorporated by reference herein:

1. Zanetti, M. Nature 355, 466-477 (1992) .

2. Ruoslahti, E. & Pierschbacher, M. Cell 44, 517-518 (1986) .

3. Hayman, E. , Pierschbacher, M. & Ruoslahti, E. , J. Cell. Biol. 100, 1948-1954 (1985) .

4. Rixon, M. , Chan, W.-Y., Davie, E. & Chung, D. Biochemiεtry 22, 3237-3244 (1983) .

5. Ginsberg, M. , Pierschbacher, M. , Ruoslahti, E. , Marguerie, G. & Plow, E. J. Biol. Chem. 260, 3931- 3936 (1985).

6. Hynes, R. Cell 69, 11-25 (1992) .

7. Ruoslahti, E. & Pierschbacher, M. Science 238, 491- 497 (1987) .

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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.