Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates generally to the field of antibodies and more specifically to the field of bifunctional antibodies which have the ability to bind to two different antigens.
• bifunctional antibodies also referred to as heterobispecific antibodies, are synthetically or genetically engineered molecules that are capable of binding to two antigenic determinants. The ability to bind to the two different antigens resides in one molecule.
• Bifunctional antibodies were first produced by fusing two different monoclonal antibody-producing hybridomas, which each recognized a different antigenic sites.
• each hybridoma is characterized by a different selectable marker, sensitivity to hypoxanthine-aminopterin-thymidine (HAT medium) and resistance to azaguanine [Milstein and Cuello Nature, 305;537-540 (1983)].
• HAT medium hypoxanthine-aminopterin-thymidine
• azaguanine Milstein and Cuello Nature, 305;537-540 (1983)].
• the fused hybrido as are capable of synthesizing two different heavy chains and two different light chains, so that theoretically there are ten different combinations which can be formed to produce an antibody containing two heavy chains associated with two light chains.
• bifunctional antibody will form an even smaller proportion of the total antibodies if the heavy chains are of different isotypes.
• a disadvantage of this method is that fused hybridomas are less stable cytogenically than the parent hybridomas and non-fused cells.
• bifunctional antibodies were used as an alternative to indirect immunocytochemi ⁇ try, since they avoid the need for direct conjugation of an indicator molecule to the antibody by chemical modification which results in a partial loss of activity and increased danger of nonspecific binding.
• bifunctional antibodies Another method for producing bifunctional antibodies has been described using heterobifunctional crosslinkers to chemically link two different monoclonal antibodies, so the aggregate will bind to two different targets [Staerz et al, Nature:314:628-631 (1985); Perez et al. Nature; 116:354-356 (1985)].
• This type of bifunctional antibody has been produced to focus a T-cell response to a chosen target such as a tumor cell or a virally infected cell [Clark and aldmann, JNCI. 79:1393- 1401 (1987) ; Gilliland et al, Proc. Natl. Acad. Sci.
• Bifunctional antibodies have also been produced by gene transfer into a hybridoma by retrovirus-derived shuttle vectors or selectable plasmids containing light and heavy chain genes [DeMonte et al, Proc. Natl. Acad. Sci.. USA. 87:2941-2945 (1990); Lenz and Weidle, Gene: j3_2:213-218 (1990)].
• This method produces a mixture of antibodies from which the bifunctional antibody must be purified.
• these transfected hybridomas are more likely to be stable than fused hybridomas.
• Bifunctional antibodies can also be produced by reduction of monoclonal antibodies to the single heavy chain associated with its single light chain (HL form) , mixing with a second monoclonal antibody followed by reoxidation to produce mixed antibodies [Staerz and
• Bifunctional antibodies produced as described above have been employed in a variety of ways.
• a bifunctional antibody with binding sites for a tumor cell surface antigen and for a T-cell surface receptor, T3 or Ti will direct the lysis of specific tumor cells by T cells [Clark and Waldman, JNCI. cited above] .
• a bifunctional antibody with specificity for fibrin and a plasminogen activator has been proposed as capable of increasing the effective concentration of the plasminogen activator in the proximity of a fibrin deposit [Haber et al, Science. 243:51-56 (1989)].
• bifunctional antibodies which are specific for gliomas and the CD3 epitope on T cells have been successfully used in treating brain tumors in human patients [T. Nitta et al, Lancet. 115:368-371 (1990)].
• the present invention provides a bifunctional antibody comprising a first selected Fab component from a first antibody characterized by a modification in its heavy chain constant region and a second selected Fab component from a second antibody characterized by a complementary modification in its heavy chain constant region.
• the two selected antibody Fab components are preferably derived from different antibodies.
• the heavy chain constant regions of these Fab components are modified to replace those areas which bring a Fab into association with another Fab to form a complete antigen-binding antibody molecule with complementary "interactive" domains.
• the interactive domains are reciprocal leucine zipper molecules.
• the complementary interactive domains are, a series of positively charged amino acids, e.g. a series of lysine residues, and a series of negatively charged amino acid residues, e.g. a series of glutamic acid residues.
• a recombinant antibody of the present invention may be associated with one or more detectable labels.
• Yet another aspect of this invention provides a method for producing the recombinant antibodies described above comprising replacing a portion of the heavy chain constant region of a first selected Fab component with a first complementary interactive domain; replacing a portion of the heavy chain constant region of a second selected Fab component with a second complementary interactive domain which will bind strongly to the first domain; and permitting the two modified Fab components to associate.
• the first and second interactive domains bring the first and second Fab components into proximity to form a single stable antibody construct.
• Recombinant techniques are employed to associate the first and second interactive domains with the first and second Fab components.
• the present invention provides a method of using the recombinant antibodies of the present invention as a diagnostic agent.
• Yet a further aspect of the present invention provides a method of using the bifunctional antibodies as therapeutic agents for the treatment of cancers and virally infected cells.
• the present invention provides novel recombinant antibodies and a method of making them, which avoid the problems associated with known bifunctional antibodies. Unlike other recombinant bifunctional antibodies and methods for making them, the antibodies and methods of this invention do not require extensive purification or strictly controlled chemical conditions.
• a bifunctional antibody or immunoglobulin molecule is obtained by modifying the constant regions of the immunoglobulin heavy chains of two selected antibody Fab fragments by replacing a portion of each heavy chain with a selected "complementary interactive domain".
• the heavy chains of an antibody molecule are ordinarily responsible for the dimeric nature of an antibody, because they hold the two Fab molecules (the antigen binding portions) of the antibody together.
• a portion of the heavy chain of each selected Fab fragment is replaced by a complementary interactive domain.
• the interactive domains thereby are associated with a selected Fab component and hold together two Fab components in a single molecule.
• the method and compositions of this invention permit Fab fragments from two different antibodies to be coupled in a single antibody construct, the bifunctional antibody construct.
• a Fab component is a portion of a selected immunoglobulin or antibody which contains one site for binding antigen.
• Each Fab fragment consists of one complete light chain and about one half of the heavy chain (the "F d " piece) of the original antibody, held together by a single disulfide bridge and noncovalent interactions.
• the "heavy chain” refers to the larger of the two polypeptide chains found in an immunoglobulin, which consists of one variable (V) domain, about three or four constant (C H ) domains, a carboxy-terminal segment, and a hinge region.
• the F d piece of the heavy chain consists of the V H , C H 1, and part of the hinge region.
• the "hinge region” lies between the heavy chain constant domains C H 1 and C H 2, and imparts flexibility to the immunoglobulin molecule.
• the bifunctional antibody of this invention provides for any two Fab molecules from selected monoclonal antibodies to be employed in the construct.
• one Fab molecule can be capable of binding a "target antigen", e.g., a tumor cell surface antigen, or a protein.
• the target antigen can be any antigen which is desired to be measured, detected or treated.
• the second Fab molecule of the construct of this invention may be an "effector antigen", e.g., a cytotoxic T cell surface antigen, a detectable label, or a therapeutic agent.
• the effector antigen is generally an antigen which achieves a desired effect in proximity to the target.
• a detectable label can be used to reveal the presence or level of the target antigen.
• a therapeutic agent can be used to neutralize the target antigen.
• a toxin, e.g., ricin, or a cytotoxic T cell can be used to lyse the target when the bifunctional antibody brings the target and effector together.
• the recombinant antibody of this invention may comprise the Fab molecules of two monoclonal antibodies directed to different epitopes located on the same cell or on different cells, or directed to antigenic determinants of the same antigen. In this manner, the recombinant antibody of the invention may be used to achieve enhanced antigen-binding ability.
• the use of a bifunctional antibody of this invention which also binds a second epitope which is present solely on the tumor cell (or is present in closer proximity to the first epitope on the tumor cell than on normal cells) , offers greater specificity than the corresponding monoclonal antibodies from which the recombinant antibody is derived.
• a "complementary interactive domain” may be defined as a peptide or polypeptide not naturally associated with a Fab component, but which is capable of binding strongly to a complementary interactive domain to hold two selected Fabs in a stable configuration mimicking that of a natural antibody.
• Each interactive domain is also characterized by an inability to bind stably to itself. However, it will bind strongly to a complementary interactive domain inserted within a portion of a second heavy chain constant region of a second Fab.
• These domains may be any interactive components which upon mixture associate only with each other.
• a presently preferred complementary interactive domain pair is selected from among known leucine zippers.
• a leucine zipper is a amino acid sequence found in DNA binding proteins [Busch et al, Trends in Genetics. 6.:36- 40 (1990) ] , in which every seventh amino acid is leucine (Leu) so that they align down one face of an ⁇ -helix and interdigitate upon dimer formation. These sequences thus form a hydrophobic region between the ⁇ helices.
• the leucine zipper regions of the rat oncogenes, c-fos and c- iun can be used in the present invention.
• lock and key interactive domain structures can be used in place of the leucine zipper.
• an amino acid sequence e.g., polyglutamic acid, which bears a negative charge
• another amino acid sequence e.g., polylysine, which bears a positive charge
• a polyglutamic acid domain associated with a first Fab molecule capable of binding one antigen would bind to a polylysine sequence associated with a second Fab molecule capable of binding a different antigen, due to the charge attraction between the domains. Because of the charge repulsion, neither domain will bind to itself.
• the interactive domain structures in the bifunctional antibodies of this invention may replace all or a portion of the heavy chain constant regions.
• the first and second complementary interactive domains completely replace a portion of the hinge region, and the CH2 and CH3 domains of the heavy chain constant regions of the naturally occurring antibodies.
• the selected Fabs are each associated directly with a complementary interactive domain. The two domains interact with each other so as to bring the first and second Fabs into association to create an antibody capable of binding two separate antigens through its two different Fab components.
• a short linking peptide may be optionally inserted between the hinge region and the interactive domain during the construction of the modified Fabs of this invention.
• the size and composition of the linking peptide may be altered, as desired. For example, the peptide may be increased in size to obtain greater hinge flexibility, if desired.
• Still another modification of the antibodies of this invention includes inserting the complementary interactive domains between the Fab fragment and another portion of the heavy chain.
• one or both of the C H 1 or C H 2 regions of the heavy chain may be added to the end of the selected interactive domain, to create another embodiment of the antibodies of this invention.
• the addition of these portions of the heavy chain may be performed utilizing the same recombinant DNA techniques described herein and in the following examples.
• the stability of the recombinant antibody depends on the binding association of the complementary interacting domains, e.g., the leucine zippers of c-fos and c-jun.
• the stability of the dimeric Fab may be further increased, if desired, by introducing cysteine residues into complementary positions of the interacting domain, e.g., the c-fos and c-jun leucine zippers, so that when the two Fabs associate, the cysteines form a -S-S- bridge upon oxidation.
• cysteine residues into complementary positions of the interacting domain, e.g., the c-fos and c-jun leucine zippers, so that when the two Fabs associate, the cysteines form a -S-S- bridge upon oxidation.
• no disulfide bridges will form upon ho odimer interaction, e.g., the c-fos with the c-fos.
• This change may be achieved by conventional site directed mutagenesis of the sequence encoding the two leucine zippers.
• Such enhanced stability may be desirable for therapeutic use of the antibody of this invention.
• a recombinant antibody of this invention may be constructed using various recombinant genetic engineering techniques known to those of skill in the art. See, e.g., Sa brook et al, "Molecular Cloning. A Laboratory Manual.” 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) .
• recombinant antibody fragments, both Fab fragments and complementary interactive domain fragments are constructed and expressed by essentially following the methods of W. D. Huse et al. Science. 246:1275-1281 (1898), which is incorporated by reference herein for further descriptions of methods and materials known to those of skill in the art.
• Conventional chemical synthesis methods may also be used in addition to genetic engineering methods in some instances.
• an embodiment of the present invention having two Fab fragments joined by a leucine zipper is prepared as follows. Plasmids containing the sequences for a desired light chain of a selected antibody and a desired interactive domain, in this case, a leucine zipper sequence, may be constructed in lambda vectors, such as those described by Huse et al, cited above. Other similar known vectors may be employed for this purpose. Desirably, these plasmids or vectors also contain appropriately placed restriction sites to permit the introduction of selected light or kappa (K) chain sequences and the F d or variable (V H ) segment of the heavy chain (V H -C H1 ) of the selected antibody donating the Fab fragment.
• K selected light or kappa
• V H variable segment of the heavy chain
• the plas id for the heavy chains preferably contains a sequence encoding a peptide for which a monoclonal antibody is available for subsequent use in immunoaffinity purification of the fused proteins.
• light and heavy chains are separately inserted into a plasmid before being transferred into an expression vector.
• the heavy chain signal sequence and two or more codons of the heavy chain from a selected monoclonal antibody are amplified and cloned into the plasmid. The same procedure is followed for the light chain. Following this, rat c-fos leucine zipper DNA
• V H -C H1 RNA sequences from the hybridomas which produce the selected antibodies are amplified by PCR using specific oligonucleotides.
• K RNA sequences are amplified by PCR. Exemplary primers are described by W. D. Huse et al, cited above.
• the PCR products of one hybridoma are inserted into a set of plasmids, one of which carries the light chain and one of which carries the heavy chain.
• the products of a second hybridoma are inserted into a similar second set of plasmids. These inserts are then excised from their respective plasmids, the ends filled in, and then ligated into a selected plasmid, which will then contain either the light or heavy chain of a selected hybridoma.
• the plasmid containing the heavy chain also contains the complementary interacting domain insert.
• the resulting plasmids encoding the light chain and heavy chain/leucine zipper regions for a selected hybridoma are co-transfected into a selected host cell, e.g., a mouse myeloma Sp2/0 cell or a human U293 cells, and selected by use of the selection marker.
• a selected host cell e.g., a mouse myeloma Sp2/0 cell or a human U293 cells
• Cultures are grown to late log phase and induced if desired, e.g., by isopropyl thiogalactoside (IPTG) for bacterial cells, harvested and resuspended in water to release the Fab from the periplasmic space by osmotic shock.
• IPTG isopropyl thiogalactoside
• This method results in the production of a Fab fragment with a leucine zipper tail.
• This same strategy may be readily repeated using the second set of plasmids for the second hybridoma to produce the second Fab fragment with the leucine zipper tail corresponding to the first Fab-leucine zipper fragment.
• the first Fab-domain fragment is collected from culture media, and may preferably be purified and concentrated by immunoaffinity chromatography and is mixed with the second Fab-domain fragment which is similarly in solution. This mixture of solutions results in the two interacting domains, e.g., the reciprocal leucine zippers, becoming associated and the formation of a recombinant antibody construct.
• the resulting recombinant antibody is tested for its ability to target the desired antigen or antigens 1.8 and for the ability of the effector Fab domain to generate the expected effect.
• this activity can be measured in contrast to the original target and effector antibodies.
• the individual Fabs would not direct the desired effect on the target cells, while the mixture of the two Fabs will have the desired effect, e.g., lysis, on the target cells, but no effect on unrelated cells.
• Expression systems including selected host cells and appropriate expression vectors, suitable for construction and expression of the Fab fragments and the entire recombinant antibody include known bacteria, yeast, insect and mammalian cell expression systems. Mammalian expression systems, including a mouse myeloma expression system, among other known mammalian systems are presently preferred. However, bacterial expression systems, particularly E. coli expression systems, may also be desirable.
• the only requirements for the suitable vector and host cells employed in this method are an efficient, preferably inducible promoter, a 5 1 untranslated region with a riboso e binding site for E.
• coli if bacterial expression is employed, or a consensus initiation signal for eukaryotes, a signal sequence for secretion, a sequence containing suitable restriction sites for inserting the PCR product, a sequence encoding an excisable intron for animal cells, but not for yeast; a poly A addition site for eukaryotic cells, and selectable marker suitable for the host cell.
• a second vector is required for eukaryotic cells containing all of the above features but with a different selectable marker.
• the host cell may be any cell capable of high levels of protein secretion. The selection of such expression systems and the components thereof are well within the skill of the art.
• the recombinant antibody produced according to present invention may be used as a diagnostic agent to detect qualitatively or quantitatively the presence of a selected target antigen in a biological sample.
• This antibody may be employed in various tissue culture diagnostic assays known to those of skill in the art.
• the recombinant antibody for such use may be provided with one or more detectable labels.
• the first antigen binding site and the second antigen binding site of the antibody may each bear a detectable label.
• the label may be one which is capable of visual detection or may be selected from systems detectable by other means, including, for example, fluorescent compounds, radioactive compounds or elements or immunoelectrodes. These and other appropriate conventional label systems are known to those of skill in- the art.
• the recombinant antibody produced by the method of this invention may be used in therapeutic regimens, such as the treatment of cancers.
• a bifunctional antibody having a binding site for a tumor cell surface antigen and for a T-cell surface receptor would be administered, by in vivo or ex vivo therapy, so that lysis of the tumor cells by T cells is enhanced.
• the bifunctional Fab is sufficiently stable to demonstrate its ability to achieve the effect, e.g., directed lysis of tumor cells by T cell clones or enhanced, specific binding of tumor cells.
• the second Fab fragment could bind a biological toxin, e.g., ricin, so that the toxin would be delivered to the site of the tumor cell by the binding action of the first Fab fragment of the bifunctional antibody.
• a recombinant antibody of the invention may be used to form an antibody conjugate by fusing toxins, radioisotope binding domains, enzymes capable of converting prodrugs to drugs, and other nucleotide sequences to the complementary interactive domains of this invention, or to other portions of the antibody of this invention.
• an antibody of this invention may be provided with enhanced tumor cell killing ability by attaching a known T cell binding site to the end of the leucine zipper, using conventional recombinant methods known to the art, and described herein.
• a recombinant antibody of the invention may comprise Fab molecules of two different antigens of the same tumor cells, a complementary interacting domain (e.g., leucine zipper construct) which keeps the Fab components in close association, and a T cell binding site.
• a complementary interacting domain e.g., leucine zipper construct
• the recombinant antibody of this invention may be further modified by attaching a gene encoding a cytokine, preferably a lymphokine, to the lower end of the interactive domain construct.
• Suitable cytokines are well known to those of skill in the art and include, among others, the interleukins (IL) 1 through 9, the interferons and tumor necrosis factor (TNF) . Attachment of the sequences encoding such proteins to the antibody constructs may be performed using the conventional techniques described below in the examples.
• a novel multicloning site plasmid facilitates the assembly of light and heavy chain cDNAs.
• Complementary oligonucleotides were synthesized to contain the following restriction sites in the following order: from the 5' end: EcoRI, Xmal, BamHI, Xbal, Spel, Sad, Sail, Xbal, Bglll, Xmal, and Hindlll..
• the oligonucleotides, after annealing, were cut with EcoRI and Hindlll and ligated to pUCl ⁇ [Bethesda Research Labs; Norrander et al, Gene.
• pMNI The heavy chain gene is assembled in pMNI before being transferred to an expression vector.
• a sequence encoding a peptide for which a monoclonal antibody is available is optionally cloned into the heavy chain gene so that the monoclonal antibody can be used for immunoaffinity of the fused proteins.
• a plasmid was rescued from the lambda vector ⁇ Hc2 [Huse et al, cited above] with an fl helper phage [J. M. Short et al, Nucl. Acids Res.. .16.:7583-7600 (1987)] and termed pHc2.
• pHc2 contains a sequence encoding a decapeptide for which a monoclonal antibody is available [J. Field et al, Mol. Cell Biol.. 8_:2159 (1988)].
• the rat c-fos leucine zipper DNA was prepared by PCR amplification using the primers 5• fosLZ and 3 ⁇ fosLZ shown in Table 2 below.
• the 5' fosLZ primer encodes from the 5' end, an Spel site, codons for three glycine residues to act as a spacer, followed by codons for the leucine zipper.
• the 3' primer contains an Xbal site which is compatible with Spel.
• the product was digested with Spel and Xbal and ligated to the plasmid pHc2 digested with Spel.
• rat c-jun leucine zipper DNA [M. Sakai et al. Cancer Res.. 49:5633-5637 (1989)] was amplified using primers 5' junLZ and 3 'junLZ (Table 3) and cloned to give pHc2/junLZ.
• pHc2/fosLZ and pHc2/junLZ From pHc2/fosLZ and pHc2/junLZ the sequence encoding the leucine zipper and the decapeptide is excised by Spel and EcoRI, and then is cloned into pMNI- Hl (containing the signal sequence) to give pMNI-H2 fos and pMNI-H2 jun.
• V H ⁇ C H1 DNA D. Synthesis and cloning of the V H ⁇ C H1 DNA
• the heavy chain primers [W. D. Huse et al, cited above] are used to amplify the V H -C H1 DNA.
• the hybridomas ME361 and OKT3 produce a 2a heavy chain, and the nucleotide sequence contains an Xhol site [P. Schreirer et al, Proc. Natl. Acad. Sci. USA. 21:4495-4499 (1981)], so that the PCR product is cut by Xhol and the V H -C H1 DNA cannot be cloned.
• ME361 [J. Thurin et al, Cancer Res. f 47:1229- 1233 (1987) ] is a hybridoma which secretes an antibody showing specificity for glycolipids abundant on human melanoma cells.
• OKT3 [E. Reinherz et al, Cell, 19:821- 827 (1980) ] is a hybridoma secreting an antibody specific for CD3 found on T cells. Therefore, a corresponding set of 5' primers were synthesized using the sequence GTC GAC, the recognition site for Sail, in place of CTC GAG, the recognition site for Xhol. In addition, the following, different 3' primer was made for ⁇ 2a:
• V H -C m region was * amplified from total RNA from hybridomas ME361 and OKT3.
• the amplified material after digestion with Sail and Spel was cloned into pMNI-H2 fos and pMNI-H2 jun digested with Sail and Spel to give pMNI-H2 fos/ME361 V H - C HI and pMNI-H2 jun/OKT3 V H -C H1 .
• the K chains were amplified using the polymerase chain reaction from total RNA extracted from the hybridomas ME361 (anti-target) and OKT3 (the anti-effector) .
• the amplified material after digestion with Sa and Xbal was cloned into pMNI-Ll digested with the same two enzymes.
• the plasmids obtained are called pMNI-Ll/ME361 and pMNI-Ll/OKT3.
• A. Cloning into an Expression Vector A variety of expression vectors suitable for expression of the recombinant proteins in different animal cells may be used for the production of the antibody of this invention.
• pHEKneo [Hendricks et al, Gene, jS4.:43-51 (1988)] for expression in mammalian mouse myeloma Sp2/0 cells.
• the plasmid pHEKneo is modified by removing the neo gene and replacing it with the gpt gene by blunt-end ligation to produce plasmid pHEKgpt.
• the light chain and heavy chain fused genes for ME361 are excised from the appropriate pMN plasmid described in the examples above by EcoRI and Hindlll, and the ends are filled in with DNA polymerase (Klenow fragment) . This ligation is performed by digesting both pHEKneo and pHEKgpt with Xhol, and filling the ends in as above.
• the blunt ended heavy chain fused gene is ligated to linearized pHEKneo, and the light chain ligated to linearized pHEKgpt, resulting in the expression vectors, pHEKneo HfosLZ/ME361 and pHEKgpt L/ME361.
• the plasmids containing OKT3 H and L sequences are transfected into mouse myeloma Sp2/0 cells. Approximately 1-20 ⁇ g Ab/mL of medium can be produced in this way.
• the supernatants containing ME361 Fab and OKT3 Fab were subjected to immunoaffinity chromatography using the monoclonal antibody 12CA5 [Wistar Institute,
• the first Fab-domain fragment is mixed with the second Fab-domain fragment which is similarly in solution, resulting in the reciprocal leucine zippers becoming associated.
• the two Fabs, Fab ME361/fosLZ and Fab OKT3/junLZ were mixed together at approximately 0.5 mg/ml to form a bifunctional antibody.
• the resulting bifunctional antibody is characterized by the first Fab (ME361) as a binding site for the human melanoma antigen and the second Fab (OKT3) as a binding site for the CD3 T cell antigen.
• the bifunctional antibody will be tested for its ability to target CD3 positive T cell clones for lysis of the melanoma cell by the technique described by Perez et al, Nature. 316:354-356 (1985); Staerz et al, Proc. Natl. Acad. Sci. USA. 81:1453-1457 (1986);
• the pMNI inserts are excised with EcoRI and Hindlll, the ends filled in with DNA polymerase (Klenow fragment) and ligated into pCDM8 [B. Seed, Nat re, 3_29 . :840-842 (1987)] cut with BstEl and the ends filled in. Constructs are checked for the correct orientation.
• Plasmids pCDM8-Ll/ME361 and pCDM8-H2/fos/ME361 are cotransfected into human U293 cells using DEAE- dextran and chloroquine [B. Seed et al, Proc. Natl. Acad. Sci. USA, 84.:3365-3369 (1987)]. After 24 hours, the cells are grown in a synthetic medium [ITS, Hybritech] which does not contain any gamma globulin. The medium is harvested for 3-4 days. In the same way the light and heavy chains for OKT3 are produced. About 100-200 ng Ab/ml medium can be produced.
• EXAMPLE 5 EXPRESSING BIFUNCTIONAL ANTIBODIES IN E. COLI This example provides an alternative method to that of Example 1 for producing the antibodies of the invention.
• Plasmids also contain appropriately placed restriction sites to permit the introduction of light or kappa (K) chain sequences and the F d segment of the heavy chain (V H -C H1 ) of the selected antibody donating the Fab fragments.
• the plasmids pHc2/fosLZ and pHC2/jun LZ were produced as described in Example 1.
• the K chains were amplified using the polymerase chain reaction from total RNA extracted from the hybridomas ME361 (anti-target) and OKT3 (the anti-effector) .
• the amplified material after digestion with Sa and Xbal was cloned into pLcl digested with the same two enzymes.
• the plasmids obtained are called pLcl-ME361K and pLcl-OKT3K.
• the K sequence is excised from pLcl and ligated into the corresponding pHc2/fosLZ and pHc2/junLZ so that both the K and V H -C H1 sequences form a bicistron with the lacZ promoter.
• the V H -C m region was amplified from total RNA from hybridomas ME361 and 0KT3.
• the amplified material after digestion with Sail and Spel was cloned into pHc2/fosLZ and pHc2/junLZ digested with Xhol and Spel to give pHc2/fosLZ/ME361 V H -C H1 and pHc2/junLZ/OKT3 V H -C H1 .
• the K chain DNA was excised from pLcl/ME361K by digestion with EcoRI and Kpnl, and the K chain DNA was force cloned into pHc2/fosLZ/ME361 V H -C H1 which had been digested with EcoRI and Kpnl.
• the OKT3 K chain DNA was cloned into pHc2/junLZ/OKT3 V H -C HI .
• These two plasmids were transfected into a bacterial host cell, ___. coli JM105, or any strain which overproduces the lac repressor.
• the transformed cells were grown in hybrid culture from mid to late log phase when IPTG (to 1 mM) was added to induce expression of the lac Z promoter. After 2-6 hours of induction, the cells were selected for ampicillin resistance, harvested by centrifugation, resuspended in a buffered sucrose solution and subjected to mild osmotic shock by dilution with 4 volumes water to release the Fab from the periplas ic space. After 30 minutes on ice, the suspension was centrifuged [Pluckthun et al. Methods of Enzvmology. 121:497-515 (1989)].
• the supernatant was subjected to immunoaffinity chromatography using the monoclonal antibody 12CA5 [Wistar Institute, Philadephia, PA] attached to the column which binds the decapeptide on the tail of the heavy chain. From the column, the Fabs were eluted in a concentrated form.
• DNA encoding polyglutamic acid is synthesized as an oligonucleotide with, at the 5* end, the nucleotide sequence for the restriction enzyme Spel, ACTAGT, followed by the codons for three glycine residues (GGN) to act as a spacer, followed by the codons for 10 glutamic acid residues (GAA/GAG) .
• GGN glycine residues
• GAA/GAG amino acids residues
• the number of glutamic acid residues is not critical, but should be sufficient to form a strong interaction with polylysines via salt bridges.
• the glutamic acid residues can be interspersed with codons for small neutral amino acids, such as alanine and serine.
• a sequence complementary to the polyGlu sequence is synthesized with the nucleotide sequence of the restriction enzyme Xbal, TCTAGA, at its 5; end, followed by nucleotides complementary to those encoding glutamic acid, followed by those for glycine.
• oligonucleotides are annealed and then ligated to pHel cut with Spel (which joins the polyGlu sequence to DNA encoding a TAG peptide in the plasmid pHcl) . Plasmids containing the oligonucleotide are checked to identify those that retain the Spel site, 5* of the DNA encoding the glutamic acids. From such a plasmid, the DNA encoding glutamic acid and the TAG peptide are excised with Spel and EcoRI and the fragment ligated to pMNI containing the heavy chain leader sequence of MaB 17-1A, also cut with Spel and EcoRI.
• Plasmid encoding the OKT3 heavy chain linked to polylysine is constructed as described above for ME361, except the codons encoding glutamic acid are replaced by those for lysine, AAA or AAG, and the V H + C H1 DNA for OKT3 are used.
• These Fabs containing polyglutamic acid and polylysine may be expressed as follows.
• the heavy chain DNA constructs are transferred into expression vectors, pCDM ⁇ or pHEk, and co-transfected with their corresponding light chains into mammalian cells, as described for the Fabs containing leucine zippers.
• Bifunctional Fabs are formed by mixing of the two different Fabs. The resulting antibodies are tested as described in the examples above.
• Fabs The elution of Fabs was followed by first concentrating the elution fractions by trichloracetic acid precipitation. The precipitates were redissolved in Laemmli sample buffer before sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS/PAGE) . Proteins were transferred to nitrocellulose and the presence of Fabs was detected by a goat anti-mouse Fab, followed by swine anti-goat IgG conjugated with alkaline phosphatase.
• SDS/PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
• modified 0KT3 Fabs prepared according to this invention was determined by FACS analysis using a fluorescein labeled anti-mouse Fab. Conventional FACS analysis was performed using a low concentration of the fluorescein labeled second antibody to obtain a low background of nonspecific binding. The results are reported in Table 5 below.
• an antibody binds 5 to 10 times that of a Fab.
• the increased binding detected with the mixture of Fabs is the expected result if the Fabs form a dimeric Fab via the leucine zipper construct of this invention.
• these results demonstrate that the hinge region of the recombinant antibody construct retains sufficient flexibility to permit the individual Fab fragments to bind in a manner similar to that of normal antibodies. If the hinge region were adversely affected by the modification of the leucine zipper, the rigidity of the Fab antibody binding regions would have reduced the ability of the dimeric recombinant antibody to bind the target.
• Binding of the c-fos LZ/Fab ME361 to human melanoma cells WM793 [M. Herlyn et al. Cancer Res.. 45_:5670 (1985)] is measured by indirect immunofluorescence analysis in the cytofluorograph [Coulter, Inc., Miami, Fl.] using fluorescemated goat anti-mouse F(ab*) 2 antibody to detect binding of LZ/Fab ME361 to the melanoma cells.
• Antibody sensitized cells are incubated first with either Mab 12CA5 specific for the tag decapeptide or rabbit anti-LZ antibody followed by the addition of fluoresceinated goat anti-mouse F(ab') 2 or goat anti-rabbit IgG antibody.
• Colon carcinoma cells SW1116 [H. Koprowski et al, Somat. Cell Genet. , 5 . :957 (1979)] are used as ME361 antigen-negative target cells.
• C-jun LZ/Fab OKT3 is a negative antibody control. The binding of c-jun LZ/Fab OKT3 to cytotoxic anti-human melanoma T cell clones [R. Somasundaram et al, AACR
• Cytotoxic Assay Melanoma cells WM793 and colon carcinoma cells SW1116 (negative control) are labeled first with siCr, then incubated with different concentrations of the LZ/Fabs of this invention, either singly or in a recombinant antibody. Concentrations may range between about 1 to about 10 ⁇ g/ml. Thereafter increasing numbers of effector cells (effector-to-target ratios of between about 5 to about 50) previously activated with 10 U/ml of recombinant IL-2 [ ] are added to the cells. Lysis of cells is the positive result in WM793 cells. Negative results are present in the SW1116 control cells for the ME361 antigen. These results are compared to measure specific antibody-directed killing.

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