CA2038911A1 - Catalytic antibody components - Google Patents

Abstract

CMS Docket No. 370068-4960 ABSTRACT OF THE DISCLOSURE
Catalytic antibody components, methods for producing catalytic antibody components, methods for using catalytic antibody components, in particular, single chain and smaller components are disclosed.
Catalytic antibody components able to promote the cleavage or formation of an amide, peptide, ester or glycosidic bond, and which are prepared from monoclonal catalytic antibodies, catalytic autoantibodies or by site-directed mutagenesis are disclosed. Methods of using catalytic antibody components alone or in combination with other antibody components or other biological moieties are disclosed.
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Description

C!IS Docket 11~. 370068-4960 ~ ~ 3 ~

FIE~D ~:rNVENTION
This invention pertains yenerally to components of ~ntibodies capable of catalytically enhancing the rate of a chemical reaction. More specifically, this invention relates to ~omponents of catalytic antibodies, e.g., heavy and light chains, which are capable of catalytically enhancing the rate of a chemical reactionO This invention also relates to methods for obtaining the catalytic components.
Several publications are refsrenced in this application by Arabic numerals within parentheses in order to more fully describe the state of the art to which this invention pertains as well as to more fully describe the invention i~self. Full citations for these references are found at the end of the specification immediately preceding the claims.
BACKGROUND OF TXE INV~NTIO~
Antibodies are well known to bind antigens and it is generally recognized that the antigen-binding segment of antibodies is composed of the variable portion of a heavy (H) and a light (L) chain. Both of these chains are thought to be important in defining the paratope conformation to one that binds antigen with high af~inity. It has recently been found that antibodies can catalytically enhance the rate of chemical reactions. In U.S. Patent No. 4,488,281, it is disclosed that catalytic antibodies can bind a substrate, cause the conversion thereof to one or more products, and release the product. The catalytic antibodies may be prepared by immunological methods wherein they are elicited to antiyens, as taught, for exampl~, in U.S. Patent No. 4,888,281.
Fab fragments o~ an antibody catalyze hydrolysis of an amide bond (1). Fv fragments, which are heterodimers consisting of the variable regions of associated light and heavy chains of an ankibody, have ~`~ '.'. ''.

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been shown to catalyze ester hydrolysis (2). These antibody components are not known to catalyze the cleavage or formation of peptide bonds, a class of reactions which is energetically more demanding.
Iverson and Lerner (3) report that while peptid~ bond cleavage is very energetically demanding, cleavage of a peptide bond by a catalytic antibody is enabled by the presence of a metal trien cofactor and will not take place without the presence of such a 10 cofactor. The trien complexes of Zn(lI), Ga(III), Fe(III), In(III), Cu(II~, Ni(II), Lu(III), Mg(II) or Mn~II) were most favored. However, a naturally occurring autoantibody able to selectively catalyze the cleavage of the peptide bond between amino acid 15 residues 16 and 17 of the neurotransmitter vasoactive intestinal peptide (VIP) without any metal cofactor, has been reported by Paul (4, 5).
It is also known that antibody binding is energetically most favored by the presence of the 20 entire H-chain and L-chain binding site (6). The VH
fra~ments of anti-lysozyme antibodies bind the antigen with an affinity of only 10% of the intact antibody (7). L-chains are also likely to participate in antigen binding int~ractions, although most studi~s 25 suggest that the contribution of L-chains is smaller than that of H-chains (8-10). It could not be expected that an antibody component smaller than an intact catalytic antibody would possess the favorable steric conformation provided by the intact catalytic antibody 30 to permit the catalysis of a peptide bond without the assistance of a metal trien cofactor as taught by Lerner and Iverson.
The reports of Fv and Fab catalysis of ester and amide bonds do not disclose that other types of 35 heterodimers catalyze any chemical reactions (8-10).
heterodimer not known or expected to catalyze chemical 230390- 17:06 . .

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reactions is the heterodimer consisting of an intact H-chain and intact L-chain linked by at least one disulfide (S-S) bond. Another heterodimer not known or ~xpected to catalyze chemical reactions is a 5 heterodimer analogous to the Fab, but consisting of the Fd- fra~ment (the Hlchain with the Fc portion removed) linked to or associated with an intact L-chain by non-covalent bonding (e.g. hydrogen bondin~, charge interaction or similar associa1ion), in contrast to the Fab which consists of the Fd fragment linked to the L-chain by at least one disulf:ide bond.
Heavy chain homodimers and light chain homodimers have heretofore not been shown to catalyze chemical reactions. It would not be expected that these homodimers would have catalytic activity because the classic binding function of antibodies is considered to require the combination of the variable regions of both a light and heavy chain, or at least a heavy chain (8, 11-13). Catalytic light and heavy chain homodimers would be advantageous because they consist of the same or similar components, and thus could be manufactured with less effort than is required to manufacture a standard antibody or a heterodimer.
There are obvious advantages that single chain proteins offer over multichain proteins (antibodies), both from the point of view of structure-function analysis as well as pharmacological and therapeutic stability. It would be advantageous if the binding and catalytic domains on an antibody were either the same or closely positioned to onP another such that the benefits of catalytic activity could be achieved by a simple protein as opposed to a multichain antibody. Heretofore, the art has not demonstrated the capability of using such components of an antibody for catalytic purposes. Similar advantages are offered by dimers formed of the several combinations of light and ~30390- 17:06 `' .

CMS Docket No. 370C68-4960 2~3~

heavy chains.
It is known to use a catalytic antibody to convert a prodrug to a drug (:L4). However, a catalytic component able to conv~rt a prodrug to a drug, or a protoxin to a toxin has special advantages, particularly when the catalyt:ic component is incorporated into a fusion or chimeric protein with a biological binding agent able to bind to cells or tissues which it is desirable to contact with the drug or toxin.
OBJ~CT~ ~F T~13 INVE~TION
It is therefore a general object of the invention to provide components of antibodies which enhance the rate of a chemical reaction.
It is a further object of the invention to identify components of catalytic antibodies which enhance the rate of a chemical reaction and which are simpler in structure than the catalytic antibodies from which they are obtained or which heretofore have been used for catalysts.
It is still a further object of the invention to provide methods for obtaining components of catalytic antibodies, which components retain the catalytic activity of the parent antibody and which can be used to enhance the rate of chemical reaction.
It is still a further object of the invention to provide a variety of methods for obtaining catalytic components of catalytic antibodies.
It is still a further object of the invention to conduct chemical reactions using catalytic components of catalytic antibodies.
~MMARY OF ~ INV~N~ION
The invention is broadly directed to components of antibodies which enhance the rate Qf a chemical reaction. The components of the antibody which have been found to be catalytic include the Fab 230390- 17: 06 -- '~

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portion of an antibody, the Fv portion thereof, a light chain, a heavy chain, a mixture of the unassociated light and heavy chains, dimers formed of the various combinations of light and hea~y chains, a variable fragment of a light chain, a viariable fragment of a heavy chain, a catalytic domain of a light chain, and a catalytic domain of a heavy chain. These components can catalyze reactions with high turnover and without themselves entering into the reactions and are advantageous over whole catalytic antibodies.
The catalytic components can be obtained in a number of different ways. Broadly, the components can be obtained from whole catalytic antibodies or autoantibodies which are created in methods known in the art, including immunological methods employing transition state analog compounds to elicit the antibodies. The catalytic components, e.g., light or heavy chains of a catalytic antibody, can be prepared by cleaving purified antibody into certain fractions and then reducing and alkylating those fractions to cleave the bonds connecting the light and heavy chains.
In still other methods, the sequence of the variable region of a catalytic antibody is determined and a gene coding for th~ variable region o~ the catalytic antibody is inserted into a cell and the variable region is then expressed in said cell.
BRIEF DEBCRIPTIO~ OF ~ DRAWING8 Fig. 1. Diagram of a prototypical IgG molecule.
Fig. 2. Reverse phase HPLC Purification of mono (125I-TYR10)-VIP
Fig. 3. VIP hydrolytic activity resides in the Fab Fragment.
Fig. 4. Identification of VIP fragments produced by IgG and comparing catalytic IgG and non-catalytic IgG.
Fig. 5. Demonstration of disaggregation to produce 230390-17:06 CMS Docket IJo. 370068-4960 2~3~

Fd- and L chains.
Fig. 6. ~IP hydrolysis by intact IgG, Fab and antibody single chains, as a function of increasing IgG, Fab and Fd/L25 concentrations, showing that progressive dissection of the antibody resulted in increased hydrolytic activity.
Fig. 7 Fortuitous preparation of catalytic dissociated L-chains of VIP-specific antibodies.
Fig. 8. VIP hydrolytic antibody synthesis by cultured EBV transformed lymphocytes.
line.
Fig. 9. Expression plasmid for Fv~ 2 fusion protein and diagram of ~xpression fusion protein.
Fig. 10 - 12. Chemical reaction pathways for production of pro-ARA-C.
DETAI~D D~CRIPTION OF T~E INVENTION
D~finition~
Chemical reaction refers to a reaction wherein at least one reactant is converted to at least one product. Such chemical reactions include chemical reactions which can be catalyzed by enzymes such as, for example, oxoreductases, transferases, hydrolases, lyases, isomPrases and ligases as well as chemical reactions for which no catalytic enzymes are Xnown, such as, for example, oxidations, reductions, additions, condensations, eliminations, substitutions, claavages and rearrangements.
The term "animal" as used herein refers to any organism with an immune system and includes mammalian and non-mammalian animals. The term "substrate" is synonymous with the reactant in the chemical reaction and can be any of a number of molecules and biomolecules including but not limited to proteins, phospholipids, carbohydrates (e.g., glycogen, glucose, etc:.), drugs (including abused substances and drugs from exogenous sources).

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Antib~dy and immunoqlobulin refer to any of several classes of structurally related proteins that function as part of the immune response of an animal, which proteins include IgG, IgD, IgE, IgA, and IgM and related proteins. Antibodies are found in plasma and other body fluids and in the membrane of certain cells.
Under normal physiological conditions (e.g. absent immun~logical dysfunction or human intervention) antibodies are produced by B cells (or the functional equivalent) of an animal in reaction to the entry of proteins or other chemical subr,tances which that animal is not immunologically tolerant of into the tissue or body fluids of that animal.
The examples of preferred embodiments of the present invention generally relate to IgG. However, the terms antibody and immunoglobulin as used herein refer to any class of antibody, including IgD, IgE, IgA, IgM and related classes and subclasses~ An antibody as described above may also be referred to as a "physiological antibody" in order to clearly distinguish an intact antibody, as is normally produced by an animal, from the antibody components of the present invention.
Autoantibodies in accordance with the invention may be naturally occurring antibodies produced by the immune system of an animal which bind to the animal's own cellular components and which are not elicited by specific immunization against a target antigen. Autoantibodies recognize a self-antigen, i.e., any antigen which the body makes using its own genetic code. Thus, self-antigans are di~tinguished from foreign antigens (e.g., bacterial, viral antigens).
The term "substrate" as defined herein can be tha same as or different from the self-antigen.
Peptide bond as used herein refers to an amida bond linking two adjacent amino acid residues and 230390-17:06 ~, , : ; :

CMS Docket ~lo. 370068-4960 2 V ~

is generically represented by the following formula An amino acid consists of a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom and a distinctive group referred to as a "side chain" (Rl and R2 in the formula above~. Amino acid as used herein includes t;he twenty naturally occurring amino acids which comprise the building blocks of proteins. It is understood ~y those sXilled in the art that when either of the adjacent amino acids is proline, the respective side chains Rl or R2 are bonded to the adjacent nitrogen atoms to ~orm the characteristic 5-membered proline ring.
The substrate containing the peptide bond or bonds to be cleaved can be any proteinaceous molecule such as, for example, a regulatory protein or a structural protein, and include6, but is not limited to, peptide hormones (e.~., insulin, growth hormone, secretin, etc.), peptide neurotransmitters and neuromodulators (e.g., vasoactive intestinal peptide, endorphins, enk phlins, bradykinins, substance P etc.) tumor proteins (e.g., oncogene products, carcinoembryonic antigens, etc.~, bacterial proteins and viral proteins (e.g., human immunodeficiency viral(HIV) gp 120, influen~a glycoproteins, etc.~.
The rate enhancement achieved by the antibody components according to the invention is either catalytic or stoichiometric. Thus, components which catalytically enhance the rate o~ the reaction are "catalytic components" and components which stoichiometrically enhance the rate of the chemical reaction are "stoichiometric components'l.

230390- 17: 06 CMS DDcket No. 37~0~8-4960 2 ~ 3 ~

A catalytic component part of an antibody in accordance with the invention is a substance which is capable of changing the rate o~ a chemical reaction, all other conditions (e.g., temperature, reactant/substrate concentration, etc.) being the same and which does not enter into the chemical reaction and therefore is not consumed in the reaction. It is also a substance which exhibits the capability of converting multiple moles of reactant/substrate per mole of catalytic component part; which, ~rom a mechanistic viewpoint, binds the reactant/substrate, effects the accelerated conversion of the reactant/substrate to the product and then r leases thP product; and which changes the rate of the chemical reaction without shifting the position of the equilibrium. The aforementioned definitions are characteristics of ideal catalysts. However, in practice, even the best of catalysts become poisoned or deactivated by contamination in the reaction system or as a result of chemical or physical destruction during the reaction process. For reasons well known in the art, the true operation of a catalyst may be obscured by components of the reaction system or by the condition o~ the reaction environment.
A stoichiometric component part in accordance with the invention enhancPs the rate of the chemical rsaction stoichiometricallyO It enhances the rate of the reaction but, unlike a catalytic component, is stoichiometrically consumed during the reaction. Thus, the term "stoichiometric enhancement" implies that the component causing the observed rate enhancement enters into the reaction as a reactant and is consumed in the process.
The art has adopted certain working definitions to express catalytic activity. These expressions are [1] kC~t, or "turnover" and [2] kCat/k~Cat, 230390- 1 7: 06 '': . ~ ' ' " ,~';. ~
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CM~ Dock*t l~o. 370068-4960 the "rate snhancement factor". Turnover indicates the number of molecules of reactant/substrate which can be converted to product per mole of catalytic component per unit time. For example, if a molecule exhibits a turnover of 103 molecules of substrate per minute and the molecule maintains its catcllytic activity for 24 hours at room temperature and at its optimal pH, each molecule of catalyst would then make a total of ~.4 x 106 conversions, indicating its catalytic bPhavior.
This total conversion is to be distinguished from the total conversion in a stoichiometric reaction, which will never exceed 1.0, no matter how long the reaction is carried out. The rate enhancement factor is a dimensionless number which expresses the rate of r~action in the presence of catalyst to the rate of reaction in the absence of catalyst, all other reaction conditions (e.g., reactant concentration, temperature, etc.) being equal.
Reference has been made to component parts of an antibody. These component parts are also correctly referred to as fragments or antib~dy fragments. These parts are defined by way of example with reference to the IgG molecule, but it will be understood by those skilled in he art that these components may be derived from any of the other antibody classes ~IgA, IgE, IgD, IgM and related classes and subclasses~. The IgG
molecul~ may be described as a "Y" shaped protein made up o~ four polypeptide chains linked together by disulfide bonds (Fig. 1). The tops of the "Y" are the N-terminals of the protein chains which comprise IgG
tetramer. ~o identical heavy chains ~also known to the art as gamma chains, hereinafter H-chains) extend from the stem of the "Y" into the arms; two identical light chains ~also known to the art as kappa or lamda chains depending on their antigenic ~tructure, hereinafter ]i-chains) are confined to the arms. Each 230390- 17: 06 ClilS Dock~t No. 3700~8-4960 2~g~ ~

polypeptide has both constant regions (C regions) and variable regions (V regions). The V regions are located in the N-terminal domains of the H and L chains. In the V region are three areas of greatest sequence variability known as the hypervariable or complementarity determining regions ("CDRs"). The H-and L-chain CDRs together form the antigen binding site. Sequence variability in ~hain CDRs underlies the range of antibody specificities that the immune system produces. All antibodies of a given type have the same constant regions, but the variable regions dif~er from onP clone of B cells to another. At the end of each arm, the L- and H-chain variable regions fold to create an antigen binding site comprising the CDRs as described above. The H-chains are about 50 kD in size, and the L-chains are about 25 kD in size.
The point at which the H-chains separate to form the top of the "Y" is known to the art as the hinge region. The IgG may cleaved by papain enzyme ?0 above the hinge region (11) into two Fab fra~ments and one Fc fragment. As described above, the top of the "Y" is known to include the variable region, which performs the specific binding function of th~ antibody.
The Fc fragment represents the bottom of the "Y" and serves complemPnt fixation and other non-~inding functions. The Fab fragment is a heterodimer cleaved from each side of the top of the "Y", and is formed of intact L-chains and of an approximately 25 kD partial H-chain known to the art as an Fd fragment.
The Fv fragment is a 25 kD heterodimer similar in structllre to the Fab fragment described above, but consisting of shorter segments of the N-terminal sequences of the H- and L-chains respectively from the top of the "Y" (15). The Fv includes the variable regions of the constituent H- and L-chain fragments.

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CMS Docket No. 37DD68-49~0 2 ~38 The Fv fragment, in turn, may be separated into two approximately 12.5 kD single chain fragments or components referred to as a variable ~ragment of an H-chain (VH) and a variable fragment oX an L-chain (VL) (15)-The H- and L-chain c~mponents may be separated from an intact antibody as complete unassociated chains, or may be produced de novo as complete unassociated chains by one of the recombinant genetic techniques known to the art from a gene coding for an L- or H-chain, or may be produced by B cells or hybridoma cells selected for the property of producing unassociated H- or L-chains. These unassociated H and ~ chains may then be used according to the present invention as non-associated catalytic chains, or may be allowed to associate by methods known to the art to produce H/L heterodimers, H/H homodimers, or L/L
homodimers.
The variable fragment of the H- or L-chain is the peptide sequence containing the variable region, and may be further reduced to a minimum peptide sequence defining the variable domain which retains the binding property inherent in that amino acid sequence.
The catalytic domain is the minimum peptide sequence which retains the catalytic property inherent in that amino acid sequence.
Each of these componants may be sequenced and expressed by recombinant methods known to the art. The catalytic domain may be usefully recombined by recombinant technology with other useful genetic sequences to produce chimeric proteins with catalytic properties. The catalytic components may be produced de novo by means of mutagenesis techniques known to the art applied to the genetic sequences of antibody H- or L-chain variable domains either before or after the genetic se~lence for a variable domain has been 230390- 17:06 CUS Dock~t ~o. 370068-44G0 2 ~

inserted in a host cell.
The catalytic component parts may be associated with molecules having functions different ~rom the component parts, or may be associated with each other.
"Associated" or "associate" when used to describe the relationship between component parts or other molec-lles means either covalent binding (for example, disulfide bonds or other chemical bonds well known to the art) or non-covalent binding (for example, hydrogen bonding, charge interaction or other non-covalent binding well known to the art)O The terms associated or associate may be qualified. For example, "associated non-covalently" xefers only to components or other molecules which are non-covalently bound. The term "unassociated" refers to components or molecules which are not linked by either covalent or non-covalent bonding.
Detail~ D~scriDt~on of the Drawin~
Fig. 1. Diagram of a prototypical IgG molecule.
Note that the COOH terminal comprises the Fc portion of the antibody. The junction of the "Y" is the hinge region. The H2N termini repre-cent the variable regions which are critical to antigen-specific binding.
Fig. 2 0 Reverse phase HPLC Purification of mono (125I-TYR10)-VIP.
Fig. 3. VIP hydrolytic activity resides in the Fab Fragment.
Fig. 3A. Silver stained SDS PAGE of Ig&, Fab VIP.
Fig. 3B. Hydrolytic activity resides in the Fab fragment.
Fig. 4. Identification of VIP ~ragments produced by IgG, illustrating cleavage at the Gln-~et bond; and comparing catalytic IgG and non-catalytic IgG. VIP was treated with immune or nonimmune IgG, extracted on C-18 cartridges and subjected to reverse phase HPLC. Most o~ the A124 absorbing material seen a~ter treatment of 230340- 17: 06 ' ' .

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CMS Docket llo. 3/0068-4960 1~

VIP with the antibody was in a peak with retention time similar to that of intact VIP (21.3 min). Peptides A
and B were missing after treatment in bu~er or nonimmune IgG. These peptides were purified by rechromatography, (4A and 4B) respectively) and identified by amino acid sequencing.
Fig. 4A. HIS~SER-ASP-AI~-VAL-PHE-THR-ASP-ASN-TYR-THR-ARG-LEU-ARG-LYS-GLN.
Fig. 4B. MET-ALA-VAL-LYS-LYS-TYR-LEU-ASB-SER-ILE-LEU-ASN.
Fig. 5. Demonstration of disaggregation to produce Fd- and L chains.
Fig. 5A. Separation of reduced, alkylated antibody single chains. Gel filtration profile (Superose 12) of reduced, alkylated Fab. A minor early eluting peak and a major peak 5retention time 31 min~ are evident.
Fig. 5R. SDS-PAGE and silver staining revealed a 59 kD band in minor peak (lane 2) and a 24kD band in the major peak (lane 3). SDS produces disaggregation, thus it is necessary to run sample under native conditions as in 5(C~ below to demonstrate that the disaggregation is due to prior treatment of the sample.
Fiy. 5C. Native PAGE ~without disaggregation induced by the separation technique) of the reduced, alkylated Fd-, L chain mixture (lane 2) and marker proteins (lane 1). Demonstrates that the prior reduct,ion and alkylation resulted in the separation of the Fd- and L-chains.
Fig. 6. VIP hydrolysis by intact IgG, Fab and antibody single chains, as a function of increasing IgG, Fab and Fd-/L25 concentrations, showing that progressive dissection of the antibody resulted in increased hydrclytic activity. This data supports the concept that catalytic components may be produced from non-catalytic antibodies by progressive dissection.
Fig. 7. Fortuitous preparation of catalytic 230390 -17: 06 , ' ~, CMS D~cket Uo. 370û68-4960 ~3~

dissociated L-chains ~f VIP-specific antibodies.
Fig. 7A. Puriication of VIP antibody L-chains.
Fig. 7B. Reducing SDS-PAGE of the protein peak purified by chromatofocusing. (retention time 26 min.) ~tained with silver (lane 2), ianti-L-chain antibody (lane 33 and anti-H-chain antibody (lane 4). Lane 1 shows silver stained marker proteins. Note lack of stain with anti-H-chain antibody.
Fig. 7C. Reducing SDS-PAGE of a control intact IgG
preparation ~affinity purified antibody from subject ~0). Land identities are as in (B).
Fig. 7D. VIP hydrolysis by purified antibody L-chains. Saturation kinetics of VIP hydrolysis by purified L-chains of VIP-autoantibodies (4 ng per assay tube). Data are fitted to the Michaelis-Menten equation.
Fig. 8. VIP hydrolytic antibody synthesis by cultured EBV transformed lymphocytes.
Fig. 9. Expression plasmid for Fv-Il-2 fusion protein and diagram of expression fusion protein. To create a single-chain recombinant plasmid is assembled (~0~ employing a DNA segment derived from a catalytic mAB, encoding the VN joined to a DNA segment encoding the VL by a 45-bp liner; VL was in turn joined to a DNA
segment encoding interleukin-2 as shown. The assembled gene is under the control of the T7 promoter.
Fig. 10 - 12. Chemical reaction pathways schemes 1, 2, and 3 for production of pro ARA-C.

230390- 17: 06 CMS Dock~t l~o. 370063-4960 ~pe~if ic Embo~ ent Several different components of antibodies are capable of catalyzing a chemical reaction. The components may be catalytic components of antibodies, which antibodies themselves have catalytic activi-ty for a given reaction, or, the catalytic components may be components of antibodies which themselves do not exhibit catalytic properties.
The several catalytic component parts include the Fab portion or an antibody, the Fd fragment of an H- hain, the Fv portion of an antibody, an L-chain, an H-chain, a mixture of an L- and H-chain wherein the chains are present as unassociated, ie free monomers, dimers which may be either heterodimers or homodimers, a variable fragment of an L-chain, a variable fragment of an H-chain, a catalytic domain of an L-shain, and a catalytic domain of an H-chain. The Fv and Fab components t as noted above, are not known for the promotion of cleavage or formation of peptide bonds, but are known for catalysis of less energetically d~manding reactions such as amide or ester hydrolysis.
It has now been shown that the catalytic activity of an antibody able to promote the cleavage or formation of peptide bonds is found to be present in, in addition to the IgG, in the Fab component thereof and also in a preparation of dissociated H and L-chains, each having a molecular mass of 25 kD. The I~G, the Pab, and the dissociated Fd and L-chains each catalytically hydroly~ed vasoactive intestinal peptide ~YIP~.
The several catalytic components of the antibody or catalytic antibody may be obtained by different methods. Catalytic properties o~ the several components may, on the one hand be present in an original antibody or catalytic antibody and be retained during the fragmentation process or, on the other hand Z3039~-17:06 .

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these properties may be created by the process of producing the component. The process of producing antibody components which are unconstrained by the complete structure of a physiologic antibody can yield a component with a catalytic property, for example, in a component produced directly or indirectly (by recombinant methods) fxom a non-catalytic antibody.
C~talytic ~utoantibo~ies A catalytic component of a catalytic autoantibody can be prepared by ~irst identifying an animal with an autoantibody to a self-antigen of the animal, isolating a serum fraction containing a plurality of antibodies, screening the serum fraction to identify an autoantibody which enhances the rate of a chemical reaction important to an autoimmune disease process, and obtaining a catalytic component of the autoantibody.
An animal with autoantibodies to a self-antigen of the animal is identified by measuring, in plasma samples or purified IgG from the animal, the saturable binding o~ the autoantibodies to the self-antigen of the animal itself, to a self-antigen of a different animal species which is identical or substantially identical to the self antigen of the animal or to a synthetic self-antigen which is identical or substantially identical to the self-antigen of the animal, using methods well known in the art. A catalytic au oantibody is identi~ied by screening autoantibodies for those which promote the catalytic cleavage or formation of a chemical bond of interest. Candidate autoantibodie~ are contacted with a sel~-antigen of the animal itself or to a self-antigen of a different animal species which is identical or substantially identical to the self-antigen of the animal, and the products o~ cleavage orformation of chemical bonds of interest of that self 230390- 17: û6 . .

C~1S Docket No. 370068~4960 ~ ~ 3 ~
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antigen, or substantially identical antigen, are detec~ed by methods well known to the art.
In another embodiment of the invention, the isolated autoantibodies are purified by standard methods and th~n ultrafiltered. The term "ultrafiltration" as used herein refers to a ~iltering process employing a membrane having pores with an average cut off molecular weight ranging from 1,000 to 10,000 Dalton~. Thus, for example, ultra~iltering an immunoglobulin with a molecular weight of 150,000 Daltons on a membrane with pores having an average cut off molecular weight of 10,000 Daltons w.ill cause molecules with mol~cular weights smaller than 10,000 Daltons to pass through the m~mbrane while the immunoglobulin will remain on the membrane~ This process activates the antibody cat lytic property and a small molecular weight inhibitor may be puri~ied from the ultrafiltrate.
The isolated autoantibodies are then screened ~or rate enhancement activity. Screening can be conveniently accomplished by treating a standardized solution of the reactant/substrate with an aliquot o~
medium containing the autoantibodies and measuring the presence of the de6ired product by conventional instrumental methods. This measurement can be readily conducted, for example, by spectrophotometric methods or by gas-liguid or high prassure liquid chromatography. By comparison with standardi~ed samples of the desired product or reactant/substrate, rates o~ reaction can be quantified.
Ratio~allY Desiqne~ Antib~e~
A catalytic component o~ a rationally designed catalytic antibody may be obtained starting with the methods taught in U.S. Patent No. 4,888,281.
According to such processes, a plurality o~ monoclonal antibodies is prepared to an antigen selected from the 230390- 17: 06 CMS Dock~t ho. 370068-4960 -` 2 0 ~

group consisting of ~i) the reactant, (ii) the raactant bound to a peptide or other carrier molecule, (iii) a reaction intermediate, (iv) an analog of the reactant, (v) an analog of the product in which the monoclonal antibody so generated is capable of binding to the reactant or a reaction intermediate, or (vi~ an analog of a reaction intermediate. The plurality of monoclonal antibodies so generated is screened to identify a monoclonal antibody which catalyses the reaction of interest and the monoclonal antibody which is desired to have the desired catalytic activity separated into its several components and those components screened for activity such that a catalytic component is obtained.
In still a further and related process, an animal is immunized with an antigen selected from the group consisting of (i) the reactant, (ii) the reactant bound to a peptide or other carrier molecule, (iii) a reaction intermediate, (iv) an analog of the reactant, (v) an analog of the product in which the monoclonal antibody so generated is capable of binding to the reactant or a reaction intsrmediate, or (vi) an analog of a reaction intermediatP, thereby generating antibody-producing l~mphocytes in said animal, antibody-producing lymphocytes are removed from the animal, those lymphocytes are fused with myeloma cells to produce a plurality of hybridoma ~ells each of which prvducas monoclonal antibodies, the plurality of monoclonal antibodies is screened to identify a monoclonal antibody which catalyzes the reaction, and, a catalytic component of that monoclonal antibody is obtained as further described below.
In yet a further related method, a catalytic antibody for a chemical reaction which is known to be catalyzed by an enzyme can be first obtained and then its components screened to identify and obtain th2 230390-17: 06 `

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Cl~15 Docket \lo. 37006~-b960 ~ o 2 ~ 3 ~

catalytic component. In this proces~, a plural1ty of monoclonal antibodies is produced to the enzyme, that plurality of monoclonal antibodies is screened to identify a monoclonal antibody which inhibits binding 5 of the reactant to the enzyme, and that first monoclonal antibody is recovered. Thereafter, a further plurality of anti-idiotype monoclonal antibodies to the ~irst antiboldy is generated, these are screened to identify a second monoclonal antibody which binds the reactant and catalytically increases the rate of reaction/ and that monoclonal antibody is reduced to its component parts which are screened to obtain a catalytic component of the monoclonal antibody.
In each of the above-described methods, if the catalytic component is known, the screening of the components may be omitted and the desired catalytic component of th~ antibody may be directly obtained.
Where the catalytic component part is an L-or H-chain of a catalytic antibody, the chain may be prepared by a process wherein catalytic antibody is purified and then cleaved into Fab and Fc ~ractions.
The Fab ~raction is then reduced and alkylated to cleave bonds connecting the ~- and H-chains, and the L-~5 and H chains are ssparated.
Where the catalytic component part is asingle,chain of a catalytic antibody, the chain may be prepared by dissociating the catalytic antibody into its component L- and H-chains and then separating those L- and H-chains. Separation may be achieved by passing the antibody through a column which is selective for molecular weight or charge. A certain percentage of tha antibody protein chains spontaneously dissociate during this process and appaar as separate single chain peaks. This appears tv be facilitated by separation after diluting the antibody to a concentration o~ less 230390- 1 7: 06 CMS Docket No. 370068-4960 2~

than 5 ~g/ml in detergent at alkaline pH.
Alternatively, L- and H-chains may be dissociated by a process wherein the antibody is reduced with a reducing agent selected from the group consisting of mercaptoethanoll dithiothreitol, and mercaptethylamine and thereafter the S~I groups on the reduced antibody are alkylated with an alkylation agent selected from the group consisting o~ iodoacetamide and iodoacetic acid.
In still a further mlethod for obtaining catalytic component parts of catalytic antibodies, an L-chain or H-chain Fd fragment thereof may be prepared by purifying the catalytic antibody, cleaving the purified antibody into Fab and Fc ~ractions, reducing and then alkylating the Fab fraction to cleave bonds connecting the L-chain and H-chain Fd fragment, contacting the L-chain and H-chain Fd fragment with a ligand or other binding molecule capable of binding to one or the other of said L- or H-chains under conditions conducive to binding, and, separating the bound L-chain or bound H-chain Fd fragment from the unbound components.
The catalytic component part may be a catalytic domain comprising a polypeptide which is a part of a variable region of a catalytic antibody or of an H- or L-chain thereof and which retains the catalytic activity. The catalytic component part may be replicated by expressing in a non-hybridoma cell a nucleic acid genetic sequence copied from the catalytic component part. For example, the component part may be replicated by inserting into a cell a fragment of a gene coding for said component part.
Alternatively, the catalytic component part may be a catalytic domain which is prepar~d by cleaving the variable region of the catalytic antibody into a series of peptide sequences, screening those peptide 230390-17:06 ' i, ~:
. ` : : :' - - ' ' '' CMS Docket ~lo. 37006a-4960 sequences to identify a peptide sequence having catalytic activity, and thereafter purifying the so-identified catalytic domain. In a preferred embodiment, the catalytic domain may be prepared in a process which includes the additional steps of claaving the peptide seguences to generate increasingly smaller peptide seguences and screening those cleaved sequences to identify those having catalytic activity. This step may then be repeated until no catalytic activity is detected in the cleavage products. The idPntified catalytic domain can then be purified. Once the catalytic domain i~ identified by determination of the peptide sequence thereof, copies may be synthesized.
The catalytic domain may also be prepared by determining the peptide sequence of the VL or V~ of the catalytic antibody, then synthesizing an overlapping series of homologous peptides representing sections of the peptide sequence of the variable region, screening the series of overlapping homologous peptides to select those with desirable catalytic properties and synthesizing the selected peptide sequence. A similar result may be achieved by determining thP nucleic acid sequence of the VL or VH f the catalytic antibody, cleaving the nucleic acid sequence into fragments or synthesizing overlapping oligonucleic acid subs~quences, and then expressing these VL or V~
subsequences in a cell line by known methods. The resulting series of subsequence peptides may then be scre~ned for desirable catalytic properties as described above.
In still another method, the catalytic domain may be prepared by determining the sequence of the variable region of the catalytic antibody, inserting into any cell (prokaryotic or eukaryotic) a gene coding ~or the variable reyion of the cataly~ic antibody, and, expressing the variable region in the cell. The 230390- 17:06 CMS Docket ~lo. 370068-b960 2~3~

inserted gene may code for a fragment of the variable region. The cell may be an animal cell, e.g. a mammalian cell, a plant cell, or a microorganism, e.g., bacteria, yeast, mold, protozoa, and fungi. If desired, the gene may bP subjected to mutagenesis before or after insertion into the cell.
In still a further method of the invention, the catalytic component part of the catalytic antibody may be produced by a process which includes inserting into any cell (prokaryotic or eukaryotic) at least one nucleic acid sequence coding for a variable region of the antibody, subjecting the nucleic acid sequence to mutagenesi~ before or after such insertion, screening the cell and its progeny ~or the presence of mutated variable regions of the antibody which demonstrate desired catalytic activity, replicating the cell, and, expressing the mutated nucleic acid sequence to produce a translati~n product with the desired catalytic activity.
In an additional process a population of cells producing antibodies, e.g. hybridomas, may be selected for those cells producing component parts of antibodies such as L- and H-chains in place of all or part of the production of intact physiological 2S antibodies.
The catalytic components of the invention may be usefully combined with or associated with one another or to molecules having other, non-catalytic, chemical, biological, or mechanical functions. The 3~ association may be non-covalent (e.g. hydrogen bonding or charge interaction or related types of association).
The association may also be covalent, utilizing any of the methods well known to the art to link the components to one another or to other molecules while retaining the desired functions o~ the componen s and the linked molecules.

230390-17:06 :' ' '~ ' C~ D~cket ~io. 37006B-4960 24 2~3~

Chimeric products may be prepared by expressing nucleic acid seguences coding for a continuous polypeptide sequence which contains a catalytic antibody component part and at least one other protein. The nucleic acid sequence thu~ may comprise a first nucleic acid se~uence coding ~or a catalytic component part of an antibody, and, at least one additional nucleic acid sequence coding for either the same catalytic component part, a different 10 catalytic component part, or at least one additional protein having a biological function different from that of the catalytic component part~ The additional protein may be a biological binding protein such as a ligand, for example, avidin, streptavidin, protein A, 15 and protein G. The additional protein may be an H-chain or L-chain of an antibody able to bind to an antigen of intPrest. Alternatively, the additional protein may be the variable region of an antibody able to bind to an antigen of interest.
Cells may be cr~ated ~hich express and secrete, in vivo, catalytic components, proteins or peptides according to the invention for therapeutic, diagnostic or industrial purposes. Cells may be taken from an animal or plant, genetically engineered to 2~ express (the protein may be designed by known methods to remain within the cell, to remain on ths cell surface or to ~e secreted from the cell) desirable catalytir components or chimeric proteins embodying one or more components, and then the cell may be 30 reintroduced into the animal or plant where thP
catalytic component will serve a desirable function e.g. a therapeutic, metabolic, immunological~ or diagnostic function.
The invention is further described in the 35 following examples.

~30390- 17:06 , CMS Docket No. 37006B-4960 ~3~

~xa~pl~ I
Puri~i¢atio~ of VIP ~pecific Catalyti~ AutoantibodieR
~rom ~uman Blood By ~inity Chro~atogrAphy Th~ IgG fractions containing VIP hydrolytic autoantibodies exhibit relatively tight binding of VIP.
This property may be used to purify specific catalytic autoantibodies on a VIP Sepharose column. Synthetic VIP ~10 mg) mixed with about 20,000 cpm (Tyr10-12sI)-VIP
was covalently coupled to 5 g CNBr-Sepharose according to the manufacturers instruction (16). Coupling e~ficiency was approximately 90~, judged by the amount of radioactivity that was immobilized. The VIP-Sepharose (4.5 ml gel) was incubated with 15 mg IgG in 3.5 ml 100 mM glycine, 50 mM Tris-HCL, pH 8.0 for ~ h at 4OC. The mixture was poured into a column, the gel washed with buffer until the A280 returned to baseline, bound IgG eluted with 0.1 M glycine-HCl, pH 2.7 and neutralized with 1 M Tris~HCl, pH 9Ø Analytical isoelectric focusing of this antibody preparation on ~HAST gels (pH gradient 3-10) followed by silver staininq revealed a ~eries of closely spaced bands with pl 6.5 to 8.5. SDS-gel electrophoresis under reducing conditions (5~ mercaptoethanol) follswed by silver staining and immunoblotting reveal~d that the antibody was composed of subunits corresponding to a 50 kD H-chain and a 25 kD L-chain. Tha af~inity puri~ied antibodies wer~ incubated with 210 pg (Tyr 10-125I)-VIP
for 3 h at 38OC~ the reaction mixture extracted on a Seppak C-18 cartridge and subjected to reverse phase HPLC (Fig. 2). An early eluting peak of radioactivity was noted ~retention time 10 min), distinct ~rom intact 1251-labeled VIP (retention time 21.0 min). This early eluting peak o~ radioactivity had a retention time identical to that of synthetic VIP(1-16) labeled with 12sI. It has previously bean shown that the unfractionated IgG cleaves VIP at the peptide bond 230390- 17:06 ~ .~
, CUS D~ckct No. 3700b8 4960 -` 2~3~

~etween residues lS and 17 (17). Data shown here indicated that the affinity purified material cleaves VIP at the same bond (Gln16-Mett7) (~ig 4).
~pl~ II
YIP ~ydroly~i~ b~ PuriL~iad Autoantibo~y A. Preparation of 12sI-labeled VIP Substrate.
The (Tyr10-125I)-VIP was prepared by known methods (29,1). Iodination of purified porcine VIP was by the chloramine T method in a sodium phosphate buf~er.
Following preliminary fractionation on a C18 cartridge, the reaction mixture was purified further by reverse phase HPLC on a Novapak C18 column using a gradient of trifluoroacetic acid in acPtonitrile for elution. Two well defined peaks of radioactivity were consistently obtained that were reactive with rabbit anti-VIP
antiserum in radioimmunoassay. Amino acid ssquPncing has shown that the early eluting peak of radioactivity (retention time 25.3 min~ corresponds to (Tyr10-l25I)VIP
and the second peak of radioactivity ~retention time 27-8 min) corresponds to di(Tyr10-125I)VIP/ Tyr22)VIP
(Fig. 2). The monoiodinated form of the peptide was preferred because it most closely corresponds in structure to unlabeled VIP.
B. Hvdrolvsis oP 125I-labeled YIP Substrate.
To evaluate the kinetics of VIP hydrolysis, 66~3 ng purified antibody was incubated with increasing concentrations of VIP in the presence of approximately 30 pM (Tyr10-i25I)-vIp The reaction was terminated with 10% trichloroacetic acid, a procedure that precipitated undegraded VIP and left the radioactive fragment (VIP 1-16) produced by antibody mediated hydrolysis in the supernatant. A plot of the reciprocals of the rate of VIP hydrolysis and the VIP
concentration was linear, suggesting conformity with Michaelis-Menton kin~tics. Xm and kC~t calculated from these data using the program ENZFITTER (Elsevier~ were 230390 17:06 .

CMS Docket Uo. 370068-4960 ~33~ ~

llO.4 nM and O.ll min-1.
Previous studies have indica~ed multiple turnovers of the autoantibody, based on the assumption that the autoantibodies detected in ~IP binding studies were responsible for hydrolysis of the peptide (17~.
Data shown here provide direct evidence ~or efficient catalysis by the autoantibodies.
~ xa~pls III
y~rolysis of VIP bY Iqt (Te~t ~ube AR~a~
A standard protocol to measurP presence of hydrolysis.
Final assay volume was set at 200 ~l but the volume of each component may vary depending on purpose of the assay.
All dilutions were made in degradation buffer (O.1 M glycine-HCl, 50 mM TRIS-HCl pH 8.0 with 0.0~5%
Tween). Sample IgG was typically be diluted to 1 mg/ml and 0.5 mg/ml starting concentration for the initial test. VIP specific antibody was be used at lower concentrations (approximately l ~g/ml).
12sI-VIP was diluted to 15,000 cpm per 50 ~l.
BSA (4~ stock) was diluted to give a final assay concentration of 0.1%. Usually, l00 ~l of 0.2%
BSA was added to the assay.
Assay tubes contained l00 ~l 0.2% BSA, 50 ~l 125I-VIP (15,000 cpm) and 50 ~l IgG sample. One se~ of tubes with buffer in the place of antibody was set aside as a control.
All tubes were capped and vortexed after all components are added.
Incubation was for 3 hr at 38C in the shaking water bath. Each tube was uncapped and l ml cold 12% TCA added to all tubes except TC and vortex.
Centrifugation was at 5800 rpm for 20 min. All tubes but the TC tube were aspirated. The pellets were counted in a gamma-counter (Fig. 4).

230390- 1 7:06 ' ' ' ' , , ~", :, ' .
`: :

CMS Docket No. 37C068-4960 2 ~

~mple IV
2rotei~ G-~pharo~e~ Puriflclltion o~_IgG ~ro~_Pln~ma The Protein G-Sepharose (Pharmacia) wa~
washed with water on a sintered glass funnel ~#36060, Pyrex) or in a column. At least 3 ml water per ml gel was used for each wash. The g~el was suspended at least 3 times on sintered glass funnlel or in column. The gel was then resuspended in 0.05 m TRIS-HCl pH 7.3 (start buffer) and packed into a column oP appropriate size (or allow to pack if using column that has been poured). One ml Protein G-Sepharose was used for each 1 ml human plasma. The column was then equilibrated with -3 column volumes of start buffer and run at 0.4 ml/min for 0.7 cm diameter column or 0.8 ml/min for 1 cm diameter column~ The sample was centriPuged (5,000 rpm, 10 min) and filtered on Millex-GS filter (0.22 ~M) and applied to the column (dialyzed ammonium sulfate precipitate of plasma) and run into the gel bed. The start buffer was added and run until A280 returns to baseline. (In the event that no peak was observed approximately 15 ml of start buffer was run.) The buffer was then changed to 0.1 M
glycine - HCl, pH 2.7 and eluted to the same flow rate.
Fractions (1 ml) were collected into tubes containing 50 Jll 1 ~ TRIS-HCl, pH 9 to minimize acid induced denaturation tthis brings pH to 7.8.). The column was re-equilibrated (2 column volumes) to skart buffer and stored in 20% ethanol at 45C.
Yc~mpl Pur~fiGatio~ O~ C~tal~tic Ch~ 0~ ~IP Autoantibodie~
The IgG from a human subject (code ~39) was subjected to afPinity chromatography on VIP Sepharose.
The affinity puriPied antibodies were then chromatographed on a mono-P column in three steps ~18).
The pH gradients used in these three chromatofocusing steps were 7.0 to 4.0, 9.0 to 6.0, and, Pinally, 10.5 230390-17: 06 CMS Dock~t Ho. 37006~-4960 29 2~3~

to 7.0~ The VIP hydrolytic antibody was recovered in the nonretained fraction during the first two chromatofocusing steps. In the third chromatofocusing StQp, a protein eluting between pH ~.3 and 7.8 possessed VIP hydrolytic activity (Fiy. 7A).
Analytical isoelectric focusillg followed by silver staining revealed a 6ingle protein band with pl 9.6 in thi~ preparation. SDS-PAGE (non-reducing) revealed two protein bands. The major banci had a molecular mass of 25 kD and was stainable with anti-human L-chain antiserum in immunoblots. The minor band had a mass of 55 kD and was also stainable with anti-L chain antiserum, suggesting that it is a L-chain dimer.
These data suggest that this preparation is composed primar.ily of L-chains derived from VIP-autoantibodies.
~n alternative method of SDS electrophoresis provided similar results. Purification of IgG antibody electrophoresed under reducing condition~ exhibited a 26 kD anti-L-chain stained band and a 61 kD anti-H
chain stained band (Fig. 7B).
The data supports the conclusion that the chromatofocused preparation was composed of L-chains free of detectable H-chain contamination. This L-chain fraction hydrolyzed VIP with kCat92.4 min~1and Km4.9~M
~Fig. 7D). The Kmvalue for the L-chain is about 45-fold larger than that of the starting IgG/ suggesting decreased binding affinity.
B~a~pl~ VI
~IP ~y~roly~is by Pur~e~ ~-Chain To evaluate the kinetics of VIP hydrolysis, about 3.7 ng puri~ied L chains were incubated with increasing concentrations of unlabeled VIP in the presence o~ approximately 30 pM (Tyr10-125I)-VIP. The reaction was terminated with 10% trichloracetic acid, a procedure that precipitated undegraded VIP and left the radioactive fragment of hydrolyzed VIP in the 230390-17: 06 ., ~, , ~
.
' ; ; ' ~
, . ,:

CMS Docket llo. 370068 4960 30 ~ 9~

supermarket. A plot of the rec.iprocals of the rate of ~IP hydrolysis and VIP concentration was linear, suggesting con~ormity with Michaelis-Menton kinekics.
Km and kC~t calculated ~rom these data using the program EN2FITTER (Elsevier) were ~.9 IlM and 40.6 minO1.
It is likely that thl~ L-chains isolated arose by the spontaneous reduction of disulfide bonds between the H and th~ L chains as a re~sult of manipulating of the antibody preparation at very dilute concentrations (less than 5 ~g/ml) and exposu:re of the antibody preparation to extreme pH values (up to pH 10.5). The data clearly shows that the dissociated L-chain of the YIP autoantibody possesses catalytic ackivity.
E~a~D 1e YI I
re~t~ent of IqG
Rith I~mobil~ Papain (Fab Pro~uctionL
Cysteine (Sigma) was added to 20 mM in 20 mM
NaH2P04with lOmM EDTA pH 7.0, to make digestion buffer.
The immobilized papain agarose gel (Pierce)(0.5 ml, equivalent to 0.25 ml settled qel volume) slurry was then added to a 13 x 100 m~l glass tube. Then, 0.75 ml settled gel was used to di~est 7.5 mg IgG. Four ml of digestion buffer was then added and mixed. This mix was centrifuged at 1000 rpm for 5 min. The buffer was discarded and the procedure repeated. The papain gel was resuspended in 0.5ml digestion buffer and trans~erred to a 25ml flask. IgG (up to lOmg IgG, usually 2-7.5mg to papain gel was added. Digestion buf~er was added to make 1.5ml total incubation volume.
Incubation was at 38~C with shaking (top speed of shaking water bath~ for 5h or overnight.
Following incu~ation 1.5ml lOmM TRIS-HCl pH 7.5 was added and the gal and ~olution was trans~erred back to a 13 x 100 mM glas~ test tube. This was centri~uged at 1000 rpm ~or 5 min.
The supernatant was then applied to 230390- 17:06 CMS Dock2t No. 37006B-4960 31 2~

equilibrated Protein A agarose c~lumn (2.5ml gel for up to 20mg papain digested IgG). Fab was separated from residual intact IgG on a Protein A agarosP column.
Protein A column purification was run the same way as the Protein G-Sepharose column of Example IV, except t~at the start buffer is 10 m~ TRIS-HCl pH 7.5, and column is regenerated with 0.1 ~ citric acid, pH 3 and storage i9 in 0.2~ sodium a~ide. The papain digested material was applied to the column (previously equilibrated with the chrom~tc,graphy buf~er).
The nonr~tained portion was tested for purity of Fab by electrophoresis (Fig. 3A). Retained portion was a mixture of nondigested IgG, Fc fra~ment and other fragments with an Fc portion.
The Fab was able to catalytically hydrolyze sI-labeled VIP using the method of Example VII (Fig.
3B)-x~mple ~III
PreparAtion of Di~sociate~ ~ixture of ~envy ~nd Li~ht Chai~ ~F~lL 25kD) To about 2 mg Fab (Example V~ t Nacl was added to 0.15 M, and mercaptoethanol was added to 0;2 M in a final volume of 5 ml of 50 mM Tris-HCl, pH 7.3. This mix~ure was incubated at 24 C for 3 h with shaking.
Than 2 ml o~ 0.5 ~ iodoacetamide was added, followed by the addition of 1 M Tris.HCl (900 ul~ to bring pH to 7.5. This mixture was incubatPd for 15 min at 24 C
with shaking. The sample was then concentrated to reduce the volume to about 1 ml on a 10 kD ultrafilter (YM10).
The resulting concentrated sample was chromatographed on Superose-12 in buffer (0.1 M
glycine-HCL, 0.05 M Tris-HCL, pH 8.0 containing 0.025%
Tween 20~. The protein peaks from the Superose-12 were analyzed by SDS-PAGE (Fig. 5B~. Pooled functions showed a molecular mass of 25-30 kD in the SDS-PAGE

230390-17:06 :, :

CMS Docket No. 370068-4960 3? ~3~

analysis. This is the Fd/L25 fraction. Note that the ~inor peak seen in lane 2 of Fig 10 is undissociated Fab and the major p~ak is unassociated 25 kD Fd/L. The Fd/L-chain mixture was able to catalyze the cleavage of VIP as illustrated in (Fig. 6).
~x~mple IX
B~PA~tiO~ 0~ oci~te~ ~xture o~ ~e~v~ Light Chai~
Into Puri~ie~ F~- ~n~ L-C~ai~
The unassociated L-chain/Fd- mixture resulting from Example VIII is subjected to further separation procedures in order to fractionate dissociated Fd, dissociated L-chains, and dimers. The first such separation procedure consists of chromatofocusinq on a Mono-P column using pH gradients of 10.5-7.0, 9.0-6.0 or 7.0-4.0, as appropriate.
Optical absorbance (280 nm) and pH of the effluent is monitored. VIP hydrolytic activity is assayed in the protein peaks. Protein peaks with VIP catalytic ~o activity are collected for ~urther analysis. The peaks with VIP catalytic actiYity have molecular weights of about 25-26 kD and about 50-60 kD. The identity of the proteins is ascertained by SDS-gel electrophoresis on Phast gels (Pharmacia, 8-25%) followed by silver ~5 staining and immunoblotting.
A. Standardized Immunoblottinq Procedure The following standard method is used to detect'Fd, ~- L-chain and other components of antibodies with high sensitivity. Gels are blotted on nitrocellulose membranes, the membranes incubated in anti-human L-chain ~kappa/lambda) or anti-H chain ~`
antibodies (Accurate)~ washed with buffer, incubated with anti-rabbit IgG conjugated with peroxidase, washed, and then stained with diamin~benzidine and H2O2.
B. Immunoblottinq Interpretation Staining of the 26 kD bands with anti-L chain and anti-H chain antibodies indicates the presence of 230390- 17:06 ' , , CHS D~cket Uo. 370068- 4960 33 2~3~

L-chains and Fd-, respectively. Staining of the 26 kD
band with one type of antiserum and not the other indicates that the preparation i~ composed o~ pur~ Fd-or L-chains. Staining of a 50~60 kD band represents homo- or heterodimers when thelse are present. Lack of staining of this band with one of the antisera in immunoblots indicates the absence of Fd-L het~rodimers.
C. A~finitY Chromato~raphy S,_paration In an alternative method, affinity chromatography is applied using specific anti-H (e.g.
anti-Fd) or anti-L-chain antibodies immobilized on a solid support.
IgG from these antisera (or ascites fluid) are purified by chromatography on protein G-Sepharose and then coupled covalently (16) to CNBr-Sepharose (Pharmacia).
To fractionate di~sociated Fd- and L-chains from the unassociated mixture, affinity chromatography using a column prepared with these immobilized antibodies is performed, using acid shock (pH 2.7) to elutP th~ retained protein. Identity of the fractionated material is confirmed by immunoblotting for Fd- and L-chains as before. NatiYe polyacrylamide gel electrophoresis and silver staining on PHAST gels 25 i6 conducted to confirm that the purified Fd- and L-chain are monomeric (as for Fig 5). Since anti~odies and antibody fragments can bP very basic, reversed polarity electrodes are used for the native PAGE, when necessary.
~A~P~ 3 Cat~ly~i~ by antibo~
a~ ~in~l~ Chai~ ~ompo~e~t~
A. Kinetic ~roperties The catalytic properties and kinetics of intact Fab component and th~ purified, dissociated Fd component and L-chain derived from Example IX are determined. Antibody concentrations suE~icient to 230390- 17: 06 CMS Dock~t ~I~. 370068-4960 3~

yield hydrolysis of about 3,000 CPM are incubated with (Tyr10125)VIP in the presence of increasing concentrations of unlabeled VIP for 3 hours at 38C.
The amount of VIP hydrolyzed i.s calculated from the amount of radioactivity rendered soluble in 10% TCA by the anti~odies. To confirm that the TCA-precipitati~n method i~ a valid indicator of VIP hydrolysis, reverse phase ~PLC of antibody treatecl (Tyr10l2s)VIP iæ
performed. The decrease in the amount of radioactivity in intact (Tyr~0~l25)VIP (retention time 25 minutes) is equivalent to the amount of radioactivity rendered TCA-soluble, when the an~ibody cleaves pPptide bonds located between rasidues 7 and 22 of VIP. The data is analyzed by the program ENZFITTER (Elsevier) and plots of rate of hydrolysis versus the substrate concentration are constructed.
The reaction kinetics are first order with respect to substrate concentration. The data is fitted to the equation V=VLS]/Km ~ [S] where V is maximal reaction velocity, Km the VIP concentration at V/w, v the initial reaction velocity, and [SJ the VIP
concentration. KC~t is obtained as [pmol VIP hydrolyzed per minute/pmol antibody or antibody single chains;
normalized for valency (intact IgG = 2; Fab = 1, H- and L-chains = 1~ and molecular mass (Fab 60 kD; Fd- and L-chains, 2S kD)].
Catalytic efficiency is computed as kCat/Km.
Increased Xm values for the dissociat~d chains indicates decreased binding affinity. Increased Km values are not detrimental to the rats of catalysis so long as the binding step is not the rate limiting step.
B. Identification of The Peptide Bonds in VIP
Cleaved by CatalYtic Antibodies and_ Catalytic Sinale Chain Components In order to determine which VIP peptide bonds are cleaved by each component, a labeled VIP is cleaved by each of type of component kestedO (Tyrl0~12s~1~)VIP

Z30390-17:06 ; "~
~:' CUS D~cket ~1~. 370068-49~0 2 ~ 3 ~

(50 ~g) or (14C-His, 13H-Asn28)VIP (50 ~g~ is treated with IgG, Fab, single Fd-chains and L-chains (a quantity sufficient to hydrvlyze at least 5~ of the peptide, based on kinetic analyses), non-immune IgG or assay diluent for 3-6 hours at 38C. The reaction mixture~
are extracted on C-18 cartridges ~Alltech), the eluates dried in v~cuo and th n subjected to reverse phase HPLC
on a Novapak-C18 column using a gradient ~
acetonitrile in trifluoroacetic acid. The absorbance 1~ of the eluate at 214 nm is mon:itored. A124 absorbing, radioactive paptides are present in reaction mixtures of VIP treated with immune IgGI but absent in VIP
treated with an equivalent quantity of non-immune IgG
or assay diluent are pooled and purified ~urther by a second round of reverse phase HPLC based on the elution behavior in the initial HPLC. Purified peptides are sequenced using an Applied Biosystems pulsed liquid phas~ sequenator with online PTH-amino acid detection.
The cleaved bonds are identified by the size and identity of the cleavage fragments.
C. Determination of the Ability of Catalytic Antibodies and Sinqle Chain Compon~nts to Cleave Peptides Unrelated to VIP
In order to determine the sequence specificity of cleavage by single chain catalytic components the hydrolytic actiYity of catalytic antibodies and single chain catalytic components is compared. The substrates are ~Z5I-labeled peptides that contain the scissile bond identified in Example IX
above, but have little sequence identity with VIP.
This minimizes the role of residues distant from the scissile bond in substrate interactions with catalytic antibodies. For Gln16-Metl7 cleaving catalytic antibodies, ;l suitabl~ substrate is pancreatic polypeptide (PP~ PP has only three sequence identities with VIP, two of which are at the potential 230390-17:06 ~',`,` ~'.
., ~ ..
- , '~

CMS Docket llo. 370068-4~60 36 2~

scissile bond (Gln 16-Met17). Substrates for other types of antibodies are chosen from the commercially available 12sI-labeled peptides: e.g., i~NP, insulin, somatostatin and endothelin, These peptides (about 100,000 cpm) are tested as sub,strates for intact or single chai~ antibodies using the experimental conditions employed to test clleavage rates for VIP as described in Example II. The reaction mixtures are extracted on C-18 cartridges and subjected to reverse 10 phase ~PLC. Appearance of radioactive peaks with retention times different from those of the intact peptides is suggestive of peptide hydrolysis by the antibodies. Substrates hydrolyzed in the preliminary screening are studied further for identification of 15 scissile bonds. The methods are similar to those employed for identifying the scissile bonds in VIP, i.eO use of low specific acti~ity l25I-labeled substrate, purification of peptide fragments by resolutive reverse phase HPLC, and identification of 20 the fragments by amino acid sequencing.
D. Determination o~ The Ability of Intact and Sinule Chain Antibodies to Cleave Analoqs of VIP
VIP analogs containing amino acid 25 substitutions at the scissile bond are synthesized for use as substra~es. The substitutions are with residues that are dissimilar to the original residues or are similar in charge or shape. For example, (Asn16, Nlel7)VIP and (Ala16, Ala17)VIP are tested as substrates 30 ~or Gln~6-Met17 cleaving catalytic antibodies. The ability of intact and single chain antibodies to cleave these substrates is tested by resolutive reverse phase HPLC. These substrates are labeled with 12sI at Tyr~, as described for VIP(1-28~ in Example II. Synthetic 35 VIP(1-16), (Asn16)VIP~1-16), (Ala16)VIP(1-16) labeled at Tyr10 with 1251 prepared by methods similar to that used in Example II, which are well known to the art, are 230390-17:06 CMS Docket Ho. 37006~-4960 used as standards. Coelution of synthetic standards with radioactive peptides produced after treatment of ~ubstrates with antibodies is constru~d as evidence for cleavage between residues 16 and 17. The relative ability of these peptides to 21Ct as subskrates for the catalytic antibodies and their single chains is determined by measuring X~ and kCat using trichloroacetic acid to distinguish between intact and fragmented VIP.
B~AMPLF XI
Cloning ~ pressi~g ~DNA
For Cataly~io Compone~t~
A. Outline of the Clonin~ Strateqy Cloning of the catalytic component cDNA may proceed by one of three approaches. In the preferred approach, mRNA from clon~l human hybridoma cell lines which produce catalytic VIP antibodies is employed as starting material. The cells are harvested and mRNA is extracted by standard methods known to the art. The cDNA is prepared by reverse transcription of the mRNA
by standard methods known to the art. Th~ cDNA for Fd-and L-chains is amplified by polymerase chain reaction ~PCR) using appropriate primers as described below.
The amplified cDNA is then ligated into expression vectors by standard methods, expressed separately in E.
25 coli., and the properties ffl the expressed single chain antibodies determined.
The second approach avoids reliance on the a~ailability of clonal antibody producing cells. The starting material is mRNA from Epstein Bar virus (EBV)-3D transformed peripheral blood lymphocytes. The cDNA isprepared and amplified by PCR as previously described, and an expression library is constructed. The cDNA
library is ~xpressed by standard methods in a mammalian cell line which is selected for the desirable property of a culture supernatant which shows a low background of VIP hydrolysis, and the resulting recombinant c~lls are then directly screened for hydrolytic VIP

230390- 17: 0~

CPIS Docket ~1~. 37006B-4960 3~ ~3~

antibodies. The cDNA showing the highest VIP catalytic activity in mammalian cells is then further cloned in E. coli using known techniques optimized for overproduction of the expressed recombinant proteins.
A third method is screening for expression of recombinant Fab, using a randomly constructed, Fd- and L-chain combinatorial library using the method o~ Huse et al. ~19).
B. Pre~aration of Antibody Secretinq Cells Stable antibody-producing hybridomas of EBV-transformed lymphocytes and mouse/human heteromyeloma are constructed by standard methods of (20, 21). The hybrids are grown in the antibiotic G-418 to stabilize the human chromosomes. Treatment with Ouabain 15 eliminates the parent EBV-lymphoblastoid cells. These heterohybrids are then screened for antibodies with VIP
hydrolytic activity.
Screening of the antibodies is performed by incubation of culture supernates with (Tyr10125~VIP for 3 20 hours, undegraded VIP is precipitated with 10% TCA, the precipitate is trapped on GF/~ filters using a Cambridge harvester, and the filters are counted for radioactivity. If the culture fluids are centrifuged (5000 x g; to remove cellular debris) and diluted two-25 fold prior to assay, their background VIP-hydrolytic activity is negligible. This method permits screening of lar~e numbers of wells in a single assay, and has been developed specifically to facilitate cloning of hydrolytic antibody producing cells. The TCA-30 precipitation method is unlikely to detect peptide bond cleavage close to the N- or C-terminii of VIP, since TCA would probably precipitate large l251-labeled peptide fragments produced by such cleavages.
The screened hybrids are then cloned by 35 limiting dilution using 0.3 cells/well with 10% Origen cloning factor (IGEN) in place of feeder layers to 230390-17:06 ,. . .
.
~' : :
. ,: .
... .
.

CMS Docket No. 370068-4960 isolate clones producing monoclonal human anti-VIP
catalytic antibodies.
C. Derivation of Primers for PCR.
Catalytic antibody is purified from the culture supernatant of monoclonal hybridoma cells by chromatography on protein G-Sepharose. Sufficient quantities (about 50 µg) of the catalytic IgG molecule are dissocaited into component H- and L-chains by reduction, alkylation and high performance gel filtration under denaturing conditions in a 6 M urea containing buffer. A minimum of 15 N-terminal residues of both the H- and L-chains are determined using an Applied Biosystems liquid phase sequenator. This amino acid sequence information is used to dervie synthetic oligonucleotides by standard methods, incorporating neccessary alternative sequences to take into account codon degeneracy, with inosine placed in codons with the highest degeneracy level. These oligonucleotides serve as 5' variable region primers for cloning the Fd-and L-chain cDNA.
An alternative to sequencing of the antibody N-terminus is to use a mixture of consensus sequence primers for the antibody variable regions (22, 23).
Primers for the constant regions are based on known sequences of the CH1 and CL domains, from Kabat and Wu' data base (24). In order to narrow down the sequence choices, the isotype of the antibody is determined using monospecific antisera directed against human IgG subclasses (Boehringer) in an ELISSA assay, and the 3' oligonucleotide for CH1 is synthesized based on the type of H-chain. Likewise, the type of L-chain (kappa or lamba) is determined and the primer for CL is designed accordingly. The 5' VN and L-primers are constructed with a Not1 restriction site and the 3' CH1 and CL primers are constructed with a translation termination codon and a convenient Not1 restriction 230390-17:06 CMS Docket No. 370068-4960 ~3~
~o site for subsequent forcad cloning. Since ~otl site is 8 bp in length, the likelihood of its presen~e in the cDNA clones of interest is low.
D. cDNA Preparation, Amplification and Sequencin~
Poly(A~)~NA is prepared from the catalytic antibody producing hybridoma cell line by the guanidinium thiocyanate/CsCl method followed by oligo(dT~-cellulose chromatog:raphy (25)o All necessary precautions are taken to minimize RNase contamination in glassware and plastic ware's. The cDNA is copied from the mRNA (5-10 ~g~ using reverse transcriptase an oligo(dT) primer and dNTP substrates as described in Example XI. The Fd- and L-chain cDNAs is then amplified using the polymerase chain reaction technique (PCR). For PCR amplification, the cDNA-RNA hybrids is then mixed with dNTPs and the 5' and 3' primers for Fd-and L-chains, Taq polymerase is added, and the sample overlaid with paxaffin oil, 25 or more cycles performed, each cycle consisting of denatur~tion (920C, 1 minute), annealing (520C, 2 minutes~ and elongation (720C, 1.5 minutes). The amplified cDNA is then ~xtracted with phenol, then with phenol/chloroform, ethanol precipitated and frozen.
For sequencing o~ Fd- and L-chain cDNA, the PCR products are purified on a 2% agarose gel, digested with Notl and ligated into a suitable vector of the pGEM series. Dideoxynucleotide chain termination sequencing is carried using T7 DNA polymerase (26~.
E. Cloninq and Expression of Amplified DNA:
Standard DNA technology is employed to construct an expression vector suitable for cloning of the amplified cDNA for Fd- and L-chains. The sequence of the oligonucleotides used to construct ~he vectox includes elements for construction, axpression and secretion of the recombinant proteins. The vector is tailored for high level expression by known methods.

230390- 17: 06 CS1S D~cket elo~ 3700~B-b960 41 2~

The vector is the pER vector which contains appropriate restric ion sites, the vX polylinker region, an ampicillin resistance gene and a strong r~n promoter ~E. coll rib~somal ~NA promot:er) under the control of the lac operator. The ribosomal RNA promoter in pER
vector is highly induced duri.ng cell growth while the lac operator confers lactose or is~propylthiogalactoside (IPTG) inducibility t~ the expression (27). The amplified cDNAs derived contain only the mature Fd or L-chain ~oding ~eguences. To facilitate sPcretion of Vu and VL into the E. coli periplasm, the leader peptide sequence for the bacterial Rel B gene is incorporated into the vector (28, 29). cDNA amplified by PCR is digested with Notl, fragments are phenol-extracted, purified on 2% agarose gels and the insert ligated to khe expression vector digested with Notl. E. coli is transformed with recombinant plasmids using calcium chloride (~
colonie~ grown with ampicillin to select successful 2~ recDmbinants incorporating a gene Por ampicillin resistance, resistant colonies toothpicked into medium containing ampicillin, and the cells grown in IPTG to induce ~xpression. Aft~r about 24 hours, the supernatant is separated from the cells, the cells are shocked hypo-osmotically to release periplasmic contents and the supernatant of the lysa~e is collected. Since the Fd- and the L-chain are secreted into the E. coli periplasm, th~ moderate level of sxpression fr4m the 1~ prom~ter should not result in toxicity due to the "jamming of membrane protein traffic." (30).
Fd- and L-chains are initially fractionated from the lysate ~nd the culture supernate by high performancle gel filtration, and ~ractions with molecular ~mass 20-30 kD are purified further by immunoaffi:nity chromatography as described in Exampl~

230340~ 17:06 ' .''", ' ' '','' ,~ .. ': , ., CMS Dock2t llo. 37DD~8 49bO
~3~

IX. Since the recombinant Fd- and L-chains contain th~
CHl and CL domains, i~mobilized monoclonal anti-human Fd (supplied by Dr. S. Rodkey) and rabbit anti-human L-chain antibodies (Accurate) are used ~or the immunoaffinity chromatography. The antibodies are purified by chromatography on protein ~-Sepharose and covalently coupled to CNBr-Sepharose using standard methods.
SDS-PAGE silver staining and immunostaining with ~pecific anti-H-chain and~ anti~-chain antibodies using a PHAST system are performed to confirm the identity and purity of the recombinant proteins as described in Example IX. Assay of (Tyr1012sl) VIP
hydrolytie activity is performed t~ monitor recovery of Fd- and L-chains during purification.
The purified Fd- and L-chains are subjected to N-terminal amino acid sequencing. Identity of the N-terminal residues of the recombinant proteins and the original antibody H- and L-chains confirms that the corrsct molecules have been cloned.
~ PL~
Clo~i~q from ~V Tranqforme~ ~ymphocytes In an alternative cloning method, cDNA for Fd- and L-chains is prepared from E8Y-transformed patient lymphocytes, ligated into an appropriate vector, ~3xpressed in e~karyotic cells and screened for VIP hydrolytic activities (31-34). Although it is a tedious matter to isolate the cDNA for catalytic antibodies from a mammalian exprPssion library, this method has advantages because the cDNA for Fd- and L-chains are short tapproximately 650 bases) and can be selectiv~ly amplified from poly(A~RNA by polymerase chain reaction~ Moreover, it is necessary to resort to screening ~or exprsssion in a mammalian cell line, ~ince high rates of background hydrolysis o~ VIP in culture sup~3rnates and lysates of E. coli transformed 230390- 17: 06 , CMS Docket Ro. 37006e-4960 43 2~

with expression vectors has been observ2d (during developmPnt of these methods). In contrast, culturP
supernates ~rom irrelevant myeloma cells, hybridoma cells and EBV-trans~ormed cel:ls show little bacXground VIP-hydrolytic actiYity (~ig. ~).
A. Primers for PCR
Since mRNA species coding for many antibody molecules are likely to be pr~esent in the starting EBV-t~n~formed cells, the "ancho:red PCR" method described by Loh and coworkers ~35) is used to ampli~y all possible Fd- and ~-chain cDNAs. The method is based on the attachment of ~ poly(dG) tail to the ~irst cDNA
strand, and the use of a complementary poly(dC) primer for second stran~ synthesis by PCR (see below). The lS poly(dC) primer for this example contains a Notl Notl restriction site for forced cloning. This primer substitutes for the V-region primers described in Example XI above, and amplifies the 5' ends of Fd- and L- chain cDNA. Primers for the constant regions are ~O based on known sequences ~f th~ CH~ and C~ domains, ~rom Kabat and Wu's data base (24). As describad in Example XI the seguence chsices are narrowed by determining the isotype of the antibodyO This is done by precipitating the hydrolytic activity present in the culture supernates o~ EBV-transformed lymphocytes with monospecific antisera directed against human IgG
subclasses (Boehringer). The oligonucleotide primers for CHl are then ~ynthesized b~sed on the type of ~-chain, incorporating necessary degeneraciesO
Likewise, the type oP L-chain ~kappa or lamba) is determinDd and the primer for CL is designed ~ccordingly. T~e 3' CHl and CL prlmers contain a transl~tion termination codon and a convenient Notl restriction site ~or subsequent forced cloning. Since the Notl 6ite i~ 8 bp in length, the likelihood of its presence in the cDNA clone of intere~t is low.

~0390-17:06 C~S Docket Uo. 370069~4960 44 ~33~

B. cDNA Preparation and Ampl~i~fication Poly(A~)RNA is prepared from the catalytic antibody producing EBV-transformed patient lymphocytes by the method described for hybridoma cells. The firs~
cDNA strand i5 synthesized uC;ing appropriate constant region primers for Fd- and L-chains. A poly(dG~ tail is then added to the first st:rand DNA by treatment with terminal deoxynucleotide transferase in dGTP f~r 1 hour. The reac~ion is stopped by heating to 70~C, and ~he DNA is recovered by ethanol precipitation. The poly(dG~ tail on the first st:rand serves as the complemen~ary sequence for the poly(dC) 5' primer durin~ second strand synthesis, catalyzed by the Taq polymerase. Twenty f~ve or more PCR cycles are performed to achieve amplification.
C. Cloninq and Expression of ~mplified DNA
The cDNA is then cloned via khe Notl site into the ma~malian expression vector H3M by known methods (36). The ~ H3M vector contains the SV40 origin o~ replication which allows template amplification in COS cells, a chimeric CMV/HIV enhancer promoter that drives the ~xpression of the cloned sequences and SV4~ small t splice and polyadenylation ~ignal. Si~ce the ~ H3M vector is ~VX/supF-based the resultant recombinant DNAs are trans~ormed into MC1061/P3 strain which is suitable for their maintenance. Bacteria are then transformed with the cDNA library. The bacterial tran~formants containing the library are ~aintained on filters. ~ransformants are divided into pools and miniprep DNA prepared for ~ransf2ction into COS cells by the calcium phosphate method (37~. The culture supernatants from tranfected cells are then assayed ~or the catalytic component using ~Tyr10l251~VIP as substrate. The D~A pool ~howing the ~ighest activity is then further screen~d until the clona for ~he optimal catalytic antibody is obtained.

230390~ 06 CMS Docket Uu. 3700~a-49~0 , ~3~

Once the cDNA has been cloned, methods ~imilar to those described in Example 12 are used to: (i) express Fd-and L-chains in bacteria, (ii) purify Fd- and L-chains, (iii) reconstitute Fab from the single chains, and (iv) determine the catalytic activity of ~ingle Fd- and L-chains, and reconstituted Fab.
~AMPL~ XIII
FO~ATION OF FAB ~ R~CO~BIN~NT Fd- ~ND ~Ç~INB
Recombinant Fd- and L-chains are mixed ~or 12 hours at pH 8.5, permitting spontaneous reconstitution of Fab (12, 14~. An increase in molecular mass, judged by gel filtration and/or native gel electrophoresis and immunoblotting with anti-H chain and anti-L-chain antiserum are evidence for formation of Fab. The catalytic properties (kC3tKm and ~pecificity) of recombinant Fd, L-chains and Fv are determined as in Example X.
~XA~P~ ~IV
~r~paratio~ of Fv ~ra~ment A. Preparation of Fab' The Fab' fragment is prepared by the ~thod of Inbar et al. (38).
one gram of catalytic antibody as derived from Xxample V, in eluting buffer (0.15 ~ NaCl, 0.01 sodium phosphate buffer ~ pH 7.4), ic adjusted to pH

4.7 by the addition of O.S M sodium acetate buffer, pH
4~5 (one tenth of total volume), and then 10 mg pepsin (in 1 ~l of 0.005 M sodium acetate, Ph 4.5~ is added.
The ~ixture is incubated for ~ix hours at 37 C and then centrifuged to remove precipitate. The supernatant is ~djusted to pH 8 and applied to a column (3 X ~4 cm) of VIP-~epharcse. The Fab' fragment i~ eluted frsm the colu~n with 0.05 M VIP-glycine in eluting buffer.
~ctivity of the purified Fab' is assayed by a kinetic analy~is of the cleavage of labelled VIP as in ~xample II.
B. PreParation of Fv ~raqment ~0390-17:06 ~:
.
:: , . :

,, , , ' ' ' ' . '~`
, ' ' ''' '', U15 Docket No. 3700~B-4960 46 ~3~ ~

The Fv fragment is preparecl from the catalytic antibody of Example V or from an Fab' fragment of ~ubsecti~n A ab~ve. Either the antibody or the Fab' fragmPnt is cleaved to the Fv fragment by the method of Hochman et al. (15).
The Fab' fragment or antibody (10 mglml in 0.15 N NaCl, 0.01 N sodium phosphate buffer at p~ 7.4) is adjusted to ph 3.8 by the addition of 1 M ~odium acetate, pH 3.7 (one tenth of total volume). To the turbid protein solution, peps~in (10 mg/ml in 0.01 M
fiodium acetate, pH 3.7) is added to give a weight ratio of 1:100 of en~yme to Fab'. After four hours at 37O C
the digestion is terminated by adjusting the pH to 7.0 with 2 M Tris~HCl, pH 8.2. Precipitate not dissolved by the rise in pH is removed by centrifugation. The supernatant is applied to a Dnp-lysine Sepharose column equilibrated and run with 0.05 M NaCl-003 N, pH 7.4).
After washing the unabsorbed fraction, the column is eluted with VIP-glycine (0.05 M, pH7.4) and the yellow fraction collected, concentrated by vacuum dialysis, and applied to a Sep~adex G~75 column, to separate FY
from undig~sted Fab' by the method of Hochman et al.
(39). For the af f inity chromatography step 1 ml of VIP-lysine is used p~r 2 mg of digest and 0.3 ml of YIP-glycine is used for elution.
Catalytic activity of the purified Fv is assayed by a kinetic analysis of the cleavage of l25I-labeled VIP as in example II. The molecular weight of the Fv is about 25 kD as measured by sPdimentation equilibrium (39).
~ANP~ XVI
~eparat~on o~ ~v ~nto VL ~d ~ Fr~a~s The heterodimer ~v i separated lnto its H-and L-chain derived components by the method of Hochman 35 et al. (39). Briefly, Fv is chromatographed in 8 M
urea at pH 9.O on DEAE-cellulose.

230390- 17:06 : ~"
: .

C~S Docket ~lo. 370068-4960 q7 AltPrnatively, the separation is performed by the method of Example VIII wherPin Fv is substituted for the Fab.
The H- and L-chain fractions produced by either method are di~tinguished by staining with anti H~chain and anti L-chain antibodies by the ~tandardlzed immunoblotting method of Example IX. Catalytic activity of the purified Fv is assayed by a kinetic analysis of ~he clea~age of l25I labeled VIP as in example II. The molecular weight of the ~eparate VL a~cl V~ chains is about 12.5 kD as det~rmined by sedimentation equillbrium (39).
~AMPL~ ~VII
Pr~p~r~tio~ of Cat~lytio ~ntibo~y Co~po~e~t nter1~Uk~lD 2 ~U~ LO~ Prot~in A fusion protein consisting o~ an interleukin 2 and an Fv catalytic antibody component able to catalytically activate a prodrug to a drug, or a protoxin to a toxin able to regulate activated T-cells is prepared by the following method.
To pr~pare a catalytic antibody fusion protein a plasmid is assembled ess~ntially as d2scribed by Chaudhaury et al. ~40), employing a DNA segment derived from a catalytic monoclonal antLbody, encoding the V~ joined to a DNA s ~ment encoding the VL by a 45-bp linker. The VL sequence is in turn joined to a DNA
se~ment encoding in~erleukin-2 (Fig, 9). The as~embled gene is under the control of the T7 promoter.
The source catalytic antibody is prepared by the methods taught by U.S. Patent No. 4,8~8,2~1. A
compound representing ~n analog to the intermediate transition ~tate of the reaction o protoxin to toxin is synthe~izedO That compound is then prepared with appropriate adjuvents and used to induce B cells to produce antibodies~ The B cells ~re ~creen~d to identify clones which produc~ an antibody able to 230390-17:0b - '~ ' ' . ' ~

CUS Docket No. 370û68-4960 48 2 ~ 3 ~

catalyze the protoxin to toxin reaction.
The DNA seguences for catalytic VH and V~
components are derived by one of several methods. One method is to prepare the cDNA by revlerse engineering (preparin~ an oligonucleotide encoding for a known peptide ~equence) by methods well known to the art, from the peptide sequences O:e VH and V~ components prepared by the methods o~ Examples XIII ~hrough XVI.
~nother method is to prepare the cDNR by reverse transcription of mXN~ isolatled from cells producing the desired protein, followed by amplification by PCR as described above in Examples XI and XII. The cDNA
sequence for interleukin 2 i~ obtained from Biotech Research Laboratories of Rockville ~aryland ~s a plasmid. The sequence encoding the V~ - {45 bp linker} - VL linked to an ampicillin resistance gene is inserted into the plasmid carrying the IL~2 gene downstream of the IPTG inducable T7 promoter by methods well known to the art.
The ~usion protein is injected into ~n animal where the IL-2 moiety causes it to selectively bind to or associate with activated T-cells. The protoxin or prodrug is then administered. The protoxin 3r prodrug which reaches the bound or associated ~usion protein is cleaved by the catalytic moiety to the active drug or toxin which kills the T cell without producing sign,ificant toxicity to other tissues. This method of treatment is useful for the treatment of a wide variety o~ di60rders, e.g., adult T-cell leukemia or autoimmune diseases or autoimmune reactions ~or which the removal o~ T cells is desirable for curative or palliative purposes.
~xampl~ X~III
~ct~vatio~ o~ ~ Pro~ru~
p8~ a ~atnl~t~o Anti~ody ~ompon~nt a~ ~ $1yoo~ide .~ntimetabolites are compounds that interfere Z30390- 17: 06 - ' ' :, , , .
.

C~IS Docket ~lo. 3700~B-49~0 3~3 ~

in either the biosynthesis, utilization, or metabolic function of normal cellular metabolites. To be succ~ssfully selective in the chemotherapy of tumors, an antimetabolite should adve3rsely a~fect one ~r more vital ~etabolic reactions in the tumor without seriously endangering normal tissues.
Some of the most successful anticancer drugs have been those based on purine or pyrimidine analogs whose activity is dependent on their ability to inhibit DNA or RNA synthesis. One such drug is arabinosyl cytosine ~I) (cytaribine, Ara C or CA) whose activity as an inhibitor of DNA ~ynthesis derive~ ~rom the presence of arabinose in place of ribose, the dif~erence being in the stereochemistry of the 2' hydroxy group. Ara C i~ administered in the ~ree 5'-hydroxyl form and only becomes activated after entry into cells by phosphorylation to the 5'-triphosphate form. Thus, it is already a prodrug, but when administered systemically, its activation can take ~0 place in any cell, tumor or normal, into which the drug enters. As a result of the wide systemic distribution of the drug, numerous sid~ effects occur, such as nausea, vomiting, alopecia, myelosuppression, etc.
It has now been found that Ara C can be ~odi~ied to a prodrug ~orm in which spontaneous intracellular activation would be reduced. First, a biological binding agent i~ selected based upon the tissue being targeted. This can be interleukin-2 as described in Example XVII if the target tissue consists of l~mphoid cells~ e.g. T4 cells, or the binding agent can be an antibody or component of an antibody selected for its ability to bind to a target tumor tissue, the binding agent being either chemically or genetically linked to a catalytic component, as described in previous examples, and able to catalytically convert the prodrug to drug. Second, the pro form of Ara C is 230390-17: 06 .
: , ~ .....
,` '-.~. :
' CMS Docket llo. 370068-4960 ` 203~

administered and its activation is then restricted to those tissues bearing the catalytic component activity.
Thus a more favorable discrimination of tumor and normal tissue results. Since Ara c has a very ~hort 5 plasma half-lie, diffusion o~E the activated drug away from the tumor site is iEollowed by rapid deactivation before significant systemic toxicity results.
SYnthesis of 5'-aalactos~l ~ra C
and its transition ætate analo~
The synthesis of the amidine galactosyl analog of Ara C [15] is outlined in Schemes 2 and 3 (iEig. 11, 12) The tribenzoylated derivative [3] from SchemP 1 ~Eig. 10) is converted to [9] by treatment with methanesulfonyl chloride in pyridine follDwed by displacement with lithium azide in the N,NM-dimethyliEormamide at 75~C. Hydrogenation o~E [9] in ethanol at 50 psi of hydro~en pres~ure in the presence of 10~ palladium on charcoal afPords the 4'amino derivative.
The 8-galactonolactam [13] is prepared by ~Eirst kreating 2,3,4,6-tetra-D-benzyl-2-d-galacto-pyranose ~ with dimethyl ~ul~oxide and acetic ~' anhydride to give 2,3,4,6,-tetra-0-benzyl-D-Galactono-1,5-lactone [12~ which is then condensed with aqueous ~5 ammonia (25% w/w) solution in the presence of trace amounts of Amberlite lR 120 H~ in dioxane for 6 hrs to affor~ tl3]. Conversion of [13] to its imido ~ster analog by treating it with trim~thyloxQnium tetrafluorobarate followed by its reaction the 5'amino derivative [10] yields the iEully protected amidine galactosyl analog [14]. Deprotection usin~
hydrogenation at 50 psi of hydrogen pressure in the presence oiE lO~ palladium on charcoal Pollowed by treatment with concentrated aqueous ammonia qives [15].
3~ The synthesis o~E the 5'-beta-D-galaotose analog oiE cytosine-beta-D-Arabinofuranoside [5] is 230390- 17: 06 CUS Docket l~o. 3700~8-4960 -51 2~3~

outlin~d in Scheme 1 (fig. 10).
Treatment of Ara C [1] with Bis (P-methoxyphenyl) phenyl methyl chloride in pyridine, followed by tribenzoylation u.sing benzoyl chloride and S then detritylation of ~2] with trichloroacetic acid in dichloromethane, affords the partially protected derivative ~3]. Dilute aqueous acid treatmen~ of beta-D-galactose pentaacetate [6~ followed by treatment with ~odium hydride and excess trichloroacetonitrile yields th~ trichloroacetimidate [8]. Coupling of [8]
with [33 in the presence of the Lewis acid boron trifluoride etherate in dichloromethase gives [(4)].
The 5'-beta-D-galactose analog of Ara C [5~ is obtained after the complate deprotection of ~4] using concentrated aqueous ammonia.
Production of antibodies and screenina for Bindinq to Transition State AnaIoqs Monoclonal antibodies to [15] in Scheme 4 (fig. 13), after conjugation to a suitable carrier, are produced essentially as described in Example 1.
Antibody-producing clones are first screened f~r their ability to bind to the Ara C analog [15] by methods similar to those described U.~. Patent No. 4,888,281.
In vitro oatalytic activity assay Those antibody ~lones displaying binding activity for [15] are screened ~or their ability to cleava the galactosyl ~oiety from the Ara C substrate ~5] by an assay es~entially as described in Koernar and Nieman ~41), but substituting galactose oxidase ~or 3~ glucose oxidase. The principle of the assay is the detection of galactose as it is released from the pro~rug by the catalytic antibody using a galactose oxidase/luminol chemiluminescence procedure.
The catalytic antibodies 6howing the desirable property are then used to produce one or more useful components as described in Examples V, VII, VIII

230390- 17:06 .: ' :

CMS Docket ilo. 37006a-4960 - ~ 0 ~

- XIV above. The use of catalytic cc)mponents instead of an intact catalytic antibody provides the advantage of a smaller molecular weight, thus permitting better tissue penetration.
In vivo assays The conversion of s~alactosyl Ara C to Ara C
by the catalytic antibody component in the presence of target cells results in inhibition of DNA synthesis and cell-killing. A si~ple assay of DN~ synthesis is carried out essentially as described in Gish et al.
(42), in which the ability of Ara C to inhibit DNA
~ynthesis in phytohaemagylutin (PHA) stimulated human lymphocytes, using a tritiated thymidine incorporation assay, is measured.

230390 -17: 06 CMS Do~ket ~o. 37006~-4960 53 ~8~1 1 REF~RE~NCE~
~. ~anda et al. Science ~ 1188-1191 (1988).
2. Baldwin, E. and Schul~z, P.G. Science ~ 1104-~107 (19~9).
3. Iverson and Lerner, ~ci~nce ~ 1184 (1989)~
4. Proceedinqs of the Symposium of Immunolo~ical Recoqnition Vol VII, published by Springer Verlag ~1989).
5. Paul, S., Volly, D.J., 13each, C.M., Johnson, D.R., Powell~ M.J., Massey, RoJ~ Sciencç 244: 115B-62 (19Bg).

6. Roholt, O., Onoue, K., and Pressman, D.
Biochemistry 51: 173 (1964).

7. Ward, E.S.I Gus60w, D. Griffiths, A.D., Jones, P.T. and Winter, G. Nature, 341: 544-546 (1989).

8. Edelman, G.M., Olins, D.E., Gally, J.A. and Zinder, N.D. Proc. Natl. Acad. Sci., 50: 753 - 761 (1963).

9. FraneX, F. and Nezlin, ~.S. ~olia Microbiol., 8:
128-130 (1963).

10. Franek, F. and Nezlin, R.S. Biokhimiya, 28: 193 (1963).

11. Porter, R.R. and Weir, R.C. ~._Cell Phvsiol., 67 ~Suppl. 1): 51-64 (1966).
25 12. Jaton, J.-C., Klinman, N.R., Givol, D. and Sela, ~. Biochemistry, 7: 4185-4195 (1968).
13. Roholt. O. Onoue, K. and Pressman, D. Proc. Natl, Acad. Sci., 51: 173-178 (1963).
14. Powell, ~.J., Massey, R.J., and Rees, A.R.
P~T/US89/01950, interna-tional publication No.
W089/10754, International publication dat~ 16 Nov.
~g89 .
15. Hochman, J. Inbar, D. and Givol, D, ~_o hemistry 12: l:L30 (1973).
35 16. Affi ity Chromatoqra~hy Principles and Methods, ~30390- 17:06 ., ~;

CMS Ducket 1~. 37006B-4960 54 ~3~

Pharmacia, ~ppsula Sweden pp. 12-18 (1986).
17. Paul, S., Volle, D.J., Beach, C.M., Johnson, D.R.
Powell, M.J. and Massey, J.J. Science, 1158 - 1162 (1989).
18. Pharmacia, Handbook, FPLC~ Ion Exchanae and Chromatofocusinq_- PrinciPles and Methods, pp. 59 to 106.
19. Huse, W.D., Sastry, L., Iverson, S.A., King, A.S., Alting-Mees, M., Burton, D.R., Benkovic, S.J. and Lerner, R.A. Science, ~6: 1275-1~81 (1989).
20. Roder, J.C., Cole, S.P.C. and Kozbor, D. Methods in Enzvmoloqy, 1~: 140-167 ~1986).
21. Kozbor, D. and Rodor, J.C. Immunolo~Y TodaY, 4:
72-79 (1983).
22. Orlandi, R., Gussow, D.H., Jones, P.T. and WintPr, G. ~roc. Natl._Acad. Sci. USA, 86: 3833-3837 ~198g) .
23. Sastry, L., Alting-Mees, M., Huse, W.D., Short, J.M., Sorg~, 3.A., Hay, B.N., Janda, K.D.
Berkovic, S.J. and Lerner, R.A. PrQc. Natl. Acad.
Sci. US~, 86: 57~8-5732, (lg89).
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- '

Claims (109)

1. A component part of an antibody which is capable of catalyzing a chemical reaction, said component part being selected from the group consisting of a light chain, a heavy chain, an Fd fragment, an unassociated mixture of a light and heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, a heterodimer consisting of a heavy chain and a light chain, a heterodimer consisting of an Fd fragment and a light chain associated non-covalently, an heavy chain homodimer, and a light chain homodimer.

2. A component part of an antibody which is capable of catalyzing the cleavage or formation of a peptide bond, said component part being selected from the group consisting of an Fab fragment and an Fv fragment.

3. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a light chain.

4. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a heavy chain.

5. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is an unassociated mixture of a light and heavy chain.

6. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a variable fragment of a light chain.

7. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a variable fragment of a heavy chain.

8. A component part of an antibody having catalytic 230390-17:06 CMS Docket No. 370068-4960 properties as recited in claim 1 wherein said component part is a catalytic domain of a light chain.

9. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a catalytic domain of a heavy chain.

10. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a light chain homodimer.

11. A component part of an antibody having catalytic properties as recited in claim 1 wherein said component part is a heavy chain homodimer.

12. A component part of an antibody having catalytic properties as recited in claim 2 wherein said component part is the Fab portion of an antibody.

13. A component part of an antibody having catalytic properties as recited in claim 2 wherein said component part is the Fv portion of an antibody.

14. A component part of an antibody having catalytic properties as recited in claim 2 wherein said component part is a heterodimer.

15. A component as recited in 1 wherein said component part is associated with at least one other molecule.

16. A component as recited in claim 1 wherein the chemical link between said component and said other molecule is covalent.

17. A component as recited in claim 1 wherein the chemical link between said component and said other molecule is non-covalent.

18. A component as recited in claim 15 wherein said other molecule is an antibody.

19. A component as recited in claim 15 wherein said other molecule is a nucleic acid.

20. A component as recited in claim 15 wherein said other component is a biological binding agent.

21. A component as recited in claim 15 wherein said 230390-17:06 CMS Docket No. 370068-4960 other molecule is an an enzyme.

22. A component as recited in claim 15 wherein said other molecule is the variable region of an antibody able to bind to an antigen of interest.

23. A component as recited in claim 15 wherein said other molecule binds to a cellular receptor.

24. A component as recited in claim 15 wherein said other molecule is a linker to a solid support.

25. A component as recited in claim 20 wherein said biological binding agent is selected from the group consisting of avidin, streptavidin, protein A, and protein G.

26. A component as recited in 1 wherein said component part is a chimeric product expressed by a nucleic acid sequence coding for a continuous polypeptide sequence which contains an antibody component part having catalytic activity, and at least one other protein, said nucleic acid sequence comprising:
(a) a first nucleic acid sequence coding for said catalytic component part; and (b) at least one additional nucleic acid sequence coding for at least one additional protein.

27. A component as recited in claim 26 wherein said additional nucleic acid sequence coding for at least one additional protein has a biological function different from that of said catalytic component part.

28. A component as recited in claim 26 wherein said additional protein is a biological binding agent.

29. A component as recited in claim 28 wherein said ligand is selected from the group consisting of avidin, streptavidin, protein A, and protein G.

30. A component as recited in claim 26 wherein said additional protein is a heavy chain of a conventional antibody able to bind to an antigen of interest.

31. A component as recited in claim 26 wherein said additional protein is a light chain of a conventional 230390- 17:06 CMS Docket No. 370068-4960 antibody able to bind to an antigen of interest.

32. A component as recited in claim 26 wherein said additional protein is the variable region of a conventional antibody able to bind to an antigen of interest.

33. A component as recited in claim 26 wherein said antibody component part having catalytic activity is able to catalytically cleave a prodrug or protoxin into a drug or toxin and said additional protein is able to bind the target cell of said prodrug or protoxin.

34. A component as recited in claim 33 wherein said additional protein is interleukin-2.

35. A method for preparing a catalytic component part of an antibody which comprises:
(a) subjecting said antibody to conditions suitable for the fragmentation of said antibody into components selected from the group consisting of a light chain, a heavy chain, an Fd fragment, an unassociated mixture of a light and heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, a heterodimer consisting of one light and one heavy chain, a heterodimer consisting of a Fd fragment and a light chain linked by non-covalent bonding, a heavy chain homodimer, and a light chain homodimer; and (b) obtaining the desired catalytic component part.

36. A method as recited in claim 35 wherein said antibody is catalytic.

37. A method as recited in claim 35 wherein said component part is a catalytic domain comprising a polypeptide which is a part of the variable region of a 230390-17:06 CMS Docket No. 370068-4960 catalytic antibody or of the heavy or light chain thereof and which retains the activity thereof.

38. A method as recited in claim 35 wherein said component part is replicated by inserting into a cell at least one fragment of at least one gene coding for said component part.

39. A method as recited in claim 38 wherein said cell is selected from the group consisting of: a bacterium, a fungus, a yeast, a mold, an animal cell, a protozoan cell, and a plant cell.

40. A method as recited in claim 37 wherein said catalytic domain is prepared by a process comprising the additional steps of:
(a) cleaving the variable region of said catalytic antibody into a series of peptide sequences;
(b) screening said peptide sequences to identify a peptide sequence having catalytic activity;
and (c) purifying said catalytic domain.

41. A method as recited in claim 40 wherein said catalytic domain is prepared by a process comprising the additional steps of:
(a) cleaving the peptide sequences obtained in step (b) of claim 40 to generate increasingly smaller peptide sequences;
(b) screening said cleaved sequences to identify those having catalytic activity;
(c) repeating steps (a) and (b) until no catalytic activity is detected in the cleavage products; and (d) purifying the so-identified domain.

42. A method as recited in claim 40 wherein said catalytic domain is prepared by a process comprising the additional steps of determining the peptide sequence of said catalytic domain and synthesizing 230390- 17:06 CMS Docket No. 370068-4960 copies of said catalytic domain.

43. A method for preparing a catalytic domain comprising the steps of:
(a) determining the sequence of the variable region of a catalytic antibody;
(b) synthesizing an overlapping series of homologous peptide sequences representing sections of the sequence of said variable region;
(c) screening said series of homologs to select a homologous peptide sequence having desirable catalytic properties, and (d) synthesizing the selected peptide sequence.

44. A method for preparing a catalytic domain comprising the steps of:
(a) determining the sequence of the variable region of a catalytic antibody;
(b) inserting into a cell a gene coding for the variable region of said catalytic antibody;
(c) expressing said variable region in said cell.

45. A method as recited in claim 44 wherein said inserted gene codes for a fragment of said variable region.

46. A method as recited in claim 38 wherein said cell is selected from the group consisting of a bacteria, a fungus, a yeast, a animal cell, a protozoan cell, and a plant cell.

47. A method as recited in claim 44 wherein said cell is selected from the group consisting of a bacteria, a fungus, a yeast, a animal cell, a protozoan cell, and a plant cell.

48. A method as recited in claim 38 wherein said gene is subjected to mutagenesis before insertion into said cell.

49. A method as recited in claim 38 wherein said gene is subjected to mutagenesis after insertion into said 230390-17:06 CMS Docket No. 370068-4960 cell.

50. A method as recited in claim 44 wherein said gene is subjected to mutagenesis before insertion into said cell.

51. A method as recited in claim 44 wherein said gene is subjected to mutagenesis after insertion into said cell.

52. A method for preparing a catalytic component part of an antibody comprising the steps of:
(a) inserting into a cell at least one nucleic acid sequence coding for a variable region of said antibody;
(b) subjecting said nucleic acid sequence to mutagenesis before insertion;
(c) screening the cell and its progeny for the presence of mutated variable regions of said antibody demonstrating desired catalytic activity;
(d) replicating said cell; and (e) expressing said mutated nucleic acid sequence to produce a translation product with the desired catalytic activity.

53. A method for producing a catalytic component part of a catalytic antibody by a process comprising the steps of:
(a) inserting into a cell at least one nucleic acid sequence coding for a variable region of said antibody;
(b) subjecting said nucleic acid sequence to mutagenesis after insertion;
(c) screening the cell and its progeny for the presence of mutated variable regions of said antibody demonstrating desired catalytic activity;
(d) replicating said cell; and (e) expressing said mutated nucleic acid sequence 230390-17:06 CHS Docket NO. 370068- 4960 to produce a translation product with the desired catalytic activity.

54. A method as recited in claim 52 wherein said cell is selected from the group consisting of a bacteria, a fungus, a yeast, a animal cell, a protozoan cell, and a plant cell.

55. A method as recited in claim 53 wherein said cell is selected from the group consisting of a bacteria, a fungus, a yeast, a animal cell, a protozoan cell, and a plant cell.

56. A method for selecting a gene fragment coding for a catalytic component part of an antibody which comprises the steps of:
(a) selecting at least one gene fragment coding for a component part, (b) inserting said gene fragment into a cell under conditions suitable for the expression of said variable region, and (c) screening said cells for those which express a catalytic component part.

57. A method as recited in claim 56 wherein said cell is selected from the group consisting of a bacteria, a fungus, a yeast, a animal cell, a protozoan cell, and a plant cell.

58. A method as recited in claim 56 wherein said gene fragment codes for a component part selected from the group consisting of a light chain, a heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, and an Fd fragment.

59. A method for the preparation of a bifunctional chimeric product comprising a catalytic component part of an antibody and a second protein by expressing a nucleic acid sequence coding for a continuous polypeptide sequence which contains an antibody 230390- 17:06 CMS Docket No. 370068-4960 component part having catalytic activity, and at least one other protein, said nucleic acid sequence comprising:
(a) a first nucleic acid sequence coding for said catalytic component part; and (b) at least one additional nucleic acid sequence coding for at least one additional protein.

60. A method as recited in claim 59 wherein said additional nucleic acid sequence coding for at least one additional protein has a biological function different from that of said catalytic component part.

61. A method as recited in claim 59 wherein said additional protein is a biological binding agent.

62. A method as recited in claim 61 wherein said biological binding agent is selected from the group consisting of avidin, streptavidin, protein A, and protein G.

63. A method as recited in claim 59 wherein said additional protein is a heavy chain of a conventional antibody able to bind to an antigen of interest.

64. A method as recited in claim 59 wherein said additional protein is a light chain of a conventional antibody able to bind to an antigen of interest.

65. A method as recited in claim 59 wherein said additional protein is the variable region of a conventional antibody able to bind to an antigen of interest.

66. A method as recited in claim 59 wherein said antibody component part having catalytic activity is able to catalytically cleave a prodrug or into a drug or toxin and said additional protein is able to bind the target cell of said prodrug or protoxin.

67. A component as recited in claim 66 wherein said additional protein is interleukin-2.

68. A method for preparing a catalytic component part of an antibody which comprises:

230390- 17:06 CMS Docket NO. 370068-4960 (a) subjecting said antibody to conditions suitable for the fragmentation of said antibody into components selected from the group consisting of a light chain, a heavy chain, an Fd fragment, an unassociated mixture of a light and heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, a heterodimer consisting of one light and one heavy chain, a heterodimer consisting of a Fd fragment and a light chain linked by non-covalent bonding, a heavy chain homodimer, and a light chain homodimer; and (b) screening said components for catalytic activity.
(c) obtaining the desired catalytic component.

69. A method for preparing a heterodimer which is capable of catalyzing the cleavage or formation of a peptide bond, said heterodimer being a component part of an antibody, comprising the steps of:
(a) identifying an antibody of interest;
(b) cleaving said antibody into at least two heterodimers; and (c) screening said heterodimers for catalytic activity.

70. A method as recited in claim 69 wherein said antibody is catalytic.

71. A method for preparing a heterodimer which is capable of catalyzing the cleavage or formation of a peptide bond, said heterodimer being a component part of an antibody, comprising the steps of:
(a) identifying an antibody-producing cell line;
and (b) screening said cell line for a cell which 230390-17:06 CMS Docket Ho. 370068 4960 expresses catalytic heterodimers.

72. A method as recited in claim 71 wherein said antibody is catalytic.

73. A method for preparing a homodimer which i capable of catalyzing a chemical reaction, said homodimer being assembled from light or heavy chains of an antibody, comprising the steps of:
(a) identifying an antibody of interest;
(b) separating the light and heavy chain components of said antibody;
(c) subjecting the light or heavy chains to conditions promoting the formation of light or heavy chain homodimers; and (d) screening said homodimers for catalytic activity.

74. A method as recited in claim 73 wherein said antibody is catalytic.

75. A method for preparing a component part of an antibody which component part is capable of catalyzing a chemical reaction, comprising the steps of:
(a) identifying an animal with an autoantibody to a self- antigen of the animal:
(b) isolating a serum fraction containing a plurality of autoantibodies;
(c) screening the serum fraction obtained in step (b) to identify an autoantibody which binds to a substrate of the said reaction;
and (d) screening components of said autoantibody to obtain a catalytic component of said autoantibody.

76. A method as recited in claim 75 wherein said catalytic component part is a component part of a catalytic autoantibody.

77. A method for preparing a component part of an antibody which is capable of catalyzing a chemical 230390- 17:06 CMS Docket Ho. 370068-4960 reaction, comprising the steps of:
(a) generating a plurality of monoclonal antibodies to an antigen selected from the group consisting of:
(i) the reactant;
(ii) the reactant bound to a peptide or other carrier molecule;
(iii)a reaction intermediate;
(iv) an analog of the reactant;
(v) an analog of the product in which the monoclonal antibody to generated is capable of binding to the reactant or a reaction intermediate; or (vi) an analog of a reaction intermediate;
(b) screening said plurality of monoclonal antibodies to identify monoclonal antibodies which bind to the substrate in the said reaction; and (c) screening components of said monoclonal antibodies to obtain a catalytic component of a said monoclonal antibody.

78. A method for preparing a component part of an antibody which component part is capable of catalyzing a chemical reaction, said component part being a component part of a catalytic antibody, comprising the steps of:
(a) generating a plurality of monoclonal antibodies to an antigen selected from the group consisting of:
(i) the reactant;
(ii) the reactant bound to a peptide or other carrier molecule, (iii) a reaction intermediate;
(iv) an analog of the reactant;
(v) an analog of the product in which the monoclonal antibody so generated is 230390- 17:06 CMS Docket No. 370068-4960 capable of binding to the reactant or a reaction intermediate; or (vi) an analog of a reaction intermediate;
(b) screening said plurality of monoclonal antibodies to identify a component which catalyzes the reaction; and (c) obtaining a catalytic component of said monoclonal antibody

79. A method for preparing a component part of an antibody which component part is capable of catalyzing a chemical reaction, said component part being a component part of a catalytic monoclonal antibody, comprising the steps of:
(a) immunizing an animal with an antigen selected from the group consisting of (i) the reactant, (ii) the reactant bound to a peptide or other carrier molecule, (iii) a reaction intermediate, (iv) an analog of the reactant, (v) an analog of the product in which the monoclonal antibody so generated is capable of binding to the reactant or a reaction intermediate, or (vi) an analog of a reaction intermediate, thereby generating antibody-producing lymphocytes in said animal;
(b) removing said antibody-producing lymphocytes from said animal;
(c) fusing said antibody-producing lymphocytes with myeloma cells and thereby producing a plurality of hybridoma cells each producing monoclonal antibodies;
(d) screening said plurality of monoclonal antibodies to identify a monoclonal antibody which catalyzes the reaction; and 230390- 17: 06 CMS Docket No. 370068-4960 (e) obtaining a catalytic component of said catalytic monoclonal antibody.

80. A method for preparing a component part of an antibody, said component part being capable of catalyzing a chemical reaction wherein said chemical reaction is known to be catalyzed by an enzyme, comprising the steps of:
(a) generating a plurality of monoclonal antibodies to said enzyme;
(b) screening said plurality of monoclonal antibodies to identify a first monoclonal antibody which inhibits binding of the reactant to the enzyme;
(c) recovering the said first monoclonal antibody;
(d) generating a plurality of anti-idiotype monoclonal antibodies to the said first antibody recovered in step (c);
(e) screening said plurality of anti-idiotype monoclonal antibodies generated in step (d) to identify a second monoclonal antibody which binds the reactant and catalytically increases the rate of the reaction; and (f) producing a quantity of the monoclonal antibody identified in step (e) by culturing a plurality of hybridoma cells, each of which products said monoclonal antibody; and (g) obtaining a catalytic component of said monoclonal antibody.

81. A method for preparing a catalytic light or catalytic heavy chain of an antibody comprising dissociating said antibody into light and heavy chains.

82. A method for preparing a catalytic light or catalytic heavy chain of a catalytic antibody comprising dissociating said antibody into light and heavy chains.

230390- 17:06 CMS Docket No. 370068-4960

83. A method as recited in claim 81 further comprising the steps of:
(a) cleaving said antibody into Fab and Fc fractions; and (b) reducing and then alkylating said Fab fraction to cleave bonds connecting light and heavy chains.

84. A method as recited in claim 82 further comprising the steps of:
(a) cleaving said antibody into Fab and Fc fractions; and (b) reducing and then alkylating said Fab fraction to cleave bonds connecting the light chain and the heavy chain Fd fragment.

85. A method as recited in claim 83 further comprising the step of separating said light and heavy chains.

86. A method as recited in claim 82 wherein said antibody is dissociated into light and heavy chains by passing said antibody through a gel column selective for a predetermined range of molecular weights.

87. A method as recited in claim 86 wherein said range of molecular weights is from 103 to 3 x 105 daltons.

88. A method as recited in claim 70 wherein said light and heavy chains are dissociated after dilution of said antibody to a concentration of less than 5 µg/ml at an alkaline pH up to pH 10.5.

89. A method as recited in claim 81 wherein said light and heavy chains are dissociated by chemical reduction of the interchain bonds.

90. A method as recited in claim 81 wherein said light and heavy chains are dissociated by enzymatic cleavage of the interchain bonds.

91. A method as recited in claim 81 wherein said light and heavy chains are dissociated by catalytic cleavage of the interchain bonds.

92. A method as recited in claim 81 wherein said light 230390- 17:06 CMS Docket No. 370068-4960 and heavy chains are dissociated in a process comprising the steps of:
(a) reducing said antibody in the presence of a reducing agent selected from the group consisting of mercaptoethanol, dithiothreitol, and mercaptethylamine; and (b) alkylating the SH groups formed in said reduction step with an alkylation agent selected from the group consisting of iodoacetamide and iodoacetic acid.

93. A method as recited in claim 81 further comprising the steps of:
(a) cleaving said antibody into Fab and Fc fractions;
(b) reducing and then alkylating said Fab fraction to cleave bonds connecting light and heavy chains;
(c) contacting said light and heavy chains with a ligand capable of binding only to said light or said heavy chain under conditions permitting said binding; and (d) separating said ligand bound light or heavy chain from said unbound light or heavy chain.

94. A component part of an antibody selected from the group consisting of a light chain, a heavy chain, an Fd fragment, an unassociated mixture of a light and heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, a heterodimer consisting of one light and one heavy chain, a heterodimer consisting of a Fd fragment and a light chain linked by non-cavalent bonding, a heavy chain homodimer, and a light chain homodimer, said component part having catalytic properties and having been prepared by:
(a) subjecting an antibody to conditions suitable 230390- 17:06 CMS Docket No. 370068-4960 for the fragmentation of said antibody into components, (b) screening said components for catalytic activity, and (c) obtaining said catalytic component.

95. A component part as recited in claim 94 wherein said antibody is catalytic

96. A catalytic domain prepared by the process comprising the steps:
(a) determining the sequence of the variable region of a catalytic antibody;
(b) synthesizing an overlapping series of homologous peptide sequences representing sections of the sequence of said variable region;
(c) screening said series of homologs to select a homologous peptide sequence having desirable catalytic properties; and (d) synthesizing the selected peptide sequence.

97. A catalytic domain prepared by the process comprising the steps:
(a) determining the sequence of the variable region of a catalytic antibody;
(b) inserting into a cell a gene coding for the variable region of said catalytic antibody;
(c) expressing aid variable region in said cell.

98. A catalytic component part of an antibody prepared by the process comprising the steps:
(a) inserting into a cell at last one nucleic acid sequence coding for a variable region of said antibody;
(b) subjecting said nucleic acid sequence to mutagenesis before insertion;
(c) screening the cell and its progeny for the presence of mutated variable regions of said antibody demonstrating desired catalytic 230390- 17:06 CMS Docket No. 370068-4960 activity;
(d) replicating said cell; and (e) expressing said mutated nucleic acid sequence to produce a translation product with the desired catalytic activity.

99. A catalytic component part of an antibody as recited in the preceding claim wherein the antibody is catalytic.

100. A bifunctional chimeric product comprising a catalytic component of an antibody and a second protein, said chimeric product having been prepared by expressing a nucleic acid sequence coding for a continuous polypeptide sequence which contains an antibody component part having catalytic activity and at least one other protein, said nucleic acid sequence comprising:
(a) a first nucleic acid sequence coding for said catalytic component part; and (b) at least one additional nucleic acid sequence coding for at least one additional protein having a biological function different from that of said catalytic component part.

101. A catalytic heterodimer prepared by a process comprising the steps of:
(a) identifying an antibody of interest;
(b) cleaving said antibody into at least two heterodimers; and (c) screening said heterodimers for catalytic activity.

102. A catalytic heterodimer prepared by a process comprising the steps of:
(a) identifying an antibody-producing cell line;
and (b) screening said cell line for a cell which expresses catalytic heterodimers.

103. A catalytic homodimer prepared by a process 20390-17:06 CMS Docket No. 370068 4960 comprising the steps of:
(a) identifying an antibody of interest;
(b) separating the light and heavy chain components of said antibody;
(c) subjecting the light or heavy chains to conditions promoting the formation of light or heavy chain homodimers; and (d) screening said homodimers for catalytic activity.

104. A catalytic component part of an antibody prepared by a process comprising the steps of:
(a) identifying an animal with an autoantibody to a self-antigen of the animal:
(b) isolating a serum fraction containing a plurality of autoantibodies;
(c) screening the serum fraction obtained in step (b) to identify an autoantibody which binds to a substrate of the said reaction;
and (d) screening components of said autoantibody to obtain a catalytic component of said autoantibody.

105. A catalytic Fv component part of an antibody able to catalyze the formation cleavage of a peptide bond, said Fv having been produced by a method comprising the steps of:
(a) selectively cleaving an antibody by contacting said antibody with pepsin to produce a mixture of fragments including the FV component part; and (b) treating the mixture such that the FV
component part is purified.

106. A process as described in claim 94 wherein said antibody is catalytic.

107. A catalytic Fab component part of an antibody able to catalyze the formation or cleavages of a peptide 230390- 17:06 CMS Docket No. 370068-4960 bond, said Fab having been produced by a method comprising the steps of:
(a) selectively cleaving an antibody by contacting said antibody with the enzyme papain to produce a mixture of fragments including the Fab component part; and (b) treating the mixture such that the Fab component part is usefully purified.

108. A method for catalyzing a chemical reaction comprising contacting a reactant with a catalytic component part of an antibody, said component being selected from the group consisting of a light chain, a heavy chain, an Fd fragment, an unassociated mixture of a light and heavy chain, a variable fragment of a light chain, a variable fragment of a heavy chain, a catalytic domain of a light chain, a catalytic domain of a heavy chain, a heterodimer consisting of one light and one heavy chain, a heterodimer consisting of a Fd fragment and a light chain associated non- covalently, a heavy chain homodimer, and a light chain homodimer.

109. A method for catalyzing the cleavage or formation of a peptide bond comprising contacting a reactant or reactants with a catalytic component part of an antibody selected from the group consisting of an Fab fragment and an Fv fragment.

230390-17:06