CA2234723A1 - Generating d-peptides: methods and compositions - Google Patents

Abstract

Methods of making and using D-antibodies and D-peptides are provided. The D-peptides or D-antibodies are analogs of ligands or receptors capable of specifically binding L-peptides, peptides containing both L-amino acids and D-amino acids, chiral and achiral non-naturally occurring peptides, and chiral and achiral non-peptide compounds. The D-peptides or D-antibodies are resistant to proteolysis in the gut and throughout the body and are less immunogenic than their L-polypeptide or L-antibody counterparts. Methods for producing such D-peptides or D-antibodies are provided.

Description

CA 02234723 l998-04-08 2 PCT~US96/163S8 GENERATING D~ ;~l~ES: METHODS AND COMPOSITIONS

INTRODUCTION

Back~round There is a growing awareness of the need to produce and identify small molecules having pharmacological activity, including for example, agonists or antagonists of various cellular acceptor molecules, such as cell-surface receptors, enzymes or antibodies. Searching for small molecules that are useful as ph~ relltir~l~ requires generating a collection of such molecules, screening thecollection for molecules with physiological activity and identifying the structure of molecules providing a positive result in the screening step. The collection of small molecules can be generated using the combinatorial library approach. However, the prior art has not recognized the .~ignifil~n~e of using D-antibodies and D-peptides to screen such combinatorial libraries of small molecules for potentialpharmacological activity and the ability to use mirror image transformations to create useful D-peptides and D-antibodies.
Nineteen of the essential twenty amino acids have the prop~lly of "chirality" or h~n~ 1n~c.~. The only achiral es~enti~l amino acid is glycine. Todescribe a chiral compound, the prefixes D and L are used to refer to the configuration of the molecule around its chiral center. The chiral center of an amino acid is the alpha carbon, and whether an amino acid is of the D
configuration or the L configuration depends upon the stereoisomeric conventionsestablished by Emil Fisher. A chiral amino acid can exist as stereoisomers, which are jclentir~l chPmi--~l structures that are mirror images of each other. Both stereoisomers are often referred to as an enantiomeric pair, and a stereoisomer is often referred to as an enantiomer, which is a nonsuperimposable mirror image ofthe other stereoisomer/enantiomer.
All of the naturally occurring chiral amino acids exist in the L
configuration, and are referred to generally as L-amino acids. The stereoisomer of each chiral amino acid in the L-configuration is referred to as a D-amino acid.

W O 97/13S22 PCTrUS96/16358 2.
A D-amino acid is one which has a configuration corresponding to the D-stereoisomer of the two stereoisomers of glyceraldehyde, L-glyceraldehyde and D-glyceraldehyde. All D-amino acid, D-polypeptide or D-peptide stereoisomers that _ave the same stereo ch~mir~l configuration as D-glyceraldehyde are de~ign~t~d as S D-, and those having the same configuration as L-glyceraldehyde are design~t~d as L-.
Recall~e the enz3~matic reactions in ribosomal translation of polypeptides are stereospecific for L-amino acids, peptides con~i~ting of all-D amino acids do not generally occur naturally. Accordingly, all naturally occurring ploteins andpolypeptides consist of L-amino acids, with the exception of certain antibiotics and bacterial cell wall pl~teins, which contain a limited number of D-amino acids introduced by specific el~ylllatic means or other post-translational modifications rather than by biosynthesis on the ribosome.
D-amino acids also occur naturally in ploleins in man as ~e result of post-translational modification by r~c~ es and as a result of spontaneous r~ce,..i,;.lion of L,lolei"s with a long in vivo lifetime. See Helfman and Bada,PNAS, 72:2891-2894 (1975). Racen~ lion is a naturally occurring process that over time, will convert naturally occurring L-amino acids into a racemic l"i~Lule of both L- and D- amino acids.
A limited portion of the structure of several approved peptide-like ~ ph~rTn~el-ti~ include a few D-arnino acids. Such products include the widely known antibiotics Valinomycin, Gramicidin A, Gramicidin S, and also the \/aSO~l~,SSill analog Desc,ll,plessi~ (RPR), Lupron (Abbott), Synarel (Syntex), Sandostatin (Sandoz), SK&-110679 (Smithklinto Reech~m), and Decapeptyl (Ipsen-Beuafor/Akzo). Such structures are small and are not solely composed of D-amino acids.
CU11~11LIY, D-peptides are made by ch~mi~al synthesis, using techniques that are well-known in the art. For example, D-peptides can be synth~i7e~l usingstepwise addition of D-amino acids in a solid-phase synthesis method involving the use of appro~?liate protective groups. Solid phase peptide synthesis techniques commonly used for L-peptides are described by Meinhofer, Hormonal Proteins and Peptides, vol. 2, (New York 1983); Kent, et al., Ann. Rev. Biochem., 57:957 CA 02234723 l998-04-08 3.
(1988); and Bodanszky et al., Peptide Synthesis, (2d ed. 1976), all of these ,erelellces are incorporated by reference herein. D-amino acids for use in the solid-phase synthesis of D-peptides can be obtained from a number of commercial sources.
D-peptides and peptides that contain mixed L- and D-amino acids are known in the art. Also, peptides cont~ining exclusively D-amino acids (D-peptides) have been synthesized. See Zawadzke et al., J. Am. Chem. Soc., 114:40024003 (1992); Milton et al., Science 256:1445-1448(1992). Ligand analogs that are known in the art are small organic molecules, L-peptides, and modified L-peptides. However, D-antibodies that specifically bind receptors or ligands or substrates have not been described in the lilel~lurt; and there remains a need for such D-antibodies and D-peptides that are analogs of ligands and receptors as well as methods for their i~lentifir~tion and production.

SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to provide methods and compositions relating to analogs of biologically active peptide and protein, including but not limited to hormones and n~u,op~plides, wherein the analogs arecomprised exclusively or es~~"l;~lly of D-amino acids and are biologically functional.
One aspect of the present invention concerns genela~ g D-peptides and D-proteins composed entirely of D-amino acids which will interact with a naturalor artificial L-peptide or L-protein target, such as a biological receptor in the body.
Novel compositions of matter comprising antibody-like entities col"~lised of D-amino acids (D-antibodies or D-peptides that are analogs of ligands or receptors) and engineered derivative forms, and a process for producing those novel antibody-like entities in such a way as to achieve required biological andph~ relltir~l functions, are provided. The general method allows production of - 30 a molecular recognition surface (such as Van der Waal's and electrostatic surface) of a D-peptide (i.e. a polypeptide or protein composed entirely or largely of D-amino acids, including D-antibodies or fragments thereof) in such a way that it -CA 02234723 l998-04-08 W O 97/13522 PCTrUS96/16358 4.
will mimic the molecular recognition surface of a natural biological ligand composed of L-amino acids, while ret~ining the advantageous ~lopclLies of D-peptides. Such D-peptides will be advantageously resistant to proteolysis in thegut, resistant to serum and tissue proteases, and are relatively immnnologicallyinert. The D-antibodies of the invention or fragments thereof, including their antigen binding loops, or reclecign~l components thereof, will have increased resistance to proteolysis in the gut and throughout the body. Compositions cu~ isillg the D-antibodies and other D-pûlypeptides of the invention are collLelllplated. D-peptide analogs of L-antibodies which preserve the binding specificity of the antigen binding loops are possible, such that relatively small structures which are no-longer antibody-like in character essçnti~lly function as analogues of natural biological ligands. Alternative screening methods to refinethe immlln-)logical approach are provided, in~hlrling the phage-generation of FAb fragments that is known in the art and is an alternative to the standard monoclonal antibody method of ~en~ldlillg an antibody to a specific antigen.
An additional advantage over the use of normal L-antibodies is that the antibody may be drastically modified including for example ret~ining only the FAb fragment in a D- analog or ret~ining the basic antigen recognition and binding loops placed on a scaffold (here, of D-amino acids) in the same orientation as in the original antibody. The design need not directly modify the antibody recognition region, but the resnlting D-antibodies or D-polypeptides may differ considerably in form from antibodies. However, D-polypeptides are usually both LallL to proteolysis and much less immnnngenic than the corresponding L-polypeptides.
In the ~l~relred embodiment, monoclonal antibodies are raised against the D-amino acid seque~res corresponding to the L-amino acid sequences of epitopes, cl-)m~in~ or whole proteins where the L-amino acid sequences of such epitopes, domains or whole proteins are previously identified by experimental or computational means. A binding site on a protein ligand would be an example of typical interest as a structure collll.lisillg the aforesaid epitope, domain, or protein.
The sequence of the monoclonal antibodies raised against the epitope domain or protein are then ~letennined, and the whole antibody or any part t'nereof which WO 97/13522 PCT~US96/16358 5.
includes the antigen binding site or part thereof is synth~i7~1 as the D-amino acid sequence corresponding to the sequenre or part of sequence in the monoclonal antibody. The resulting antibodies or subfragments thereof which are composed entirely of D-amino acids generally interact with the original natural, biological L-S forms of the above mentioned epitopes, ~lnmzlin~ or whole proteins. Unlikehllm~ni7~-1 antibodies, heavy modification of the D-polype?tides of the invention, including reduced size of the peptide chain is possible without typically requiring design to m~int~in hllm~ni7~tion, and the resl-lting D-polypeptide may be longerlasting in vivo than ph~rm~rologically functional analogs of p~ eills, such as hormones and neuropeptides and natural antibodies.
In another aspect, the invention comprises the met~od described above, wherein the synthesis of a D-peptide corresponding to the nonoclonal antibody comprises d~ ion of the binding site of the monoclonal antibody and the L-amino acid sequence of said binding site and ~,ylllhesis of ~ D-peptide that corresponds to the L-amino acid sequence or seqllenres of the binding sites of the monoclonal antibody, i.e. the D-peptide has the same amiro acid sequence as the L-amino acid sequence of the binding site, except that the D-peptide has D-aminoacids in place of the L-amino acids of the monoclonal antibody binding sites.
In yet another aspect, the invention provides synthr iz~cl D-antibodies comprising polypeptides or peptides comprised of a D-amino acid sequenre that corresponds to an L-amino acid sequence of an L-antibody consisting of L-amino acids. The D-antibodies of the invention are comprised exclusively or essentially of D-amino acids or the corresponding enantiomers of amino acid analogs. Also, the D-antibodies of the invention may contain one or more of the achiral glycineamino acid residues.
The D-antibodies of the invention can include a receptor, a substrate binding site on an en7yme, an epitope of a receptor that i,l ~lreles with ligandbinding when an antibody is bound to the receptor, a ligand binding site of a receptor, a co-factor binding site on an en_yme and a suga- binding site on a - 30 protein. In another embodiment, the D-peptides can incluce a ligand for a receptor, a substrate for an en_yme binding site, a peptide hormone for a receptor, a non-peptide hormone for a receptor, a neurotr~n~ lel for a receptor, a co-CA 02234723 l998-04-08 W O 97/13522 PCTrUS96/16358 6.
factor for a co-factor binding site on an enzyme and a sugar for a sugar bindingsite on a protein.
As yet another aspect, methods of screening and molecular activity and processes which are the equivalent to producing a recognition surface in this manner whether involving biological manipulation or ch~mi~l synthetic methods, such as mass or combinatorial screening, are also within the scope of the invention. As described below, L-antibodies that are generated in response to D-peptide antigens can be produced using the known methods of monoclonal antibody production and phage-generation of FAb fr~gm~nt~.
DETAILED DESCRIPTION
Generally, the method of generating a D-peptide that binds to a ligand or receptor entails creating a D-version of the ligand or receptor, probing or screening a library made of L-peptides with the D-version of the ligand or receptor, tletpcting "hits" or L-peptides that bind to the D-version of the ligand or receptor and then synth~ci7ing a D-version of the L-peptides. The D-version of the L-peptide is capable of binding to the L-version of the ligand or receptor.
Such D-versions of the L-peptide are often referred to herein as D-peptides or D-antibodies. These steps can be conrlllcted sequentially or repeated (such as thescreening step) before procee-ling to the next step. Further it will be apparel-L that completion of only some of the steps will facilitate the design of active molecules, such as therapeutics.
Usually, the first step is to choose a protein target (or non-peptide ligand), such as a receptor, or enzyme to which one wishes to design a novel D-peptide ligand. Typically, such a ligand D-peptide will provide either inhibition of thenormal function as an antagonist, or in some cases of the normal function activation as an agonist. The target prol~h-s or protein components (such as a ligand binding site) are synth~o~i7Pd in their mirror image form, by making them of D-amino acids (D-polypeptides). The res~llting molecules may be referred to D-polypeptides, such as D-receptors, D-enzymes, or D-hormones, parts of which correspond in sequence to normal L-receptors, L-enzymes and L-hormones, respectively. Usually the amino acid sequences of the D-version and the L-version L

W O 97/13522 PCTrUS96116358 7.
of such molecules are identical except for the fact that they are mirror images of each other. The term D-receptor will be used to refer to all protein targets whether D-receptors, D-enzymes, D-hormone or any other D-protein, or to parts of such ~lo~eills, for which an agonist, antagonist, or any other novel ligand is desired. As one typical embodiment, a ligand binding domain of a D-receptor would be made by first looking at the amino acid sequence of the ligand binding domain of the natural receptor, and then one may re-synthesize the same sequenceusing D-amino acids instead of L-amino acids. Ligand binding sites with molecular recognition surfaces cont~cting at least two surfaces of the ligand are pl~r~llcd, especially when the recognition surfaces are less than 100 amino acids apart in the amino acid sequence of the protein.
Usually, the second step is to perform combinatorial screening of an L-peptide library by probing with the D-receptor or D-polypeptide. Somrtimtos itwill be desirable not to use the first step if the target is already known.
Combhlato,ial screening ~ iL~ the creation of a diverse set of peptides that canbe then converted into D-peptides. ~lt~rn~tively~ imml-nr,logical production of antibodies or recombinant production of antibodies can be used to create a diverse set of L-peptides that can be converted into the corresponding D-peptides.
Synthetic peptide libraries can be prepared combinatorially in advance, and thenscreened against the D-receptor. One advantage of synthetic peptide libraries isthat they provide greater ch~mic~l diversity when non-naturally occurring amino acids are used, especially amino acids with the same charge as naturally occurring amino acids but differing in the distance that the charge is located from the peptide backbone. Alterations in tli~t~nre can be accomplished with 1, 2, or 3 atom extenders of the negative or positive charge, preferably carbon atoms are used.
Recombinant peptide libraries can be used as well, as known in the art.
One type of p,crellcd combinatorial library is the phage display D-peptide approach. Typically~ one would first prepare a bacteriophage display with normalL-peptides using methods known in the art. Alternatively, other recombinant - 30 libraries could be used that rely on plasmids to produce L-peptides. Here the combinatorial ~cscllLaLion of many dirrclellL peptides or proteins is implemented indirectly, by random mutation or partly random mutation or combinatorial CA 02234723 l998-04-08 W O 97/13522 PCT~US96/16358 8.
selection of nucleotide segments of the DNA expressing the plo~ills. Preferably in recombinant libraries, such as phage display libraries, a short section of DNA
encodes a mllt~ttorl region that will become part or all of the L-peptide.
Preferably, the mllt~tt~d region is not randomly mllr~t-od and does not contain all possible amino acid sequences for a 3 to 10 or a 5 to 8 amino acid mllt~tPd region.
Instead mutations are selective and usually introduce at predçtermin,od amino acid positions, conservative mutations, such as swapping polar amino acids for dirre~polar amino acids, negatively charged amino acids for dirrel~,llL negatively charged amino acids and hydrophobic amino acids for different hydrophobic amino acids.
Such mllt;~ted regions be used with synthetic peptide libraries and can are preferably used with D-antibody phage display.
Bacteriophage libraries are inexpensive and easy to manipulate, Bacteriophage display or "phage display" is widely used. It was originally developed as "fusion phage" technology (S.F. Parmley and G.P. Smith, Gene, 1~ 73:305-318, 1988, herein incorporated by reference) to describe the chimeric nature of coat proteins displaying random amino acids at their N-termini. The earliest phage display libraries, developed as a technology for more general screening purposes, appeared in a number of laboratories in 1990 and were typically implemented by cloning a synthetic piece of DNA into gene III of an fdor M13 fil~m~ntQus bacteriophage (J.K. Scott and G.P. Smith, Science 249:386-390, 1990; J.L. Devlin, L.C. p~ng~nihAn, P.E. Devlin, Science 249:404~06, 1990; S.E. Cwirla, E.A. Peters, R.W. Barrett, W.J. Dower, Proc. Nat. Acad.
Sci. USA 87:6378-6382, 1990). In these cases one may also display, not just combinatorially generated peptide segments but regions on protein domains from other sources to stabilize the protein or aid in its display. The gene or gene segment for such regions is inserted into the bacteriophage DNA and expressed asa domain with variable sequence content at the surface of the bacteriophage protein. Polymerase Chain Reaction amplification of the bacteriophage plaque followed by DNA sequencing identifies the genes which have generated peptide sequence binding to a target receptor.
A further advantage of this combinatorial screening is to increasingly refine the screening with further cycles of screening. Preferably, additional cycles of W O 97/13522 PCT~US96/16358 9.
screening offer higher stringency conditions to select for tighter binding, i.e. a lower ~pa~ l Kd. Higher stringency conditions can be accomplished by increasing the t~ p~ture, lowering or increasing the ionic strength, increasing the concentration of chaotropic agents and the like, or a combination thereof. For , S example, one can infect cells with an affinity purified population of phage so that the next selection by affinity uses up to a million copies of the original selection allowing a bacteriophage to be retrieved and amplified. In such a typical case, the method allows access to at least 108 dirr~lell~ peptides in a tube of approximately 1 ml volume.
Usually, there is a third step where the L-peptides are fl~otectçd and then sequenced or identified as to which sequence they represent using a detection oridentification means, many of which are known in the art for combinatorial libraries. In using combinatorial peptide libraries, the binding or enriched peptides respectively may be subjected to amino acid sequencing e.g. by Edman degradation, or iclentified by other means. For example, "hits" may i~1entified by position on a grid on which the combin~ti~-n~ were generated in a controlled amlel, or by chemically labeled tags including radio-labeled tags, by unique linked nucleic acid labels, by their final mass as determined by mass spectrometry (see PCT/US95/03355, which is herein incorporated by reference), by iterative resynthesis and screening of smaller subpools or submixtures (see 71lc~erm~nn etal., J. of Medicinal Chem., 37(17): 2678-2685 (1994)(incorporated by reference herein), or by any combination of these or other known techniques. In using bacteriophage display, the sequences are usually ~ ucerl by amplification and inspection of the DNA sequence coding for the peptide of interest.
Usually, there is a fourth step where the L-peptide or L-protein sequences are noted and the D-peptides or D-proteins are then made with the same amino acid sequence, using D-amino acids rather than L-amino acids. These D-peptides or D-proteins correspond to the mirror images of the selected combinatorial peptides. Such molecules can now interact with the original L-target receptor having the amino acid sequence corresponding to the D-receptor that was made forthe screen. Such receptors again include for present purposes receptors proper, W O 97/13522 PCTrUS96/16358 10.
enzymes, hormones and other plot~ s against which one may wish to make a D-peptide ligand.
An important aspect of the invention is the display of L-antibodies or parts thereof such as Fab fragments by cloning into the phage, not only to refine the binding site (recognition loops) to the target, but to refine the frame, particularly the Fab fragment bearing the antigen binding site. Particularly but not exclusively, all the peptides and protein displayed on the phage will contain atleast the antigenic binding loops of the monoclonal antibodies raised against the original D-antigen, and which will carry the recognition in D-form to interact with the original L-form of the antigen, say a natural receptor in the body. As stated above the invention includes that these loops may be modified by phage display for refinement purposes, and this also includes that a degree of design may be carried out so that l~ ,..f nt~ of the antigenic binding site sequences are raised in the phage. The modification envisaged by phage display include extensive modification of the frame, repl~remtont by non-antibody frames (folds of other ploL~ills) to carry the recognition loops, and a high degree of reduction in size including removal of the frame possibly including insertion of Cystine cross links. Modification of antibody components and selection by phage display is known in the state of the art but without regard to D-antibodies, and is known as use of "semisynthetic antibody libraries".(C. Barbas et al., Proc. Nat. Acad. Sci. USA, 89,44~7,1992).It is important to note that these modifications and the ability to perform them with beneficial effect on the final product have a special and unprecedented si~ni~lr~nre in regard to this present invention described herein which results in assigning a required recognition surface to a D-peptide or D-protein. As described above theD-Antibodies or fragments thereof will be resistant to proteolysis and less immnnogenic, an effective form of hllm~ni7~tion. Thus, the antibodies will retain hnm~ni~:3tion in D-form even when drastically modified. This includes any forms generated by phage display and made of D-amino acids. This natural hnm~ni7~tion is not the case for drastic modification in the usual application of phage display of semisynthetic antibody libraries. Hence this present invention allows that the Fab frame can be progressively reduced in size with mutations included and screened so that the activity is selected to be retained. Reduction of size in a single jump W O 97/13S22 PCT~US96/16358 11.
might well lose important interactions with the frame, causing the binding loops to become distorted. It was noted in a co~ .uL~l simulation study (V.P.Collura, P.J.Greany and B.Robson, Protein F~n~ pe~ g~ 7, 2211-223, 1992), of the antigen recognition loop H2 from Fab fragment McPC603, that there are important hlle~aclions between the antibody recognition loops and the rest of the Fab fr~gment ~ ;vely, the antigen recognition loops themselves, being three on the heavy chain and three on the light chain, may be presented and refined for binding, to be ~y../~ d as D-peptides without a supporting molecular scaffold, individually or joined, for example with oligoglycine spacers. .Alle, ..~ively they may be ~l~,s~ d on a scaffold other than an Fab fragment or other antibody component, in which the active loops are replaced by three selected antibody recognition loops. Typically these will be the loops identified in binding studies as binding most strongly to the target or its analog, but are ~l.eel~cl to be the three loops of the heavy chain as in most antibodies where studies have been carried out, these have more ~le~,ive interaction with the ~ntigen Similarly, the two larger loops of the heavy chain may be selected on occasion without the third smaller loop. In seeking a protein scaffold, engi~peli~lg may be required in~ln~ling using one or more loops in the retro (backwards) sequence direction. In all the above examples, the initial proposed structures are typically to be refined by phage display.
Optionally, these reagents may need refinement as a drug, by rational design by exploring a structure-activity relationship of analogues, or by modeling studies at the receptor where this is of known structure, and may optionally include for design and refnt?ment purposes coll~palison with rcLlvillvel~o forms of known natural or discovered or ~e~ignPcl L-peptide ligands and also optionally by colll~alison with the receptor sequence. Further refinPrnPnt may be required forenh~nre-l oral delivery and availability and hllpl~v~d pharmacokinetic properties.
~, Further chPmie~l mo~lifi~tiQn including added groups including for example extension at either termimls with ~ ition~l amino acid residues may be required.~ 30 One may also require a ~lcs~llLa~ion mP-linm such as a liposome system to be used. Peptide extensions for targeting and cell entry can include for example L-amino acid based antibodies and ~hormones preferably of long half life.

CA 02234723 l998-04-08 W O 97/13S22 PCT~US96/16358 12.
Peptide and protein extensions and additions which may be made of D-amino acids and still be active in D-forms include m~g~inin~, cecl~hls and other lytic peptides, viral cell entry peptides, endosome escape peptides of viral origin, peptides of for example lgG3 or milk protein origin which may cross the blood brain barrier.
In one embodiment antibodies composed exclusively or essenti~lly of D-amino acids interact with peptides and pluLeills made of L-amino acids in effectively the same way as the corresponding L-antibodies made of L-amino acidswill interact with peptides or proL~ s made of D-amino acids. By preparing monoclonal antibodies against the D-peptide or D-protein sequences otherwise i(lentir~l to L-peptide or L-protein sequences of the required ph~rm~r,ological target, and then making the D-antibody corresponding to the monoclonal antibody,matter is effectively reflected twice through the plane of a hypothetical mirror, leaving the binding site of the D-antibody as a molecular recognition analog of the endogenous or natural ph~ rological target.
The ph~rm~rological target can be any natural endogenous or ot_er biological or non-natural ligand or receptor, including a hormone, n~ul~pc;~lide, virus particle, other biologically active peptide, or enzyme. L-polypeptide or L-protein sequences of the target may l~l~,S~llL an epitope or set of epitopes in or near the binding site of a receptor or other protein target, or the subdomain ordomain of the receptor or other target, or the whole or part of the receptor or other target. The L-polypeptide or L-protein amino acid sequences are used to generate the coll~onding D-amino acid sequence, which is the same sequence as that of the L-polypeptide or L-protein sequence of the target amino acids differing only in en~ntiomPric form and conl~lisillg D-amino acids. Not all amino acids need to be converted from L to D form so long as they do not alter the antigenicity and immllnogenicity of the synth~ci7-ocl D-peptide antigen and do not alter the specificities of the resulting D-antibody.

W O 97/13522 PCT~US96/16358 M ETHOD OF ~IAK~NG D-A~BODIES AND D-PEPTrDES THAT ARE ~iNALOGS OF
LIGA~DS OR E~ECEPTORS
The present invention provides a method for id~ ifyillg a D-antibody or a D-peptide that is an analog of a ligand or a receptor comprising: selecting the ligand or a receptor; determinin~ the L-amino acid sequence comprising either the ligand or the ligand binding site of the receptor; synth~ci7in~ a D-peptide corresponding to the L-peptide or L-polypeptide co~ isillg the ligand or the ligand binding site of the receptor; ~r~a~illg a monoclonal antibody to said D-peptide; synth~i7ing a D-antibody co~ ,lisillg a D-amino acid sequence corresponding to an L-amino acid seq~en~e of the monoclonal antibody; and assaying said D-antibody for specific binding to the ligand or the ligand binding site of the receptor. The D-peptide or D-polypeptide corresponding to the L-peptide or L-polypeptide has the same amino acid seq ~t?nre from the carboxy tf ~ Illillll~; to the amino te- ~~ s as the L-peptide or L-polypeptide, except that the L-amino acids are replaced with their corresponding D-stereoisomers.
This method can be used for any ligand or receptor, so long as the molecule has a stereochemical configuration and the enantiomer of the molecule can be prepared. Thus, the ligands can be non-peptides, non-naturally occurring ligands, and peptides comprising a mixture of D- and L-amino acids. In general, any epitope of a ligand or receptor might be synthPsi7P~l using D-amino acids and attached to a carrier molecule to render it immunogenic. In such a case, the D-epitope may be regarded as a hapten. A D-protein in excess of approximately 3~
residues may not always be immnnt)genic without such a carrier molecule, since it is unlikely to be cleaved an presented on major histompatibility antigen to the T-cell receptor. The D-peptides or ploteills made from antibodies raised against the D-epitopes may for example include activation of a receptor by inducing dimerization of receptor p.oteins or oligomerization generally.
In the case of non-peptide ligands or receptors for which mirror-image analogs are desired, the method is modified such that the enantiomer of the non-- 30 peptide ligand or receptor is syn~hPsi7Pcl and used to generate an antibody that specifically binds to it. Then, the L-amino acid sequence of the antibody or a CA 02234723 l998-04-08 W O 97/13S22 PCT~US96/16358 14.
portion thereof, such as its Fab fr~gm~?nt, is determined, and from the L-amino acid sequence the corresponding D-antibody is synthesized.
The method can also be applied to achiral ligands or receptors or molecules. For achiral ligands or receptors or molecules, the method comprises using the achiral ligand, receptor or other molecule to generate an L-antibody;
deterrninin~ the amino acid seql-~nre of the L-antibody or a portion thereof, such as the Fab fragment; synthesizing the corresponding D-antibody; and assaying theD-antibody for specific binding to the achiral ligand, receptor or other molecule.
The selection of the chiral or achiral, natural or non-naturally occurring, ligand or receptor or other molecule to which a D-antibody is desired for binding, is accomplished using infonnation about such molecules in the li~ ul~ as well asany information regarding the amino acid sequence, if any, of such molecules.
The selection of a ligand binding site of an L-polypeptide or naturally occurring protein is accomplished by lc:r~ ce to the literature regarding àmino acid 1~ sequences of known ligand binding sites, receptors or ligands. If any suchmolecules are peptides that have not yet been sequenced, one of ordinary skill can sequence the peptides using well-known peptide sequencing methods.
The synthesis of a D-polypeptide antigen that corresponds to an L-amino acid sequence of a peptide ligand, receptor or other molecule, is accomplished using ch~.mi~l synthesis. The D-polypeptide antigen is synth~i7~ preferably using known solid phase stepwise Merrifield-type peptide synthesis techniques developed for L-peptide synthesis, but in this case using D-amino acids or protected D-amino acids in the stepwise synthesis. Other methods of synthPsi7ingD-polypeptide antigens are contemplated, including covalent bonding of monomers that were produced via stepwise synthesis. The means of synthesizing the D-polypeptide antigens and other D-polypeptides or D-antibodies of the invention are known in the art, and the invention can be practiced using methods of D-polypeptide synthesis that are yet to be developed.
Generally, the solid-phase synthesis of D-peptides comprises: ~tt~ching a protected D-amino acid to an inert solid support through the unprotected carboxyl or amino group of the D-amino acid; selectively removing the protecting group onthe amino or carboxyl group of the first D-amino acid; introducing the next D--CA 02234723 l998-04-08 W O 97/13522 PCT~US96/16358 15.
amino acid having the ~ropliate amino or carboxyl group protected and reacting it under conditions that permit formation of an amid linkage between the second D-amino acid and the first D-amino acid already attached to the solid support.
The protecting group on the amino or carboxyl group of the second D-amino acid is then selectively removed. This procedure is repeated for each successive addition of a D-amino acid to the synth~ (l D-peptide. After the D-peptide has been completely synth~osi7~1, protective groups if any rem~ining on the D-peptide are removed, and the chemically synth.-si7~-1 D-peptide is cleaved from the solid support. In order to make a large, D-peptide, the chtomie~lly synth~i7~od D-peptides can be c11~.mir~lly ligated using methods known in the art.
Once the D-polypeptide antigen is synthesized, it is used to produce antibodies. The antibodies to be produced can be either monoclonal antibodies orphage-generated FAb fragments, both methods are known in the art. As rli~c~ e above, a carrier molecule may be required to render the D-peptide or protein immnnogenic, particularly since it may not be able to cleave the D-molecule for p,l,i,e,ll~tion as major histocompatibility complex antigens to the T-cell. Suchmolecules are available commercially as kits already primed with chemical groupsto join to the peptide epitope. Proteins typically available include Keyhole Limpet Hemocyanin and Bovine Serum Albumin. The preparation of monoclonal antibodies to the D-polypeptide or D-peptide is accomplished using standard methods for production of monoclonal antibodies specific for a desired antigen.
Phage-generated peptides are described in PCT/US91/04384 (Dower et al., filed June 19, 1991); PCT/US91/02989 (Dower et al., filed May 1, 1991);
PCT/US92/08879 (Schatz et al., filed Oct. 15, 1992); PCT/US94/05796 (Aldwin et al., filed May 23, 1994); U.S. Patent No. 5,491,074 (Aldwin et al., filed May24, 1994); and PCT/FR95/00127 (Sodoyer et al., filed Feb. 2, 1995), all of whichare incorporated herein by reference. The monoclonal antibodies or phage-generated FAb fragment~ are screened using methods known in the art for specificbinding to the D-polypeptide antigen. The selected antibody or a portion of the antibody, such as the modified Fab fragment or a single-chain Fv or ~ lfi~le-bonded Fv fragment is then isolated and sequenced. Fv fragments are the ~m~ ost functional moieties of antibodies required for binding of an antigen. Reiter et al., CA 02234723 l998-04-08 W O 97/13522 PCT~US96/16358 16.
Protein Engineering, 7(5):697-704 (199 ). The L-amino acid sequence of the antibody or portion lc~lc;sellLil,g a binding site, Fab fragment, single-chain Fv fragment or ~ llfi~le-bonded Fv fragment is then used to determine the corresponding D-amino acid seqllenre of a D-antibody of the invention. The D-antibody of the invention, which includes D-amino acid sequences corresponding to an entire L-antibody or portions thereof, including the Fab fragment, the single-chain Fv fragment and the ~ fi~le-bonded Fv fragments, is synthf?ci7ed as described above.
In the case of the use of phage display of antibody, it is known that antibodies are or parts of antibodies displayed can be refined by phage display in their binding recognition loops and in part of the ~l~olLhlg antibody fold; suchantibodies are termed "semisynthetic" (C.F.Barbas et al., Proc. Nat. Acad. Sci.
USA 89-44~7, 1992). Normally mo~lific~ti- n to the frame of the antibody or Fab fragment confers no advantage in clinical use since hllm~ni7~tion will most typically be lost. However, as described above the principle significance of theinvention is that the protease-resi~L~"L properties and reduced immlmogenicity of D-antibodies implies a degree of built-in hllm~ni7~tion, such that modification to the frame is not in general a restriction. Consequently, the invention allows for all such modifications to the frame such as would normally risk loss of hllm~ni7~tion, including reduction of size of the frame as ~ c~lsse-l above, in the practice ofphage display, and including progressive reduction of the size of the antibody frame in phage display. This includes reduction in steps with enrichment by passing the cloned Fab head with randomized amino acids by phage display on progressively smaller frames, such that screening is performed at each size reduction step to retain activity. In contrast, too large a size reduction in a single step would risk total loss of activity due to too large a change of interactionsbetween loop and frame.
This invention greatly broadens the scope of application of D-proteins by readily allowing production of biologically functional forms. By taking the sequence, or portions thereof of genes which express proteins as the starting point, the invention also provides a means for more direct conversion of ~-lrol.llation in the human genome into ph~rrn~reutic ~l products.

W O 97113522 PCTrUS96/16358 17.
The D-antibodies of the invention, which include both D-analogs of complete antibodies as well as D-analogs of portions of antibodies, can be used in therapeutic compositions, as agonists or antagonists or catalysts. Also, the D-antibodies can be used to specifically bind target cells that express specific antigens, such as viral or bacterial antigens. The D-antibodies, which are not only D-antibodies but also D-peptide analogs of Fab fragments or other portions of L-antibodies, have several advantages over the corresponding L-antibodies. First, because biological systems and ~)lO~illS and enzymes are generally stereospecific for L-polypeptides or L-amino acids, the D-antibodies of the invention are resistant to proteolysis in the gut and throughout the body. Second, since D-peptides are generally less immnnogenic than their corresponding L-peptides, theD-antibodies of the invention are less likely to cause an immlln.~ response in ahuman host, which is an important characteristic for therapeutic compositions (U.S. Provisional Application 60/005,508 filed October 10, 1995 and U.S.
Provisional Application 60/014,433, are herein incorporated by ler~ ce).

D-PEPTnDES THAT A~RE ~NALOGS OF LIGANDS OR RECEPTORS
The D-antibodies of the invention are essentially D-peptides that are analogs of ligands or receptors that are capable of binding to peptide or non-peptide, natural or non-naturally occurring, chiral or achiral molecules.
The D-antibodies can be ~ecign~-l to bind to either receptors or ligands or any chiral or achiral, natural or non-naturally occurring molecules. When a D-antibody capable of specifically binding to a ligand is desired, then it is produced according to the methods described herein, using the ligand as either the template from which an amino acid sequence is obtained (if the ligand is a polypeptide) or as the antigen against which L-antibodies, whether monoclonal or phage-geneldt~d, are raised. T.ig~n(1s include naturally occurring polypeptides or peptides, non-peptides such as hormones, non-naturally occurring peptides or non-peptides, andchiral or achiral compounds. When a D-antibody that specifically binds a receptor - 30 is desired, it is produced according to methods described herein. In one embodiment, the receptor or a portion thereof is used as the original polypeptide from which an L-amino acid sequence can be determined and the corresponding CA 02234723 l998-04-08 W O 97/13522 PCTrUS96/16358 18.
D-polypeptide antigen synth~si7ed. Use of combinatorial libraries are well knownin the art. See PCT/IB95/00560 (Hodges et al., filed June 13, 1995) and PCT/US95/03355 (Benkovic et al., filed March 23, 1995) both of which are incorporated by reference herein.
Ligand generally refers to a member of a ligand binding pair, i.e. a ligand and a receptor. A portion of the ligand or surface of the ligand specifically binds a portion of a receptor or a surface of a receptor. Most often a ligand can exert a biological effect, i.e. a biological ligand. Generally, the ligand's overall structure or molecular weight is smaller than the receptor, but this is not a n.ocess~ry condition. A ligand is often a L-peptide or L-polypeptide, but other non-peptidemolecules, including steroids, co-factors, n~ulv~ "~ ulot~ r analogs, non-peptide hormones, non-peptide hormone analogs, and nucleotides, nucleosides and sugars and modified forms of non-peptide molecllles are contemplated as ligands. Non-naturally occurring and achiral ligands are also contemplated. Usually, ligands will not include D-polypeptides or D-amino acids.Usually, the affinity of the ligand for the receptor (apl?alellL Kd at a relevant temperature, ionic strength, and pH, such as at a human physiological condition) is less than lmM, preferably 1 pM to 100 ,ILM or less than 100 ,uM, more preferably10 pM to 1 ,.IM or less than 1 ,uM, and most preferably 100 pM to 100 nM or lessthan 100 nM. A D-ligand refers to a ligand with a D- configuration, usually a non-peptide.
Receptor generally refers to a member of a ligand binding pair. A receptor need not be considered a biological receptor with a specific biological function.
Instead, receptor refers to a member of the ligand binding pair with a molecularrecognition surface that binds, usually non-covalently, to the ligand. The receptor will have at least one ligand binding site with such a surface. Generally, the ligand binding site will be solvent acces~ihle, preferably water a-~ces~ible. The ligand binding site will often be composed of L-amino acids linked together by peptide bonds, such as a biological receptor for a peptide hormone. In other in~t~nreS, the receptors might be an organic molecule, such as crown ether, or anucleic acid, such as DNA.

W O 97113~22 PCTrUS96tl6358 19.
D-antibody refers to an antibody or fragment thereof that includes a D-amino acid sequence, including modifications to m~int~in the fragment or separate fragments in the conformation that they would have in the entire D-antibody (andhence also the mirror image to the conformation that the corresponding L-peptidesegments would have in the monoclonal L-Antibody). The fr~gm.ont~ will typicallybe the binding recognition loops of the antibodies that are involved in direct interaction with antigen, there being three such loops on the heavy chain and three such loops on the light chain of the antibody. For example, it has been noted that the conformation of the antigen-combining loop H2 from Fab McPC603 V.P.
Colura, P. G. Greany, and B.Robson, Protein Fn~in~ering 7:221-233, 1994) depends critically on interaction with the rest of the Fab antibody fragment.
Modifications can be of various types. Generally, a D-antibody amino acid sequence will be entirely made of D-amino acids. In some instances it will be preferable to include L-amino acids in the amino acid sequence of a D-antibody.
In particular, after reading the methods of the invention herein, it will be appalellL
that the molecular recognition surface of a D-antibody will be primarily, if notentirely, composed of D-amino acids, while the other regions of a D-antibody, ifpresent can be primarily, if not entirely, composed of L-amino acids, such as the non-variable regions of a monoclonal antibody. Additionally, D-antibodies can include single chain FAbs, or any subfragments or analogues thereof, where the heavy and light chains are joined end-to-end via a flexible linker, and mini-bodies, where the heavy and light chains are joined, preferably by a ~ llfi~le or similar bond by the introduction of two residues like cysteines, one in each chain. See Reiter et al., supra. Preferably the ligand binding site will be made of at least 90% D-amino acids, more preferable at least 95%, and most preferably 96% to 100% or 100%; wherein the percentage is calculated as the number of D-amino acids divided by the total number of amino acids in a sequence, multiplied by 100.
A D-amino acid sequence is normally used for making the protein (or peptide) ligand or receptor in the process of making a D-antibody that resembles~ 30 the receptor or the ligand. When an analog to a protein ligand is desired, the D-antibody is made using the D-amino acid sequence of the protein ligand. When an analog to a protein receptor is desired, the D-antibody is made using the D-amino CA 02234723 l998-04-08 W O 97/13522 PCT~US96/16358 20.
acid sequen~e of the protein receptor. Such D-amino acid ligands and receptors can be considered a D-polypeptide antigen. Usually the D-amino acid sequence of either the protein receptor or ligand is composed entirely of D-amino acids, butsubstitution of L-amino acids is tolerated in regions of the protein that do notadversely alter the Kd by more than two orders of m~gnitllde and more preferablyone order of m~nit~tle compared to an all D-amino acid protein ligand or rece,L)lol .
D-antigen refers to antigen with a D- configuration.
L-antibody refers to antibody made from L-amino acids, including those found in nature, made by illllllUl~ illg m~mm~l~, made by phage and other methods known in the art for making trlmr~te~1 or modified antibodies, minibodies, including, but not limited to single chain antibodies or imml-noreactive fragments thereof.

DETECTION METHODS
The invention also provides for detection methods. As the antibodies and peptides of the invention provide for peptidase resistant molecules, such compounds of the invention are particularly suited for detection of analytes.
Generally, the method of detecting an analyte comprises cont~ting the analyte with a D-antibody, and dettocting a complex of the analyte and D-antibody. Many variations in the method of detection can be accomplished with the antibodies ofthe invention. The antibodies of the invention can be used for instance in ELISAassays and separation methods can be applied comprising an additional step of separating the complex from unbound D-antibody. Washing steps are not required if complementation assays, as known in the art, are used that change the rate ofproduction of a detect~hle signal. Such assays can be con-lu-~ted using a kit cont~inin~ the n~cess~ry assay components.
A myriad of analytes can be ~letected using the antibodies of the invention and affinity selection for purification of the desired analyte(s). Primarily analytes will be those for which an antibody can be g~llel~led by methods known in the art at the time of the filing of this application and those methods later discovered.
Generally, the analyte is a ligand for a receptor, a substrate for a binding site on CA 02234723 l998-04-08 W O 97tl3522 PCTAUS96/16358 21.
an enzyme, a peptide hormone~ a non-peptide hormone, a neuloLl,.n~ itt~or, a co-factor, or a sugar.
The antibodies of the invention can be labelled if so desired by covalent or non-covalent means to facilitate detection. Such labels include enzymes capable of generating a ~letect~hle signal, fluorescent compounds (including FITC), radioactive atoms (including C14 and I125), biotin, and avidin. Preferably, labels are ~tt~rht?d at a region of the antibody that does not include the ligand binding site or at the C- or N- terminus. Labels can also include toxins to kill cells or inhibit cell proliferation.
The antibodies of the invention can also be used as biosensors in either using enzymatic or electrical ~letectiQn methods or a combination of the two.
Ligand specific electrodes can be generated using the antibodies of the invention as taught in the art for other biological molecules that bind ligands, particularlyantibodies.

Co~posrrIoNs The invention also includes pharm~l~e~lti~ compositions and methods of mo~ ting biological conditions. For instance antibodies of the invention can be used to inhibit ligand binding to a receptor by contacting an effective amount of D-antibody to a receptor, thus blocking the ligand from binding to the receptor.Usually, the amount will vary from 1 ,ug to lOOmgl more preferably from 10 ~L~g to lOmg or more than .01 mg, and most preferably from .1 mg to 10 mg.
Ph~ e~lti~l compositions comprise a physiologically suitable carrier and the compounds of the invention. Such carriers include buffers and coatings as known in the art.
All publications and patent applications mentioned in this specification are herein incorporated by lcfelcnce to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The invention now being fully described, it will be ~arcllL to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims (82)

22.
WHAT IS CLAIMED IS:

1. A D-antibody comprising a polypeptide comprised of a D-amino acid sequence that corresponds to an L-amino acid sequence of an L-antibody consisting of L-amino acids.

2. The D-antibody of claim 1 wherein said L-antibody is a monoclonal antibody or a phage generated antibody fragment that specifically binds a D-antigen made from D-amino acids or D-ligand.

3. The D-antibody of claim 2 wherein said D-antigen is a D-polypeptide selected from the group consisting of a receptor, a substrate binding site on anenzyme, an epitope of a receptor that interferes with ligand binding when a receptor antibody is bound, ligand binding site of a receptor, a co-factor binding site on an enzyme and a sugar binding site on a protein.

4. The D-antibody of claim 2 wherein said D-antigen is selected from the group consisting of a ligand for a receptor, a substrate for a binding site on an enzyme, a peptide hormone, a non-peptide hormone, a neurotransmitter, a neurotransmitter analog, a steroid, a steroid analog, a co-factor, and a sugar.

5. The D-antibody of claim 2 wherein said monoclonal antibody is a modified FAb fragment.

6. The D-antibody of claim 5 wherein said monoclonal antibody is comprised entirely of D-amino acids.

7. The D-antibody of claim 3 wherein said monoclonal antibody preferentially recognizes an epitope on a cancer cell compared to a non-cancer cell.

8. The D-antibody of claim 3 wherein said monoclonal antibody recognizes an epitope on a virus.

23.

9. The D-antibody of claim 6 wherein said monoclonal antibody has a label attached.

10. The D-antibody of claim 9 wherein said label kills cancer cells.

11. A method of making a D-peptide that is an analog of a ligand comprising:

1) selecting a L-amino acid ligand binding site or a L-ligand, 2) synthesizing a D-amino acid polypeptide that corresponds to said ligand binding site or a D-ligand, 3) preparing monoclonal antibodies to said D-amino acid polypeptide or said D-ligand, and 4) synthesizing a D-antibody comprising a D-amino acid sequence corresponding to at least a portion of said monoclonal antibody.

12. The method of claim 11 wherein said preparing comprises determining an amino acid sequence of said monoclonal's antigen binding site known to bind an antigen.

13. The method of claim 12 wherein said preparing further comprises injecting subcutaneously or administering orally said D-amino acid polypeptide into an appropriate mammal.

14. A method for identifying a D-peptide that is an analog of a peptide or polypeptide comprising:
1) selecting a L-amino acid ligand binding site, 2) synthesizing a D-amino acid polypeptide that corresponds to said ligand binding site, 3) preparing monoclonal antibodies to said D-amino acid polypeptide, 4) synthesizing a D-antibody comprising a D-amino acid sequence corresponding to at least a portion of said monoclonal antibody, and 24.
5) testing said D-antibody for specific binding activity of a receptor of said peptide or polypeptide.

15. The method of claim 13 wherein said testing is selected from the group consisting of competitive binding assays, cell culture competitive binding assays, steroid receptor binding assays using gene activation, and displacement assays.

16. A method of detecting an analyte comprising:
1) contacting said analyte with a D-antibody, and 2) detecting a complex of said analyte and said D-antibody.

17. The method of claim 15 further comprising an additional step of separating said complex from unbound D-antibody.

18. The method of claim 15 wherein said analyte is selected from the group consisting of a ligand for a receptor, a substrate for binding site on an enzyme, a peptide hormone, a non-peptide hormone, a neurotransmitter, a co-factor, and a sugar.

19. A complex comprising a D-antibody and an analyte.

20. The complex of claim 19 wherein said analyte is a L-polypeptide antigen.

21. A method of inhibiting ligand binding to a receptor comprising contacting an effective amount of D-antibody to a receptor when said ligand is present.

22. A method of identifying an L-peptide corresponding to a D-peptide that is an analog of a ligand to an L-amino acid ligand binding site comprising:
1) screening at least one L-peptide with a D-polypeptide that corresponds to said L-amino acid ligand binding site, and 2) determining the binding of said at least one L-peptide to said D-polypeptide.

25.

23. The method of claim 22, further comprising the step of:
3) synthesizing a D-peptide comprising a D-amino acid sequence that corresponds to at least a portion of an L-amino acid sequence of said at least one L-peptide, wherein said at least one L-peptide binds to said D-polypeptide.

24. The method of claim 22, wherein said L-amino acid ligand binding site is a biological receptor.

25. The method of claim 22, wherein the amino acid sequence of said L-amino acid ligand binding site is identical to the amino acid sequence of said D-polypeptide, except that said D-polypeptide is made entirely of D-amino acids.

26. The method of claim 23, wherein said D-peptide is a D-antibody.

27. The method of claim 22, wherein said at least one L-peptide is at least one L-FAb fragment.

28. The method of claim 27, wherein said at least one L-FAb fragment is phage display library of L-FAb fragments.

29. The method of claim 28, wherein said phage library of L-FAb fragments contains fragments of progressively smaller L-FAb fragments, wherein no F-Ab fragment is less than 4 amino acids and each fragment is identical in amino acidsequence to at least one portion of at least one L-Ab fragment.

30. The method of claim 29, wherein said L-amino acid ligand binding site is a biological receptor.

31. The method of claim 30, wherein L-FAb fragments have a mutated region 3 to 10 amino acids in length containing selective amino acid replacements.

26.

32. The method of claim 22, wherein said at least one L-peptide is at least ten L-peptides.

33. The method of claim 32, wherein said at least one L-peptide is a combinatorial library.

34. The method of claim 32, wherein said combinatorial library is a phage display library.

35. The method of claim 32, wherein said phage display library encodes peptides 4 to 20 amino acids in length and each peptide has a mutated region.

36. The method of claim 35, wherein said D-polypeptide is selected from the group consisting of a receptor, a substrate binding site on an enzyme, an epitope of a receptor that interferes with ligand binding when a receptor antibody is bound, ligand binding site of a receptor, a co-factor binding site on an enzyme and a sugar binding site on a protein.

37. The method of claim 33, wherein said combinatorial library comprises L-peptides ranging from 20 to 120 amino acids in length and no more than 50,000 peptides in complexity.

38. The method of claim 37, wherein said screening step comprises repeating said screening step at a higher stringency conditions to select for higher affinity binding of said L-peptides to said D-polypeptide.

39. The method of claim 38, wherein said L-peptides have an apparent Kd of 10 µM or less.

40. The method of claim 39, wherein said L-peptides have an apparent Kd of .01 µM or less.

27.

41. The method of claim 40, further comprising the step of:
3) synthesizing a D-peptide comprising a D-amino acid sequence that corresponds to at least a portion of an L-amino acid sequence of said at least one L-peptide, wherein said at least one L-peptide binds to said D-polypeptide.

42. A method of generating a L-peptide corresponding to a D-peptide that is an analog of a receptor to an L-ligand comprising:
1) screening at least one L-peptide with a D-ligand that corresponds to said L-ligand, and 2) determining the binding of said at least one L-peptide to said D-ligand.

43. The method of claim 42, further comprising the step of:
3) synthesizing a D-peptide comprising a D-amino acid sequence that corresponds to at least a portion of an L-amino acid sequence of said at least one L-peptide, wherein said at least one L-peptide binds to said D-ligand.

44. The method of claim 42, wherein said L-ligand is a non-peptide with a chiral center.

45. The method of claim 42, wherein said L-ligand is the mirror image of said D-ligand.

46. The method of claim 43, wherein said D-peptide is a D-antibody.

47. The method of claim 42, wherein said at least one L-peptide is at least one L-FAb fragment.

48. The method of claim 47, wherein said at least one L-FAb fragment is phage display library of L-FAb fragments.

28.

49. The method of claim 48, wherein said phage library of L-FAb fragments contains fragments of progressively smaller L-FAb fragments, wherein no F-Ab fragment is less than 6 amino acids and each fragment is identical in amino acidsequence to at least one portion of at least one L-Ab fragment.

50. The method of claim 49, wherein said L-ligand has a molecular weight of a peptide no more than 50 amino acids in length.

51. The method of claim 50, wherein L-FAb fragments have a mutated region 5 to 8 amino acids in length containing selective amino acid replacements.

52. The method of claim 51, wherein said at least one L-peptide is at least 100 L-peptides.

53. The method of claim 42, wherein said at least one L-peptide is a combinatorial library.

54. The method of claim 52, wherein said combinatorial library is a phage display library.

55. The method of claim 52, wherein said phage display library encodes peptides 4 to 20 amino acids in length and each peptide has a mutated region.

56. The method of claim 55, wherein said D-ligand is selected from the group consisting of a ligand for a receptor, a substrate for a binding site on an enzyme, a peptide hormone, a non-peptide hormone, a neurotransmitter, a neurotransmitter analog, a steroid, a steroid analog, a co-factor, and a sugar.

57. The method of claim 53, wherein said combinatorial library comprises L-peptides no more than 10 amino acids in length and no more than 10,000 peptides in complexity.

29.

58. The method of claim 57, wherein said screening step comprises repeating said screening step at a higher stringency conditions to select for higher affinity binding of said L-peptides to said D-ligand.

59. The method of claim 58, wherein said L-peptides have an apparent Kd of 1 µM or less.

60. The method of claim 59, wherein said L-peptides have an apparent Kd of .1 µM or less.

61. The method of claim 60, further comprising the step of:
3) synthesizing a D-peptide comprising a D-amino acid sequence that corresponds to at least a portion of an L-amino acid sequence of said at least one L-peptide, wherein said at least one L-peptide binds to said D-ligand.

62. A product by the process of one of the claims of 22, 23, 41, 42, 43, or 61.

63. A compound comprising:
D-peptide that has a mirror image that binds to D-polypeptide.

64. The compound of claim 63, wherein said D-peptide can bind to an L-amino acid ligand binding site.

65. The compound of claim 64, wherein said L-amino acid ligand binding site is a biological receptor.

66. The compound of claim 64, wherein the amino acid sequence of said L-amino acid ligand binding site is identical to the amino acid sequence of said D-polypeptide, except that said D-polypeptide is made entirely of D-amino acids.

67. The compound of claim 64, wherein said D-peptide is a D-antibody.

30.

68. The compound of claim 67, wherein said D-peptide an L-FAb fragment.

69. The compound of claim 68, wherein said L-FAb fragment is six to 200 amino acids in length.

70. The compound of claim 64, wherein said D-polypeptide is selected from the group consisting of a receptor, a substrate binding site on an enzyme, an epitope of a receptor that interferes with ligand binding when a receptor antibody is bound, ligand binding site of a receptor, a co-factor binding site on an enzyme and a sugar binding site on a protein.

71. The compound of claim 70, wherein said D-peptide is 6 to 20 amino acids in length.

72. The compound of claim 71, wherein said L-peptides have an apparent Kd of .1 µM or less.

73. A compound comprising:
D-peptide that has a mirror image that binds to D-ligand.

74. The compound claim 73, wherein said D-peptide can bind to an L-ligand.

75. The compound claim 74, wherein said L-ligand is a non-peptide with a chiral center.

76. The compound of claim 75, wherein said L-ligand has a molecular weight of a peptide no more than 10 amino acids in length.

77. The compound of claim 74, wherein said D-peptide is a D-antibody.

78. The compound of claim 77, wherein said D-peptide is an L-FAb fragment.

31.

79. The compound of claim 78, wherein said L-FAb fragment is 10 to 100 amino acids in length.

80. The compound of claim 74, wherein said D-ligand is a ligand for a receptor, a substrate for a binding site on an enzyme, a peptide hormone, a non-peptide hormone, a neurotransmitter, a neurotransmitter analog, a steroid, asteroid analog, a co-factor, and a sugar.

81. The compound of claim 70, wherein said D-peptide is 5 to 30 amino acids in length.

82. The method of claim 72, wherein said L-peptides have an apparent Kd of .01 µM or less.