Note: Descriptions are shown in the official language in which they were submitted.
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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.
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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
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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
-
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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
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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-
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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
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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
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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
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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,
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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
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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.
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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.
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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
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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-
-
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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.,
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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.
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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
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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.
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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
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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
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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.
