Peptides which function as hinge regions in protein

Details Peptides which function as hinge regions in protein Peptides which function as hinge regions in protein

2000-03-17

2003-11-04

Gambel, Phillip (Department: 1644)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

11 to 14 amino acid residues in defined sequence

C530S300000, C530S350000, C530S387100, C530S387300, C530S391100, C530S391300, C530S391500, C530S391700, C530S391900, C530S402000, C530S866000, C530S867000

Reexamination Certificate

active

06642356

ABSTRACT:

This invention relates to peptides which function as hinge regions in proteins, to proteins containing such hinge regions and to the use of said proteins in medicine.
In clinical antibody therapy and imaging applications the avidity of a dimeric antibody species is often required to achieve an effective antibody affinity in vivo, but without the effector functions or lengthy serum permanence conferred by the Fc domain (ref. 1-3—for the literature referenced by number herein see the list “References” hereinafter). F(ab′)2 molecules meet this requirement and can be produced by proteolytic cleavage of monoclonal IgG of appropriate isotypes, or by use of recombinant immunoglobulin derived domains produced in
E. coli.
The ability to secrete antibody fragments to the oxidising periplasm of
E. coli
has led to rapid advances in the engineering of grafted, highly expressed Fab's. Importantly, the use of
E. coli
also enables the cost effective and rapid production of the amounts of antibody material required to supply a large market (ref. 4, 5). Engineered Fab′ is often expressed with only one hinge-cysteine. This cysteine can be used for attachment of other Fab's to make a F(ab′)
2
or attachment of therapeutic effector molecules such as radionuclides, enzymes, or toxins (ref. 6).
Several protein engineering approaches for producing divalent antigen binding species in vivo in
E. coli
have been reported, using both modified scFvs and Fab's. Simple hinge modifications do not give substantial yields in vivo of dimeric species from
E. coli
(ref. 7-9). Techniques for increasing dimerisation in vivo are well characterised, but these often use large non-immunoglobulin dimerisation motifs which are potentially immunogenic and can cause severe reductions in the level of expression of soluble protein (ref. 10-13). The simplest route to production of dimeric antigen binding species remains the direct disulphide or chemical cross-linking of Fab's in vitro (ref. 2, 7, 14, 15). The choice of covalent linkage between the two Fab's is an important one. If the F(ab′)
2
is cleaved in vivo then the resulting Fab′ molecules generated suffer both from loss of avidity and very rapid clearance from the circulation (ref. 1, 2). Single disulphide bonds are known to be more susceptible to cleavage in vivo than protected disulphides, sulphide, or thioether bonds (ref. 2, 9, 16). However, two disulphides as found in the hinge region of F(ab′)
2
isolated from proteolytic cleavage of IgG1 have previously been found to be as robust as one thioether bond, as judged by serum permanence (ref. 9).
There is a need for non-immunogenic dimeric antibody species which overcome the problem of facile in vivo cleavage while still being efficient to manufacture and couple to other effector molecules. We have now found a peptide, which when part of a larger protein such as a Fab′ fragment efficiently generates dimers and yields dimeric material which is highly resistant to chemical reduction in vitro and has long serum permanence times in vivo. Advantageously, the peptide is well tolerated in
E. coli
and in our tests to date has been shown to be non-immunogenic.
Thus according to one aspect of the invention we provide a peptide of formula (1) (SEQ ID NO:1):
N
TCPPCPXYCPPCPA
C
  (1)
wherein X and Y, which may be the same or different, is each a neutral aliphatic L-amino acid residue, and protected and reactive derivatives thereof.
In formula (1) and the other peptides described herein conventional single letter abbreviations are used to represent amino acid residues except where otherwise indicated. The superscripts “N” or “C” are used to indicate respectively the N- or C-terminal residue of a peptide.
Neutral L-amino acid residues represented by each of the groups X and Y include glycine, alanine, valine, leucine, isoleucine, serine and threonine residues.
Protected derivatives of the peptides of formula (1) include N- and C-terminal protected derivatives in which the N-terminal amino group or the C-terminal carboxyl group is linked to a protecting group. N-protected derivatives include for example optionally substituted benzyloxy-carbonylamino, allyloxycarbonylamino, cycloalkyloxycarbonylamino, t-butoxycarbonylamino, trifluoroacetylamino, phthalylamino, aralkylamino, e.g. benzylamino, diphenylmethylamino or triphenylmethylamino, tosyl-amino or formylamino derivatives. C-protected derivatives include for example esters, such as optionally substituted alkyl, e.g. methyl, ethyl or t-butyl, aralkyl, e.g. benzyl or benzhydryl, silyl, e.g. trimethylsilyl and phthalimidomethyl esters, and esters with polymers, for example functionalised styrene-based resins.
Reactive derivatives of peptides of formula (1) include those in which the C-terminal carboxyl group is functionalised, and is, for example, an acyl halide such as an acyl chloride, an aryl cyanide or azide, an anhydride, an ester, for example a N-hydroxy succinimide, p-nitrophenyl or pentachloro-phenyl ester, a N-acyl heterocycle such as a N-acyl imidazole, pyrazole or triazole or an activated acid formed by addition of a carbodiimide or isoxazolium reagent.
Particularly useful peptides of formula (1) include those wherein X is an alanine residue, In another preference, Y in particular is a threonine residue. An especially useful peptide according to the invention has the formula (2) (SEQ ID NO:2):
N
TCPPCPATCPPCPA
C
  (2)
and protected and reactive derivatives thereof.


REFERENCES:
patent: 5041533 (1991-08-01), Wunsch et al.
patent: 5635371 (1997-06-01), Stout et al.
patent: 284 898 (1988-03-01), None
Crasto et al. Protein Engineering 2000; 13(5):309-312.*
Better et al.,PNAS USA, 90:457-61 (1993).
Humphreys et al,J. Immunol. Methods, 209:193-202 (1997).
Kaku et al.,General pharmacology, 27:435 (1996).
King et al.,Biochemical J., 281:317 (1992).
Lyons et al.,Protein Engineering, 3:703 (1990).
Pack et al.,Biochem., 31:1579 (1992).
Rodrigues et al.,J. Immunology, 151:6954-61 (1993).

Affiliated with

Also associated with

Rating

Peptides which function as hinge regions in protein does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Peptides which function as hinge regions in protein, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Peptides which function as hinge regions in protein will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3184810