EP-4741422-A2 - SERUM ALBUMIN BINDING PROTEINS

EP4741422A2EP 4741422 A2EP4741422 A2EP 4741422A2EP-4741422-A2

Abstract

Inventors

DOMBRECHT, BRUNO
SCHOTTE, PETER
VERVERKEN, Cedric Jozef Néotére

Assignees

Ablynx NV

Dates

Publication Date: 20260513
Application Date: 20120614

Claims (12)

Use of the amino acid sequence Alb-23 as set out in SEQ ID NO: 1 or a variant of Alb-23 as a moiety, binding unit or fusion partner in order to increase the half-life of one or more therapeutic moieties, wherein said variant of Alb-23 comprises: (i) the amino acid motif GP on positions 44 and 45; (ii) the amino acid motif SKN on positions 74 to 76; (iii) a CDR1 that is the amino acid sequence SFGMS as set out in SEQ ID NO:29; (iv) a CDR2 that is the amino acid sequence SISGSGSDTLYADSVKG as set out in SEQ ID NO:30; (v) a CDR3 that is the amino acid sequence GGSLSR as set out in SEQ ID NO:31; and that preferably also comprises (vi) a G at position 16; and/or (vii) an R at position 83 and preferably both a G at position 16 and an R at position 83; wherein said variant of Alb-23 further comprises between 1 and 7, such as between 1 and 5 further amino acid differences with the sequence given in SEQ ID NO:1.
Use according to claim 1, wherein said amino acid sequence of Alb-23 as set out in SEQ ID NO: 1 or said variant of Alb-23 are suitably linked to said one or more therapeutic moieties either directly or optionally via one or more suitable linkers or spacers.
Use according to claim 1 or 2, wherein said one or more therapeutic moieties are selected from a polypeptide, protein, compound, small molecule or other therapeutic entity.
Use according to any of claims 1 to 3, wherein said one or more therapeutic moieties are one or more immunoglobulin single variable domains.
Use according to any of claims 1 to 4, wherein said one or more therapeutic moieties are one or more VHHs, humanized VHHs or camelized VHs.
Use according to claim 4 or 5, in which at least one of the immunoglobulin single variable domains comprises at least two disulphide bridges.
Use according to claim 5 or 6, in which at least one of the VHHs, humanized VHHs, camelized VHs or immunoglobulin single variable domains is a VHH of class I.
Use according to any of claims 4 to 7, in which at least one of the immunoglobulin single variable domains is directed against c-met.
Use according to claim 8, in which at least one of the immunoglobulin single variable domains directed against c-met comprises two disulphide bridges.
Use according to claim 9, in which said immunoglobulin single variable domain against c-met is 4E09 as set out in SEQ ID NO: 12 or a variant of 4E09.
Use according to any of claims 8 to 10, in which said immunoglobulin single variable domain against c-met is A00790105 as set out in SEQ ID NO: 19.
Use according to any of the preceding claims, wherein the half-life is defined as the time taken for the serum concentration of the polypeptide to be reduced by 50% in vivo.

Description

The present invention relates to amino acid sequences that are capable of binding to serum albumin; to proteins and polypeptides comprising or essentially consisting of such amino acid sequences; to nucleic acids that encode such amino acid sequences, proteins or polypeptides; to compositions, and in particular pharmaceutical compositions, that comprise such amino acid sequences, proteins and polypeptides; and to uses of such amino acid sequences, proteins and polypeptides. Other aspects, embodiments, advantages and applications of the invention will become clear from the further description herein. If any terms are not specifically defined herein, these terms have the meaning given to them in WO 2009/068627 or WO 06/122787. If any terms used herein are not specifically defined herein or in WO 2009/068627/WO 06/122787, then they have their usual meaning in the art, for which reference is for example made to the standard handbooks. Amino acid sequences that are capable of binding to human serum albumin and uses thereof in polypeptide constructs in order to increase the half-life of therapeutically relevant proteins and polypeptides are known in the art. For example, WO 04/041865 by applicant describes Nanobodies directed against serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other Nanobodies directed against a desired target) in order to increase the half-life of said protein. The international application WO 06/122787 describes a number of Nanobodies against (human) serum albumin. These Nanobodies include the Nanobody called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787). [Nanobody® and Nanobodies® are trademarks of Ablynx N.V.]. Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties. As of the date of first filing of the present application, the use of Nanobodies against (human) serum albumin for extending the half-life of therapeutic moieties such as Nanobodies has been validated by means of clinical trials. For example, the safety, tolerability, immunogenicity and pharmacokinetics (PK) of ALX-0141, a protein construct that comprises two Nanobodies against RANK-L and one Nanobody against human serum albumin, has been confirmed in phase I clinical trials (data presented by Ablynx N.V. on May 27, 2011 at the Annual European Congress of Rheumatology (EULAR) in London). Also, numerous published patent applications of Ablynx N.V. give examples of constructs with increased half-life that comprise one or more Nanobodies against a therapeutic target and one or more Nanobodies against serum albumin (such as Alb-8). Reference is for example made to WO 04/041862, WO 2006/122786, WO 2008/020079, WO 2008/142164, WO 2009/068627 and WO 2009/147248. Although it has been established that the use of Nanobodies against (human) serum albumin (such as those described in WO 04/041865 and WO 06/122787, and in particular the humanized variants of Alb-1 described in WO 06/122787) provide a good and broadly applicable methodology for extending the half-life of Nanobodies and of other therapeutic moieties and entities, this does not mean that the skilled person would not benefit from having further improved Nanobodies against human serum albumin at his disposal for this purpose. The present invention provides such an improved Nanobody that is directed against human serum albumin (called "Alb-23"; see SEQ ID NO:1), as well as a number of variants of this Nanobody (also referred to herein as "Alb-23 like sequences" or "Alb-23 variants", see SEQ ID NO's 3 to 11 for some non-limiting examples), as well as as compounds, polypeptides and other (protein) constructs comprising the same (as further described herein). The advantages that the improved Nanobody of SEQ ID NO:1 and its variants can provide over the Nanobodies described in WO 04/041865 and WO 06/122787 will become clear from the further description herein. For example and without limitation, these advantages may include improved stability (such as improved thermal stability as determined by measuring the Tm); and/orand/or improved storage stabilty, as for example measured in the SEC experiment described in Example 5); and/ora reduced tendency to form dimers under certain formulation conditions (for example, at high concentrations in certain aqueous formulation buffers - see again for example Example 5). In addition, it has been found that the improved Nanobody of SEQ ID NO:1 and its variants are particularly suited for extending the half-life of immunoglobulin single variable domains that contain more than one disulphide bridge, such as VHH's and Nanobodies belonging to the "VHH-1 class" (which as further described herein may comprise two or even three disulphide bridges). For example and without limitation, it has been found that polypeptides that comprise one or