JP2009511032A - Nanobodies and polypeptides for EGFR and IGF-IR - Google Patents

Abstract

The present invention relates to polypeptides and Nanobodies against epidermal growth factor receptor (EGFR) and / or insulin growth factor-I receptor (IGF-IR). The invention also includes nucleic acids encoding the Nanobodies and polypeptides; methods for preparing the Nanobodies and polypeptides; host cells that express or are capable of expressing the Nanobodies and polypeptides; the Nanobodies, polypeptides, Also relates to compositions comprising nucleic acids and / or host cells, in particular pharmaceutical compositions; and the use of said Nanobodies, polypeptides, nucleic acids, host cells and / or compositions, especially for prophylactic, therapeutic or diagnostic purposes. .
[Selection figure] None

Description

  The present invention relates to polypeptides and Nanobodies (Nanobody ™) for epidermal growth factor receptor and / or insulin growth factor-I receptor (“EGFR” and “IGF-IR”, respectively) [Note: Nanobody ( Trademark), Nanobodies (TM) and Nanoclone (TM) are trademarks of Ablynx.

  The invention also includes nucleic acids encoding the Nanobodies and polypeptides; methods for preparing the Nanobodies and polypeptides; host cells that express or are capable of expressing the Nanobodies and polypeptides; the Nanobodies, polypeptides, Compositions comprising nucleic acids and / or host cells, in particular pharmaceutical compositions; and said Nanobodies, polypeptides, in particular for prevention, treatment or diagnostic purposes, such as for the prevention, treatment or diagnostic purposes described herein Also, the use of nucleic acids, host cells, and / or compositions.

  Other aspects, embodiments, advantages and applications of the present invention will become apparent upon further description herein.

International patent applications WO 05/044858 and WO 04/041867 by the present applicant include Nanobodies against EGFR, polypeptides comprising the same, compositions comprising the Nanobodies and polypeptides, and the Nanobodies, poly Methods for preparing and using peptides and compositions are described.
International Patent Application No. 05/044858 Pamphlet International Patent Application No. 04/041867 Pamphlet

  In general, Nanobodies to EGFR, which are alternatives to, and preferably have improved properties compared to, the Nanobodies and polypeptides described in WO 05/044858 and WO 04/041867 It is an object of the present invention to provide a polypeptide.

  Specifically, it is an object of the present invention to provide various improved Nanobodies and polypeptides for EGFR. For example, in one preferred but non-limiting embodiment, the present invention relates to Nanobodies against EGFR that bind to the extracellular domain EGFR and antagonize EGF on EGFR, a TGFα binding site, but not an erbitux binding site. I will provide a. In another preferred but non-limiting embodiment, the present invention provides Nanobodies that bind to the extracellular domain EGFR and antagonize EGF, erbitux, and TGFα binding sites on EGFR. In yet another preferred but non-limiting embodiment, the present invention provides Nanobodies that bind to the extracellular domain EGFR and antagonize a TGFα binding site on EGFR but not an EGF or erbitux binding site To do. As detailed herein, different (therapeutic or diagnostic) applications can be found for all these different types of Nanobodies and polypeptides, depending on the properties desired for use.

  In general, Nanobodies and polypeptides described herein, particularly improved Nanobodies to EGFR described herein, have been disclosed in these publications, as described in WO 05/044858 and WO 04/041867. It can be used for all uses and uses for Nanobodies and polypeptides. Thus, for example, the Nanobodies described herein can be found in WO 05/044858 and WO 04/041867 in place of Nanobodies against EGFR described in WO 05/044858 and WO 04/041867. It can be used in the preparation of the described polypeptides and Nanobody constructs. The Nanobodies and polypeptides described herein can also be formulated and used in the same manner as described in WO 05/044858 and WO 04/041867.

  In particular, the Nanobodies, polypeptides and compositions described herein are EGFR and / or IGF-IR related diseases and disorders, in particular EGFR and / or IGF-IR (ie in a tissue or cell). It can be used for the prevention, diagnosis and / or treatment of diseases and disorders associated with or characterized by overexpression. Examples of such diseases and disorders will be apparent to those skilled in the art and include various forms of cancer and tumors, such as those described herein, and certain skin inflammatory diseases.

  The anti-IGF-IR Nanobodies described herein can themselves be used to treat cancer and other diseases and disorders associated with IGF-IR (overexpression), but any anti-EGFR therapy ( Promotes treatment with anti-EGFR compounds, anti-EGFR antibodies (including antibody fragments), or treatment with anti-EGFR Nanobodies or polypeptides) and reduces the amount of anti-EGFR compound or antibody used in the treatment (eg, for example, , Reduce the side effects associated with them), and / or can be used to prevent or reduce resistance to anti-EGFR therapy. Examples of such anti-EGFR treatment will be apparent to those skilled in the art, for example from the relevant prior art literature cited herein. See also, for example, Friess et al., Clin. Cancer Res, 11 (14), 2005, 5300 and Thaker et al., Clin. Cancer Res., 11 (13), 2005, 4923. Several types of mouse monoclonal antibodies such as IMClone systems IMC-C225 (erbitux or cetuximab), Merck EMD7200 (matuzumab), Abgenix ABX-EGF (panitumumab), and Genmab 2F8 It has been successfully introduced in preclinical and clinical applications.

  Also, in general, the in vitro and / or in vivo activity and / or efficacy of Nanobodies and polypeptides described herein can be determined by in vitro assays, in vivo assays, cell-based assays, or WO 05/044858 and WO It can be determined using one or more of the animal models described in 04/041867. See also Roovers et al., “Efficient inhibition of EGFR signaling and of tumor growth by antagonisitic anti-EGFR Nanobodies”. Other assays, models, and techniques for assessing the effectiveness of the Nanobodies, polypeptides and compositions described herein, particularly in treating tumors and / or inhibiting tumor cell growth or proliferation, include, for example: It will be apparent to those skilled in the art from the prior art literature relating to EGFR and IGF-IR described herein.

  It is also an object of the present invention to provide Nanobodies against IGF-IR and polypeptides comprising them.

  The role of the IGF-I receptor in tumors and strategies for targeting IGF-IR in the treatment of cancer are described, inter alia, by Hofman and Garcia-Echeverria, Drug Discovery Today, Vol. 10, 15, August 2005, 1041, Papa et al., Cancer Res., 1993, August 15, 53 (16): 3736-40; Arteaga et al., J. Clin. Invest., Vol. 84., November 1989, 1418-1423; Maloney Cancer Research 63, 5073-5083, 2003, Baehr and Groner, Growth Factors, March 2005, 23 (1) 1-14; Ulfarsson et al., Clin. Cancer Res., 2005, 11 (13), 2005, Burtrum et al., Cancer Research, 63, 8912-8921 (2003), Miyamoto et al., Clin. Cancer Res., 2005, 11 (9), 2005, Hailey et al., Molecular Cancer Therapeutics, Vol. 1, 1349-1353, 2002 See December.

  It has also been reported that the IGF-I receptor is involved in acquiring resistance to Herceptin (Altundag et al., Mol. Cancer Ther., 2005, (4) 7, 1136, Monier et al., Bull. Cancer. , 2004, Sep; 91 (9): 685-94, Chakravarti et al., Cancer Research 62, 200-207, 2002). Regarding the synergistic effect of EGF and IGF-I, for example, Faisal et al., Journal of Surgical Research, 69, 354-358 (1997), Adams et al., Growth Factors, June 2004, Vol. 22 (2), 89 -95, and Knowlden, Endocrinology, 2005, 21 July (electronic publication before printing). Enhancement of EGFR signaling by IGF is described in Chong et al., Biochem. Biophys. Res. Commun., 2004, 322 (2): 535-41. The relationship between EGFR and IGF-IR expression in breast cancer is described, for example, in van den Berg et al., Br. J. Cancer, 1996 ,, 73 (4): 477-81. Combination therapy for cancer using a monoclonal antibody against IGF-IR is described, for example, in Cohen et al., Clinical Cancer Research Vol. 11, 2063-2073 (2005). Lu et al., JBC, Vol. 279., No. 4, 2856-2865 (2004) describe the effect of simultaneous blocking of EGFR and IGF-IR on the growth of cancer cells using bispecific monoclonal antibodies. is doing. Manes et al., Endocrinology, Vol. 138, No. 3, 905 describes epitope mapping of IGF-IR.

  In the present invention, these objects are generally achieved by using Nanobodies and polypeptides provided in the present invention.

  Thus, specifically, for each of EGFR or IGF-IR from a warm-blooded animal, more specifically for each of EGFR or IGF-IR from a mammal, in particular, for each of human EGFR or IGF-IR. Providing superior Nanobodies against EGFR and / or polypeptides and Nanobodies against IGF-IR, and providing proteins and polypeptides comprising or consisting essentially of at least one Nanobody One purpose.

  Specifically, the Nanobodies and the proteins and / or polypeptides are provided that are suitable for preventive, therapeutic, and / or diagnostic purposes in warm-blooded animals, specifically mammals, more specifically humans. This is the object of the present invention.

  More specifically, in warm-blooded animals, specifically mammals, more specifically humans, one or more types associated with and / or mediated by EGFR or IGF-IR, respectively. The Nanobody and the protein and / or polypeptide that can be used for the prevention, treatment, alleviation and / or diagnostic purposes of a disease, disorder, disease (such as those described herein), or symptoms It is an object of the present invention to provide.

  It is also associated with and / or mediated by EGFR or IGF-IR, respectively, in warm-blooded animals, specifically mammals, more specifically humans (as described herein). The Nanobody and the protein and / or the protein and / or protein, which can be used for the preparation of a medicament or veterinary pharmaceutical composition for the treatment and / or prevention of one or more diseases, disorders or symptoms (such as diseases, disorders and symptoms) It is also an object of the present invention to provide polypeptides.

  One specific but non-limiting object of the present invention is the conventional antibody against each of EGFR or IGF-IR, or Fab ′ fragment, F (ab ′) 2 fragment, scFv construct, “diabody”. Compared to other fragments of (single) domain antibodies such as “dAb” reported by Ward et al., Supra, and also WO 05/044858 and WO Improved therapeutic and / or pharmacological properties and / or (eg, improved ease of preparation and / or reduced product cost) compared to Nanobodies and polypeptides against EGFR described in 04/041867 Nanobodies, proteins and / or polypeptides for each of EGFR or IGF-IR having other advantageous properties (such as It is to provide a. These improved advantageous properties will become apparent from further description herein.

  These objectives are achieved by the Nanobodies, proteins and polypeptides described herein. These Nanobodies are also referred to herein as “Nanobodies of the invention” and these proteins and polypeptides are also collectively referred to herein as “Polypeptides of the invention”.

  Accordingly, in a first aspect, the present invention provides an excellent Nanobody against EGFR, specifically an excellent Nanobody against EGFR from warm-blooded animals, more specifically an excellent Nanobody against EGFR from mammals, particularly humans Regarding the Nanobody against the derived EGFR, the excellent Nanobody is as defined below.

  In another aspect, the invention relates to a binding protein for IGF-IR having a molecular weight of less than 15 kDa. In one embodiment, the binding protein can inhibit the interaction of IGF-I with IGF-IR. In a preferred embodiment, the polypeptide is a single domain antibody, a domain antibody, “dAb”, VH, VHH, or Nanobody.

  Accordingly, the present invention preferably provides Nanobodies to IGF-IR, specifically Nanobodies to IGF-IR from warm-blooded animals, more specifically Nanobodies to IGF-IR from mammals, particularly human-derived IGFs. -Relates to Nanobodies against IR.

  In another aspect, the invention relates to a protein or polypeptide comprising, or consisting essentially of, Nanobodies for each of the EGFR or IGF-IR.

  According to another embodiment, the present invention provides Nanobodies and polypeptides against EGFR, and Nanobodies against EGFR, particularly for use in combination therapy for the treatment of cancer. In the combination therapy, Nanobodies and polypeptides against IGF-IR as described herein may be combined with anti-EGFR Nanobodies and polypeptides as described in WO 05/044858 and WO 04/041867, and / or herein. May be used with the anti-EGFR Nanobodies and polypeptides described in.

  According to one specific but non-limiting embodiment, the polypeptides described herein comprise at least one Nanobody for EGFR and at least one Nanobody for IGF-IR. In the bispecific Nanobody construct, Nanobodies and polypeptides against IGF-IR described herein are one or more anti-EGFR Nanobodies and polypeptides described in WO05 / 044858 and WO04 / 041867. Peptides and / or may be mixed with one or more anti-EGFR Nanobodies and polypeptides described herein.

  For pharmaceutical use, the Nanobodies and polypeptides of the invention are preferably directed to human EGFR or IGF-IR, respectively, and for use as veterinary medicine, the Nanobodies and polypeptides of the invention It will be apparent to those skilled in the art that are directed against EGFR or IGF-IR, respectively, preferably derived from the species to be treated.

  In addition, according to the present invention, Nanobodies and polypeptides having a directivity to each of EGFR or IGF-IR derived from the first species of warm-blooded animals can be EGFR derived from one or more other warm-blooded animals or It may or may not show cross-reactivity with IGF-IR. For example, Nanobodies and polypeptides directed against human EGFR or IGF-IR, respectively, may or may not be cross-reactive with EGFR or IGF-IR from one or more primates, and / or Or in animal models for diseases, in particular animal models for diseases and disorders associated with EGFR or IGF-IR, respectively (such as the species and animal models discussed herein) (eg, mouse, It may or may not be cross-reactive with EGFR or IGF-IR from one or more animals (such as rats, rabbits, pigs, or dogs). In this embodiment, if cross-reactivity is present, the cross-reactivity allows the Nanobodies and polypeptides against human EGFR or IGF-IR, respectively, to be tested in the disease model, so However, it will be apparent to those skilled in the art that it has advantages from a pharmaceutical development perspective.

  More generally, Nanobodies and / or polypeptides that target one animal's EGFR or IGF-IR, respectively, where the Nanobodies and / or polypeptides provide the desired effect on the species to be treated ( It is also within the scope of the present invention to use Nanobodies and polypeptides that target human EGFR or IGF-IR, respectively, for the treatment of other types of animals.

  In the broadest sense, the present invention relates to a specific antigenic determinant, epitope, site, domain, subunit or confirmation of EGFR or IGF-IR targeted by the Nanobodies and polypeptides of the present invention (where applicable) Is not particularly limited by or defined by these. However, specifically, the Nanobodies and polypeptides of the present invention may be directed to epitopes exposed on the cell surface.

  Where applicable, the Nanobodies of the invention may be able to bind to each antigenic determinant, epitope, site, domain, subunit or confirmation of two or more EGFR or IGF-IR, Included within the scope of the present invention. In such cases, the antigenic determinant, epitope, site, domain, subunit of each EGFR or IGF-IR to which the Nanobody and / or polypeptide of the invention binds may be the same (eg, EGFR or IGF Each -IR may contain repeating structural motifs or be present as multimers, and may be different (in the latter case, the Nanobodies and polypeptides of the invention may have the same or different affinities and / or Or may bind with specificity to the different antigenic determinants, epitopes, sites, domains, subunits). Also, for example, if EGFR or IGF-IR, respectively, exists in active and inactive conformations, the inventive Nanobodies and polypeptides will either attach to these conformations, or these conformations. May bind to both (ie, with affinity and / or specificity that may be the same or different). Also, for example, the Nanobodies and polypeptides of the invention may bind to the respective conformation of EGFR or IGF-IR, which is bound to the appropriate ligand, or not bind to the appropriate ligand, each of EGFR or IGF-IR. It may bind to a conformation and may bind to both such conformations (with affinity and / or specificity that may be the same or different again).

  In addition, the Nanobodies and polypeptides of the present invention generally include all natural or synthetic analogs, variants, mutants, alleles, portions and fragments of EGFR or IGF-IR, respectively, or EGFR or IGF-IR, respectively. It is also expected to bind to at least the analogs, variants, mutants, alleles, portions and fragments. Each contains one or more antigenic determinants or epitopes that are substantially identical to the antigenic determinant or epitope that the Nanobody and polypeptide of the invention bind to EGFR or IGF-IR, respectively (eg, wild-type EGFR or Each of IGF-IR). Also in that case, the Nanobodies and polypeptides of the invention have the same or different (high or low) affinity and the same as when the Nanobodies of the invention bind to (wild type) EGFR or IGF-IR, respectively. It can bind to the analogs, variants, mutants, alleles, moieties and fragments with specificity. It is also within the scope of the present invention that the Nanobodies and polypeptides of the present invention bind to certain analogs, variants, mutants, alleles, portions and fragments, but not others. .

  Where each EGFR or IGF-IR is present as a monomer, and one or more multimers, the Nanobodies and polypeptides of the invention are only against each EGFR or IGF-IR present as a monomer. When bound, or when the Nanobodies and polypeptides of the invention further bind to one or more of the multimers, they are included within the scope of the invention. In addition, when each of EGFR or IGF-IR can associate with other proteins or polypeptides to form a protein complex, the Nanobody and the polypeptide of the present invention can be EGFR or IGF-IR, respectively, in an unassociated state. And EGFR or IGF-IR in association with each other, or both, are also encompassed within the scope of the present invention. In all of these cases, the Nanobodies and polypeptides of the invention have the same or different (high or low) affinities when bound to EGFR or IGF-IR, respectively, in monomeric or unassociated state. And / or with specificity, the Nanobodies and polypeptides of the invention may be associated with the multimer or associated protein complex.

  In general, the Nanobodies and polypeptides of the present invention are at least in the most relevant form (such as monomers, multimers, or aggregates) from a biological and / or therapeutic standpoint, as will be apparent to those skilled in the art. Will combine.

  When using and / or comprising, or consisting essentially of, parts, fragments, analogs, mutants, variants, alleles, and / or derivatives of Nanobodies and polypeptides of the invention The use of peptides is also included within the scope of the present invention as long as it is suitable for the applications envisioned herein. Such portions, fragments, analogs, mutants, variants, alleles, derivatives, proteins, and / or polypeptides are further described herein.

  As discussed in more detail herein, the Nanobodies of the invention generally comprise a single amino acid chain, which is a “framework sequence” or “FR” (reviewed herein) and “ It is thought to consist of “complementarity determining regions” or CDRs. Some preferred CDRs present in the Nanobodies of the present invention are those described herein.

More generally, referring to the definitions further provided herein for Nanobodies to IGF-IR, the CDR sequences present in the Nanobodies of the invention can be obtained / obtained by a method comprising the following steps: it can:
a) (i) immunizing a mammal belonging to the family Camelidae with IGF-IR or a part or fragment thereof, and improving an immune response against IGF-IR and / or an antibody (more specifically, a heavy chain antibody); (Ii) obtaining a biological sample containing a heavy chain antibody sequence and / or a VHH sequence directed against IGF-IR from the immunized mammal as described above; and (iii) IGF from the biological sample. -Directed by methods generally including obtaining (eg, isolating) heavy chain antibody and / or VHH sequences directed against IR and / or (i) directed against IGF-IR or at least a portion or fragment thereof. Screening a library comprising a heavy chain antibody sequence and / or a VHH sequence having sex, and (ii) from the library, IG Providing at least one VHH domain directed against IGF-IR by a method generally comprising obtaining (eg, isolating) a heavy chain antibody sequence and / or VHH sequence directed against IR. ;
b) A heavy chain antibody sequence and / or VHH sequence for IGF-IR is obtained in this way to increase the affinity and / or specificity of the heavy chain antibody sequence and / or VHH sequence for IGF-IR. Heavy chain antibody sequences and / or VHH sequences against IGF-IR can be subjected to affinity maturation, mutagenesis (eg, random mutagenesis or site-directed mutagenesis), and / or any other technique. , In some cases, a process to be provided;
c) determining the sequence of the CDRs of the heavy chain antibody sequence and / or VHH sequence obtained in this way against IGF-IR; and optionally d) at least one, preferably at least two, more preferably Providing a Nanobody, wherein all three CDRs (CDR1, CDR2 and CDR3, specifically at least CDR3) have the sequence determined in step c).

  Usually, in step d), all CDR sequences present in the Nanobody of the invention are derived from the same heavy chain antibody or VHH sequence. However, in the broadest sense, the present invention is not limited to these. For example, in a Nanobody of the invention (which is often less preferred), a heavy chain antibody to two or three different IGF-IRs or CDRs derived from a VHH sequence may be appropriately bound and / or In the Nanobodies of the invention, one or more CDRs derived from heavy chain antibodies or VHH sequences (specifically at least CDR3) are replaced by one or more CDRs derived from different sources (eg synthetic CDRs or humans) The antibody or CDR derived from the VH domain) may be bound appropriately.

  According to a non-limiting but preferred embodiment of the present invention, the CDR sequence in the Nanobody of the present invention is 10-5 to 10-12 mol / liter or less, preferably 10-7 to 10-12 mol / liter or less. , More preferably with a dissociation constant (KD) of 10 −8 to 10 −12 mol / liter or less, and / or at least 107 M−1, preferably at least 108 M−1, more preferably at least 109 M−1, such as at least 1012 M− With a binding affinity of 1 and / or with an affinity of less than 500 nM, preferably less than 200 nM, preferably less than 10 nM, preferably less than 10 nM, for example less than 500 pM, respectively, To join. The affinity of the Nanobodies of the present invention for each of EGFR or IGF-IR can be determined by methods known per se, such as, for example, the assays described herein.

In a preferred but non-limiting embodiment, the present invention relates to a Nanobody for EGFR (as defined herein), consisting of four framework regions (respectively FR1-FR4) and three complementarity determining regions (respectively CDR1-CDR3). ) Concerning:
(A) CDR1 is an amino acid sequence selected from the group consisting of:
TYTMA [SEQ ID NO: 42]
SYGMG [SEQ ID NO: 43]
GFAMG [SEQ ID NO: 44]
SNNMG [SEQ ID NO: 45]
GDVMG [SEQ ID NO: 46]
SYVVG [SEQ ID NO: 47]
DYNMA [SEQ ID NO: 48]
TYTMA [SEQ ID NO: 49]
NNAMA [SEQ ID NO: 50]
SYVMG [SEQ ID NO: 51]
SYAMG [SEQ ID NO: 52]
A [SEQ ID NO: 53]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only two or one “amino acid difference” (defined herein):
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) the amino acid sequence preferably comprises only amino acid substitutions, compared to the amino acid sequence above Does not include amino acid deletions or insertions;
And / or:
(B) CDR2 is an amino acid sequence selected from the group consisting of:
GISRSDGGTYDADSVKG [SEQ ID NO: 54]
GISWRGDSTGYADSVKG [SEQ ID NO: 55]
AISWSGGSLLYVDSVKG [SEQ ID NO: 56]
AIGWGLETHYSDSVGKG [SEQ ID NO: 57]
GFSRSTSTTHYADSVKG [SEQ ID NO: 58]
GIAWGDGITYYADSVKG [SEQ ID NO: 59]
HISWLGGRTYYRDSVKG [SEQ ID NO: 60]
GFSGSGGATYYAHSVEG [SEQ ID NO: 61]
AISWRGGSTYYADSVKG [SEQ ID NO: 62]
AINWSSGSTYADSVKG [SEQ ID NO: 63]
TIAWDGSTYYADSVKG [SEQ ID NO: 64]
GLSWSADSTYYADSVKG [SEQ ID NO: 65]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having three, two or only one “amino acid difference” (defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
(C) CDR3 is an amino acid sequence selected from the group consisting of:
ASVKLVYVNPRNRYSY [SEQ ID NO: 66]
AAGSAWYGTLYEYDY [SEQ ID NO: 67]
AAGSTWYGTLYEYDY [SEQ ID NO: 68]
MVGPPPRSLDYGLGNNHYEYDY [SEQ ID NO: 69]
SSTRVIVITLPRMYNY [SEQ ID NO: 70]
NSRSSWVIFTIKGQYDR [SEQ ID NO: 71]
RPGMIITTIQATYGF [SEQ ID NO: 72]
GSPYGTELLPYTRIEQYAY [SEQ ID NO: 73]
ANEYWVYVNPRNRYTY [SEQ ID NO: 74]
RRKSGEVVFTIPARYDY [SEQ ID NO: 75]
VYRVGAISEYSGTDYYTDEYDY [SEQ ID NO: 76]
GYQINSGNYNFKDYEYDY [SEQ ID NO: 77]
SYNVYYNNYYYPISRDEYDY [SEQ ID NO: 78]
HRRPFASVFTTRMYDY [SEQ ID NO: 79]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having three, two or only one “amino acid difference” (defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions And no amino acid deletions or insertions.

In another preferred but non-limiting embodiment, the present invention relates to a nanobody against IGF-IR (this As defined in the description):
(A) CDR1 is an amino acid sequence selected from the group consisting of:
FNAMG [SEQ ID NO: 94]
INVMA [SEQ ID NO: 95]
NYAMG [SEQ ID NO: 96]
RTAMA [SEQ ID NO: 97]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only two or one “amino acid difference” (defined herein):
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
(B) DR2 is an amino acid sequence selected from the group consisting of:
VIISGGSTHYVDSVKG [SEQ ID NO: 98]
EITRSGRTNYVDSVKG [SEQ ID NO: 99]
AINWNSRSTYYADSVKG [SEQ ID NO: 100]
TITWNSGTTRYADSVKG [SEQ ID NO: 101]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having three, two or only one “amino acid difference” (defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
(C) CDR3 is an amino acid sequence selected from the group consisting of:
KKFGDY [SEQ ID NO: 102]
IDGSWREY [SEQ ID NO: 103]
SHDSDYGGTNANLYDY [SEQ ID NO: 104]
TAAAAVITPTRGYNY [SEQ ID NO: 105]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having three, two or only one “amino acid difference” (defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions And no amino acid deletions or insertions.

  Thus, some particularly preferred but non-limiting CDR sequences and CDR sequence combinations present in the Nanobodies of the present invention are as shown in Table A-1 for EGFR or IGF-IR, respectively.

Table A-1: Nanobodies for EGFR and IGF-IR

  Accordingly, in the Nanobodies of the present invention, at least one of the CDR1, CDR2 and CDR3 sequences present is selected from the group consisting of each of CDR1, CDR2 and CDR3 listed in Table A-1 for EGFR or IGF-IR, respectively. Or at least one of each of CDR1, CDR2, and CDR3 listed in Table A-1 for each of EGFR or IGF-IR, and at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably Each CDR1 selected from the group consisting of CDR1, CDR2 and CDR3 sequences having at least 99% “sequence identity” (as defined herein) and / or listed in Table A-1 for EGFR or IGF-IR, respectively. , C R2 and at least one of the CDR3, 3 pieces, are selected from two or "difference in amino acid" in one only group consisting respectively CDR1, CDR2 and CDR3 sequences having (as defined herein).

  In particular, in the Nanobody of the invention, at least the CDR3 sequence present is selected from the group consisting of the CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, or Table A- for EGFR or IGF-IR, respectively. Selected from the group consisting of at least one CDR3 sequence listed in 1 and a CDR3 sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity; And / or selected from the group consisting of at least one of the CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, and a CDR3 sequence having only 3, 2, or 1 amino acid difference.

  Preferably, in the Nanobody of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are selected from the group consisting of the respective CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively. Or at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least one of each of the CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively. Are each selected from the group consisting of CDR1, CDR2 and CDR3 sequences having at least 99% sequence identity and / or CDR1, CDR2 respectively listed in Table A-1 for each of EGFR or IGF-IR Preliminary least one of the CDR3 sequences, three, selected from the group consisting of each of CDR1, CDR2 and CDR3 sequences with differences 2 or one only of the amino acid.

  In particular, in the Nanobody of the invention, at least the CDR3 sequence present is selected from the group consisting of the CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, or Table A- for EGFR or IGF-IR, respectively. Selected from the group of CDR3 sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with at least one of the CDR3 listed in 1; At least one of the CDR1 and CDR2 sequences to be selected from the group consisting of the respective CDR1 and CDR2 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, or to Table A-1 for EGFR or IGF-IR, respectively. Enumerated Selected from the group of CDR1 and CDR2 sequences each having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity with at least one of the CDR1 and CDR2 sequences, respectively And / or consist of at least one of the CDR1 and CDR2 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, and each of the CDR1 and CDR2 sequences with only 3, 2, or 1 amino acid difference Selected from the group.

  Most preferably, in the Nanobody of the invention, all three CDR1, CDR2 and CDR3 sequences present are selected from the group consisting of the respective CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively. Or at least one of each of the CDR1, CDR2, and CDR3 sequences listed in Table A-1 for each of EGFR or IGF-IR, and at least 80%, preferably at least 90%, more preferably at least 95%, and even more Preferably each CDR1, CDR2 and CDR selected from the group of CDR1, CDR2 and CDR3 sequences each having at least 99% sequence identity and / or listed in Table A-1 for EGFR or IGF-IR respectively. At least one of the sequences, three, selected from the group consisting of each of CDR1, CDR2 and CDR3 sequences with differences 2 or one only of the amino acid.

  Even more preferably, in the Nanobody of the invention, at least one of the CDR1, CDR2 and CDR3 sequences present is a group consisting of the CDR1, CDR2 and CDR3 sequences respectively listed in Table A-1 for EGFR or IGF-IR, respectively. Selected from. Preferably, in this embodiment, one of the other two CDR sequences, or preferably both, is at least one of each of the corresponding CDR sequences listed in Table A-1 for EGFR or IGF-IR respectively; Selected from corresponding CDR sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity and / or a table for each of EGFR or IGF-IR. Selected from the group consisting of at least one of the corresponding sequences listed in A-1 and a CDR sequence having only 3, 2 or 1 amino acid difference.

  In particular, in the Nanobodies of the present invention, at least the CDR3 sequence present is selected from the group consisting of CDR3 listed in Table A-1 for each of EGFR or IGF-IR. Preferably, in this embodiment, at least one of the CDR1 and CDR2 sequences present, or preferably both, are at least 80% of the respective CDR1 and CDR2 sequences listed in Table A-1 for each of EGFR or IGF-IR. Selected from the group of CDR1 and CDR2 sequences each having a sequence identity of preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, and / or Table A for EGFR or IGF-IR respectively. Selected from the group consisting of at least one of the CDR1 and CDR2 sequences listed in -1, respectively, and each of the CDR1 and CDR2 sequences having only three, two or only one amino acid difference.

  Even more preferably, in the Nanobody of the invention, at least two of the CDR1, CDR2 and CDR3 sequences present are each a group consisting of the CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively. Selected from. Preferably, in this embodiment, the remaining CDR sequences present are at least 80%, preferably at least 90%, with at least one of each of the corresponding CDR sequences listed in Table A-1 for EGFR or IGF-IR, respectively. %, More preferably at least 95%, even more preferably at least 99% of sequence identity, selected from the group of CDR sequences and / or listed in Table A-1 for each of EGFR or IGF-IR Selected from the group consisting of CDR sequences having at least one of the sequences to be matched and only three, two or only one amino acid difference.

  In particular, in the Nanobodies of the invention, at least the CDR3 sequence is selected from the group consisting of the CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively, and either CDR1 or CDR2 sequence is EGFR or IGF-IR Each is selected from the group consisting of the respective CDR1 and CDR2 sequences listed in Table A-1. Preferably, in this embodiment, the remaining CDR sequences present are at least 80%, preferably at least 90% more than at least one of the corresponding CDR sequences listed in Table A-1 for EGFR or IGF-IR, respectively. A corresponding CDR sequence selected from the group of CDR sequences, preferably having at least 95%, even more preferably at least 99% sequence identity, and / or listed in Table A-1 for EGFR or IGF-IR, respectively Selected from the group consisting of CDR sequences with 3, 2 or only 1 amino acid difference.

  Even more preferably, in the Nanobodies of the invention, all CDR1, CDR2 and CDR3 sequences present are selected from the group consisting of the respective CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively. .

  In general, combinations of CDRs listed in Table A-1 for EGFR or IGF-IR (described in the same row of Table A-1) are preferable. Accordingly, in the present invention, one CDR in the Nanobody is a CDR sequence shown in Table A-1, or at least 80% of the CDR sequences listed in Table A-1 for each of EGFR or IGF-IR, Table A-1 is preferably selected from the group of CDR sequences having at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity and / or for each of EGFR or IGF-IR. And at least one of the other CDRs, preferably both are selected from the group consisting of at least one of the CDR sequences listed in 1 and a CDR sequence having only 3, 2 or 1 amino acid difference. CDR sequences belonging to the same combinations listed in Table A-1 (described in the same row of Table A-1) Selected from the group of CDR sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with CDR sequences belonging to the same combination or belonging to the same combination And / or selected from the group consisting of CDR sequences belonging to the same combination and CDR sequences having only 3, 2 or 1 amino acid differences. The other preferred cases shown in the above paragraph also apply to the CDR combinations shown in Table A-1.

  Thus, by way of non-limiting example, Nanobodies of the present invention are shown in Table A-1, for example, CDR1 sequences having greater than 80% sequence identity with one of the CDR1 sequences shown in Table A-1 ( However, it can include one of the CDR2 sequences (which belong to different combinations), a CDR2 sequence having only 3, 2 or only one amino acid difference, and a CDR3 sequence.

  Certain preferred Nanobodies of the invention may comprise, for example: (1) a CDR1 sequence having greater than 80% sequence identity with one of the CDR1 sequences shown in Table A-1; shown in Table A-1 One of the CDR2 sequences (but belonging to different combinations) and a CDR2 sequence with only three, two or only one amino acid difference; CDR3 as shown in Table A-1 (but belonging to different combinations) A CDR3 sequence having more than 80% sequence identity with one of the sequences; or (2) a CDR1 sequence having more than 80% sequence identity with one of the CDR1 sequences shown in Table A-1; a CDR2 sequence; One of the CDR3 sequences shown in Table A-1 for each of EGFR or IGF-IR; or (3) a CDR1 sequence; greater than 80% sequence identity with one of the CDR2 sequences shown in Table A-1 CDR2 sequences having; shown and in Table A-1, and CDR3 sequences belonging to the same combination and CDR2 sequences, three, CDR3 sequences with differences 2 or one only of the amino acid.

  Some particularly preferred inventive Nanobodies may include, for example: (1) a CDR1 sequence having greater than 80% sequence identity with one of the CDR1 sequences shown in Table A-1; Table A- CDR2 sequences belonging to the same combination shown in 1 and CDR2 sequences having a difference of 3, 2 or 1 amino acid; CDR3 sequences belonging to the same combination shown in Table A-1 and 80% CDR3 sequences having sequence identity greater than: (2) CDR1 sequences; CDR2 sequences listed in Table A-1 for EGFR or IGF-IR, respectively; and CDR3 listed in Table A-1 for EGFR or IGF-IR, respectively Sequence (CDR2 and CDR3 sequences may belong to different combinations).

  Some even more preferred inventive Nanobodies may include, for example: (1) a CDR1 sequence having greater than 80% sequence identity with one of the CDR1 sequences shown in Table A-1; CDR1 sequences listed in Table A-1 for each IGF-IR and belonging to the same combination; and CDR3 sequences belonging to different combinations shown in Table A-1; or (2) CDR1 sequences shown in Table A-1 A CDR2 sequence as shown in Table A-1 and belonging to the same or different combination and a CDR2 sequence having a difference of only 3, 2 or 1 amino acids; and Table A-1 for EGFR or IGF-IR respectively. CDR3 sequences listed and having greater than 80% sequence identity with CDR3 sequences belonging to the same or different combinations.

  Particularly preferred Nanobodies of the invention include, for example, the CDR1 sequences shown in Table A-1; the CDR2 sequences shown in Table A-1 that have more than 80% sequence identity with the CDR2 sequences belonging to the same combination; The CDR3 sequences shown in A-1 and belonging to the same combination may be included.

  In the most preferred Nanobodies of the invention, the CDR1, CDR2 and CDR3 sequences present are selected from one of the combinations of CDR1, CDR2 and CDR3 sequences listed in Table A-1 for EGFR or IGF-IR, respectively.

Preferably, the CDR sequence is at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% with one of the CDR sequences listed in Table A-1 for EGFR or IGF-IR, respectively. Selected from the group of CDR sequences having the sequence identity (defined herein) and / or three CDR sequences listed in Table A-1 for EGFR or IGF-IR, respectively, and 3 When selected from the group consisting of CDR sequences with two or only one amino acid difference,
i) all amino acid substitutions are preferably conservative amino acid substitutions (as defined herein), and / or
ii) The amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the CDR sequences listed in Table A-1 for EGFR or IGF-IR, respectively.

  According to a non-limiting but preferred embodiment of the present invention, the CDR sequences in the Nanobodies of the present invention are defined as above and are also 10-5 to 10-12 mol / liter or less, preferably 10-7. With a dissociation constant (KD) of -10-12 mol / liter or less, more preferably 10-8 to 10-12 mol / liter or less, and / or at least 107 M-1, preferably at least 108 M-1, more preferably at least Nanobodies of the invention with a binding affinity of 109 M-1, for example at least 1012 M-1, and / or with an affinity of lower than 500 nM, preferably lower than 200 nM, preferably lower than 10 nM, such as lower than 500 pM Binds to EGFR or IGF-IR, respectively. The affinity of the Nanobodies of the invention for EGFR or IGF-IR, respectively, can be determined by methods known per se, for example using the assays described herein.

  According to another preferred but non-limiting embodiment of the invention, (a) CDR1 is 1-12 amino acid residues, usually 2-9 amino acid residues, eg 5, 6, or 7 Has a length of amino acid residues, and / or (b) CDR2 is 13-24 amino acid residues, usually 15-21 amino acid residues, eg, 16 or 17 amino acid residues long And / or (c) CDR3 has a length of 2 to 35 amino acid residues, usually 3 to 30 amino acid residues, for example 6 to 23 amino acid residues. Yes.

  The Nanobody having the CDR sequence preferably has a framework sequence as further defined herein.

  In another aspect, the invention relates to at least 80%, preferably 90%, more preferably 95%, from the group consisting of SEQ ID NOs: 80-93 or with one or more of the amino acid sequences of SEQ ID NOs: 80-93, More preferably, it relates to a Nanobody having an amino acid sequence selected from the group consisting of amino acid sequences having 99% sequence identity (as defined herein).

  In another embodiment, the present invention provides at least 80%, preferably 90%, more preferably 95%, from the group consisting of SEQ ID NOs: 106-109 or with one or more of the amino acid sequences of SEQ ID NOs: 106-109, More preferably, it relates to a Nanobody having an amino acid sequence selected from the group consisting of amino acid sequences having 99% sequence identity (as defined herein).

  According to a specific but non-limiting embodiment, the latter amino acid sequence has been “humanized” as further described herein.

  The polypeptides of the present invention comprise or consist essentially of the Nanobodies of the present invention. Some preferred but non-limiting examples of polypeptides of the invention are shown in SEQ ID NOs: 110-143.

  In general, a protein or polypeptide comprising or consisting essentially of a single Nanobody (such as a Nanobody of the invention) is referred to herein as a “monovalent” protein or polypeptide, or “monovalent construct”. Proteins and polypeptides comprising or consisting essentially of more than two Nanobodies (such as at least two Nanobodies of the invention, or at least one Nanobody of the invention and at least one other Nanobody) are , Referred to herein as “multivalent” proteins or polypeptides, or “multivalent constructs”, which provide some advantage over the corresponding monovalent Nanobodies of the present invention. Some non-limiting examples relating to the multivalent construct will become clear from the further description herein.

  According to one specific but non-limiting embodiment, the polypeptide of the invention comprises or essentially consists of at least two inventive Nanobodies, such as two or three inventive Nanobodies. It consists of these. As further described herein, the multivalent construct comprises a single Nanobody or, for each of EGFR or IGF-IR, as compared to a protein or polypeptide of the invention consisting essentially of these. Resulting in some advantage, such as a much improved affinity and / or specificity.

  According to another specific but non-limiting embodiment, the polypeptide of the invention comprises at least one Nanobody of the invention and at least one other Nanobody (ie another epitope, antigen, target , Which is directed to a protein or polypeptide). The protein or polypeptide is also referred to herein as a “multispecific” protein or polypeptide, or “multispecific construct”, which provides some advantage over the corresponding monovalent Nanobody of the invention . Some non-limiting examples regarding the multispecific construct will also be apparent from further description herein.

  According to yet another specific but non-limiting embodiment, the polypeptide of the invention comprises at least one Nanobody of the invention, optionally one or more other Nanobodies, and the invention. Comprising or essentially consisting of at least one other amino acid sequence (such as a protein or polypeptide) that confers at least one desired property to the Nanobody and / or the resulting fusion protein. The fusion protein also provides some advantage over the corresponding monovalent nanobody of the invention. Some non-limiting examples regarding the amino acid sequences and fusion constructs will also be apparent from further description herein.

  For example, to obtain a trivalent bispecific construct comprising two Nanobodies of the present invention and one other Nanobody and optionally one or more other amino acid sequences, Two or more can be combined. Other non-limiting examples of such constructs and some constructs that are particularly preferred in the context of the present invention will become apparent from further description herein.

  In the above construct, one or more Nanobodies and / or other amino acid sequences may be directly linked or may be linked via one or more linker sequences. Some preferred but non-limiting examples of such linkers will become clear from the further description herein.

  In one preferred embodiment of the invention, the polypeptide of the invention comprises one or more (eg two, or preferably one) that allows the resulting polypeptide of the invention to cross the brain blood barrier. ) (In some cases through one or more suitable linker sequences) one or more (eg two or preferably one) Nanobodies of the invention. Specifically, the one or more amino acid sequences that allow the resulting polypeptide of the present invention to cross the brain blood barrier are one or more (eg, Nanobodies described in WO 02/057445) There may be two, or preferably one) amino acid sequences, of which FC44 (SEQ ID NO: 35) and FC5 (SEQ ID NO: 36) are some preferred non-limiting examples.

  In another preferred embodiment of the invention, the polypeptide of the invention has one or more (eg two or preferably one) amino acids that increase the in vivo half-life of the resulting polypeptide of the invention. It includes one or more (eg, two, or preferably one) Nanobodies of the present invention linked to a sequence (possibly via one or more suitable linker sequences). Specifically, the amino acid sequence that increases the in vivo half-life of the resulting polypeptide of the present invention is one or more (eg, two or preferably one) Nanobodies, and particularly human serum albumin, etc. Nanobodies directed against human serum proteins of PMP6A6 ("ALB? 1", SEQ ID NO: 32), ALB? 8 (humanized ALB? 1, SEQ ID NO: 33), and PMP6A8 ("ALB ? 2 ", SEQ ID NO: 34) are some preferred non-limiting examples. Other examples of preferred Nanobodies for mouse or human serum albumin are described in the following applicants' patent applications:

  In yet another preferred embodiment of the invention, the polypeptide of the invention comprises one or more (eg two or preferably one) Nanobodies of the invention and the resulting polypeptide of the invention is a blood brain barrier. One or more (eg, two, or preferably one) amino acid sequences that allow it to pass through, and one or more (eg, two) that increase the in vivo half-life of the resulting polypeptide of the invention. Or (preferably one) amino acid sequence (optionally linked via one or more suitable linker sequences). Even in this case, the one or more amino acid sequences that allow the resulting polypeptide of the present invention to cross the blood brain barrier are one or more (eg, Nanobodies described in WO 02/057445) (for example, May be two, or preferably one) Nanobodies (described herein), wherein the amino acid sequence that increases the in vivo half-life of the resulting polypeptide of the invention is one or more (eg two Or preferably 1) Nanobodies (described herein).

  According to a preferred but non-limiting embodiment of the present invention, the polypeptide of the present invention is preferably 10-5 to 10-12 mol / liter or less, preferably 10-7 to 10-12 mol / liter or less. , More preferably with a dissociation constant (KD) of 10 −8 to 10 −12 mol / liter or less, and / or at least 107 M−1, preferably at least 108 M−1, more preferably at least 109 M−1, such as at least 1012 M− A polypeptide of the invention with an affinity of 1 and / or with an affinity of EGFR or IGF-IR of less than 500 nM, preferably less than 200 nM, preferably less than 10 nM, for example less than 500 pM. Combine to each. The affinity of a polypeptide of the invention for each of EGFR or IGF-IR can be determined by methods known per se, for example using the assays described herein.

Some preferred but non-limiting examples of polypeptides of the invention are the polypeptides of SEQ ID NOs: 110-143:
-SEQ ID NOs 134-135 are some examples of multivalent (specifically divalent) polypeptides of the invention against IGF-IR:
-SEQ ID NOs: 110-133 and 141-143 are some examples of multivalent (specifically bivalent) polypeptides of the invention for EGFR:
-Of these, SEQ ID NOs: 141-143 are some examples of multivalent (specifically bivalent) biparatopic polypeptides of the invention for EGFR:
-SEQ ID NOs: 136-140 are some examples of bispecific polypeptides of the invention comprising one Nanobody for EGFR and one Nanobody for IGF-IR.

  Other polypeptides of the invention are, for example, one or more of the amino acid sequences of SEQ ID NOs: 110 to 143 and greater than 80%, preferably greater than 90%, more preferably greater than 95%, such as It may be selected from the group consisting of amino acid sequences having “sequence identity” (defined herein) of greater than 99%, and the Nanobodies contained in said amino acid sequences are preferably defined herein To do.

  In another aspect, the invention relates to a nucleic acid encoding a Nanobody of the invention and / or a polypeptide of the invention. The nucleic acid is also referred to herein as “the nucleic acid of the present invention”, and may be, for example, a gene construct as defined herein.

  In another aspect, the invention relates to a host or host cell that expresses or is capable of expressing a Nanobody of the invention and / or a polypeptide of the invention and / or comprising a nucleic acid of the invention. Some preferred but non-limiting examples for the host or host cell will become clear from the further description herein.

  The invention further comprises at least one Nanobody of the invention, at least one polypeptide of the invention, and / or at least one nucleic acid of the invention, depending on the intended use of the composition, It relates to a product or composition optionally containing one or more other ingredients of the composition known per se. The product or composition may be, for example, a pharmaceutical composition (defined herein), a veterinary pharmaceutical composition, or a diagnostic product (also defined herein) or composition. Some preferred but non-limiting examples of said product or composition will become clear from the further description herein.

  The invention further relates to methods of making or producing the Nanobodies, polypeptides, nucleic acids, host cells, products and compositions described herein. Some preferred but non-limiting examples of the method will become clear from the further description herein.

  The present invention further prevents and / or prevents uses and uses of the Nanobodies, polypeptides, nucleic acids, host cells, products and compositions described herein, and diseases and disorders associated with EGFR and IGF-IR, respectively. It relates to a method of treatment. Some preferred but non-limiting applications and uses will become apparent from further description herein.

  Other aspects, embodiments, advantages and applications of the present invention will become apparent from the further description below.

Detailed Description of the Invention The above and other aspects, embodiments, and advantages of the present invention will become apparent from the following further description, wherein:
a) Unless otherwise indicated or defined, all terms used have their ordinary meaning in the art and will be apparent to those skilled in the art. For example, Sambrook et al., “Molecular Cloning: A Laboratory Manual” (2nd. Ed.), VoIs. 1-3, Cold Spring Harbor Laboratory Press (1989), edited by F. Ausubel et al., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987), Lewin, "Genes II", John Wiley & Sons, New York, NY, (1985), Old et al., "Principles of Gene Manipulation: An Introduction to Genetic Engineering", 2nd edition , University of California Press, Berkeley, CA (1981), Roitt et al., “Immunology” (6th. Ed.), Mosby / Elsevier, Edinburgh (2001), Roitt et al., Roitt's Essential Immunology, 10th Ed. Blackwell Publishing, UK ( 2001) and Janeway et al., “Immunobiology” (6th Ed.), Garland Science Publishing / Churchill Livingstone, New York (2005), and standard handbooks such as general background literature cited therein. I want to be.
b) Unless otherwise indicated, whether the term “immunoglobulin sequence” is used herein to indicate either heavy chain antibodies or conventional four chain antibodies, full-length antibodies, their individual molecular chains , And any portion, domain or fragment thereof (including, but not limited to, an antigen binding domain, or a VHH domain or a VH / VL domain, respectively). Further, the term “sequence” is used herein (eg, in terms such as “immunoglobulin sequence”, “antibody sequence”, “variable region sequence”, “VHH sequence”, or “protein sequence”). If so, it is generally to be understood that both the relevant amino acid sequence and the nucleic acid or nucleotide sequence that encodes it are included, where there is no need for a limited interpretation from the context.
c) Unless otherwise indicated, all methods, steps, techniques and operations not specifically described in detail can be carried out by methods apparent to those skilled in the art. Again, reference is made to the standard handbooks and general background art documents listed herein, and other references cited therein.
d) Amino acid residues are represented using the usual 3 letter or 1 letter amino acid abbreviations shown in Table A-2.

Table A-2: One-letter or three-letter amino acid abbreviations

note:
(1) It may be considered as an amino acid having no polar charge.
(2) It may be considered as an amino acid having no non-polar charge.
(3) The fact that amino acid residues are shown in this table as having a charge at pH 6.0-7.0 or as having no charge, as will be apparent to those skilled in the art, is 6. The amino acid residues shown in the table do not reflect in any way the charge that the amino acid has at a pH below 0 and / or a pH above 7.0, as will be apparent to those skilled in the art. May or may not have a charge at such a high pH or low pH.
(4) As is well known in the art, the charge of His residues is also highly dependent on slight pH changes, but His residues are usually essentially charged at pH around 6.5. You can think that you do not have.

e) For comparison of two or more nucleotide sequences, [number of nucleotides in the first nucleotide sequence that are identical to nucleotides in corresponding positions in the second nucleotide sequence] [first nucleotide sequence By dividing [total number of nucleotides in] and multiplying by [100%], the percentage of “sequence identity” between the first and second nucleotide sequences can be calculated, where: Each nucleotide deletion, insertion, substitution, or addition in a second nucleotide sequence compared to the first nucleotide sequence is considered a difference in a single nucleotide (position).
Alternatively, the percent sequence identity between two or more nucleotide sequences can be calculated using known computer algorithms for sequence alignment, such as NCBI Blast v2.0 using normal settings. it can.
For several other techniques, computer algorithms and settings for determining percent sequence identity, see, for example, WO 04/037999, European Patent 0 967 284, European Patent 1 085 089, WO No. 00/55318, WO 00/78972, WO 98/49185, and British Patent Application No. 2 357 768.
Usually, to determine the percentage of “sequence identity” between two nucleotide sequences by the computational method outlined above, the nucleotide sequence with the highest number of nucleotides is the “first” nucleotide sequence and another nucleotide sequence Is the “second” nucleotide sequence.
f) For the comparison of two or more amino acid sequences, [the number of amino acid residues identical in the first amino acid sequence to the amino acid residues at corresponding positions in the second amino acid sequence] Divide by the total number of amino acid residues in one amino acid sequence] and multiply by [100%] to calculate the percentage of “sequence identity” between the first amino acid sequence and the second amino acid sequence Where the deletion, insertion, substitution, or addition of an amino acid residue in the second amino acid sequence compared to the first amino acid sequence is each a difference in a single amino acid residue (position) And is defined herein as "amino acid difference".
Alternatively, the percent sequence identity between two amino acid sequences can also be calculated using known computer algorithms for the determination of nucleotide sequence identity using conventional settings.
Usually, in order to determine the ratio of “sequence identity” between two amino acid sequences by the calculation method outlined above, the amino acid sequence having the largest number of amino acid residues is the “first” amino acid sequence, The amino acid sequence is the “second” amino acid sequence.
Also, in determining sequence identity between two amino acid sequences, one skilled in the art will recognize that amino acid residues have a similar chemical structure and are insignificant in the function, activity, or other biological properties of the polypeptide. Substitutions of so-called “conserved” amino acids substituted with other amino acid residues that have only little or no effect can be considered. Substitution of the conserved amino acid is, for example,
It is well known to those skilled in the art from WO 04/037999, UK Patent Application Publication No. 2 357 768, WO 98/49185, WO 00/46383, and WO 01/09300, and the substitution ( Preferred) types and combinations can be selected based on relevant teachings by WO 04/037999, and WO 98/49185 and other references cited herein.
The conservative substitution is preferably a substitution in which one amino acid belonging to the following groups (a) to (e) is replaced by another amino acid belonging to the same group: (a) a small aliphatic A nonpolar or slightly polar group: Ala, Ser, Thr, Pro and Gly; (b) a polar, negatively charged group or its (non-charged) amide: Asp, Asn, Glu and Gln; (c) polar and positively charged groups: His, Arg and Lys; (d) large aliphatic nonpolar groups: Met, Leu, He, Val and Cys; and (E) Aromatic groups: Phe, Tyr and Trp.
Particularly preferred conservative substitutions are: Ala to Gly or Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser Substitution of Gln to Asn; Glu to Asp; Gly to Ala or Pro; His to Asn or Gln; Ile to Leu or Val; Leu to Ile or Val Substitution of Lys for Arg, GIn or Glu; substitution of Met for Leu, Tyr or Ile; substitution of Phe for Met, Leu or Tyr; substitution of Ser for Thr; substitution of Thr for Ser; Substitution of Trp to Tyr; substitution of Tyr to Trp; and / or substitution of Phe to Val, Ile or Leu .
Arbitrary amino acid substitutions applied to the polypeptides described herein are amino acid variations between homologous proteins from different species, developed by Schulz et al., Principles of Protein Structure, Springer-Verlag, 1978. Frequency analysis, structure formation potential analysis developed by Chou and Fasman, Biochemistry 13: 211, 1974, and Adv. Enzymol., 47: 45-149, 1978, and Eisenberg et al., Proc. Nad. Acad Sci. USA 81: 140-144, 1984, Kyte and Doolittle; J Molec. Biol. 157: 105-132, 198 1, and proteins developed by Goldman et al., Ann. Rev. Biophys. Chem. 15: 321-353, 1986 May be based on the analysis of hydrophobic patterns in the literature, all of which are incorporated herein by reference in their entirety. Information regarding the primary structure, secondary structure, and tertiary structure of Nanobodies can be found in the description herein and in the general background literature cited above. For this purpose, for example, Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996), Spinelli et al., Natural Structural Biology (1996); 3, 752-757, and Decanniere et al., Structure, Vol. 7, 4, 361 (1999) describes the crystal structure of the llama-derived VHH domain. Other information regarding amino acid residues that form the VH / VL interface and possible substitutions for these proteins in conventional VH domains can be found in WO 94/04678, WO 96/34103. WO 03/035694, Muyldermans et al., Protein Eng. 1994 Sep; 7 (9): 1129-3, Davies and Riechmann (1994 and 1996).
g) Amino acid and nucleic acid sequences are said to be “completely homologous” if they have 100% sequence identity (as defined herein) over their entire length.
h) When comparing two amino acid sequences, the term “amino acid difference” refers to an insertion, deletion or substitution of a single amino acid at the position of the first amino acid compared to the second amino acid sequence; It is understood that two amino acid sequences may contain one, two or more amino acid differences.
i) a nucleic acid sequence or amino acid sequence, eg, other natural nucleic acids, other coexisting in the natural or natural resources and / or the reaction medium or medium from which they are obtained, When separated from at least one other component, such as a protein / polypeptide, other biological component or macromolecule, or at least one contaminant, impurity, or minor component, “almost isolated (in form It is believed that there is. Specifically, when a nucleic acid sequence or amino acid sequence is purified at least 2-fold, specifically at least 10-fold, more specifically at least 100-fold, and up to 1000-fold or more, “almost isolated” Conceivable. Nucleic acid or amino acid sequences in “substantially isolated form” are preferably substantially uniform when determined by a suitable technique, such as a suitable chromatographic technique such as polyacrylamide gel electrophoresis.
j) As used herein, the term “domain” generally refers to a polypeptide comprising a globular region of an antibody molecular chain, specifically a globular region of a heavy chain antibody, or almost exclusively of the globular region. Typically, the domain comprises peptide loops (eg, 3 or 4 peptide loops) stabilized, for example, as a sheet or by disulfide bonds.
k) The term “antigenic determinant” refers to an antigen-binding molecule (such as a Nanobody or polypeptide of the invention), specifically an epitope on an antigen that is recognized by the antigen-binding site of the molecule. The terms “antigenic determinant” and “epitope” can be used interchangeably herein.
l) Amino acid sequences that can bind to specific antigenic determinants, epitopes, antigens or proteins (or at least one of these sites, fragments or epitopes) and have affinity and / or specificity (Nanobodies of the invention, Antibodies, polypeptides, or antigen binding proteins or polypeptides or fragments thereof) are said to be “directed against” or “directed against” these antigenic determinants, epitopes, antigens or proteins.
m) The term “specificity” refers to the number of different types of antigens or antigenic determinants to which a particular antigen binding molecule or antigen binding protein (such as a Nanobody or polypeptide of the invention) can bind. . The specificity of the antigen binding protein can be determined based on affinity and / or binding. The affinity expressed by the equilibrium constant (KD) of dissociation between an antigen and an antigen-binding protein is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding protein, and the smaller the numerical value of KD, The binding strength between the antigenic determinant and the antigen-binding molecule is strong (or affinity is also expressed by an affinity constant (KA) that is 1 / KD). As will be apparent to those skilled in the art (eg, from other disclosures herein), affinity can be determined by methods known per se, depending on the specific antigen of interest. Binding is a measure of the strength of binding between an antigen binding molecule (such as a Nanobody or polypeptide of the invention) and an appropriate antigen. Binding is related both to the affinity between the antigenic determinant and the antigen binding molecule on that antigen binding molecule, and the number of suitable binding sites present on the antigen binding molecule. Usually, the antigen-binding protein (such as Nanobody or polypeptide of the present invention) is 10-5 to 10-12 mol / liter or less, preferably 10-7 to 10-12 mol / liter or less, more preferably 10-. With a dissociation constant (KD) of 8-10-12 mol / liter or less and / or with a binding affinity of at least 107 M-1, preferably at least 108 M-1, more preferably at least 109 M-1, for example at least 1012 M-1. Join. Any KD value greater than 10-4 liters / mole is considered a measure of non-specific binding. Preferably, the Nanobodies or polypeptides of the invention bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, preferably less than 10 nM, for example less than 500 pM. Specific binding of an antigen binding protein to an antigen or antigenic determinant is, for example, Scatchard analysis and / or radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich antagonism assay and those known per se to those skilled in the art Can be determined by any suitable method known per se, such as an antagonistic binding assay such as various variants of
n) As further described herein, the amino acid sequences and structures of Nanobodies include, but are not limited to, four framework regions or “FRs”, as described in the art and herein. Are called “framework area 1” or “FR1”, “framework area 2” or “FR2”, “framework area 3” or “FR3”, and “framework area 4” or “FR4”, respectively. , The framework region is blocked by three complementarity determining regions or “CDRs” and are referred to in the art as “complementarity determining region 1” or “CDR1”, “complementarity determining region 2” or “ It is called “CDR2”, “complementarity determining region 3” or “CDR3”.
o) Similarly, as further described herein, the total number of all amino acid residues in the Nanobody may range from 110 to 120, preferably 112 to 115, and most preferably 113. However, Nanobody portions, fragments, analogs or derivatives (described herein) meet other requirements outlined by these portions, fragments, analogs or derivatives, preferably as described herein. They are not limited to these lengths and / or sizes as long as they are suitable for the purpose.
p) The amino acid residues of Nanobodies are according to Kabat et al. (see “Richmann and Muyldermans” cited herein (see, eg, FIG. 2 of the above reference) for VHH domains from camelids. Sequence of proteins of immunological interest ”, US Public Health Services, NIH Bethesda, MD, Publication No. 91). According to this numbering, Nanobody FR1 contains amino acid residues 1-30, Nanobody CDR1 contains amino acid residues 31-35, and Nanobody FR2 region contains amino acids 36-49. Nanobody CDR2 contains amino acid residues 50-65, Nanobody FR3 contains amino acid residues 66-94, Nanobody CDR3 contains amino acid residues 95-102 The FR4 region of Nanobody contains amino acid residues at positions 103-113. [In this embodiment, as is well known in the VH and VHH domains, the total number of amino acid residues in each of the CDRs may vary, and may be changed to the total number of amino acid residues indicated by Kabat numbering. May not correspond (ie, one or more positions by Kabat numbering may not be filled in the actual sequence, or the actual sequence may be more than the number allowed by Kabat numbering. It may contain many amino acid residues). This generally means that Kabat numbering may or may not correspond to the actual amino acid residue numbering in the actual amino acid sequence. However, in general, according to Kabat numbering, position 1 by Kabat corresponds to the start position of FR1 and vice versa regardless of the number of amino acid residues in the CDR, and position 36 by Kabat is the start of FR2. Corresponding to the position and vice versa, Kabat position 66 corresponds to the start position of FR3 and vice versa, and Kabat position 103 corresponds to the start position of FR4 and vice versa. ]
Other methods for numbering amino acid residues of VH domains that are also applicable to camelid VHH domains and Nanobodies have been reported by Chothia et al. (Nature 342, 877-883 (1989)), These are so-called “AbM definitions” and so-called “contact definitions”. However, in this specification, claims, and drawings, unless otherwise indicated, follow the Kabat numbering applied to the VHH domain by Riechmann and Muyldermans.
q) Drawings, sequence listings, and experimental parts / examples are provided solely for the purpose of further illustrating the present invention and, in the meaning of the present specification, unless expressly indicated otherwise herein. And should not be construed or understood to limit the scope of the claims.

  For a general description of heavy chain antibodies and their variable regions, see in particular the following references, which are mentioned as general background art: WO 94 / by Vrije Universiteit Brussel. 04678, WO 95/04079 and WO 96/34103; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO 00/43507 by Unilever. No. 65057, WO 01/40310, WO 01/44301, European Patent No. 1143231 and WO 02/48193; WO 97 by Vlaams Instituut voor Biotechnologie (VIB) / 49805, WO 01/21817, WO 03/03 5694, WO 03/054016 and WO 03/055527; WO 03/050531 by Algonomics and the present applicant, WO 01/90190 by the Canadian National Research Association, WO No. by the Antibody Research Institute, Inc. 03/025020 (= European Patent No. 1 433 793), as well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863 and WO 04 04/065511 and another published patent application, Hamers-Casterman et al., Nature June 3, 1993; 363 (6428): 446-8, Davies and Riechmann, FEBS Lett. February 21, 1994; 339 ( 3): 285-90, Muyldermans et al., Protein Eng. September 1994; 7 (9): 1129-3, Davies and Riechmann, Biotechnology (NY) May 1995; 13 (5): 475-9, Gharoudi 9th Forum of Applied B iotechnology, Med. Fac. Landbouw Univ. Gent. 1995; 60 / 4a part I: 2097-2100, Davies and Riechmann, Protein Eng. June 1996; 9 (6): 531-7, Desmyter et al., Nat Struct Biol. September 1996; 3 (9): 803-11; Sheriff et al., Nat Struct Biol. September 1996; 3 (9): 733-6, Spinelli et al., Nat Struct Biol. September 1996; 3 ( 9): 752-7, Arabi Ghahroudi et al., FEBS Lett. 15 September 1997; 414 (3): 521-6, Vu et al., MoI Immunol. November-December 1997; 34 (16-17): 1121-31, Atarhouch et al., Journal of Camel Practice and Research 1997; 4: 177-182, Nguyen et al., J. Mol. Biol. January 23, 1998; 275 (3): 413-8, Lauwereys et al., EMBO. J. July 1, 1998; 17 (13): 3512-20, Frenken et al., Res Immunol. 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  According to the terminology used in the above references, a variable region present in a naturally occurring heavy chain antibody is a heavy chain variable region present in a conventional four chain antibody (hereinafter referred to as the present specification). Is also referred to as a “VHH domain” to distinguish it from a light chain variable region present in a conventional four chain antibody (hereinafter referred to as “VL domain” herein). .

  As described in the prior art documents cited above, VHH domains are isolated VHH domains (based on said domains and share these structural features and functional properties with naturally occurring VHH domains. The same applies to Nanobodies) and many unique structural features and functional properties that make them very advantageous for use as functional antigen binding domains or proteins. Specifically, without limitation, a VHH domain (designed to be functionally bound to an antigen by nature in the absence of a light chain variable region and interaction therewith). And Nanobodies can act as a single, relatively low molecular weight, functional antigen-binding structural unit, domain, or protein. In this respect, VHH domains are generally not suitable alone for actual application as a single antigen binding protein, and to provide a function as an antigen binding unit (eg, Fab fragments etc. Differentiated from conventional antibody fragments, VH and VL domains of conventional four-chain antibodies that need to be bound to some form or other molecule (such as scFv fragments consisting of a VH domain covalently linked to a VL domain).

Because of these unique properties, the use of a VHH domain or Nanobody as a single antigen binding protein or antigen binding domain (as part of a larger protein or polypeptide) There are various significant advantages over the use of conventional antibody fragments (such as Fab or F (ab ′) 2 fragments), scFv fragments or:
-Since only a single domain is required to bind the antigen with high affinity and high selectivity, the need to have two separate domains also makes these two domains special (like scFv) It is not necessary to be present in the correct spatial conformation and configuration (by using a linker designed in
-VHH domains and Nanobodies can be expressed from a single gene and do not require post-translational folding or modification;
-VHH domains and Nanobodies can be easily modified into multivalent and multispecific forms (discussed further herein);
-VHH domains and Nanobodies are highly soluble and do not tend to aggregate (similar to antigen-binding domains derived from mice described in Ward et al., Nature, Vol. 341, 1989, p. 544) is there);
-VHH domains and Nanobodies are very stable to heat, pH, proteolytic enzymes, and denaturing agents or conditions (see, eg, Ewert et al., Supra);
-VHH domains and Nanobodies are easy and relatively inexpensive to prepare, even on the scale required for production. For example, VHH domains, Nanobodies, and proteins / polypeptides comprising them can be produced using microbial cultures (eg, as detailed below) and are required, for example, in conventional antibody fragments. Does not require the use of such mammalian expression systems;
-VHH domains and Nanobodies are of lower molecular weight (about 15 kDa and about 1/10 of conventional IgG) compared to conventional four chain antibodies and their antigen binding fragments, and therefore Compared to conventional four-chain antibodies and their antigen-binding fragments, they exhibit higher penetrability into tissues, including but not limited to solid tumors and other high density tissues;
-VHH domains and Nanobodies exhibit so-called pore-binding properties (especially due to the extended CDR3 loop compared to conventional VH domains), so that conventional four-chain antibodies and their antigen-binding fragments Can access targets and epitopes that cannot be accessed. For example, VHH domains and Nanobodies have been shown to inhibit enzymes (see, eg, WO 97/49805, Transue et al., (1998), supra, Lauwereys et al., (1998), supra). Wanna)

  As noted above, the present invention generally includes or consists essentially of Nanobodies that are directed against each of EGFR or IGF-IR, and are described herein. It relates to polypeptides that can be used for the prevention, treatment and / or diagnostic purposes.

  As also described further herein, the present invention further includes nucleic acids encoding the Nanobodies and polypeptides, methods of preparing the Nanobodies and polypeptides, host cells that express the Nanobodies and polypeptides, The present invention relates to compositions comprising Nanobodies, polypeptides, nucleic acids, host cells, and uses of the Nanobodies, polypeptides, nucleic acids, host cells or compositions.

  It should be noted that, in general, the term Nanobody is used herein in the broadest sense and is not limited to a particular biological resource or a particular method of preparation. For example, as discussed in detail below, the Nanobodies of the present invention can usually be obtained by: (1) by isolating the VHH domain of a naturally occurring heavy chain antibody; (2) naturally By expressing a nucleotide sequence encoding an existing VHH domain; (3) “humanizing” (described herein) a naturally occurring VHH domain and expressing a nucleic acid encoding the humanized VHH domain (4) “camelize” (described herein) or encode a camelized VH domain from any animal species, particularly a mammalian VH domain such as a human. By expressing the nucleic acid; (5) “domain antibody” or “camelized” of “Dab” described by Ward et al. (Supra), or encoding the camelized VH domain. (6) by using synthetic or semi-synthetic techniques for the preparation of proteins, polypeptides, and other amino acid sequences known per se, (7) nucleic acid synthesis known per se And / or (8) any combination of one or more of the above methods by preparing a nucleic acid encoding a Nanobody by the procedure of and expressing the resulting nucleic acid; Suitable methods and techniques for performing the above methods will be apparent to those skilled in the art from the disclosure herein and include, for example, the methods and techniques detailed herein.

  One preferred type of Nanobody is that corresponding to the VHH domain of a naturally occurring heavy chain antibody that is directed against EGFR or IGF-IR, respectively. As further described herein, the VHH sequence is transmitted to camelid species by EGFR or IGF-IR, respectively (immune response and / or heavy chain antibody directed against EGFR or IGF-IR, respectively). Immunize appropriately (to increase), collect a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from the camelid, and use any known technique, By generating a VHH sequence having directivity for each of EGFR or IGF-IR from the sample, it can be generated or obtained. Such techniques will be apparent to those skilled in the art and / or are further described herein.

  Alternatively, the naturally occurring VHH domain that is directed against EGFR or IGF-IR, respectively, is obtained from a naive library of camelid VHH sequences using, for example, one or more known screening methods. Can be obtained by screening using EGFR or IGF-IR, respectively, or at least a part, fragment, antigenic determinant, or epitope, respectively. Such libraries and procedures are described, for example, in WO 99/37681, WO 01/90190, WO 03/025020 and WO 03/035694. Alternatively, an improvement from a naive VHH library, such as a VHH library obtained from a naive VHH library, by techniques such as random mutagenesis and / or CDR shuffling, as described in WO 00/43507, for example. Synthetic or modified semi-synthetic libraries may be used.

  Yet another approach for obtaining VHH sequences that are directed against EGFR or IGF-IR, respectively, is to use heavy chain antibodies (to increase immune response and / or heavy chain antibodies to EGFR or IGF-IR, respectively) Appropriately immunizing a transgenic animal capable of expressing, and taking a suitable biological sample (blood sample, serum sample, B cell sample, etc.) from the transgenic animal, and any The method includes a step of generating a VHH sequence having directivity with respect to EGFR or IGF-IR from the sample using a known technique. For example, mice expressing heavy chain antibodies and other methods and techniques described in WO 02/085945 and WO 04/049794 can be used for this purpose.

  Although it corresponds to the amino acid sequence of the naturally occurring VHH domain, one or more amino acid residues in the amino acid sequence of the naturally occurring VHH sequence (particularly the framework sequence) are converted into a conventional human four-chain antibody Particularly preferred types of Nanobodies of the present invention include Nanobodies having amino acid sequences that have been “humanized” by substitution by one or more amino acid residues found at corresponding positions in the derived VH domains It is out. This can be done in a manner known per se which will be clear to the skilled person, for example on the basis of the further description herein and the prior art relating to humanization cited herein. Even in this case, the humanized Nanobody of the present invention can be obtained by using any appropriate method known per se (methods shown in the above (1) to (8)), and therefore exists in nature. Note that the polypeptide obtained by using a polypeptide containing a VHH domain as a starting material is not strictly limited.

  Corresponds to the amino acid sequence of a naturally occurring VH domain, but one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain derived from a conventional four-chain antibody are represented in the VHH domain of a heavy chain antibody. Another particularly preferred class of Nanobodies of the present invention includes Nanobodies having an amino acid sequence that have been “camelized” by substitution by one or more amino acid residues found at the position. This can be carried out, for example, in a manner known per se which will be clear to the skilled person on the basis of the further description herein. The “camelized” substitution preferably forms a VH-VL interface or is a position of an amino acid present in the VH-VL interface and / or as defined herein (eg, WO 94/04678). Davies and Riechmann (1994 and 1996), supra), inserted at the position of the so-called camelization feature sequence. Preferably, the VH sequence used as a starting material or starting point for the generation or design of camelid Nanobodies is preferably a VH sequence from a mammal, more preferably a VH sequence such as a VH3 sequence from a human. However, the camelized Nanobodies of the present invention can be obtained using any suitable method known per se (methods shown in the above (1) to (8)), and thus naturally occurring VH domains can be obtained. Note that the polypeptide obtained by using the containing polypeptide as a starting material is not strictly limited.

  For example, as described further herein, both “humanized” and “camelized” both provide nucleotide sequences that encode naturally occurring VHH domains or VH domains, respectively, and generate new May be carried out by altering one or more codons in said nucleotide sequence in a manner known per se so that the nucleotide sequence encodes a “humanized” or “camelized” Nanobody of the invention, respectively. it can. In order to obtain the desired Nanobody of the present invention, this nucleic acid can be expressed by a method known per se. Alternatively, each amino acid sequence of the desired humanized or camelized Nanobody of the present invention is designed based on the amino acid sequences of naturally occurring VHH domains or VH domains, respectively. Can be synthesized. Also, nucleotide sequences encoding each of the desired humanized or camelized Nanobodies of the present invention are designed based on the naturally occurring VHH domain or amino acid sequence or nucleotide sequence of each VH domain, and nucleic acid synthesis known per se The nucleic acid thus obtained can be expressed by a method known per se in order to obtain a novel synthesis using the technique and then to obtain the desired Nanobody of the present invention.

  Other suitable methods and techniques for obtaining the Nanobodies of the invention and / or nucleic acids encoding them, starting from naturally occurring VH sequences, or preferably VHH sequences, will be apparent to those skilled in the art, for example In order to obtain the Nanobody of the present invention, or the nucleotide sequence or nucleic acid sequence encoding it, one or more sites of one or more naturally occurring VH sequences (one or more FR sequences and / or CDR sequences, etc.) ), One or more sites of one or more naturally occurring VHH sequences (such as one or more FR and / or CDR sequences), and / or one or more synthetic or semi-synthetic sequences in a suitable manner. May be included.

According to one preferred but non-limiting aspect of the invention, Nanobodies can be defined in the broadest sense as polypeptides that generally include:
(A) an amino acid sequence consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences, wherein the amino acid residue at position 108 according to Kabat numbering is Q;
And / or;
(B) an amino acid (as defined herein) or cysteine consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences and having an amino acid residue at position 45 according to Kabat numbering. An amino acid sequence which is a residue and is preferably E at position 44;
And / or;
(C) consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences, the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S; In particular an amino acid sequence selected from the group consisting of R and S.

Thus, in a first preferred but non-limiting aspect, the Nanobody of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) the amino acid residue at position 108 according to Kabat numbering is Q;
And / or:
(B) the amino acid residue at position 45 according to Kabat numbering is a charged amino acid or cysteine, and position 44 according to Kabat numbering is preferably E;
And / or:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S, in particular selected from the group consisting of R and S;
and:
(D) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein.

Specifically, in the broadest sense, Nanobodies can generally be defined as polypeptides that include:
(A) an amino acid sequence consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences, wherein the amino acid residue at position 108 according to Kabat numbering is Q;
And / or:
(B) consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences, wherein the amino acid residue at position 44 according to Kabat numbering is E, and the amino acid at position 45 according to Kabat numbering An amino acid sequence in which the residue is R;
And / or:
(C) consisting of four framework regions / sequences blocked by three complementarity determining regions / sequences, the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S; In particular an amino acid sequence selected from the group consisting of R and S.

Thus, in one preferred but non-limiting aspect, Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) the amino acid residue at position 108 according to Kabat numbering is Q;
And / or:
(B) the amino acid residue at position 44 according to Kabat numbering is E and the amino acid residue at position 45 according to Kabat numbering is R;
And / or:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S, in particular selected from the group consisting of R and S;
and:
(D) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein.

Specifically, a Nanobody for each of EGFR or IGF-IR according to the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively.
(A) the amino acid residue at position 108 according to Kabat numbering is Q;
And / or:
(B) the amino acid residue at position 44 according to Kabat numbering is E and the amino acid residue at position 45 according to Kabat numbering is R;
And / or:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S, in particular selected from the group consisting of R and S;
and:
(D) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein.

Specifically, according to one preferred but non-limiting aspect of the present invention, Nanobodies are generally from four framework regions / sequences generally blocked by three complementarity determining regions / sequences. Defined as a polypeptide comprising the amino acid sequence
(A-1) the amino acid residue at position 44 according to Kabat numbering is from the group consisting of A, G, E, D, G, Q, R, S, L, preferably from G, E, or Q And (a-2) the amino acid residue at position 45 according to Kabat numbering is selected from the group consisting of L, R, or C, preferably selected from the group consisting of L or R And (a-3) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of W, R, or S, preferably selected from W or R, most preferably W;
(A-4) the amino acid residue at position 108 according to Kabat numbering is Q;
Or:
(B-1) the amino acid residue at position 44 according to Kabat numbering is selected from the group consisting of E and Q;
(B-2) the amino acid residue at position 45 by Kabat numbering is R; and (b-3) the amino acid residue at position 103 by Kabat numbering is selected from the group consisting of W, R and S Preferably W;
(B-4) the amino acid residue at position 108 according to Kabat numbering is selected from the group consisting of Q and L; preferably Q;
Or:
(C-1) the amino acid residue at position 44 according to Kabat numbering is from the group consisting of A, G, E, D, Q, R, S and L; preferably from the group consisting of G, E and Q And (c-2) the amino acid residue at position 45 according to Kabat numbering is selected from the group consisting of L, R and C; preferably, selected from the group consisting of L and R; and (c -3) the amino acid residue at position 103 by Kabat numbering is selected from the group consisting of P, R and S; preferably selected from the group consisting of R and S; and (c-4) Kabat number The amino acid residue at position 108 is selected from the group consisting of Q and L; preferably Q;
And (d) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein. .

Thus, in another preferred but non-limiting aspect, the Nanobody of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) Position 44 according to Kabat numbering is selected from the group consisting of A, G, E, D, G, Q, R, S, L; preferably from the group consisting of G, E, or Q;
and:
(B) the amino acid residue at position 45 according to Kabat numbering is selected from the group consisting of L, R, or C; preferably selected from the group consisting of L or R;
and:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of W, R or S; preferably selected from W or R, most preferably W;
And (d) the amino acid residue at position 108 according to Kabat numbering is Q;
and:
(E) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

Thus, in another preferred but non-limiting aspect, the Nanobody of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively.
(A) Position 44 according to Kabat numbering is selected from the group consisting of E and Q;
and:
(B) the amino acid residue R at position 45 according to Kabat numbering;
and:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of W, R and S; preferably W;
and:
(D) the amino acid residue at position 108 according to Kabat numbering is selected from the group consisting of Q and L; preferably Q;
and:
(E) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively.
(A) Position 44 according to Kabat numbering is selected from the group consisting of A, G, E, D, Q, R, S and L; preferably selected from the group consisting of G, E and Q;
and:
(B) the amino acid residue at position 45 according to Kabat numbering is selected from the group consisting of L, R and C, preferably from the group consisting of L and R;
and:
(C) the amino acid residue at position 103 according to Kabat numbering is selected from the group consisting of P, R and S, preferably from the group consisting of R and S;
and:
(D) the amino acid residue at position 108 according to Kabat numbering is selected from the group consisting of Q and L, preferably Q;
and:
(E) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

Two particularly preferred but non-limiting groups of Nanobodies of the present invention are those according to a) above, those according to (a-1) to (a-4), those according to b), (b-1) ) To (b-4), c) and / or (c-1) to (c-4),
a) amino acid residues 44 to 47 according to Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein), and the amino acid residue at position 108 is Q or L Yes;
and:
b) the amino acid residues 43-46 according to Kabat numbering form the sequence KERE or KQRE (or a KIRE-like sequence as defined herein), and the amino acid residue at position 108 is Q or L Yes, preferably Q.

Thus, in another preferred but non-limiting aspect, the Nanobody of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, and CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively:
(A) The amino acid residues 44 to 47 according to Kabat numbering form the sequence GLEW (or a GLEW-like sequence as defined herein), and the amino acid residue at position 108 is Q or L Is;
and:
(B) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, and CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively:
(A) the amino acid residues 43-46 according to Kabat numbering form the sequence KERE or KQRE (or a KIRE-like sequence as defined herein), and the amino acid residue at position 108 is Q Or L, preferably Q;
and:
(B) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

  In the Nanobodies of the present invention in which the amino acid residues 43-46 according to Kabat numbering form the sequence KERE or KQRE, the amino acid residue at position 37 is preferably F. In the Nanobody of the invention in which the amino acid residues 44 to 47 according to Kabat numbering form the sequence GLEW, the amino acid residue at position 37 is a group consisting of Y, H, I, L, V or F V is most preferably V.

Therefore, the Nanobody of the present invention is generally classified based on the following three groups based on the amino acid residues present at the above positions, without being limited to the description in the present specification in any sense. it can:
a) "GLEW group": Nanobodies having the amino acid sequence GLEW at positions 44-47 according to Kabat numbering and Q or L at position 108 according to Kabat numbering. As further described herein, nanobodies belonging to this group typically have V at position 37 and may have W, P, R, or S at position 103, preferably at position 103. Has W. The GLEW group also includes several GLEW-like sequences as shown in Table A-3 below;
b) “KERE group”: Nanobodies having the amino acid sequence KERE or KQRE at positions 43 to 46 according to Kabat numbering and Q or L at position 108 according to Kabat numbering. As described herein, Nanobodies belonging to this group typically have F at position 37, L or F at position 47, and W, P, R, or S at position 103. And preferably has W at position 103;
c) “103 P, R, S group”: Nanobody having P, R or S at position 103. These Nanobodies can have the amino acid sequence GLEW at positions 44-47 according to Kabat numbering, or the amino acid sequence KERE or KQRE at positions 43-46 according to Kabat numbering, the latter being most preferred Has a combination of F at position 37 and L or F at position 47 (as defined for the KERE group), and can have Q or L at position 108 according to Kabat numbering, Preferably it can have Q.

  Thus, in another preferred but non-limiting aspect, the Nanobodies of the invention are Nanobodies belonging to the GLEW group (defined herein), in which case CDR1, CDR2 and CDR3 are defined herein as Preferably, it is defined in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein.

  In another preferred but non-limiting embodiment, the Nanobodies of the invention are Nanobodies belonging to the KERE group (defined herein), in which case CDR1, CDR2 and CDR3 are preferably As defined in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein.

  Thus, in another preferred but non-limiting aspect, the Nanobodies of the invention are Nanobodies belonging to the 103 P, R, S group (as defined herein), in which case CDR1, CDR2 and CDR3 are Preferably, it is defined herein as one of the preferred embodiments herein, and more preferably as one of the more preferred embodiments herein.

  Also more generally, in addition to the 108Q, 43E / 44R, and 103P, R, S residues described above, the Nanobodies of the present invention form (part of) a VH / VL interface in a conventional VH domain. One or more amino acid residues having a charge greater than one of the naturally occurring amino acid residues present at the same position in the corresponding naturally occurring VH sequence at one or more positions, specifically (Table A It may contain one or more charged amino acids (as described in -2). Such substitutions include GLEW-like sequences referred to in Table A-3 below, and WO 00 /, for example, to so-called “microantibodies” to obtain Nanobodies having Q at position 108 with KLEW at positions 44-47 Although substitution described in 29004 is mentioned, it is not limited to these. Other possible substitutions at these positions will be apparent to those skilled in the art based on the disclosure herein.

  In one embodiment of the Nanobody of the invention, the amino acid residue at position 83 is selected from the group consisting of L, M, S, V and W;

  Also, in one embodiment of the Nanobody of the invention, the amino acid residue at position 83 is selected from the group consisting of R, K, N, E, G, I, T and Q; most preferably K or E ( Either for Nanobodies corresponding to naturally occurring VHH domains); or R (for “humanized” Nanobodies as described herein). The amino acid residue at position 84 is in one embodiment selected from the group consisting of P, A, R, S, D, T, and V, most preferably P (Nanobody corresponding to the naturally occurring VHH domain). Or R (for “humanized” Nanobodies as described herein).

  Further, in one embodiment of the Nanobody of the invention, the amino acid residue at position 104 is selected from the group consisting of G and D;

  Collectively, amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108 in the Nanobody are also referred to herein as “characteristic residues”. Characteristic residues and amino acid residues at the corresponding positions of VH3, the most closely related human VH domain, are summarized in Table A-3.

  Some combinations of particularly preferred but non-limiting feature residues found in naturally occurring VHH domains are shown in Table A-4. For comparison, the corresponding amino acid residue of human VH3 called DP-47 is shown in italics.

Table A-3: Characteristic residues in Nanobodies

note:
(1) In particular, but not limited to, with KERE or KQRE at positions 43-46.
(2) Usually as GLEW in the 44th to 47th positions.
(3) Usually as KERE or KQRE at positions 43-46, for example, as KEREL, KEREF, KQREL, KQREF or KEREG at positions 43-47. Alternatively, sequences such as TARE (eg, TEREL), KECE (eg, KECEL or KECER), RRE (eg, REREG), QERE (eg, QEREG), KGRE (eg, KGREG), and the like are also possible. Other sequences that are possible but less preferred include DECKL and NVCEL, for example.
(4) With both GLEW at positions 44-47 and KERE or KQRE at positions 43-46.
(5) At positions 83-84 of naturally occurring VHH domains, often as KP or EP.
(6) In particular, but not limited to, with GLEW in positions 44-47.
(7) The GLEW group includes GLEW-like sequences such as GVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER and ELEW in positions 44 to 47, for example.

Table A-4: Some preferred but non-limiting combinations of characteristic residues in naturally occurring Nanobodies See here for the humanization of these combinations.

  In the Nanobody, each amino acid residue at a position other than the characteristic residue may be any amino acid residue that is naturally present at the corresponding position of the naturally occurring VHH domain (according to Kabat numbering).

  The amino acid residue will be apparent to those skilled in the art. Tables A-5 to A-8 show some non-limiting residues that can be present at each position (according to Kabat numbering) of FR1, FR2, FR3, and FR4 of naturally occurring VHH domains. . For each position, the amino acid residue most frequently found at each position of the naturally occurring VHH domain (which is also the most preferred amino acid residue for said position of the Nanobody) is shown in bold and the other amino acids preferred for each position Residues are underlined (Note: The number of amino acid residues present in positions 26-30 of the naturally occurring VHH domain is such that the amino acid residues at these positions already form part of CDR1. And supports the hypothesis underlying Chothia's (see above) numbering).

  Tables A-5 to A-8 also show some non-limiting residues that can be present at each position of the human VH3 domain. Again, for each position, the amino acid residues that are most frequently found at each position of the naturally occurring human VH3 domain are shown in bold, and the preferred other amino acid residues are underlined.

  For reference only, Table A-5 also includes VHH entropy ("VHH Ent") and VHH variation ("VHH Var") data at each amino acid position for a representative 1118 VHH sequence sample. (Data provided courtesy of David Lutje Hulsing of Utrecht University and Prof. Theo Verrips). The values of VHH entropy and VHH variation give an indication of the variation and extent of amino acid residue conservation among the analyzed 1118 VHH sequences, and low numbers (<1, eg <0.5) indicate amino acid residues Is very conserved between VHH sequences (small variation). For example, G at position 8 and G at position 9 have VHH entropy values of 0.1 and 0, respectively, which are very conserved and have little variation in these residues. (At position 9, it is G in all of the analyzed 1118 sequences), but in the case of residues that form part of the CDR, values generally above 1.5 Are observed (data not shown). (1) The amino acid residues listed in column 2 of Table A-5 are based on a larger number of samples than the 1118 VHH sequences analyzed to determine the VHH entropy and VHH variation shown in the last two columns. (2) The data presented below support the hypothesis that amino acid residues 27 to 30 and possibly amino acids 93 and 94 also form part of the CDR region ( It should be noted that the present invention is not limited to a particular hypothesis or explanation, but as noted above, Kabat numbering is used herein). See Oliveira et al., PROTEINS: Structure, Function and Genetics, 52: 544-552 (2003) for a general description of sequence entropy, sequence variation, and methodologies for determining them.

Table A-5: Non-limiting examples of amino acid residues in FR1 (see footnote in Table A-3 for footnote)

Table A-6: Non-limiting examples of amino acid residues in FR2 (see footnotes in Table A-3 for footnotes)

Table A-7: Non-limiting examples of amino acid residues in FR3 (see footnote in Table A-3 for footnote)

Table A-8: Non-limiting examples of amino acid residues in FR4 (see footnote in Table A-3 for footnote)

Thus, in another preferred but non-limiting aspect, the Nanobody of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) characteristic residues are as defined herein;
and:
(B) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) FR1 is selected from the group consisting of the following amino acid sequences:

[1] QVQLQESGGGXVQAGGSLRRLSCAASG [26] [SEQ ID NO: 1]

Or from the group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above. Selected:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions when compared to the amino acid sequence described above, and does not contain amino acid deletions or insertions;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; And / or
ii) the amino acid sequence preferably contains only amino acid substitutions when compared to the amino acid sequence described above, and does not contain amino acid deletions or insertions;
and:
(B) FR2 is selected from the group consisting of the following amino acid sequences:

[36] WXRQAPGKXXEXVA [49] [SEQ ID NO: 2]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) The substitution of an amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions when compared to the amino acid sequence described above, and does not contain amino acid deletions or insertions;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequences described above;
and:
(C) FR3 is selected from the group consisting of the following amino acid sequences:

[66] RFTISRDNAKNTVYLQMNSLXXEDTAVYYCAA [94] [SEQ ID NO: 3]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions when compared to the amino acid sequence described above, and does not contain amino acid deletions or insertions;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequences described above;
and:
(D) FR4 is selected from the group consisting of the following amino acid sequences:

[103] XXQGTXVTVSS [113] [SEQ ID NO: 4]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions when compared to the amino acid sequence described above, and does not contain amino acid deletions or insertions;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequences described above;
(E) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, more preferably in one of the more preferred embodiments herein;
Characteristic residues are indicated by “X” and are as defined above, and the numbers in parentheses indicate the position of the amino acid according to Kabat numbering.

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, and CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively:
(A) FR1 is selected from the group consisting of the following amino acid sequences:

[1] QVQLQESGGGLVQAGGSLRRLSCAASG [26] [SEQ ID NO: 5]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) The substitution of an amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; and / Or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) the characteristic residue at position 11 is as shown in the sequence above;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; And / or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) the characteristic residue at position 11 is as shown in the sequence above;
and:
(B) FR2 is selected from the group consisting of the following amino acid sequences:
[36] WFRQAPGKERELVA [49] [SEQ ID NO: 6]
[36] WFRQAPGKEREFVA [49] [SEQ ID NO: 7]
[36] WFRQAPGKEREGA [49] [SEQ ID NO: 8]
[36] WFRQAPGKQRELVA [49] [SEQ ID NO: 9]
[36] WFRQAPGKQREFVA [49] [SEQ ID NO: 10]
[36] WYRQAPGKGLEWA [49] [SEQ ID NO: 11]
Or consisting of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined in the specification) with one of the above amino acid sequences Selected from the group,
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably includes only amino acid substitutions when compared to the amino acid sequence described above and includes amino acid deletions or insertions; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in each of the above sequences;
And / or selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in each of the above sequences;
and:
(C) FR3 is selected from the group consisting of the following amino acid sequences:

[66] RFTISRDNAKNTVYLQMNLSLKPEDTAVYYCAA [94] [SEQ ID NO: 12]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 83 and 84 are as shown in the sequence above;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; And / or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 83 and 84 are as shown in each of the above sequences;
and:
(D) FR4 is selected from the group consisting of the following amino acid sequences:

[103] WGQGTQVTVSS [113] [SEQ ID NO: 13]
[103] WGQGTLVTVSS [113] [SEQ ID NO: 14]

Or a group consisting of amino acid sequences having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with the amino acid sequence described above Selected from:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; and / Or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in the sequence above;
And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in each of the above sequences;
and:

(E) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to FR4 are framework regions 1 to 4, respectively, and CDR1 to CDR3 are complementarity determining regions 1 to 3, respectively:
(A) FR1 is selected from the group consisting of the following amino acid sequences:

[1] QVQLQESGGGLVQAGGSLRRLSCAASG [26] [SEQ ID NO: 5]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) the characteristic residues at position 11 are as shown in each of the above sequences;
and:
(B) FR2 is selected from the group consisting of the following amino acid sequences:

[36] WFRQAPGKERELVA [49] [SEQ ID NO: 6]
[36] WFRQAPGKEREFVA [49] [SEQ ID NO: 7]
[36] WFRQAPGKEREGA [49] [SEQ ID NO: 8]
[36] WFRQAPGKQRELVA [49] [SEQ ID NO: 9]
[36] WFRQAPGKQREFVA [49] [SEQ ID NO: 10]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in each of the above sequences;
and:
(C) FR3 is selected from the group consisting of the following amino acid sequences:

[66] RFTISRDNAKNTVYLQMNLSLKPEDTAVYYCAA [94] [SEQ ID NO: 12]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 83 and 84 are as shown in each of the above sequences;
and:
(D) FR4 is selected from the group consisting of the following amino acid sequences:

[103] WGQGTQVTVSS [113] [SEQ ID NO: 13]
[103] WGQGTLVTVSS [113] [SEQ ID NO: 14]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in each of the above sequences;
and:
(E) CDR1, CDR2 and CDR3 are as defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

In another preferred but non-limiting embodiment, the Nanobody of the invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively.
(A) FR1 is selected from the group consisting of the following amino acid sequences:

[1] QVQLQESGGGLVQAGGSLRRLSCAASG [26] [SEQ ID NO: 5]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) the characteristic residues at position 11 are as shown in each of the above sequences;
and:
(B) FR2 is selected from the group consisting of the following amino acid sequences:

[36] WYRQAPGKGLEWA [49] [SEQ ID NO: 11]

And / or selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having two or only one “amino acid difference” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in each of the above sequences;
and:
(C) FR3 is selected from the group consisting of the following amino acid sequences:

[66] RFTISRDNAKNTVYLQMNLSLKPEDTAVYYCAA [94] [SEQ ID NO: 12]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 83 and 84 are as shown in each of the above sequences;
and:
(D) FR4 is selected from the group consisting of the following amino acid sequences:

[103] WGQGTQVTVSS [113] [SEQ ID NO: 13]

And / or selected from the group consisting of one of the amino acid sequences described above and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence described above; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in each of the above sequences;
and:
(E) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.
Several other framework sequences that can be present in the Nanobodies of the present invention can be found in the above-mentioned EP 656 946 (see also US Pat. No. 5,759,808 to the same invention). Wanna)

In another preferred but non-limiting aspect, the Nanobodies of the present invention can have the following structure:

FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

Here, FR1 to 4 represent framework regions 1 to 4, respectively, and CDR1 to CDR3 represent complementarity determining regions 1 to 3, respectively:
(A) FR1 is selected from the group consisting of FR1 present in Nanobodies of SEQ ID NOs: 80 to 93 or 106 to 109, respectively.
Or consisting of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of said FR1 Selected from the group:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; and / Or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR1 sequence; and
iii) the characteristic residue at position 11 is as shown in said FR1 sequence;
And / or selected from the group consisting of one of said FR1 sequences and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-5; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR1 sequence; and
iii) the characteristic residue at position 11 is as shown in said FR1 sequence;
and:
(B) FR2 is selected from the group consisting of FR2 present in Nanobodies of SEQ ID NOs: 80 to 93 or 106 to 109, respectively.
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of said FR2. Selected from the group:
i) The substitution of an amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; and / Or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the FR2 sequence; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in the FR2 sequence;
And / or selected from the group consisting of one of said FR2 sequences and an amino acid sequence having only 3, 2 or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-6; And / or
ii) the amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the FR2 sequence; and
iii) The characteristic residues at positions 37, 44, 45 and 47 are as shown in the FR2 sequence;
and:
(C) FR3 is selected from the group consisting of FR3 present in Nanobodies of SEQ ID NOs: 80-93 or 106-109,
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of said FR3. Selected from the group:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; and / Or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR3 sequence; and
iii) The characteristic residues at positions 83 and 84 are as shown in the FR3 sequence;
And / or selected from the group consisting of one of said FR3 sequences and an amino acid sequence having only 3, 2 or 1 “amino acid differences” (as defined herein):
i) the substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-7; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR3 sequence; and
iii) The characteristic residues at positions 83 and 84 are as shown in the FR3 sequence;
and:
(D) FR4 is selected from the group consisting of FR4 present in Nanobodies of SEQ ID NOs: 80-93 or 106-109,
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of said FR4. Selected from the group:
i) Amino acid substitution at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; and / Or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR4 sequence; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in the FR3 sequence;
And / or selected from the group consisting of one of said FR4 sequences and an amino acid sequence having only 3, 2, or 1 “amino acid differences” (as defined herein):
i) substitution of any amino acid at any position other than the characteristic residue is preferably either a conservative amino acid substitution (as defined herein) and / or an amino acid substitution as defined in Table A-8; And / or
ii) the amino acid sequence preferably comprises only amino acid substitutions and no amino acid deletions or insertions when compared to the FR4 sequence; and
iii) The characteristic residues at positions 103, 104, and 108 are as shown in the FR4 sequence;
and:
(E) CDR1, CDR2 and CDR3 are defined herein, preferably in one of the preferred embodiments herein, and more preferably in one of the more preferred embodiments herein.

Some Nanobodies of the invention that are particularly preferred can be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 80-93 or 106-109, respectively, or at least 80%, preferably one of said amino acid sequences Can be selected from the group consisting of amino acid sequences having at least 90%, more preferably at least 95%, and even more preferably at least 99% sequence identity (as defined herein);
i) Characteristic residues are as shown in Table A-3 above,
ii) Any amino acid substitution at any position other than the characteristic residue is preferably any of conservative amino acid substitutions (as defined herein) and / or amino acid substitutions as defined in Tables A-5 to A-8. And / or
iii) The amino acid sequence preferably contains only amino acid substitutions and no amino acid deletions or insertions when compared to the amino acid sequence.

Some more particularly preferred Nanobodies of the invention can be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 80-93 or 106-109, respectively, or at least 80% with one of said amino acid sequences, Preferably can be selected from the group consisting of amino acid sequences having at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein);
(1) The characteristic residues are as shown in the corresponding sequences derived from SEQ ID NOs: 80 to 93 or 106 to 109,
(2) Arbitrary amino acid substitutions at any position other than the characteristic residue are preferably conservative amino acid substitutions (defined herein) and / or amino acid substitutions as defined in Tables A-5 to A-8. And / or (3) the amino acid sequence preferably comprises only amino acid substitutions when compared to the corresponding sequence selected from SEQ ID NOs: 80-93 or 106-109, and lacks amino acids. Does not include lost or inserted.

  Some of the most preferred inventive Nanobodies for EGFR and IGF-IR, respectively, can be selected from the group consisting of the amino acid sequences of SEQ ID NOs: 80-93 or 106-109, respectively.

  Preferably, the CDR and FR sequences in the Nanobody of the present invention are 10-5 to 10-12 mol / liter or less, preferably 10-7 to 10-12 mol / liter or less, more preferably 10-8 to 10-10. With a dissociation constant (KD) of -12 mol / l or less and / or with a binding affinity of at least 107 M-1, preferably at least 108 M-1, more preferably at least 109 M-1, for example at least 1012 M-1. Alternatively, it binds a Nanobody of the invention to EGFR or IGF-IR, respectively, with an affinity lower than 500 nM, preferably lower than 200 nM, preferably lower than 10 nM, for example lower than 500 pM. The affinity of the Nanobodies of the invention for EGFR or IGF-IR, respectively, can be determined by methods known per se, for example using the assays described herein.

  According to one non-limiting aspect of the present invention, Nanobodies may be defined herein, but specifically when compared to the corresponding framework regions of a naturally occurring human VH domain, As long as it has at least one amino acid difference (as defined herein) in at least one framework region as compared to 47 corresponding framework regions. More specifically, according to one non-limiting aspect of the invention, Nanobodies may be defined herein, but when compared to the corresponding framework regions of a naturally occurring human VH domain, Specifically, at least one “residue difference” in at least one characteristic residue (such as at positions 108, 103, and / or 45) when compared to the corresponding framework region of DP-47 ( As defined herein). Usually, Nanobodies are present in at least one of FR2 and / or FR4, specifically at least one of the characteristic residues in FR2 and / or FR4 (again in positions 108, 103 and / or 45) At least one amino acid difference from the naturally occurring VH domain.

  A humanized Nanobody of the present invention may also be defined herein, but when compared to the corresponding framework region of a naturally occurring VHH domain, at least “1” in at least one of the framework regions. In the condition of having “amino acid differences” (as defined herein). More specifically, according to one non-limiting aspect of the present invention, the humanized Nanobody of the present invention may be defined herein as a corresponding framework region of a naturally occurring VHH domain and When compared, provided that at least one of the characteristic residues (including the characteristic residue at positions 108, 103, and / or 45) has at least a “one amino acid difference” (as defined herein) It is. Usually Nanobodies are present in at least one of FR2 and / or FR4, specifically at least one of the characteristic residues in FR2 and / or FR4 (again in positions 108, 103 and / or 45). At least one amino acid difference from the naturally occurring VHH domain.

  As is apparent from the disclosure herein, natural or synthetic analogs, mutants, variants, alleles, homologs and orthologs of the Nanobodies of the invention as defined herein (herein “analog” And specifically the use of analogs of Nanobodies of SEQ ID NOs: 80-93 or SEQ ID NOs: 106-109 are also within the scope of the present invention. Thus, according to one embodiment of the invention, the term “nanobody of the invention” broadly includes these analogs.

  In general, one or more amino acid residues may be substituted, deleted, and / or added in the analog when compared to the Nanobodies of the invention as defined herein. The substitutions, insertions or deletions may be made in one or more of the framework regions and / or for one or more of the CDRs. Where the substitution, insertion or deletion is made in one or more framework regions, substitutions, insertions or deletions at characteristic residues are usually less preferred (appropriate humanized substitutions described herein) (Except in some cases) they may be performed at one or more of the characteristic residues and / or at one or more of the other positions of the framework residues.

  By way of non-limiting example, a substitution can be, for example, a conservative substitution (as defined herein) and / or an amino acid residue is another amino acid naturally occurring at the same position in other VHH domains. Although it may be substituted with residues (see Tables A-5 to A-8 for some non-limiting examples of such substitutions), the invention is generally limited to these Not. Thus, improving the properties of the Nanobodies of the present invention, or at least detracting from desirable properties, or combinations or balances of desirable properties of the Nanobodies of the present invention (to the extent that the Nanobodies of the present invention are not suitable for the intended use). Any one or more optional substitutions, deletions or insertions, or any combination thereof are included within the scope of the present invention. In general, one of ordinary skill in the art will consider these effects on the properties of the Nanobodies thus obtained based on the disclosure herein and, in some cases, for example, making a limited number of possible substitutions After performing a few routine experiments, etc., suitable substitutions, deletions or insertions can be determined and selected.

  For example, depending on the host organism used to express the Nanobody or polypeptide of the invention, one or more sites for post-translational modification (one or more glycosylation sites) are within the ability of one skilled in the art. The deletions and / or substitutions may be designed to be removed. Alternatively, for example, one or more binding sites of a functional group (described herein) can be introduced to allow substitution or insertion to allow position-specific PEGylation (also described herein). Can be designed.

  As can be seen from the data on VHH entropy and VHH variation shown in Tables A-5 to A-8 above, some amino acid residues in the framework regions are more conservative than those in other regions. In general, the invention is not broadly limited to these, but any substitutions, deletions or insertions are preferably made at less conservative positions. In general, amino acid substitutions are preferred over amino acid deletions or insertions.

  The analog preferably has a dissociation constant of 10 −5 to 10 −12 mol / liter or less, preferably 10 −7 to 10 −12 mol / liter or less, more preferably 10 −8 to 10 −12 mol / liter or less. KD) and / or with a binding affinity of at least 107 M-1, preferably at least 108 M-1, more preferably at least 109 M-1, such as at least 1012 M-1, and / or less than 500 nM, preferably less than 200 nM Which preferably binds Nanobodies of the invention to EGFR or IGF-IR, respectively, with an affinity of less than 10 nM, for example less than 500 pM. The affinity of the Nanobodies of the invention for EGFR or IGF-IR, respectively, can be determined by methods known per se, for example using the assays described herein.

  The analogs are also preferably such that they retain the preferred properties of the Nanobodies described herein.

  Also, according to certain preferred embodiments, the analog comprises at least 70%, preferably at least 80%, more preferably at least 90%, such as at least one of the Nanobodies of SEQ ID NOs: 80-93 or SEQ ID NOs: 106-109. Have greater than 95% or 99% sequence identity and / or at most 20, preferably at most 10, even more preferably at most 5, such as 4, 3, 2, or 1 It has only one amino acid difference (as defined herein).

  Also, the analog framework sequences and CDRs are preferably such that the analogs are in accordance with the preferred embodiments described herein. More generally, as described herein, the analog has (a) a Q at position 108 and / or (b) a charged amino acid or cysteine residue at position 45; Preferably, it has E at position 44, more preferably E at position 44, R at position 45, and / or (c) P, R, or S at position 103.

  One preferred class of Nanobodies analogs of the invention comprises humanized Nanobodies (as compared to naturally occurring Nanobodies of the invention). As described in the background art documents cited herein, the humanization is usually performed by replacing one or more amino acid residues present in the sequence of naturally occurring VHH with a human such as a human VH3 domain. Substitution with other amino acids found at the same position in the VH domain. Possible humanized substitutions or combinations of humanized substitutions exist naturally, for example, with the possible humanized substitutions shown in the tables herein, the background art literature cited herein, and / or the sequences of Nanobodies. Comparison to the sequence of the human VH domain will be apparent to those skilled in the art.

  It is necessary to select humanized substitutions so that the resulting humanized Nanobody retains the preferred properties of the Nanobodies described herein, and more preferably, the resulting humanized Nanobody is described for the analog in the above paragraph. It needs to be like that. Typically, those of ordinary skill in the art will, based on the disclosure herein, and in some cases, introduce, for example, a limited number of possible humanized substitutions to affect the properties of the Nanobodies thus obtained. After performing a few routine experiments, such as examining the effects of, it would be possible to determine and select a suitable humanized substitution or combination of humanized substitutions.

  In general, as a result of humanization, the Nanobodies of the invention can be made more “human-like” while retaining the favorable properties of the Nanobodies of the invention described herein. As a result, the humanized Nanobody has several advantages such as lower immunogenicity compared to the corresponding naturally occurring VHH domain. In this case as well, after performing a few routine experiments based on the disclosure herein, one of ordinary skill in the art would appreciate the favorable properties brought about by humanized substitution on the one hand and the natural on the other hand. It would be possible to select a humanized substitution or a combination of suitable humanized substitutions that optimizes or achieves the desired or preferred balance between the preferred properties of the VHH domains present.

  Humanized analogs and other analogs, and the nucleic acid sequences encoding them, can be provided by any method known per se. For example, an analog prepares a nucleic acid encoding a naturally occurring VHH domain and replaces the codon for one or more amino acid residues to be replaced with the corresponding codon for the desired amino acid residue (eg, position specific The resulting nucleic acid and / or nucleotide sequence is expressed in a suitable host or expression system, and thus modified by mutagenesis or PCR using primers with appropriate mismatches). The resulting analog can be obtained by isolation and / or purification, as the case may be, since it is in an approximately isolated form (eg, as described herein). This can generally be performed using methods known per se, such as, for example, the handbooks and references cited herein, the background art documents cited herein, and / or Or it will be apparent to those skilled in the art from the further description herein. Alternatively, a nucleic acid encoding a desired analog can be synthesized using a method known per se (for example, using an automatic synthesizer for nucleic acid sequences having a predetermined amino acid sequence), and then the present specification. It can be expressed according to the description in the manual. Yet other methods combine one or more naturally occurring and / or synthetic nucleic acid sequences, each encoding a portion of the desired analog, and then combining the combined nucleic acid sequences according to the description herein. Expression may be included. Analogs can also be provided by chemical synthesis of the corresponding amino acid sequence using per se known peptide synthesis techniques such as those described herein.

  In this embodiment, for example, the sequence of the Nanobody of the present invention is derived from a human VH sequence (amino acid sequence or corresponding nucleotide sequence) such as a human VH3 sequence such as DP-47, DP-51, or DP-29. In order to provide and / or confer the preferred properties of Nanobodies to the sequences thus obtained, one or more camelid substitutions (one or more amino acid residues in said human VH sequence may be replaced with a VHH domain) Those skilled in the art will also be able to design and / or prepare Nanobodies of the present invention (including these analogs) by introducing a naturally occurring amino acid residue at the corresponding position in). It will be obvious. Again, this can generally be done using the various methods and techniques described in the previous paragraph, using the amino acid sequence and / or nucleotide for the human VH sequence as a starting point.

  Some preferred but non-limiting examples of camelid substitutions can be derived from Tables A-5 to A-8. It will be apparent to those skilled in the art that camelid substitutions at one or more characteristic residues generally have a greater effect on the desired properties than substitutions at one or more other amino acid positions, Any suitable combination of these is included within the scope of the present invention. For example, introducing one or more camelizing substitutions already imparted with at least one desired property and then introducing other camelizing substitutions that improve said properties and / or confer other favorable properties You can also Even in this case, those skilled in the art will generally introduce a limited number of possible camelid substitutions based on the disclosure herein and in some cases, for example (compared to the original VH domain). ) After a few routine experiments such as examining whether the favorable properties of Nanobodies have been obtained or improved, suitable camelid substitutions or combinations of camelid substitutions can be determined and selected .

  In general, however, the camelid substitution preferably has at least (a) an amino acid or cysteine residue having a Q at position 108 and / or (b) a charged position at position 45, preferably 44 E in position, more preferably E in position 44, R in position 45 and / or (c) P, R or S in position 103 and one or more other camelizations Such substitution is optionally further included. More preferably, camelid substitutions are substitutions that result in these analogs (described herein), such as Nanobodies and / or humanized analogs of the invention, and / or preferably analogs as defined in the paragraph above. It is.

  Similarly, as will be apparent from the disclosure herein, a portion or fragment of a Nanobody of the invention as defined herein, or two or more portions or fragments, specifically SEQ ID NOs: 80- It is also within the scope of the present invention to use 93, or portions or fragments of Nanobodies of SEQ ID NOs: 106-109. Thus, according to one embodiment of the invention, the term “a Nanobody of the invention” broadly encompasses the part or fragment.

  In general, a portion or fragment of the Nanobody of the present invention (including these analogs) has one or more N-terminal amino acid residues, C, compared to the corresponding full-length Nanobody of the present invention (or these analogs). One or more terminal amino acid residues, one or more adjacent internal amino acid residues, or any combination thereof has an amino acid sequence deleted and / or removed.

  The portion or fragment is preferably 10-5 to 10-12 mol / liter or less, preferably 10-7 to 10-12 mol / liter or less, more preferably 10-8 to 10-12 mol / liter or less. At a constant (KD) and / or with a binding affinity of at least 107 M-1, preferably at least 108 M-1, more preferably at least 109 M-1, for example at least 1012 M-1, and / or lower than 500 nM, preferably The Nanobodies of the invention are preferably bound to EGFR or IGF-IR, respectively, with an affinity lower than 200 nM, preferably lower than 10 nM, for example lower than 500 pM. The affinity for EGFR or IGF-IR, respectively, can be determined by methods known per se, for example using the assays described herein.

  Any portion or fragment preferably comprises at least 10 contiguous amino acid residues, preferably at least 20 contiguous amino acid residues of the corresponding amino acid sequence of the full-length Nanobody of the present invention, and more Preferably it contains at least 30 contiguous amino acid residues, for example at least 40 contiguous amino acid residues.

  In addition, the optional portion or fragment is preferably one containing at least one of CDR1, CDR2 and / or CDR3, or at least a part thereof (specifically, at least CDR3 or at least a part thereof). is there. More preferably, any portion or fragment is preferably at least one of CDRs (preferably at least CDR3 or a portion thereof) and other CDRs joined by an appropriate framework sequence or at least a portion thereof. Including at least one (CDR1 or CDR2) or at least a portion thereof. More preferably, any portion or fragment is preferably at least one of the CDRs (preferably at least CDR3 or a portion thereof), and the rest, also joined by an appropriate framework sequence or at least a portion thereof. Including two CDRs or at least a portion thereof.

  According to another particularly preferred but non-limiting embodiment, said part or fragment is at least one of the corresponding full-length nanobodies of the invention, as described, for example, in WO 03/0505051 (Lasters et al.). Includes CDR3, eg, FR3, CDR3, and FR4.

  As noted above, providing an analog (as defined herein) of the Nanobody of the invention and / or providing other portions or fragments (as defined herein) of the Nanobody of the invention In order to do this, it is also possible to combine two or more such parts or fragments (derived from the same or different Nanobodies of the invention). It is also possible, for example, to bind one or more parts or fragments of the Nanobodies of the invention to one or more parts or fragments of the human VH domain.

  According to one preferred embodiment. The portion or fragment comprises at least 50%, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, for example at least one of the Nanobodies of SEQ ID NOs: 80-93, or 106-109. It has 90%, 95%, 99% or more sequence identity.

  Portions and fragments, and the nucleic acid sequences encoding them, can be provided by any method known per se, optionally in combination. For example, the portion or fragment is expressed by a method known per se (such as the method described herein) by inserting a stop codon into the nucleic acid encoding the full-length nanobody of the present invention. Can be obtained. Alternatively, the nucleic acid encoding the portion or fragment can be obtained by appropriately limiting the nucleic acid encoding the full-length nanobody of the present invention or by synthesizing the nucleic acid by a method known per se. Portions or fragments can also be provided using peptide synthesis techniques known per se.

  The invention also includes, in its broadest sense, the nanobody derivatives of the invention. The derivatives are generally chemically and / or biologically (such as by enzymes) by modification of the Nanobody of the invention and / or one or more amino acid residues that form the Nanobody of the invention. It can be obtained by modification.

  Examples of such modifications, and examples of amino acid residues (which may be on the protein backbone but preferably on the side chain) in the Nanobody sequence that can be modified by the method, to apply the modification The methods and techniques that can be used, as well as the possibilities and advantages of utilizing the modifications, will be apparent to those skilled in the art.

  For example, the modification may involve the introduction of one or more functional groups, residues or moieties into the Nanobody of the invention (eg, by covalent bonding or other suitable method), specifically one or more desired properties or It may include the introduction of one or more functional groups, residues or moieties that impart functionality to the Nanobodies of the present invention. Examples of such functional groups will be apparent to those skilled in the art.

  For example, the modification increases the half-life, solubility, and / or absorption of the Nanobodies of the invention, reduces the immunogenicity and / or toxicity of the Nanobodies of the invention, and any undesirables of the Nanobodies of the invention Remove or reduce side effects and / or impart other advantageous properties to the Nanobodies and / or polypeptides of the invention and / or reduce undesirable properties, or provide a combination of two or more of the foregoing It may include the introduction of one or more functional groups (eg, by covalent bonding or other suitable method). Examples of such functional groups and techniques for introducing them will be apparent to those skilled in the art, and in general, all the functional groups and techniques mentioned in the general background literature cited in the above description of this specification, and pharmaceutical Functional modifications and methods known per se for protein modifications, in particular antibody or antibody fragment modifications (such as scFv and single domain antibodies) may be included, for example as described in Remington's Pharmaceutical Sciences, See 16th edition, Mack Publishing Co., Easton, PA (1980). The functional group may be attached to the Nanobody of the invention either directly (eg, by a covalent bond) or optionally via a suitable linker or spacer, as will also be apparent to those skilled in the art.

  One of the most widely used techniques for increasing the half-life and / or reducing immunogenicity of pharmaceutical proteins is a suitable pharmacologically acceptable such as poly (ethylene glycol) (PEG) Conjugating a polymer or a derivative thereof (such as methoxypoly (ethylene glycol) or mPEG). In general, any suitable form of PEGylation may be used such as PEGylation used in the art such as antibodies and antibody fragments (including but not limited to (single) domain antibodies and scFv). For example, Chapman, Nat. Biotechnol., 54, 531-545 (2002), Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003), Harris and Chess, Nat. Rev. See Drug. Discov., 2, (2003), as well as WO 04/060965. Various reagents for PEGylation of proteins are commercially available, for example, from Nektar Therapeutics, USA.

  Preferably, position-specific PEGylation is used, specifically via a cysteine residue (see eg Yang et al., Protein Engineering, 16, 10, 761-770 (2003)). For this purpose, for example, PEG may be attached to a naturally occurring cysteine residue in the Nanobody of the invention, all using protein engineering techniques known per se to those skilled in the art. The Nanobody of the present invention may be modified in order to suitably introduce one or more cysteine residues for binding, or an amino acid sequence comprising one or more cysteine residues for binding PEG It may be fused to the N-terminus or C-terminus of the Nanobody.

  Preferably, for the Nanobodies and proteins of the invention, the molecular weight is less than 100,000, such as those having a molecular weight of greater than 5000, a molecular weight of greater than 10,000 and less than 200,000, such as from 20,000 to Use PEG such as those in the 80,000 range.

  Another, usually less preferred modification, is an N-linked type, usually performed during translation and / or as part of post-translational modifications, depending on the host cell used to express the Nanobody or polypeptide of the invention. Or contains O-linked glycosylation.

  Yet another modification may include the introduction of one or more detectable labels or signal generating groups or moieties depending on the intended use of the labeled Nanobody. Suitable labels and methods for their binding, use and detection will be apparent to those skilled in the art, such as fluorescent labels (fluorescein, isocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, and fluorescamine) , And fluorescent metals such as 152 Eu or other metals belonging to the lanthanide series), phosphorescent label, chemiluminescent label or bioluminescent label (luminal, isoluminol, telomatic acridinium ester, imidazole, acridinium salt, oxalic acid Ester, dioxetane, or GFP and analogs thereof), radioisotopes (3H, 125I, 32P, 35S, 14C, 51Cr, 36Cl, 57Co, 58Co, 59Fe, and 75Se, etc.), metals, metal oxides Or other metal suitable for diagnostic or imaging in vivo, in vitro or in situ, such as 99mTc, 123I, 111In, 131I, 97Ru, 67Cu, 67Ga, and 68Ga Metal cations (such as 157Gd, 55Mn, 162Dy, 52Cr, and 56Fe), and chromophores and enzymes (malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, α-glycerophosphate Dehydrogenase, triose phosphate isomerase, biotin avidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, β-galactosidase, ribonuclease Over Ze, urease, catalase, glucose-6-phosphate phosphatase, glucoamylase and acetylcholinesterase, etc.), but are not limited to. Other suitable labels will be apparent to those skilled in the art and include, for example, sites that can be detected using NMR or ESR spectroscopy.

  The labeled Nanobodies and polypeptides of the present invention may depend on the specific label chosen, for example, in vivo, in vitro or in situ assays (such as ELISA, RIA, EIA, and other “sandwich assays”). And immunoassays known per se), and in vivo diagnostic and imaging purposes.

  As will be apparent to those skilled in the art, another modification may include, for example, the introduction of a chelating group to chelate one of the metals or metal cations. Suitable chelating groups include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

  Yet another modification may include the introduction of a functional group that is one site of a specific binding pair, such as a biotin- (strept) avidin binding pair. The functional group can be used to bind the Nanobody of the present invention to another protein, polypeptide, or compound that binds to the other of the binding pairs through the formation of a binding pair. For example, a Nanobody of the invention can be complexed with biotin and bound to another protein, polypeptide, compound or carrier complexed with avidin or streptavidin. For example, the complexed Nanobody can be used as a reporter, for example, in a diagnostic system in which a detectable signal generator is complexed with avidin or streptavidin. The binding pair can also be used, for example, to bind a Nanobody of the invention to a carrier, such as a carrier suitable for pharmaceutical use. One non-limiting example is the liposomal formulation reported in Cao and Suresh, Journal of Drug Targeting, 8, 4, 257 (2000). The binding pair can also be used to bind the Nanobody of the present invention to a drug having a therapeutic effect.

  Kill some cells that express the target targeted by the Nanobodies of the invention (eg, in the treatment of cancer) or inhibit or slow the growth and / or proliferation of the cells in some applications For purposes intended, the Nanobodies of the invention may be conjugated to toxins or toxic residues or sites. For example, examples of toxic sites, compounds or residues that can be conjugated to the Nanobodies of the present invention to provide compounds with cytotoxicity will be apparent to those skilled in the art, for example, the preceding references cited above. It can be found from the technical literature and / or further description herein. An example is the so-called ADEPT ™ technique described in WO 03/055527.

  Other possible chemical and enzymatic modifications will be apparent to those skilled in the art. The modifications can be introduced for research purposes (eg, examining the relationship between function and activity). See, for example, Lundblad and Bradshaw, Biotechnol. Appl. Biochem., 26, 143-151 (1997).

  Preferably, the derivative has a dissociation constant of 10 −5 to 10 −12 mol / liter or less, preferably 10 −7 to 10 −12 mol / liter or less, more preferably 10 −8 to 10 −12 mol / liter or less. KD) and / or with a binding affinity of at least 107 M-1, preferably at least 108 M-1, more preferably at least 109 M-1, such as at least 1012 M-1, and / or less than 500 nM, preferably less than 200 nM The Nanobodies of the invention are bound to EGFR or IGF-IR, respectively, with a low affinity, preferably less than 10 nM, for example less than 500 pM. The affinity of the Nanobodies of the present invention for each of EGFR or IGF-IR can be determined by methods known per se, such as, for example, the assays described herein.

  As mentioned above, the present invention also relates to a protein or polypeptide consisting of approximately at least one Nanobody of the present invention. By the term “consisting essentially of”, the amino acid sequence of the polypeptide of the present invention is exactly the same as the amino acid sequence of the Nanobody of the present invention, or the amino terminal side, the carboxy terminal side, or the amino terminal And 1 to 20 amino acid residues, for example 1 to 10 amino acid residues, preferably 1, 2, 3, 4, 5, or 6 amino acid residues added to both the carboxy terminal side and the carboxy terminal side It means to correspond to the amino acid sequence of the Nanobody of the present invention having a limited number of amino acid residues such as 1 to 6 amino acid residues such as a group.

The amino acid residues may or may not have a charge, may modify (biological) properties of the Nanobody, or may be influenced by other methods, adding other functionality to the Nanobody. May be. For example, the amino acid residue is:
a) may include a Met residue at the N-terminus, eg, as a result of expression in a heterologous host cell or host organism;
b) When synthesized in a host cell, it may form a signal sequence or leader sequence that secretes the Nanobody. Suitable secretory leader peptides will be apparent to those skilled in the art and may be further described herein. Usually, the leader sequence is attached to the N-terminus of the Nanobody, but the invention is not broadly limited thereto;
c) directing and / or penetrating or infiltrating Nanobodies into specific organs, tissues, cells or cell sites or compartments and / or cell layers such as cell membranes, epithelial cell layers, tumors such as solid tumors, or blood- A biological barrier such as the brain-barrier may form a sequence or signal that allows the Nanobody to enter or penetrate. The amino acid sequence will be apparent to those skilled in the art. Some non-limiting examples include WO 03/026700 and Temsamani et al., Expert Opin. Biol. Then, 1, 773 (2001), Temsamani and Vidal, Drug Discov. Today, 9, 1012 (2004), And Rousselle, J. Pharmacol. Exp. Then, 296, 124-131 (2001), and low molecular weight peptide vectors ("Pep-trans vectors"), and Zhao et al. The described membrane transport sequence. C-terminal and N-terminal sequences for intracellular targeting of antibody fragments are described, for example, in Cardinale et al., Methods, 34, 171 (2004). Other suitable techniques for intracellular targeting include the expression and / or use of so-called “intracellular antibodies” comprising the Nanobodies of the invention, as described below;
d) A “tag” may be formed, such as an amino acid sequence or residue that enables or facilitates purification of Nanobodies, for example, by affinity techniques that are directed to such sequences or residues. The sequence or residue may then be removed (chemical or enzymatic cleavage) (for this purpose, the tag is attached or cleavable to the Nanobody via an optionally cleavable linker sequence) May be included). Some preferred but non-limiting examples of said residues are myc-tags such as multiple histidine residues, glutathione residues and AAAEQKLISEEDLNGAA [SEQ ID NO: 31];
e) It may be one or more amino acid residues that are functionalized and / or can act as a binding site for a functional group. Suitable amino acid residues and functional groups will be apparent to those skilled in the art and include, but are not limited to, the amino acid residues and functional groups described herein for the derivatives of Nanobodies of the present invention.

  According to another embodiment, the polypeptide of the present invention provides an amino-terminal, carboxy-terminal, or amino acid to provide a fusion protein comprising said Nanobody of the present invention and one or more other amino acid sequences. It comprises a Nanobody of the invention fused to at least one other amino acid sequence both at the terminal and carboxy terminal side. This fusion is also referred to herein as a “Nanobody fusion”.

  The one or more other amino acid sequences may be any suitable and / or desired amino acid sequence. Other amino acid sequences may or may not have a charge, may alter the (biological) properties of the Nanobody, or may be influenced by other methods, and may affect the Nanobody or polypeptide of the invention. Other functions may or may not be added. Preferably, the other amino acid sequence is such as to confer one or more desirable properties to the Nanobody or polypeptide of the invention.

  Examples of such amino acid sequences will be apparent to those skilled in the art and generally include all amino acid sequences used for peptide fusions based on conventional antibodies and their fragments, including but not limited to scFv and single domain antibodies. May contain. See, for example, Nature Biotechnology, 23, 9, 1126-1136 (2005), a review by Holliger and Hudson.

  For example, the amino acid sequence increases the half-life, solubility, or absorption of the polypeptide of the invention when compared to the Nanobody of the invention itself, reduces immunogenicity or toxicity, eliminates unwanted side effects or It may be an amino acid sequence that reduces and / or imparts other advantageous properties and / or reduces undesirable properties. Some non-limiting examples of said amino acid sequences are serum proteins such as human serum albumin (see eg WO 00/27435) or haptenic molecules (eg recognized by circulating antibodies). (For example, see WO 98/22141).

  Other amino acid sequences may provide a second binding site, which can be any desired protein, polypeptide, antigen, antigenic determinant, or epitope (those targeted by the Nanobodies of the present invention). And directed to the same protein, polypeptide, antigen, antigenic determinant or epitope, or different protein, polypeptide, antigen, antigenic determinant or epitope). Good. For example, another amino acid sequence provides a second binding site directed against a serum protein (eg, another serum protein such as human serum albumin or IgG) to increase half-life in serum. May be. For example, European Patents 0 368 684, WO 91/01743, WO 01/45746 and WO 04/003019 (referenced to various serum proteins), WO 06/040153, and See Harmsen et al., Vaccine, 23 (41); 4926-42.

  According to another embodiment, the one or more amino acid sequences comprise one or more portions, fragments or domains of conventional four chain antibodies (specifically human antibodies) and / or heavy chain antibodies. Good. For example, although usually less preferred, the Nanobodies of the present invention can be converted to a conventional (preferably human) VH or VL domain, or to a natural or synthetic analog of a VH or VL domain, again in this case, optionally with a linker sequence ( And other (single) domain antibodies such as, but not limited to, dAbs reported by M. et al.

  At least one Nanobody may be linked to one or more of the (preferably human) CH1, CH2 and / or CH3 domains, optionally via a linker sequence. For example, a Nanobody bound to a suitable CH1 domain is similar to a conventional Fab fragment or F (ab ′) 2 fragment, for example, with a suitable light chain, but one of the conventional VH domains (F (ab ') In the case of two fragments), or one or both can be used to form antibody fragments / structures substituted by the Nanobodies of the invention. Two Nanobodies can also be attached to the CH3 domain (possibly via a linker) to provide a construct with increased in vivo half-life.

  According to a specific embodiment of the polypeptide of the present invention, one or more Nanobodies of the present invention can confer one or more effector functions and / or one or more of the polypeptides of the present invention. It may be attached to one or more antibody portions, fragments, or domains that may confer the ability to bind to the Fc region of. For example, for this purpose, without limitation, one or more other amino acid sequences are more preferably derived from one or more of a heavy chain antibody (described herein), etc. It may contain the CH2 and / or CH3 domain of a conventional human four chain antibody-derived antibody and / or forms (part of), for example, an IgG-derived, IgE-derived, or another human Ig-derived Fc region. You may do it. For example, WO 94/04678 includes a camelid VHH domain or a humanized derivative thereof (Nanobody), each comprising a Nanobody and a human CH2 and CH3 domain (but not a CH1 domain). The camelid CH2 and / or CH3 domains are human CH2 to provide an immunoglobulin that consists of chains and has the effector functions provided by the CH2 and CH3 domains and can function even in the absence of any light chains. And heavy chain antibodies substituted in the CH3 domain have been described. Other amino acid sequences that can be suitably coupled to the Nanobodies of the invention to provide effector functions will be apparent to those skilled in the art and can be selected based on the desired effector function. See, for example, WO 04/058820, WO 99/42077 and WO 05/0117148 and the reviews by Holliger and Hudson, supra. Binding the Nanobody of the present invention to an Fc site can also increase the half-life compared to the corresponding Nanobody of the present invention. For some applications, it is preferred to use Fc sites and / or constant regions (CH2 and / or CH3 domains) that increase half-life without having biologically significant effector functions. Further preferred. Other suitable constructs that include one or more Nanobodies and one or more constant regions and have increased in vivo half-life will be apparent to those of skill in the art, eg, attached to the CH3 domain, optionally via a linker sequence 2 nanobodies may be included. In general, any fusion protein or derivative with increased half-life preferably has a molecular weight greater than 50 kD, which is a cut-off value for renal absorption.

  When other amino acid sequences are synthesized from a host cell (eg, depending on the host cell used to express the polypeptide of the invention, the pre-form, pro-form, or pre-pro-form of the polypeptide of the invention). A signal sequence or leader sequence that secretes the Nanobody or polypeptide of the present invention (to provide the above) may be formed.

  Other amino acid sequences also guide and / or penetrate or invade Nanobodies into specific organs, tissues, cells, or cell sites or compartments, and / or cell layers such as cell membranes, epithelial cell layers, solid tumors, etc. Or a biological barrier such as the blood-brain-barrier may form a sequence or signal that allows nanobodies or polypeptides to enter or permeate. Suitable examples of such amino acid sequences will be apparent to those skilled in the art, for example expressing the “Peptrans” vector, sequences reported by Cardinale et al., And Nanobodies and polypeptides of the invention as so-called “intracellular antibodies”. For example, WO 94/02610, WO 95/22618, US Pat. No. 6,0049,440, WO 03/014960, WO 99/07414, WO 05/01690, EP 1 512 696, and Cattaneo, A. & Biocca, S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag, and Kontermann, Methods 34, (2004), 163-170, and other references described therein, including amino acid sequences and antibody fragments known per se, Not a constant.

  Kill some cells that express the target targeted by the Nanobodies of the invention (eg, in the treatment of cancer) or inhibit or slow the growth and / or proliferation of the cells in some applications For applications intended for, the Nanobodies of the invention may be conjugated to (cyto) toxic proteins or polypeptides. For example, examples of such toxic proteins or polypeptides that can be conjugated to the Nanobodies of the present invention to provide the cytotoxic polypeptides of the present invention will be apparent to those of skill in the art, for example, the preceding references cited above. It can be found from the technical literature and / or further description herein. An example is the so-called ADEPT ™ technique described in WO 03/055527.

  According to one preferred but non-limiting embodiment, the one or more further amino acid sequences comprises at least 2, for example 3, 4, 5 or more Nanobodies, said Nanobodies optionally Includes at least one other Nanobody to provide a polypeptide of the invention linked via one or more linker sequences (as defined herein). Polypeptides of the present invention comprising more than two Nanobodies, at least one of which is a Nanobody of the present invention, also referred to herein as a “multivalent” polypeptide of the present invention, in which Nanobodies present are also referred to herein as being in “multivalent form”. For example, a “bivalent” polypeptide of the present invention comprises two Nanobodies, optionally linked via a linker sequence, and a “trivalent” polypeptide of the present invention optionally via two linker sequences. The same applies to tetravalent or higher, including at least one Nanobody present in the polypeptide, and at most all Nanobodies present in the polypeptide are Nanobodies of the present invention. is there.

  In a multivalent polypeptide of the invention, more than two Nanobodies may be the same or different, eg the same antigen or antigen (for the same part or epitope or for different parts or epitopes) Determinants may be directed, or may be directed to different antigens or antigenic determinants, and any suitable combination thereof. For example, the bivalent polypeptide of the present invention comprises (a) two identical Nanobodies, (b) a first Nanobody directed against the first antigenic determinant of the protein or antigen, and the protein or antigen. A second Nanobody that is directed to the same antigenic determinant and different from the first Nanobody, (c) a first Nanobody directed to the first antigenic determinant of the protein or antigen, and the protein or A second Nanobody directed against other antigenic determinants of the antigen, or (d) a first Nanobody directed against the first protein or antigen, and a second (different from the first antigen) A second Nanobody directed against a protein or antigen may be included. Similarly, the trivalent polypeptide of the present invention includes, but is not limited to, for example, (a) three identical Nanobodies, (b) two having directivity to the first antigenic determinant of the antigen The same Nanobody, a third Nanobody directed against another antigenic determinant of the same antigen, (c) two identical Nanobodies directed against the first antigenic determinant of the antigen, and A third Nanobody directed to a second antigen different from the first antigen, (d) a first Nanobody directed to a first antigenic determinant of the first antigen, and the first antigen A second Nanobody having directivity to the second antigenic determinant, a third Nanobody having directivity to a second antigen different from the first antigen, or (e) directing to the first antigen A first Nanobody having a second antigen different from the first antigen. A second Nanobody sex, third may include a Nanobody directed against a third antigen different from said first and second antigens.

  In a preferred embodiment, the invention provides a bivalent polypeptide comprising or consisting essentially of two identical Nanobodies to EFGR. Non-limiting examples of the bivalent polypeptide are shown in SEQ ID NOs: 122-123. In another preferred embodiment, the present invention provides a bivalent polypeptide comprising or consisting essentially of two identical Nanobodies against IGF-IR. Non-limiting examples of the bivalent polypeptide are shown in SEQ ID NOs: 134-135.

  Including at least two Nanobodies, wherein at least one Nanobody is directed against a first antigen (ie, against EGFR or IGF-IR, respectively) and at least one Nanobody is a second antigen A polypeptide of the invention that is directed to (different from EGFR or IGF-IR, respectively) is also referred to as a “multispecific” polypeptide of the invention, and the Nanobodies present in the polypeptide are defined herein. Is also referred to as being in “multivalent form”. Thus, for example, a “bispecific” polypeptide of the invention comprises at least one Nanobody directed against a first antigen (ie, EGFR or IGF-IR, respectively), and (EGFR or IGF-IR). A polypeptide comprising at least one other Nanobody directed against a second antigen (different from each other), wherein the “trispecific” polypeptide of the invention comprises the first antigen (EGFR or IGF-IR) At least one Nanobody directed to each) and at least one other Nanobody directed to a second antigen (different from EGFR or IGF-IR, respectively), and (EGFR or IGF-IR, respectively), And a polypeptide comprising at least one other Nanobody directed against a third antigen (different from the second antigen) and others The same applies to the case of.

  Thus, in its simplest form, the bispecific polypeptide of the invention comprises a first Nanobody targeting EGFR or IGF-IR, respectively, and a second Nanobody directed against a second antigen. The first and second Nanobodies are bivalent polypeptides of the invention (as defined herein), optionally linked via a linker sequence (as defined herein), and the invention In its simplest form, the trivalent polypeptide of the first Nanobody directed against EGFR or IGF-IR respectively, the second Nanobody directed against the second antigen, and the third antigen. A first nanobody having directivity, wherein the first, second and third nanobodies are optionally linked via one or more, specifically two linker sequences. A trispecific polypeptide of the present invention, which may be had (as defined herein).

  However, as will be clear from the above description, the multispecific polypeptide of the invention comprises at least one Nanobody for each of EGFR or IGF-IR, and one or more antigens different from each of EGFR or IGF-IR. The present invention is not limited to these in the sense that any number of nanobodies having directivity may be included.

  According to one specific but non-limiting embodiment, the polypeptides described herein are Nanobodies against at least one EGFR, optionally linked using one or more suitable linkers. And at least one Nanobody against IGF-IR. In the bispecific Nanobody construct, the Nanobodies and polypeptides described herein for IGF-IR are Nanobodies and polypeptides for one or more EGFR described in WO 05/044858 and WO 04/041867. And / or may be associated with Nanobodies and polypeptides against one or more EGFR described herein. Non-limiting examples of the bispecific Nanobody construct include SEQ ID NOs: 136-140.

  Bispecific polypeptides that contain two binding sites, each binding site specific for a tumor-associated antigen (an antigen expressed on tumor cells, also referred to as a “tumor marker”) are very useful in tumor targeting. Is advantageous. The bispecific polypeptide can target two tumor-associated antigens simultaneously, resulting in increased tumor specificity. Most tumor-associated antigens are not truly tumor specific and are known to be found in normal tissues or cells (although at low levels in most cases). Thus, monospecific binding sites, Nanobodies or polypeptides for only one tumor marker will capture or kill cells non-specifically as a result of also recognizing their normal tissues or cells. Polypeptides that have specificity for more than two markers on one or more tumor cells have a much higher tumor specificity, resulting in more specific binding. Thus, they simultaneously inhibit activation of multiple receptors and downstream signaling, leading to better inhibition, capture and killing of tumor cells.

  Accordingly, the present invention comprises or consists essentially of at least two binding sites, wherein the at least two binding sites are respectively bispecific or multispecific polypeptides directed against a tumor associated antigen or epitope. Also related. In certain embodiments, each binding site is directed against a different epitope on the same tumor-associated antigen (also referred to as a double paratope binding site). In another embodiment, each binding site is directed against a different tumor associated antigen. Each binding site may be directed against a different tumor associated antigen on a single or adjacent tumor cell. In a preferred embodiment, said at least two binding sites have a low or moderate affinity for their individual tumor-associated antigens or epitopes, and thus normal tissues expressing one of the tumor-associated antigens. Or it is only slightly retained on the cells. However, their at least two binding sites preferably target tumor cells that express both antigens recognized by the bispecific or multispecific polypeptide (have high binding).

  The binding site according to the present invention may be any peptide or nucleotide comprising a site having a known binding affinity for at least one antigen. The binding site may be a protein, polypeptide, protein fragment (such as an antibody fragment), or one or more subunits of any protein. Typical examples of binding sites are enzymes, receptors or transport proteins. The binding site may be a carrier protein such as albumin or an antibody. The binding site may be or contain a DNA or RNA sequence.

  In a preferred embodiment, one or more binding sites of the bispecific or multispecific polypeptide of the invention is a polypeptide of less than 15 kDa. In an even more preferred embodiment, the one or more binding sites of the bispecific or multispecific polypeptide of the invention are VH, VHH, domain antibody, single domain antibody, “dAb”, or Nanobody. In the most preferred embodiment, the binding site of the bispecific or multispecific polypeptide of the invention is a Nanobody.

  For example, various components of the EGFR family (EGFR, HER2, HER3, HER4) are overexpressed on tumors in breast cancer, colon cancer, ovarian cancer, lung cancer, and head and neck cancer. By targeting two of these tumor-associated antigens, or different epitopes on one or more of these tumor-associated antigens simultaneously, a much more selective and / or increased targeting is obtained.

  Thus, in a preferred embodiment, the invention also relates to a bispecific polypeptide comprising or consisting essentially of a Nanobody directed against EGFR and a Nanobody directed against another member of the EGFR family. provide. The polypeptides of the present invention may comprise or consist essentially of Nanobodies directed to EGFR and Nanobodies directed to HER2. In another embodiment, a polypeptide of the invention may comprise or consist essentially of a Nanobody directed against EGFR and a Nanobody directed against HER3. In another embodiment, the polypeptides of the present invention may comprise or consist essentially of Nanobodies directed against EGFR and Nanobodies directed against HER4. In a further embodiment, the polypeptides of the present invention may comprise or consist essentially of Nanobodies directed against HER2 and Nanobodies directed against other components of EGFR. The polypeptides of the present invention may comprise or consist essentially of Nanobodies directed to HER2 and Nanobodies directed to HER3. In another embodiment, the polypeptides of the present invention may comprise or consist essentially of Nanobodies directed against HER2 and Nanobodies directed against HER4. In another further embodiment, the polypeptide may comprise or consist essentially of a Nanobody directed against HER3 and a Nanobody directed against another component of EGFR. The polypeptides of the present invention may comprise or consist essentially of Nanobodies directed to HER3 and Nanobodies directed to HER4.

  In another preferred embodiment, the invention comprises a bispecific comprising, or consisting essentially of, Nanobodies directed against one specific epitope of EGFR and Nanobodies directed against other specific epitopes of EGFR. A polypeptide is provided. Non-limiting examples of the bispecific / dual paratope polypeptide are SEQ ID NOs: 141-143.

  Another non-limiting example is CD138 and CD38 tumor markers expressed on multiple myeloma cells. The simultaneous targeting of CD138 and CD38 provides a much more selective targeting for those multiple myeloma cells.

  Tumor markers that can be co-targeted through the bispecific or multispecific polypeptides of the invention include EGFR, IGF-IR, HER2, HER3, HER4, CEA, VEGF, CD38, and CD138. However, it is not limited to these.

  In another preferred embodiment, the present invention has, or consists essentially of, at least three binding sites, two of said at least three binding sites directed to tumor associated antigens or epitopes, and other binding sites Relates to trispecific or multispecific polypeptides that are directed against other targets or antigens. Preferably, the target or antigen is a molecule that increases the in vivo half-life (defined herein) of the polypeptide, or a molecule having an effector effect, such as a CD3, Fc receptor, or complement protein. In certain embodiments, two binding sites directed against a tumor associated antigen or epitope are directed against different epitopes on the same tumor associated antigen (also referred to as a double paratope binding site). In another embodiment, the two binding sites directed against a tumor associated antigen or epitope are directed against different tumor associated antigens. Each of these binding sites may be directed against a different tumor associated antigen on a single or adjacent tumor cell.

  In preferred embodiments, the one or more binding sites on the trispecific or multispecific polypeptide of the invention is a polypeptide of less than 15 kDa. In an even more preferred embodiment, the one or more binding sites of the trispecific or multispecific polypeptide of the invention are VH, VHH, domain antibody, single domain antibody, “dAb”, or Nanobody. In the most preferred embodiment, the binding site of the trispecific or multispecific polypeptide of the invention is a Nanobody.

  In certain embodiments, the invention includes Nanobodies to EGFR, Nanobodies to IGF-IR, and Nanobodies to human serum albumin, such as non-limiting examples SEQ ID NOs: 138-140, etc. A trispecific polypeptide is provided. In another embodiment, the present invention includes, but is not limited to, Nanobodies for specific epitopes on EGFR, Nanobodies for other specific epitopes on EGFR, and Nanobodies for human serum albumin. Illustrative trispecific polypeptides such as SEQ ID NOs: 142-143 are provided.

  Furthermore, the specific order or arrangement of the various Nanobodies in the polypeptides of the invention can be determined by the final properties of the polypeptide of the invention (EGFR or IGF-IR, respectively, or affinity for one or more other antigens). Are included within the scope of the present invention, but in general the order or arrangement is not critical, including, but not limited to, sex, specificity, or binding. In some cases, those skilled in the art can make a suitable selection after performing a few routine experiments based on the disclosure herein. Thus, when referring to a particular multivalent or multispecific polypeptide of the present invention, this should encompass any order or arrangement of related Nanobodies, unless expressly indicated otherwise. You must be careful.

  Also, bispecific comprising or consisting essentially of at least two Nanobodies, wherein one of the at least two Nanobodies increases or decreases the affinity for the antigen when the other Nanobody binds to the antigen. Sex or multispecific polypeptides are also encompassed within the scope of the present invention. The binding is referred to as “conditional bispecific or multispecific binding”. Said bispecific or multispecific polypeptide is also referred to as "conditional bispecific or multispecific polypeptide of the invention".

  Binding of the first of the at least two Nanobodies to the antigen may modulate, eg, promote, reduce, or inhibit binding of the second of the at least two Nanobodies to the antigen. it can. In one embodiment, binding of the at least two Nanobodies to the antigen by the first stimulates binding of the second of the at least two Nanobodies to the antigen. In another embodiment, binding of the first of the at least two Nanobodies to the antigen at least partially inhibits binding of the second of the at least two Nanobodies to the antigen. In another embodiment, binding of the first of the at least two Nanobodies to the antigen inhibits binding of the second of the at least two Nanobodies to the antigen. In such embodiments, the polypeptide of the invention becomes bound to its second target antigen, eg, through binding to a protein that increases the half-life of the polypeptide, and dissociates from the protein that increases the half-life. Until then, it may be maintained in vivo in the host organism.

  The regulation of binding in the above situation is achieved as a result of the structure of the antigen binding sites of the Nanobody being close to each other. The structural proximity is achieved, for example, by the nature of the structural component that binds two or more antigen binding sites by introducing a linker with a relatively stiff structure that holds the antigen binding sites in close proximity. be able to. Advantageously, two or more antigen binding sites can regulate binding to an antigen at one site by a process involving steric hindrance and / or conformational changes within the polypeptide. Are physically close to each other.

Accordingly, in certain embodiments, the present invention also relates to a method for producing a conditional binding bispecific or multispecific polypeptide of the present invention comprising the following steps:
a) selecting the first Nanobody by its ability to bind to the first epitope;
b) selecting the second Nanobody by its ability to bind to the second epitope;
c) binding Nanobodies, optionally via a linker sequence, and d) the conditional binding bispecific or multispecificity of the present invention, depending on the ability to bind to said first epitope and said second epitope. Selecting a polypeptide.

  In one embodiment, the conditional binding bispecific or multispecific polypeptide of the invention can be selected by its ability to bind to the first epitope and the second epitope, wherein binding to one of the epitopes is Increase binding to another epitope. Advantageously, the bond is increased by 25% or more, advantageously 40%, 50%, 60%, 70%, 80%, 90% or more, preferably 100% or more.

  In another embodiment, the conditional binding bispecific or multispecific polypeptide of the present invention may be selected for its ability to bind to said first epitope and said second epitope and to one of said epitopes Binding reduces the binding to other epitopes. In a preferred aspect of this embodiment, the conditional binding bispecific or multispecific polypeptide of the present invention is selected according to its ability to bind to said first epitope and said second epitope, said first and second Cannot bind to both epitopes simultaneously. In this case, the first and second Nanobodies antagonize binding to the epitope. Advantageously, the binding is 25% or more, advantageously 40%, 50%, 60%, 70%, 80%, 90% or more, preferably up to 100% so that the binding is completely inhibited, Or it can be reduced to a value close to that. Binding to the epitope can be measured by a conventional antigen binding assay such as ELISA, by a technique using fluorescence such as FRET, or by a technique such as surface plasmon resonance that measures the mass of the molecule.

  In a further embodiment of the above method, an additional step of selecting a third further Nanobody by its ability to bind to a third further epitope is provided. Thus, the multispecific polypeptide produced contains more than two Nanobodies. In this aspect of the invention, at least two of the Nanobodies have conditional bispecific binding (binding of the antigen by the first of the at least two Nanobodies to the first of the at least two Nanobodies. Modulation of, eg, increase, decrease or inhibit, binding to the antigen by the two. One or more other Nanobodies can also provide conditional binding (also called conditional multispecific binding) or can freely bind independently of the epitope.

  In a preferred embodiment, the bispecific conditional binding polypeptide comprises a first Nanobody that binds to a target molecule and a molecule or group that increases the half-life of the polypeptide (examples of such molecules or groups are described below). And a second nanobody that binds. In certain embodiments, the first Nanobody can only bind to the target molecule when a molecule or group that increases half-life is attached to the second Nanobody. In another embodiment, the first Nanobody can only bind to the target molecule when a molecule or group that increases half-life is detached from the second Nanobody. Thus, the bispecific conditional binding polypeptide continues to circulate in the subject's bloodstream by bulky molecules such as HSA. When in close proximity to the target molecule, antagonism between the binding sites of the bispecific conditional binding polypeptide results in dissociation of the HSA and binding to the target.

  Finally, it is within the scope of the present invention that the polypeptide of the present invention comprises two or more Nanobodies and one or more other amino acid sequences (described herein).

  For multivalent and multispecific polypeptides comprising one or more VHH domains and formulations thereof, see Conrath et al., J. Biol. Chem., Vol. 276, 10. 7346-7350, 2001, and, for example, WO See also 96/34103 and WO 99/23221. Some other examples of some specific multispecific and / or multivalent polypeptides of the invention can be found in the applicant's applications described herein.

  One preferred but non-limiting example of a multispecific polypeptide of the invention comprises at least one Nanobody of the invention and at least one Nanobody that results in an increased half-life. Some preferred but non-limiting examples of the Nanobodies include human serum albumin, thyroxine binding protein, (human) transferrin, fibrinogen, immunoglobulins such as IgG, IgE or IgM, or listed in WO 04/003019. Nanobodies against serum proteins such as one of the other serum proteins that have been produced.

  For example, Nanobodies directed against mouse serum albumin (MSA) can be used for experiments in mice, and Nanobodies directed against human serum albumin can be used for pharmaceutical applications.

  In another embodiment of the invention, the at least one (human) serum protein is any (human) serum albumin, (human) serum immunoglobulin, (human) thyroxine binding protein, (human) transferrin, (human ) A polypeptide construct as described herein, such as fibrinogen.

According to a specific but non-limiting embodiment of the present invention, the polypeptide of the present invention comprises at least one Nanobody against human serum albumin in addition to one or more Nanobodies of the present invention. These Nanobodies for human serum albumin may be those generally described in the applicant's application cited above (see, eg, WO 04/062551), but are particularly preferred but not limited According to a specific embodiment, said Nanobody against human serum albumin comprises four framework regions (respectively FR1-FR4) and three complementarity determining regions (respectively CDR1-CDR3);
i) CDR1 is an amino acid sequence selected from the group consisting of:
SFGMS [SEQ ID NO: 15]
LNLMG [SEQ ID NO: 16]
INLG [SEQ ID NO: 17]
NYWMY [SEQ ID NO: 18]
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having two or only one “amino acid difference” (as defined herein):
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
and:
ii) CDR2 is an amino acid sequence selected from the group consisting of:
SISSGSSDTLYADSVKG [SEQ ID NO: 19]
TITVGDSTNYADSVKG [SEQ ID NO: 20]
TITVGDSTSYADSVKG [SEQ ID NO: 21]
SINGRGDDTRYADSVKG [SEQ ID NO: 22]
AISADSSTKNYADSVKG [SEQ ID NO: 23]
AISADSSDKRYADSVKG [SEQ ID NO: 24]
RISTGGGGYSYYADSVKG [SEQ ID NO: 25]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
iii) CDR3 is an amino acid sequence selected from the group consisting of:
DREAQVDTLDDY [SEQ ID NO: 26]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
Or an amino acid sequence selected from the group consisting of:
GGSLSR [SEQ ID NO: 27]
RRTWHSEL [SEQ ID NO: 28]
GRVSRS [SEQ ID NO: 29]
GRGSP [SEQ ID NO: 30]
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions And no amino acid deletions or insertions.

In another aspect, the present invention includes four framework regions (respectively FR1-FR4) and three complementarity determining regions (respectively CDR1-CDR3), each of the combinations of CDR1, CDR2, and CDR3 described below: With respect to Nanobodies against human serum albumin selected from the group consisting of Nanobodies having one of:
-CDR1: SFGMS; CDR2: SISSGSSDTLYADSVKG; CDR3: GGSLSR;
-CDR1: LNLMG; CDR2: TITVGGDSTNYADSVKG; CDR3: RRTWHSEL;
-CDR1: INLLG; CDR2: TITVGDSTSYADSVKG; CDR3: RRTWHSEL;
-CDR1: SFGMS; CDR2: SINGRGDDTRYADSVKG; CDR3: GRVSRSS;
-CDR1: SFGMS; CDR2: AISADSSDKRYADSVKG; CDR3: GRGSP;
-CDR1: SFGMS; CDR2: AISADSSDKRYADSVKG; CDR3: GRGSP;
-CDR1: NYWMY; CDR2: RISTGGGGYSYYADSVKG; CDR3: DREAQVDTLFDY.

In a Nanobody of the invention comprising a combination of the CDRs, each CDR has at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity with the CDRs ( Substituting with a CDR selected from the group consisting of amino acid sequences, having a definition as defined herein;
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or substitution with a CDR selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having three, two or only one “amino acid difference” (as defined herein) Is possible;
(1) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (2) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions And no amino acid deletions or insertions.

  However, among the Nanobodies of the present invention comprising a combination of the above CDRs, Nanobodies comprising one or more of the CDRs listed above are particularly preferred; Nanobodies comprising two or more of the CDRs listed above are particularly preferred; Nanobodies comprising the three listed CDRs are particularly most preferred.

  In these Nanobodies against human serum albumin, the framework regions FR1-FR4 are preferably as defined above for the Nanobodies of the present invention.

  Some preferred but non-limiting examples of Nanobodies directed against human serum albumin that can be used in the polypeptides of the invention are listed in Table A-9 below. ALB-8 is a humanized ALB-1.

Table A-9: Preferred but non-limiting examples of albumin binding Nanobodies

  In general, any derivative and / or polypeptide of the present invention with increased half-life (eg, PEGylated Nanobody or polypeptide of the present invention, EGFR or IGF-IR, all as described herein) Multispecific Nanobodies, (human) serum albumin or Nanobodies fused to the Fc region) at least 1.5 times, preferably at least 2 times, eg at least 5 times, eg, at least 5 times the corresponding Nanobody of the invention Has a half-life greater than at least 10 or 20 times.

  Also, any derivative or polypeptide of the present invention with increased half-life is preferably longer than 1 hour, preferably longer than 2 hours, more preferably longer than 6 hours, such as longer than 12 hours. Although it has a long half life, for example about 1 day, 2 days, 1 week, 2 weeks or 3 weeks, and preferably more than 2 months, the latter is not necessarily critical.

  Half-life is generally defined in vivo as the time required for the serum concentration of a polypeptide to be reduced to 50%, eg, due to degradation of the ligand and / or elimination or capture of the ligand by natural mechanisms. Pharmacokinetic analysis and half-life determination are well known to those skilled in the art. Details are described in Kenneth, A, et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and Peters, et al., Pharmacokinete analysis: A Practical Approach (1996). See also "Pharmacokinetics", M Gibaldi and D Perron, Marcel Dekker, 2nd edition (1982).

  According to one aspect of the invention, the polypeptide can be conjugated to one or more molecules that increase the half-life of the polypeptide in vivo.

  The polypeptides of the present invention are stabilized in vivo, and their half-life is increased by binding to molecules that are resistant to degradation and / or excretion or capture. Usually, the molecule is a naturally occurring protein that itself has a long half-life in vivo.

  Another preferred but non-limiting example of a multispecific polypeptide of the present invention is at least one Nanobody of the present invention, and a polypeptide of the present invention in a particular organ, tissue, cell, or cell site or compartment And / or allows the polypeptide of the present invention to penetrate or invade specific organs, tissues, cells, or cell sites or compartments, and / or nanobodies to cell layers such as cell membranes, epithelial cell layers, solid tumors, etc. Or at least one Nanobody that penetrates or passes through biological barriers such as the blood-brain-barrier. Examples of said Nanobodies include cell surface proteins, markers or epitopes specific to the desired organ, tissue or cell (eg, cell surface markers associated with tumor cells) and the single domain described in WO 02/057445 Nanobodies that are directed to antibody fragments that are directed to the brain of these are FC44 (SEQ ID NO: 35) and FC5 (SEQ ID NO: 36).

Table A-10: Sequence list of FC44 and FC5

  In the polypeptides of the invention, one or more Nanobodies and one or more other polypeptides may be directly linked to each other (eg as described in WO 99/23221) and / or Alternatively, they may be linked via one or more suitable spacers or linkers, or any combination thereof.

  Suitable spacers or linkers for use in multivalent and multispecific polypeptides will be apparent to those skilled in the art and may generally be any linker or spacer used in the art to join amino acid sequences. Preferably, the linker or spacer is suitable for use to construct a protein or polypeptide intended for pharmaceutical use.

  Some particularly preferred spacers include spacers and linkers used in the art to attach antibody fragments or antibody domains. These include, for example, linkers used in the art to construct diabodies or scFv fragments together with the linkers described in the general background literature cited above (but in this respect diabody and scFv In the fragment, the linker sequence used has a length, a degree of flexibility, and other properties such that the associated VH and VL domains can be combined to form a complete antigen binding site. It should be noted, however, that each Nanobody itself forms a complete antigen binding site, so there is no particular limitation on the length or flexibility of the linker used in the polypeptides of the invention. Wanna)

  For example, the linker may be a suitable amino acid sequence, specifically an amino acid sequence of 1-50, preferably 1-30, such as 1-10 amino acid residues. Some preferred examples of the amino acid sequences include (glyxserly) z-type gly-ser linkers, such as (gly4ser) 3 or (gly3ser2) 3 described in WO 99/42077), naturally occurring Hinge-like regions, such as the hinge region of heavy chain antibodies, or similar sequences (such as those described in 94/04678).

  Some other particularly preferred linkers are polyalanine (such as AAA) and the linkers listed in Table A-11, of which AAA, GS-7 and GS-9 are particularly preferred.

Table A-11: List of linker sequences

  Another suitable linker generally comprises an organic compound or polymer, particularly an organic compound or polymer suitable for use in pharmaceutical proteins. For example, poly (ethylene glycol) residues have been used to attach antibody domains, see, eg, WO 04/081026.

  The length of the linker used, the degree of flexibility and / or another property (although not as critical as is usually the case with linkers used for scFv fragments), either EGFR or IGF-IR, respectively, or The scope of the present invention is that it may have some effect on the properties of the final polypeptide of the present invention, including but not limited to affinity, specificity, or binding to one or more other antigens. Is contained within. Based on the disclosure herein, a person of ordinary skill in the art will be able to determine suitable linkers for use with a particular polypeptide of the present invention after performing some limited routine experimentation. Let's go.

  For example, in a multivalent polypeptide of the invention comprising a Nanobody directed against a multimeric antigen (such as a multimeric receptor or another protein), the linker is preferably present in the polypeptide of the invention Each Nanobody has such a length and flexibility that it can bind to an antigenic determinant present on each subunit of the multimer. Similarly, directed against two or more different antigenic determinants on the same antigen (eg directed to different epitopes of the antigen and / or directed to different subunits of multimeric receptors, channels or proteins) In multispecific polypeptides of the invention comprising Nanobodies), the linker preferably has a length and flexibility to attach each Nanobody to the antigenic determinant of interest. Even in this case, based on the disclosure herein, after carrying out some limited routine experiments, those skilled in the art will recognize suitable linkers for use with the specific polypeptides of the invention. Will be able to determine.

  The linker used imparts one or more other desirable properties or functions to the polypeptide of the invention and / or formation of a derivative (as described herein for the derivatives of Nanobodies of the invention) and / or Providing one or more sites for attachment of functional groups is also encompassed within the scope of the present invention. For example, a linker comprising one or more charged amino acids (see Table A-2 above) provides excellent hydrophilicity, but forms or contains a low molecular weight epitope or tag Can be used for detection, identification and / or purification purposes. Even in this case, based on the disclosure herein, after carrying out some limited routine experiments, those skilled in the art will recognize suitable linkers for use with the specific polypeptides of the invention. Would be able to determine.

  Finally, when more than two linkers are used in the polypeptides of the invention, these linkers may be the same or different. Even in this case, based on the disclosure herein, after carrying out some limited routine experiments, those skilled in the art will recognize suitable linkers for use with the specific polypeptides of the invention. Would be able to determine.

  Usually, for ease of expression and production, the polypeptides of the invention are linear polypeptides. However, the present invention is not limited to these in the broadest sense. For example, if the polypeptide of the present invention contains more than three Nanobodies, a linker having more than three “arms”, each “arm” attached to the Nanobody, to obtain a “star” construct. It is also possible to combine them by use. Although usually less preferred, it is also possible to use a circular construct.

  The present invention also includes derivatives of the polypeptides of the present invention, which may be essentially similar to the derivatives of the Nanobodies of the present invention, as described herein.

  The invention also includes proteins or polypeptides "consisting essentially of" the polypeptides of the invention (the term "consisting essentially of" has essentially the same meaning as indicated above. Have).

  According to one embodiment of the invention, the polypeptide of the invention is in essentially isolated form, as defined herein.

  The Nanobodies, polypeptides and nucleic acids of the present invention are known per se and can be prepared in a manner apparent to those skilled in the art from the following description herein. For example, the Nanobodies and polypeptides of the present invention can be any known per se for the production of antibodies, specifically antibody fragments, including but not limited to (single) domain antibodies and scFv fragments. It can be prepared using the method. Some preferred but non-limiting methods for producing Nanobodies, polypeptides and nucleic acids include the methods and techniques described herein.

As will be apparent to those skilled in the art, one method that is particularly useful for the production of Nanobodies and / or polypeptides of the present invention typically comprises the following steps:
In a suitable host cell or host organism (also referred to herein as “host of the invention”) or a nucleic acid encoding said Nanobody or polypeptide of the invention (also referred to herein as “nucleic acid of the invention”) ) In another expression system suitable for expressing:
-The step of isolating and / or purifying the Nanobody or polypeptide of the present invention thus obtained. Specifically, the method may comprise the following steps:
-Culturing and / or maintaining the host of the invention under conditions such that the host of the invention expresses and / or produces at least one Nanobody and / or polypeptide of the invention; :
-Isolating and / or purifying the Nanobody or polypeptide of the present invention thus obtained.

  The nucleic acids of the invention can take the form of single-stranded or double-stranded DNA or RNA, preferably in the form of double-stranded DNA. For example, the nucleotide sequence of the present invention may be genomic DNA, cDNA or synthetic DNA (such as DNA using codons that are specifically adapted for expression in the intended host cell or host organism).

  According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form, as described herein.

  The nucleic acids of the present invention may take the form of, be part of and / or be part of a vector such as a plasmid, cosmid, or YAC, for example. Can take a separated form.

  The nucleic acids of the present invention can be prepared or obtained by methods known per se, based on information regarding the amino acid sequences of the polypeptides of the present invention described herein, and / or simply from suitable natural resources. Can be separated. In order to obtain an analog, the nucleotide sequence encoding the naturally occurring VHH region can be subjected to, for example, site-directed mutagenesis to obtain the nucleic acid of the present invention encoding the analog. Also, as will be apparent to those skilled in the art, to prepare the nucleic acids of the invention, for example, at least one nucleotide sequence encoding a Nanobody, and several such as, for example, a nucleic acid encoding one or more linkers. The nucleotide sequences can be linked in any suitable manner.

  Techniques for generating the nucleic acids of the invention will be apparent to those of skill in the art, for example, automated DNA synthesis, site-directed mutagenesis, more than two natural and / or synthetic sequences (or two of these) In order to generate cassettes and / or sites that can be easily digested and / or cleaved using appropriate restriction enzymes (eg, to introduce a mutation that expresses the cleaved gene product). These include the introduction of one or more restriction sites and / or the introduction of mutations by PCR reactions using one or more “mismatch” primers and using the sequence of a naturally occurring GPCR as a template. It is not limited. These and other methods will be apparent to those skilled in the art, see the usual handbooks by Sambrook et al. And Aubusel et al.

  The nucleic acids of the present invention take the form as part of, exist in, and / or may be part of a genetic construct, as will be apparent to those skilled in the art. The gene construct is generally one or more per se, such as, for example, one or more suitable regulatory elements (such as a suitable promoter, enhancer, terminator, etc.) and another element of the gene construct described herein. It comprises at least one nucleic acid of the invention optionally linked to an element of a known genetic construct. Said gene construct comprising at least one nucleic acid of the invention is also referred to herein as “gene construct of the invention”.

  The gene construct of the present invention may be DNA or RNA, and is preferably double-stranded DNA. Moreover, the gene construct of the present invention is in a form suitable for transformation of the target host cell or host organism, in a form suitable for integration into the genomic DNA of the target host cell or host organism, or the target host cell. Alternatively, it can take a form suitable for replication, maintenance and / or passage alone in the host organism. For example, the gene construct of the present invention can take the form of a vector such as a plasmid, cosmid, YAC, viral vector or transposon, for example. Specifically, the vector may be an expression vector that is a vector provided for expression in vitro and / or in vivo (eg, a suitable host cell, host organism and / or expression system).

In one preferred but non-limiting embodiment, the genetic construct of the invention comprises
a) at least one nucleic acid of the invention operably linked to b);
b) including one or more control elements, such as a promoter, and possibly a suitable terminator,
Optionally further c) including one or more other elements of a gene construct known per se;
Here, the terms “regulatory element”, “promoter”, “terminator”, and “operably linked” have their usual meanings in the art (as detailed herein). The “other elements” present in the gene construct are, for example, 3′- or 5′-UTR sequences, leader sequences, selectable markers, expression markers / reporter genes, and / or transformation or integration (efficiency) ) May be promoted or increased. These and other elements suitable for the gene construct will be apparent to those skilled in the art, such as the type of construct used, the intended host cell or host organism, the method by which the subject nucleotide sequences of the invention are expressed (eg, , Through constitutive expression, transient expression, inducible expression, etc.) and / or depending on the transformation technique used. For example, control sequences, promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but not limited to (single) domain antibodies and scFv) can be obtained in a similar manner. Can be used.

  Preferably, in the genetic construct of the present invention, said at least one nucleic acid of the present invention, and said control element, and optionally said one or more other elements are "operably linked" to each other, Thereby, they are usually intended to be in a functional relationship with each other. For example, if a promoter is able to control / regulate the transcription and / or expression of a coding sequence by initiation or otherwise (understand that the coding sequence is “controlled” by the promoter) It is thought to be “operably linked” to the coding sequence. In general, when two nucleotide sequences are operably linked, they have the same orientation and are usually within the same reading frame. Usually, though not necessarily, they are essentially adjacent.

  Preferably, the control elements and other elements of the gene construct of the present invention are such that they can confer a desired biological function in the intended host cell or host organism.

  For example, a promoter, enhancer or terminator must be “operable” in the intended host cell or host organism so that (for example) the promoter is transformed and / or (as defined above). In addition, transcription and / or expression of nucleotide sequences, such as coding sequences, operably linked can be initiated or otherwise controlled / regulated.

  Some particularly preferred promoters are promoters known per se for expression in the host cells mentioned herein, in particular those mentioned here and / or used in the examples below. Promoters for expression in bacterial cells, such as but not limited to.

  A selectable marker is a host cell and / or host organism that has been (successfully) transformed with a nucleotide sequence of the present invention under appropriate selection conditions. It must be distinguishable from Some preferred but non-limiting examples of such markers include genes that confer resistance to antibiotics (such as kanamycin or ampicillin), genes that confer temperature resistance, non-transformed cells or organisms survive There are genes that allow a host cell or organism to survive in the absence of certain factors, compounds and / or (food) ingredients in the medium that are essential above.

  A leader sequence is a sequence that allows the desired post-translational modification in the intended host cell or host organism and / or directs the transcribed mRNA to a desired site or organelle of the cell. The leader sequence may also cause the expression product to be secreted from the cell. As such, the leader sequence may be any pro-sequence, pre-sequence, or pre-pro sequence operable in the host cell or host organism. The leader sequence need not be expressed in bacterial cells. For example, leader sequences that are known per se for the expression and production of antibodies and antibody fragments (including but not limited to single domain antibodies and scFv fragments) can be used in much the same way. .

  The expression marker or reporter gene must be such that the expression of the gene construct (the presence of the gene or nucleotide sequence) can be detected in the host cell or host organism. An expression marker may optionally localize the expression product to a particular site or organelle of a cell and / or to a particular cell, tissue, organ or site in a multicellular organism. The reporter gene may be expressed as a fusion protein with the amino acid sequence of the present invention. Some preferred but non-limiting examples include fluorescent proteins such as GFP.

  Some preferred but non-limiting examples of suitable promoters, terminators, and other elements include those that can be used for expression in the host cells described herein, and specifically herein. And / or those suitable for expression in bacterial cells such as those used in the examples below. For other promoters, selectable markers, leader sequences, expression markers, and other elements that may be present / used in the gene constructs of the invention, such as terminators, transcriptional and / or translational enhancers and / or integration factors For some non-limiting examples, see Sambrook et al., Handbook by Aubusel et al., And WO 95/07463, WO 96/23810, WO 95/07463, WO 95/21191. No. WO 97/11094, WO 97/42320, WO 98/06737, WO 98/21355, US Pat. No. 6,207,410, US Pat. No. 5,693,492, and See what is shown in EP 1 085 089. Other examples will be apparent to those skilled in the art. See also references on the above general background art and references described below.

  The gene construct of the present invention is suitably linked to one or more of the above other elements, typically using, for example, the methods described in the general handbooks by Sambrook et al. And Ausubel et al. Can be obtained.

  In many cases, the gene construct of the present invention is obtained by inserting the nucleotide sequence of the present invention into a suitable (expression) vector known per se. Some preferred but non-limiting examples of suitable expression vectors include those used in the examples below and those described below.

The nucleic acids of the invention and / or the gene constructs of the invention can be used to transform host cells or host organisms to express and / or produce the Nanobodies or polypeptides of the invention. Suitable hosts or host cells will be apparent to those skilled in the art, such as any suitable fungus, prokaryotic, or eukaryotic cell or cell line, or any suitable fungus, prokaryotic, or eukaryotic organism, eg, as described below. For example:
-Escherichia coli strains, Proteus strains such as Mirabilis varieties, Gram-negative strains such as Pseudomonas strains such as fluorescent bacteria, Bacillus strains such as Bacillus subtilis or Brevis, Streptomyces genus such as Streptomyces lividans Bacterial strains such as, but not limited to, strains of Staphylococcus such as Staphylococcus carnosus, and strains of the genus Lactococcus such as Lactococcus lactis-Trichoderma genus such as Trichoderma reesei, Fungal cells such as, but not limited to, cells from the genus Neurospora such as Neurospora crassa, the genus Soldaria such as Soldaria macrospora, the Aspergillus genus such as Aspergillus niger or Aspergillus soja, or another filamentous fungus The genus Saccharomyces, Schizosaccharo Schizosaccharomyces spp. Yeast cells including, but not limited to, cells derived from the genus Yarrowia such as -Amphibian cells such as Xenopus oocytes -Cells / cell lines derived from lepidoptera such as Scitrophia SF9 and SF21 cells, or Schneider Cells and cell lines derived from insects such as cells / cell lines such as cells and Kc cells-Plants or plant cells such as tobacco, and / or-CHO cells, BHK cells (BHK-21 cells, etc.) ), And HeLa cells, COS cells ( COS-7 cells, etc.), and PER. Mammalian cells or cell lines and antibodies and antibody fragments ((single), including but not limited to cells or cell lines derived from humans, including but not limited to cells or cell lines derived from humans such as C6 cells. Other hosts or host cells known to express and produce domain antibodies and scFv fragments (including but not limited to) may be apparent to those of skill in the art. References on general background art cited above and, for example, WO 94/29457, WO 96/34103, WO 99/42077, Frenken et al. (1998), supra, Riechmann and Muyldermans, (1999). ), Supra, van der Linden, (2000), supra, Thomassen et al. (2002), supra, Joosten et al., (2003), supra, Joosten et al., (2005), supra, and herein. See also cited references.

  The Nanobodies and polypeptides of the present invention can be introduced and expressed in one or more cells, tissues, or organs of a multicellular organism for prevention and / or treatment (eg gene therapy). To this end, the nucleotide sequence of the present invention is, for example, a gene thus derived (for example using liposomes) or a suitable gene (eg derived from a retrovirus such as an adenovirus, an adeno-associated virus, etc. parvovirus). After insertion into a therapeutic vector, it can be introduced into a cell or tissue using any suitable method. As will be apparent to those skilled in the art, the gene therapy involves administering a nucleic acid of the invention or a suitable gene therapy vector encoding a nucleic acid of the invention to a patient or a particular cell or a particular tissue or organ of the patient. Can be performed in vivo and / or in situ within the patient's body, or is suitable (often a patient to be treated, such as explanted lymphocytes, bone marrow aspirate or tissue biopsy sample) Cells) can be treated with the nucleotide sequences of the invention in vitro and then suitably (re) introduced into the patient's body. All of these are Culver, KW, “Gene Therapy”, 1994, p. Xii, Mary Ann Liebert, Inc., Publishers, New York, NY), Giordano, Nature F Medicine 2 (1996), 534-539, Schaper, Circ. Res. 79 (1996), 911-919, Anderson, Science 256 (1992), 808-813, Verma, Nature 389 (1994), 239, Isner, Lancet 348 (1996), 370-374, Muhlhauser, Circ Res. 77 (1995), 1077-1086, Onodera, Blood 91; (1998), 30-36, Verma, Gene Ther. 5 (1998), 692-699, Nabel, Ann. NY Acad. Sci .: 811 (1997), 289-292, Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51, Wang, Nature Medicine 2 (1996), 714-716, WO 94/29469, WO 97/00957. US Pat. No. 5,580,859, US Pat. No. 5,589,466, or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, provides gene therapy vectors, techniques, and delivery systems well known to those skilled in the art. What to do with It can be. For example, in situ expression of scFv fragments (Afanasieva et al., Gene Ther., 10, 1850-1859 (2003)) and diabody (Blanco et al., J. Immunol, 171, 1070-1077 (2003)) has been reported. ing.

  For the expression of Nanobodies in cells, these are described, for example, in WO 94/02610, WO 95/22618, and US Pat. No. 6,0049,040, WO 03/014960, Cattaneo, A. and Biocca. , S. (1997) Intracellular Antibodies: Development and Applications. Landes and Springer-Verlag; and in Kontermann, Methods 34, (2004), 163-170.

  For production, the Nanobody or polypeptide of the present invention is introduced into the breast milk of a transgenic mammal such as a rabbit, cow, goat or sheep breast milk (for general techniques for introducing transgenes into mammals, see eg US Pat. No. 5,741,957, US Pat. No. 5,304,489, and US Pat. No. 5,849,992), or plants or leaves, flowers, fruits, seeds, roots, It can be produced in a part of a plant such as a tuber (eg tobacco, corn, soy or alfalfa), but is not limited thereto or can be produced, for example, in silkworm moths.

  Furthermore, the Nanobodies and polypeptides of the present invention may be expressed and / or produced in cell-free expression systems, suitable examples of which will be apparent to those skilled in the art. Some preferred but non-limiting examples include expression in wheat malt systems, rabbit reticulocyte lysates, or systems using E. coli by the Zubay method.

  As noted above, one of the advantages of using Nanobodies is that polypeptides based thereon can be prepared by expression in appropriate bacterial systems, and appropriate bacterial expression systems, vectors, host cells, regulatory elements Etc. will be apparent to those skilled in the art, for example, from the references described above. However, it should be noted that the present invention is not broadly limited to expression in bacterial systems.

  Preferably, the present invention uses an expression system (in vivo or in vitro) such as a bacterial expression system that provides the polypeptide of the present invention in a form suitable for pharmaceutical use, which expression system will also be apparent to those skilled in the art. As will be apparent to those skilled in the art, polypeptides of the invention suitable for pharmaceutical use can also be prepared using peptide synthesis methods.

  In industrial scale production, preferred heterologous hosts for (industrial) production of nanobodies or protein therapeutics containing nanobodies include large scale expression / production / culture, particularly large scale pharmaceuticals. Examples include Escherichia coli, Pichia pastoris, and budding yeast strains suitable for expression / production / culture. Suitable examples of the strain will be apparent to those skilled in the art. Such strains and production / expression systems are commercially available from companies such as Biovitrum (Uppsala, Sweden).

Alternatively, mammalian cell lines, particularly Chinese hamster ovary (CHO) cells, can be used for large scale expression / production / culture, particularly large scale pharmaceutical expression / production / culture. The strains and production / expression systems are also commercially available from several companies.

  The choice of a particular expression system depends in part on certain post-translational modifications, more specifically glycosylation. Production of recombinant proteins containing Nanobodies where glycosylation is desirable or necessary requires the use of mammalian host cells that have the ability to glycosylate the expressed protein. In this connection, it will be apparent to those skilled in the art that the resulting glycosylation pattern (type, number, and position at which the residues bind) depends on the cell or cell line used for expression. Preferably, a human cell or cell line (giving a protein having an essentially human glycosylation pattern), or essentially and / or functionally identical to at least human glycosylation pattern, or at least human glycosylation Another mammalian cell or cell line that provides a mimicked glycosylation pattern is used. In general, prokaryotic cells such as E. coli do not have the ability to glycosylate proteins, and the use of lower prokaryotic cells such as yeast usually results in glycosylation patterns that differ from human glycosylation. Nevertheless, it should be understood that the above host cells and expression systems can be used in the present invention, depending on the desired Nanobody or protein to be obtained.

  Thus, according to one non-limiting embodiment of the invention, the Nanobody or polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the present invention, the Nanobodies and polypeptides of the present invention are not glycosylated.

  According to one preferred but non-limiting embodiment of the invention, the Nanobodies or polypeptides of the invention are in bacterial cells, in particular in bacterial cells such as the above cell lines suitable for the manufacture of drugs on a large scale. To produce.

  According to another preferred but non-limiting embodiment of the present invention, the Nanobody or polypeptide of the present invention is a yeast cell, such as the above cell line suitable for the manufacture of drugs on a large scale, in particular large scale. Produce in.

  According to yet another preferred but non-limiting embodiment of the present invention, the Nanobody or polypeptide of the present invention is in mammalian cells, specifically human cells or human cell lines, more specifically large scale. In human cells or human cell lines such as the above cell lines suitable for production of drugs in

  Where expression in a host cell is used to produce the Nanobodies and proteins of the invention, the Nanobodies and proteins of the invention are produced intracellularly (eg, in the cytoplasm, in the periplasm, or in inclusion bodies) and then It may be isolated from the host cell and optionally further purified, or produced extracellularly (eg, in the medium in which the host cell is cultured) and then isolated from the medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred as it further greatly facilitates further isolation and downstream processing of the resulting Nanobodies and proteins. Normally, the above bacterial cells such as E. coli strains do not secrete proteins outside the cell, except for some proteins such as toxins and blood toxins. Secretory production in E. coli refers to the production of secretions through the inner membrane into the periplasmic space. It means to transfer. Periplasmic production has several advantages over cytoplasmic production. For example, the N-terminal amino acid sequence of the secreted protein may be identical to the natural genetic product after cleavage of the secretory signal sequence by a specific signal peptidase. Also, it appears that protease activity is much lower in the periplasm than in the cytoplasm. Furthermore, in the periplasm, since there are few contaminating proteins, protein purification is easier. Another advantage is that the periplasm provides a more oxidative environment than the cytoplasm and can form disulfide bonds in the correct positions. Proteins overexpressed in E. coli are often found in insoluble aggregates called inclusion bodies. These inclusion bodies can be present in the cytoplasm and in the periplasm, and the recovery of bioactive proteins from these inclusion bodies requires a denaturation / refolding process. Many recombinant proteins, such as proteins with therapeutic effects, are recovered from inclusion bodies. Alternatively, as would be apparent to one of skill in the art, recombinant strains of bacteria that have been genetically modified to secrete the desired protein, specifically the Nanobodies or polypeptides of the invention can be used.

  Thus, according to a non-limiting embodiment of the present invention, a Nanobody or polypeptide of the present invention is produced intracellularly and isolated from a host cell, specifically from bacterial cells or inclusion bodies in bacterial cells. Nanobody or polypeptide. According to another non-limiting embodiment of the present invention, a Nanobody or polypeptide of the present invention may be a Nanobody or polypeptide that is produced extracellularly and isolated from a medium in which host cells are cultured.

Some preferred but non-limiting examples of promoters for use in these host cells include:
-For expression in E. coli: lac promoter (these derivatives such as lacUV5 promoter), arabinose promoter, left (PL) and right (PR) promoters of lambda phage, trp operon promoter, hybrid lac / trp promoter ( tac and trc), a T7-promoter (more specifically, a promoter for T7-phage gene 10), and another T-phage promoter, a promoter for a Tn10 tetracycline resistance gene, and the above promoter comprising one or more foreign regulatory operator sequences A recombinant variant of;
-For expression in budding yeast: ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 (cytochrome c isomerase 1), GAPDH (glyceraldehyde-3-phosphate dehydrase), PGK1 (Phosphoglycerate kinase), PYK1 (pyruvate kinase) constitutive promoter, GAL1,10,7 (galactose metabolizing enzyme), ADH2 (alcohol dehydrogenase 2), PHO5 (acid phosphatase), CUP1 (copper metallothionein) Regulatory promoter, CaMV (cauliflower mosaic virus 35S promoter) foreign promoter;
-For expression in Pichia pastoris: AOX1 promoter (alcohol oxidase I)
-For expression in mammalian cells: human cytomegalovirus (hCMV) immediate early enhancer / promoter, before human cytomegalovirus (hCMV) containing two tetracycline operator sequences so that the promoter can be controlled by the Tet repressor Early promoter variants, herpes simplex virus thymidine kinase (TK) promoter, Rous sarcoma virus terminal repeat (RSV LTR) enhancer / promoter, elongation factor 1α (hEF-1α) promoter from human, chimpanzee, mouse, or rat, Examples include SV40 early promoter, HIV-1 terminal repeat promoter, and β-actin promoter.
Some preferred but non-limiting examples of vectors for use in these host cells include:
-Vectors for expression in mammalian cells: pMAMneo (Clontech), pcDNA3 (Invitrogen), pMC1neo (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 37110), pdBPV-MMTneo (342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460) and 1ZD35 (ATCC 37) -Based expression systems such as those based on-Vectors for expression in bacterial cells: pET vector (Novagen) and pQE vector (Qiagen)
-Vectors for expression in yeast or another fungal cell: pYES2 (Invitrogen) and Pichia expression vector (Invitrogen)
-Vectors for expression in insect cells: pBlueBacII (Invitrogen) and other baculolus vectors-vectors for expression in plants or plant cells: eg cauliflower mosaic virus or tobacco mosaic virus, agrobacterium Or a vector based on a Ti-plasmid.
Some preferred but non-limiting examples of secretory sequences for use in these host cells include:
-For expression in bacterial cells such as E. coli: PelB, Bla, OmpA, OmpC, OmpF, OmpT, StII, PhoA, PhoE, MalE, Lpp, LamB, etc., TAT signal peptide, hemolysin C-terminal secretion signal- For expression in yeast: α-mating factor prepro sequence, phosphatase (pho1), invertase (Suc)
-For expression in mammalian cells: When the target protein is derived from a eukaryotic cell, specific signals, mouse Igκ chain V-J2-C signal peptide and the like can be mentioned.

  Suitable techniques for transformation of a host or host cell of the invention will be apparent to those skilled in the art and may depend on the intended host cell / host organism and the genetic construct used. See also the above handbooks and patent applications.

  After transformation, a step of detecting and selecting a host cell or host organism that has been successfully transformed with the nucleotide sequence / gene construct of the present invention can be performed. This step can be, for example, a step of selecting based on a selectable marker contained in the gene construct of the present invention, or a step comprising detecting the amino acid sequence of the present invention using, for example, a specific antibody.

  A transformed host cell (which can take the form of a stable cell line) or host organism (which can take the form of a stable mutant line or strain) forms another aspect of the present invention.

  Preferably, these host cells or host organisms express the amino acid sequences of the invention (in the case of host organisms, in at least one cell, site, tissue or organ thereof) or (at least) (eg (Under suitable conditions). The invention also includes further generations, progeny and / or subsequent generations of the host cell or host organism of the invention, which are obtained, for example, by cell division or sexual or asexual reproduction. Good.

  In order to express / obtain the expression of the amino acid sequences of the present invention, the transformed host cell or host organism is maintained, maintained and / or under conditions such that the (desired) amino acid sequence is expressed / produced. Incubate. Suitable conditions will be apparent to those skilled in the art and typically depend on the host cell / host organism used and the regulators that control the expression of the (relevant) nucleotide sequences of the invention. Again, reference is made to the handbooks and patent applications described in the above paragraph relating to the gene constructs of the present invention.

  In general, suitable conditions include the use of a suitable medium, the presence of a suitable diet and / or a suitable nutrient source, the use of a suitable temperature, and optionally the presence of a suitable inducer or compound (e.g., the present invention). All of which can be selected by one skilled in the art. Again, under such conditions, the amino acid sequences of the present invention can only be expressed if continuously, transiently or properly derived.

  It will be apparent to those skilled in the art that the amino acid sequences of the present invention may be (first) expressed as an immature form (above) and then post-translationally modified depending on the host cell / host organism used. Again, the amino acid sequences of the present invention may be glycosylated depending on the host cell / host organism used.

  The amino acid sequences of the present invention can then be obtained from the host cell / host organism and / or from the medium in which the host cell or host organism is cultured, by (preparative) chromatography and / or electrophoresis, fractional precipitation. Known per se, such as affinity methods (eg, using a specific cleavable amino acid sequence fused to a polypeptide of the invention) and / or preparative immunization methods (using antibodies to the amino acid sequence to be isolated) The protein can be isolated using isolation and / or purification techniques.

  Usually, for pharmaceutical use, the polypeptide of the invention comprises at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient, and / or adjuvant. It can be formulated as a pharmaceutical that optionally contains one or more other pharmacologically active polypeptides and / or compounds. By way of non-limiting example, the formulation is for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection, or intravenous infusion), for systemic administration, inhalation, transdermal patch, It can take a form suitable for administration by implants, suppositories and the like. The suitable dosage forms may be solid, semi-solid or liquid depending on the method of administration and the method and carrier for use in the formulation and will be apparent to those skilled in the art and are further described herein. .

  Thus, in another aspect, the invention comprises at least one Nanobody of the invention or polypeptide of the invention and at least one suitable carrier, diluent or excipient (suitable for pharmaceutical use). , And in some cases, pharmaceutical compositions comprising one or more other active substances.

  In general, the Nanobodies and polypeptides of the present invention can be formulated and administered by any suitable method known per se, for example the general background literature cited above (in particular, WO 04/041862, WO 04/041863, WO 04/041865 and WO 04/041867), and Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, US Patent A (1990), Or see standard handbooks such as Remington, The Science and Practice of Pharmacy, 21th Edition, Lippincott Williams and Wilkins (2005).

  For example, the Nanobodies and polypeptides of the invention can be formulated and administered by methods known per se for conventional antibodies and antibody fragments (such as scFv and diabodies) and other pharmacologically active proteins. The formulation and methods of preparing it will be apparent to those skilled in the art, for example, parenteral (eg, intravenous, intraperitoneal, subcutaneous, intramuscular, intraluminal, intraarterial or intrathecal) or topical ( Suitable formulations are for transdermal or intradermal administration.

  Formulations for parenteral administration may be, for example, sterile solutions, suspensions, dispersions or emulsions suitable for infusion or injection. Suitable carriers or diluents for the formulation include, but are not limited to, sterile water and buffered aqueous solutions, and solutions such as phosphate buffered saline, Ringer's solution, dextrose solution, and Hank's solution, aqueous oils Glycols such as glycerol, ethanol, propylene glycol, or mineral oils, animal oils and vegetable oils such as peanut oil, soybean oil, and suitable mixtures thereof. Usually, an aqueous solution or a suspension is preferred.

  The invention further relates to the use of the Nanobodies, polypeptides and compositions described herein for the diagnosis, treatment or prevention of cancer. The present invention provides single-domain antibodies, more precisely Nanobodies directed against tumor-specific or tumor-associated antigens such as EGFR (EGFR) and insulin growth factor receptor (IGF-IR). The invention further relates to their use in diagnosis and therapy. The antibody may have a framework sequence having high homology to the human framework sequence. A composition containing antibodies against IGF-IR and EGFR alone or in combination with other agents is described.

  EGFR is part of the ERBB receptor family and four closely related components are EGFR (ErbB-1), HER2 (ErbB-2 or Neu), HER3 (ErbB-3), and HER4 (ErbB -4). Each of these components consists of an extracellular ligand binding region, a transmembrane region and an intracellular tyrosine kinase region (Yarden et al., 2001, Nature Rev. Mol. Cell Biol. 2, 127-137).

  The first step in epithelial cell division stimulation is the specific binding of a ligand such as epidermal growth factor (EGF) or transforming factor α (TGFα) to a membrane glycoprotein known as EGFR (EGF receptor). Binding (Carpenter et al., 1979, Epidermal Growth Factor, Annual Review Biochem., Vol. 48, 193-216). The mature EGF receptor consists of 1186 amino acids divided by an extracellular part of 621 residues and a cytoplasmic part of 542 residues connected by a single transmembrane segment of 23 residues (Ullrich et al., 1986). , Nature, Vol. 309, 418-425).

  The extracellular region of the EGF receptor can be subdivided into 4 domains. It has been demonstrated that domains I and III flanked by cysteine-rich regions may contain the receptor's EGF binding site (Ogiso et al., 2002. Crystal structure of the complex of human epidermal growth factor Cell 110, 775-787, Garrett et al., 2002. Crystal structure of a truncated EGFR extracellular domain bound to transforming growth factor alpha. Cell 110, 763-773.). If the ligand is not bound to the receptor, the receptor remains inactive and linked by domain II-domain IV intramolecular interactions (Ferguson KM, Berger MB, Mendrola JM, Cho HS, Leahy DJ, Lemmon MA 2003. EGF activates its receptor by removing interactions that autoinhibit ectodomain dimerization. Mol. Cell. 11: 507-17).

  Binding of the monovalent ligand to domains I and III of EGFR exposes domain II, resulting in receptor dimerization by intermolecular contact with adjacent receptor domain II. Activation of tyrosine kinases in the cytoplasmic region requires a receptor dimerization state. This results in phosphoryl transfer of tyrosine residues in the intracellular region and initiates a myriad of signaling cascades, resulting in DNA synthesis and even cell proliferation and differentiation. Activation of EGFR is a series of events in which a number of adapter proteins associate in a structure known as clathrin-coated pits, resulting in endoplasmic reticulum called clathrin-coated endoplasmic reticulum (CCV) after cell membrane invagination and budding. To start.

  The activated receptors are then distributed into endosomes and ultimately into lysosomes for proteolysis, resulting in receptor down-regulation or encapsulation (Vieira AV, Lamaze C, Schmid SL, 1996 Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274: 2086-2089). In normal cells, the feedback mechanism controls abnormal signaling by the receptor.

  Abnormal activation of EGFR is involved in processes involved in tumor growth and progression, such as tumor cell proliferation, angiogenesis, metastasis, inhibition of apoptosis, and resistance to radiation or chemotherapy (Gruenwald V, Hidalgo). M 2003. Developing inhibitors of the EGFR for cancer treatment. J. Natl. Cancer Inst. 95: 851-867, and cited references). EGF receptor dysregulation can be due to constitutive receptor signals due to i) overexpression of ligands or receptors (eg, after gene duplication), or ii) generation of heterodimers or EGFR variants such as EGFRvIII. It is the result of transmission. For example, EGFR-HER2 heterodimer has a characteristic that its signaling activity is more persistent compared to EGFR homodimer (Arteaga CL 2001. The EGFR: from mutant oncogene in nonhuman Cancers to therapeutic target in human neoplasia. J. Clin. Oncol. 19: 32S-40S).

  EGFR is more than 40% of NSCLC (Non-Small Cell Lung Cancer), more than 95% of head and neck cancer, more than 30% of pancreatic cancer, more than 90% of kidney cancer, more than 35% of ovarian cancer, more than 40% of glioma, And is expressed in a wide range of epithelial-derived tumors such as more than 31% of bladder cancer (Salomon et al., 1995. Crit. Review Oncol. Hematol, 19, 183-232). Breast cancer cells show a positive correlation between EGF receptor density and tumor size and a negative correlation with the degree of differentiation (Sainsbury et al., 1985, EGFRs and Oestrogen Receptors in Human Breast Cancer, Lancet, Vol. 1, 364-366, Sainsbury et al., 1985, Presence of EGFR as an Indicator of Poor Prognosis in Patients with Breast Cancer, J. Clin. Path., Vol. 38, 1225-1228, Sainsbury et al., 1987. Epidermal-Growth- Factor Receptor Status as Predictor of Early Recurrence and Death From Breast Cancer, Lancet, Vol. 1, 1398-1400). High level of EGFR expression correlates with disease progression, increased metastasis, and poor prognosis, which is strong for the development of effective EGFR target antigens for the treatment of various solid tumors Provide grounds.

  Since synovial fibroblasts and keratinocytes are also EGF receptor-expressing cell types, these cells are candidate target cells for the treatment of inflammatory arthritis and psoriasis, respectively. EGFR is also known as inflammatory arthritis (US Pat. No. 5,906,820, US Pat. No. 5,614,488), pharyngeal papilloma (Johnston D, Hall H, DiLorenzo TP, Steinberg BM 1999. Elevation of the EGFR and dependent signaling in human papillomavirus- Cancer Res. 59: 968-74.) and excessive secretion of mucus in the lung (Barnes PJ, Hansel TT 2003. Prospects for new drugs for chronic obstructive pulmonary disease. Lancet 364: 985-996, US Pat. No. 6,663,324) And U.S. Pat. No. 655 1989).

  Identification of MAbs that inhibit EGFR is an approach that has been used in clinical development targeting aberrant EGFR signaling in malignant neoplasms. Examples of antibodies targeting the EGFR include IMC-C225 (Erbitux, Imclone), EMD72000 (Merck, Darmstadt), ABX-EGF (Abgenix), h-R3 (theraCIM, YM Biosciences) and humax-EGFR ( Genmab). The mechanism of action of these antibodies relies on binding of the ligand to the receptor and subsequent inhibition by inhibiting the receptor's phosphotransfer and downstream signaling cascades. Mab225 (Erbitux is a chimeric derivative of this), F (ab ′) 2 fragment derived from 225 is only for internalization of EGFR and moderate receptors after prolonged incubation with EGFR-expressing cells. Capture can be induced. However, the monovalent Fab ′ fragment derived from 225 induces only receptor downregulation after preincubation with rabbit anti-mouse antibody (Fan Z, Mendelsohn J, Masui H, Kumar R 1993. Regulation of EGFR). in NIH3T3 / Her-14 cells by antireceptor monoclonal antibodies. J. Biol. Chem. 268: 21073-21079, Fan Z, Lu Y, Wu X, Mendelsohn J 1994. Antibody-induced EGFR dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J. Biol. Chem. 269: 27595-27602). These antibodies show antitumor activity against a broad panel of human tumor xenografts (reviewed in Grunwald V, Hidalgo M 2003. Developing inhibitors of the EGFR for cancer treatment. J. Natl. Cancer Inst. 95 : 851-867).

  The main goal of tumor treatment is to kill all tumor cells. A therapeutic agent that kills cells is defined as cytotoxic. A therapeutic that prevents cell replication without killing the cell is defined as cytostatic. Since known therapeutic agents based on antibodies that bind to the EGF receptor only suppress cell replication, the conventional antibodies act as cytostatic agents (European Patent No. 667165, European No. 359282, US Pat. No. 5,844,093). However, none of these antibodies are currently available as low molecular weight pharmaceuticals, are not completely effective in treating tumors, and most have limitations of high toxicity. Furthermore, developing a new compound (NCE) with sufficient activity and selectivity for the target sequence is a very difficult and time consuming process. Antibodies offer great potential as drugs because they have excellent selectivity for their target and low intrinsic toxicity. Furthermore, development time can be significantly reduced compared to the development of new compounds (NCE).

  There are several uses of antibodies derived from sources such as mice, sheep, goats, rabbits, etc., and their humanized derivatives as therapeutic agents for cancers that require cytotoxic or cytostatic effects. There is a problem for a reason. Because conventional antibodies are not stable at room temperature, they cannot depart from the need for a continuous cold chain (from manufacturing to patient treatment) that adds extra costs for development and therapeutic use. In developing countries, refrigeration may not be possible. Furthermore, the mammalian cell lines required to express intact, functional antibodies require a high level of assistance from a time and equipment standpoint, and the yield is very low so that the production of said antibodies or on a small scale The production of is expensive. Conventional antibodies often exhibit functional antigen binding activity only within a limited pH window. Therefore, they are unsuitable for applications in environments with extreme pH conditions such as in the digestive tract. Furthermore, conventional antibodies are unstable at low and high pH and are therefore not suitable for oral administration. Furthermore, conventional antibodies are unsuitable for use in assays or kits performed outside of the bioactive temperature range (eg, 37 ± 20 ° C.) because the binding activity is temperature dependent.

  Polypeptide therapeutics, specifically antibody-based therapeutics, have great potential as drugs because of their excellent selectivity for their targets and low intrinsic toxicity. However, antibodies obtained for therapeutically useful targets need to be further modified to avoid undesirable immune responses in human individuals to be formulated for human therapy when administered. This is known by those skilled in the art. The modification process is commonly referred to as “humanization”. It is known to those skilled in the art that antibodies obtained from species other than humans need to be humanized in order to make the antibodies therapeutically useful in humans ((1) CDR grafting: Protein Design Labs: US Pat. No. 6,180,370, US Pat. No. 5,693,761; Genentech US Pat. No. 6,054,297; Celltech: US Pat. No. 460167, European Patent No. 626390, US Pat. No. 5,859,205; No. 5,869,619, US Pat. No. 5,766,886, US Pat. No. 5,821,123). There is a need for methods of producing antibodies that avoid the need for fundamental humanization or that completely eliminate the need for humanization. There is a need for new types of antibodies that have well-defined framework regions or amino acid residues and can be administered to human subjects with virtually no or no humanization required. Yes.

  An important drawback of conventional antibodies is that they are complex and large molecules and are therefore relatively unstable and susceptible to degradation by proteolytic enzymes. This means that oral, sublingual, topical, nasal, vaginal, rectal, or inhalation administration of conventional antibody drugs does not have low pH tolerance at these sites, Means that it is impossible due to the action of proteolytic enzymes in these sites or in the blood and / or their large size. In order to overcome some of these problems, they must be administered by injection (intravenous, subcutaneous, etc.). Administration by injection requires expert training to use the hypodermic syringe or needle accurately and safely. It further requires a sterile instrument, a liquid formulation of the therapeutic polypeptide, a sterile stable form of the outer polypeptide vial container, and a suitable subject site to pierce the needle. In addition, subjects typically experience physical and psychological stress before and while receiving injections. Thus, there is a need for a method for delivering therapeutic polypeptides that is simpler and more comfortable for the subject, as well as avoiding the need for injections and saving costs and time.

  A solid tumor consists of cells that are dense and highly proliferative. For the treatment of solid tumors, it is very important that the therapeutic antibody penetrates to the deepest part of the tumor and is rapidly and uniformly distributed to prevent tumor recurrence. Complete conventional antibody (150 kDa) formed by gene fusion of F (ab ′) 2, Fab ′ and scFv fragments (gene segments encoding heavy and light chain variable regions and separated by short linker sequences) ScFv was shown to exhibit the most rapid and uniform distribution in the tumor mass (Yokota T, Milenic DE, Whitlow M) compared to the derived immunoglobulin forms (such as the smallest antigen binding unit) , Schlom J 1992. Rapid tumor penetration of a single-chain Fv and comparison with other immunoglobulin forms).

  These and other objects are achieved through the use of the Nanobodies of the present invention.

  Nanobodies derived from camelid heavy chain antibodies described in the present invention are known to have a tendency to bind to cavities (WO 97/49805, Lauwereys et al., EMBO J. 17, 5312, 1998). Thus, the Nanobodies are inherently suitable for recognizing ligand binding sites on the receptor and binding epitopes that are less sensitive to conventional antibodies, and thus function through different mechanisms of action, Can cause cytotoxic effects on tumor cells.

  Heavy chain antibodies do not contain light chains. Thus, in contrast to the binding regions of conventional antibodies that require functional folding of the heavy and light chain variable regions (domains), the binding region is driven by a single domain, VHH (approximately 15 kDa). Is formed. Since only a single gene segment is required for the expression of functional VHH, these VHHs are suitable as components (Conrath KE, Lauwereys M, Wyns L, Muyldermans S 2001. Camel single-domain antibodies). as modular building units in bispecific and bivalent antibody constructs. J. Biol. Chem. 276: 7346-7350). The heavy chain antibody fragment (or multimeric derivative) can easily be “all at once” in a fermenter using an inexpensive expression system, such as yeast, E. coli, or other microorganisms. Can be produced (European Patent No. 0 698 097).

  Furthermore, camelid nanobodies have been shown to have unexpectedly high thermal stability and resistance to harsh conditions such as extreme pH and denaturants in the Tm range of 60 ° C to 80 ° C (Ewert S et al., Biochemistry 2002 Mar 19; 41 (11): 3628-36, Perez et al., Biochemistry, 2001, 40: 74-83) and therefore they are suitable for delivery by oral administration. This allows for superior labeling efficiency, especially compared to conventional antibodies and their derivatives (such as scFv). As such, higher specific activity can be obtained that results in superior imaging results or therapeutic efficiency. Furthermore, camelized Nanobodies are more storage stable (Perez et al., Biochemistry, 40, 74, 2001). Since VHH is the smallest antigen binding site (15 kDa), they are very suitable for obtaining an optimal distribution in the solid tumor mass. Since VHH is derived from naturally occurring heavy chain antibodies that have already undergone maturation in vivo, it does not require any in vitro affinity maturation that requires time and effort. A polypeptide comprising one or more single domain antibodies (particularly Nanobodies) that bind to tumor-specific or tumor-associated antigens such as EGFR (EGFR) and insulin growth factor receptor (IGF-IR), homologues of said polypeptides, and It is an object of the present invention to provide a functional site. Said polypeptide i) inhibits binding of a natural ligand to the receptor and / or ii) induces receptor down-regulation and / or iii) inhibits receptor homo- and hetero-dimerization. And / or iv) tumors that induce IGF-IR and / or EGFR by inducing apoptosis in human cells, thereby altering the biological activity of EGFR upon binding, and v) using labeled polypeptides It is possible to detect the cell and vi) have a cytotoxic effect on the cell by binding to its tumor antigen. The polypeptide may or may not bind in the ligand binding groove of EGFR. The polypeptide is a single domain antibody.

  It is another object of the present invention to provide nanobodies or polypeptides that bind to compounds having therapeutic effects, such as anti-cancer agents, or bound to contrast agents suitable for visualization in MRI or CAT-scan.

  One embodiment of the invention is an anti-EGFR or anti-IGF-IR polypeptide comprising at least one single domain antibody directed to EGFR or IGF-IR, respectively.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, wherein at least one single domain antibody is a heavy chain variable region.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, wherein at least one single domain antibody is VHH or Nanobody.

  Another embodiment of the present invention is the above anti-EGFR or anti-antigen wherein at least one single domain antibody is VHH substituted with one or more amino acid residues without substantially altering antigen binding capacity. IGF-IR polypeptide.

  Another embodiment of the present invention is that at least one single domain antibody is specifically camelized in which one or more amino acid residues are substituted without substantially altering antigen binding capacity. Said anti-EGFR or anti-IGF-IR polypeptide, which is VH.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, wherein at least one single domain antibody is a humanized VHH.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-, wherein said at least one single domain antibody is a homologous sequence, functional site, or functional site of a homologous sequence of a full length single domain antibody. It is an IR polypeptide.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, further comprising at least one covalently linked or recombinantly fused substance to increase half-life.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein the substance for increasing the half-life is polyethylene glycol or serum protein.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein said substance is a single domain antibody directed against serum proteins.

  Another embodiment of the present invention is the anti-EGFR or anti-IGF-IR polypeptide, wherein the serum protein is any of serum albumin, serum immunoglobulin, thyroxine binding protein, transferrin or fibrinogen.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein said serum protein is derived from human.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein said single domain antibody is a homologous sequence, functional site, or functional site of a homologous sequence of a full-length single domain antibody. is there.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, wherein the polypeptide is a homologous sequence, functional site, or functional site of a homologous sequence of a full-length single domain antibody.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein the number of single domain antibodies directed against EGFR or IGF-IR is at least two.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein said at least two single domain antibodies are head-to-tail bound without a linker sequence.

  Another embodiment of the present invention is the above anti-EGFR, wherein said at least one single domain antibody is capable of binding to EGFR and internalizing the receptor but not colocalizing with transferrin. Or an anti-IGF-IR polypeptide.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, further comprising one or more radioisotopes.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, wherein the at least one radioisotope is either 188Re, 131I or 211At.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, further comprising one or more anticancer agents.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR, wherein said at least one anticancer agent is any of anthracycline, methotrexate, vindesine, cisplatin, lysine, calicheamicin, or a cytokine. It is a polypeptide.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, further comprising one or more enzymes capable of activating a prodrug.

  Another embodiment of the invention is a nucleic acid encoding the anti-EGFR or anti-IGF-IR polypeptide.

  Another embodiment of the present invention is the anti-EGFR or anti-IGF-IR polypeptide, or a nucleic acid encoding the polypeptide, for treating and / or preventing and / or alleviating a disease associated with an inflammatory process. .

  Another embodiment of the present invention is the anti-EGFR or anti-IGF-IR polypeptide, or a nucleic acid encoding the polypeptide, for treating and / or preventing and / or alleviating a disease associated with a malignant tumor. .

  Another embodiment of the present invention encodes the above-mentioned anti-EGFR or anti-IGF-IR polypeptide or the polypeptide for preparing a medicament for treating and / or preventing and / or alleviating a disease associated with a malignant tumor Use of nucleic acids.

  In another embodiment of the present invention, the tumor is breast cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, cancer of the central nervous system, head and neck cancer, kidney cancer, bone Said anti-EGFR polypeptide, anti-IGF-IR polypeptide or nucleic acid selected from the group consisting of cancer, blood and lymphoid cancer.

  Another embodiment of the present invention is a composition comprising the anti-EGFR or anti-IGF-IR polypeptide, or a nucleic acid encoding said polypeptide, and a suitable excipient.

Another embodiment of the present invention is a method of diagnosing a disease characterized by EGFR disease signaling or the presence of IGF-IR comprising the following steps:
(A) contacting the sample with the anti-EGFR or anti-IGF-IR polypeptide,
(B) a step of detecting the binding of the polypeptide to the sample, and (c) a disease characterized in that the difference in binding compared to the sample is an abnormal signaling of EGFR or the presence of IGF-IR, respectively. Comparing the standard useful for the diagnosis of with the binding detected in step (b).

  Another embodiment of the present invention is a kit for screening for the above diseases using the above method.

  Another embodiment of the present invention is a kit for screening for the above-mentioned diseases comprising the isolated anti-EGFR or anti-IGF-IR polypeptide.

  Another embodiment of the present invention is for inhibiting the interaction between EGF and one or more EGFR receptors, respectively, or between IGF-IR and one or more IGF-IR receptors, respectively. Use of the anti-EGFR or anti-IGF-IR polypeptide.

Another embodiment of the present invention is a method of producing the above anti-EGFR or anti-IGF-IR polypeptide comprising the following steps:
(A) culturing a host cell containing a nucleic acid capable of encoding the anti-EGFR or anti-IGF-IR polypeptide under conditions for expressing the polypeptide, and (b) culturing the produced polypeptide. The process of collecting more.

  Another embodiment of the present invention is the above method, wherein said host cell is a bacterium or yeast.

  Another embodiment of the present invention is a kit for screening for cancer comprising the anti-EGFR or anti-IGF-IR polypeptide.

  Another embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide as described above, further comprising one or more contrast agents.

  Another embodiment of the present invention is the above anti-EGFR or anti-IGF-IR polypeptide, wherein the contrast agent is any of a radioactive label, an enzyme label, a magnetic resonance paramagnetic chelate, and / or an optical dye.

  Another embodiment of the invention is the use of the above anti-EGFR or anti-IGF-IR polypeptide for imaging.

  Another embodiment of the invention is a method of imaging an EGFR or IGF-IR target comprising the step of administering the anti-EGFR or anti-IGF-IR polypeptide.

  Use of an anti-EGFR or anti-IGF-IR polypeptide as described above for preparing a composition, more preferably a diagnostic composition for imaging.

  Another embodiment of the present invention is the above method, wherein the number of anti-EGFR or anti-IGF-IR single domain antibodies is at least two.

  Another embodiment of the present invention is a method as described above, wherein said polypeptide is administered to a subject in microparticles, ultrasound bubbles, microspheres, emulsions or liposomes.

  Another embodiment of the invention comprises on the respective receptor of the anti-EGFR or anti-IGF-IR polypeptide comprising the step of fusing said polypeptide to one or more single domain antibodies directed against serum proteins. This is a method for increasing the residence time.

  Another embodiment of the present invention is the above method, wherein said anti-EGFR or anti-IGF-IR polypeptide is said polypeptide.

Another embodiment of the invention is a method of identifying an agent that modulates the binding of the anti-EGFR or anti-IGF-IR polypeptide to EGFR or IGF-IR comprising the following steps:
(A) the above-mentioned polypeptide is converted to EGFR or IGF-IR, or a combination thereof under conditions that allow binding between the polypeptide and the corresponding target in the presence or absence of a candidate modulator. Contacting said target with a fragment, and (b) reducing binding in the presence of said candidate modulator relative to binding in the absence of said candidate modulator, said candidate modulator is said anti-EGFR or Measuring the binding of the anti-IGF-IR polypeptide to the target of step (a), identified as an agent that modulates binding to EGFR or IGF-IR, respectively.

Another embodiment of the present invention provides EGFR via binding of the anti-EGFR or anti-IGF-IR polypeptide or anti-IGF-IR polypeptide, respectively, to EGFR or IGF-IR, comprising the following steps: Or a method of identifying an agent that modulates a disease mediated by IGF-IR:
(A) the above-mentioned polypeptide is converted to EGFR or IGF-IR, or a combination thereof under conditions that allow binding between the polypeptide and the corresponding target in the presence or absence of a candidate modulator. Contacting said target with a fragment, and (b) reducing the binding in the presence of said candidate modulator relative to binding in the absence of said candidate modulator, said candidate modulator is EGFR or IGF- Measuring the binding of the polypeptide identified as an agent that modulates an IR-mediated disease to the target of step (a).

Another embodiment of the present invention provides an agent that modulates the binding of epidermal growth factor to its receptor through binding of the above-mentioned anti-EGFR or anti-IGF-IR polypeptide to EGFR, comprising the following steps: Is a method to identify:
(A) the anti-EGFR or anti-IGF-IR polypeptide described above is EGFR, under conditions that allow binding between the polypeptide and the corresponding target in the presence or absence of a candidate modulator. Or a contact thereof with a target that is a fragment thereof, or a homologous sequence thereof, and (b) a decrease in binding in the presence of said candidate modulator relative to binding in the absence of said candidate modulator. Measuring the binding of the polypeptide to the target of step (a), wherein the candidate modulator is identified as an agent that modulates the binding of a natural ligand of EGFR.

  Another embodiment of the present invention includes EGFR mediated or anti-IGF-IR mediated, comprising the anti-EGFR or anti-IGF-IR polypeptide and EGFR or IGF-IR, respectively, or a fragment thereof. A kit for screening for a drug that regulates a disease.

  Another embodiment of the invention is an unknown agent that modulates the binding of the polypeptide to EGFR or IGF-IR and is identified by the method.

  Another embodiment of the invention is an unknown agent that modulates an EGFR mediated or anti-IGF-IR mediated disease and is identified by the above method.

  Another embodiment of the present invention is the above unknown agent, wherein the disease is one or more of cancer, rheumatoid arthritis, psoriasis, or mucus hypersecretion in the lung.

  Another embodiment of the invention is to treat and / or prevent and / or prevent diseases susceptible to modulation by delivering an EGFR or IGF-IR antagonist that can pass through the gastric environment without inactivation. Or an anti-EGFR or anti-IGF-IR polypeptide for alleviation.

  Another embodiment of the present invention is to treat, prevent, and treat symptoms of diseases that are susceptible to modulation by delivering EGFR or IGF-IR antagonists that can pass through the gastric environment without inactivation. Use of the above anti-EGFR or anti-IGF-IR polypeptide to prepare a medicament for alleviation.

  Another embodiment of the present invention is to treat and / or prevent and / or prevent symptoms of diseases susceptible to modulation by delivering an EGFR or IGF-IR antagonist to the vagina and / or gastrointestinal tract without inactivation. The above-mentioned polypeptide for alleviation.

  Another embodiment of the present invention treats, prevents and / or alleviates the symptoms of disease susceptible to modulation by delivering an EGFR or IGF-IR antagonist to the vagina and / or gastrointestinal tract without inactivation. Use of the polypeptide for the preparation of a medicament for

  Another embodiment of the present invention is for treating and / or preventing and / or reducing diseases susceptible to modulation by delivering an EGFR or IGF-IR antagonist to the upper respiratory tract or lung without inactivation. Said polypeptide.

  Another embodiment of the present invention is the use of the above polypeptide for the preparation of a medicament for treating, preventing and / or alleviating the symptoms of a disease that requires delivery of a therapeutic compound to the upper respiratory tract or lung. is there.

  Another embodiment of the invention is to treat and / or prevent diseases that increase the permeability of the intestinal mucosa that are susceptible to modulation by delivering an EGFR or IGF-IR antagonist to the intestinal mucosa without inactivation. And / or the above-mentioned polypeptide for alleviation.

  Another embodiment of the invention is to treat, prevent and / or treat symptoms of diseases that increase intestinal mucosal permeability that are susceptible to modulation by delivering EGFR or IGF-IR antagonists without inactivation. Use of the polypeptide to prepare a medicament for alleviation.

  Another embodiment of the present invention provides a method for treating and / or preventing and / or alleviating diseases susceptible to modulation by delivering an EGFR or IGF-IR antagonist to sublingual tissue without inactivation. It is a polypeptide.

  Another embodiment of the invention is an agent for treating, preventing and / or alleviating the symptoms of a disease susceptible to modulation by delivering an EGFR or IGF-IR antagonist to the sublingual tissue without inactivation Use of the above polypeptide for the preparation of

  Another embodiment of the present invention provides a polypeptide as described above for treating and / or preventing and / or alleviating a disease susceptible to modulation by delivering an EGFR or IGF-IR antagonist through the skin without inactivation. It is.

  Another embodiment of the present invention is to prepare a medicament for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by delivering an EGFR or IGF-IR antagonist through the skin without inactivation. For the use of the above polypeptide.

  Another embodiment of the present invention relates to the polypeptide, nucleic acid or drug, use of the polypeptide, nucleic acid or drug, the polypeptide, the disease is cancer, rheumatoid arthritis, psoriasis, or hypersecretion of mucus in the lung. Use of certain of the above polypeptides.

Another embodiment of the invention is the use of the above polypeptide to inhibit the interaction between EGF and one or more EGFR.
Another embodiment of the present invention is a therapeutic composition comprising:
(A) VHH that inhibits the growth of human tumor cells by binding to the EGFR or IGF? IR of the tumor cells
(B) Antitumor agent

  Another embodiment of the present invention is the above therapeutic composition for separate administration of the components.

  The present invention relates to an anti-EGFR (EGFR) polypeptide comprising at least one single domain antibody directed against EGFR. The invention also relates to an anti-IGF-IR receptor comprising at least one single domain antibody directed against IGF-IR. The present invention also relates to a nucleic acid capable of encoding the polypeptide.

  EGFR is overexpressed on the surface of many cancer cells, and this overexpression is associated with poor prognosis, progression, reduced survival, and resistance to chemotherapy or hormonal therapy. EGFR overexpression also alters cell cycle regulation (increases tumor cell proliferation), inhibits apoptosis and promotes angiogenesis. Thus, blocking EGFR-related signaling in tumor cells results in (i) inhibition of tumor cell growth, (ii) inhibition of the cell cycle, (iii) induction of apoptosis, and (iv) inhibition of angiogenesis. .

  The pharmaceutical composition of the present invention blocks EGF binding to a receptor, inhibits receptor tyrosine kinase activation induced by EGF binding (inhibits tyrosine phosphorylation), Potent anti-angiogenic activity that inhibits cell growth by EGF in vitro cell cultures, has a pro-apoptotic effect, down-regulates VEGF production and reduces the number of microvessels Have. These effects are increased when the pharmaceutical composition is combined with other anti-tumor therapies.

  One embodiment of the invention relates to a pharmaceutical composition comprising at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent or excipient.

  According to a preferred but non-limiting embodiment, the pharmaceutical composition is suitable for oral administration.

  According to one aspect of the invention, the Nanobody is a polypeptide derived from a heavy chain antibody and its framework regions and complementarity determining sites are part of a single domain polypeptide. Examples of such heavy chain antibodies include, but are not limited to, naturally occurring immunoglobulins lacking a light chain. Such immunoglobulins are disclosed, for example, in WO 94/04678.

  The antigen binding site of this unique type of heavy chain antibody has a unique structure that includes a single variable region. For reasons of clarity, variable regions derived from heavy chain antibodies that naturally lack a light chain are known herein as VHH or VHH domains or Nanobodies. The VHH domain peptide can be derived, for example, from antibodies obtained from camelid species such as camels, dromedaries, llamas, alpaca, and guanacos. Other species other than camelids (eg, sharks, pufferfish) may produce functional antigen-binding heavy chain antibodies that naturally lack light chains. VHH domains derived from the heavy chain antibody are included within the scope of the present invention.

  Camelid antibodies express unique and extensive functional heavy chain antibodies lacking light chains. VHH molecules derived from camelid antibodies are the smallest of the known complete binding regions (about 15 kDa, or 10 times smaller than conventional IgG), so they are delivered to dense tissues and high It is very suitable for reaching the limited space between molecules.

  Other examples of Nanobodies include the conventional 4 modified by substitution of one or more amino acid residues with residues unique to camelids (so-called camelization of heavy chain antibodies, WO 94/04678). Nanobodies derived from the VH domain of this chain antibody. The position can occur preferentially in VH-VL interfaces, including positions 37, 44, 45, 47, 103, and 108, and in so-called camelid feature residues (WO 94/04678).

  Nanobodies represent a small, robust and efficient recognition unit formed by a single immunoglobulin (Ig) region.

  The anti-EGFR or anti-IGF-IR polypeptides and their derivatives disclosed herein not only have advantageous features over conventional antibodies such as low toxicity and high selectivity, but also exhibit other properties. They are more soluble and therefore can be stored and / or administered at higher concentrations compared to conventional antibodies.

  Conventional antibodies are not stable at room temperature, must be refrigerated for preparation and storage, require laboratory equipment that requires cooling, storage and transport, and contribute to time and expense. The anti-EGFR or anti-IGF-IR polypeptides of the present invention are stable at room temperature so that they can be prepared, stored and / or transported without the use of refrigeration equipment, reducing cost, time and environmental burden can do. Furthermore, conventional antibodies are not suitable for use in assays or kits that are performed outside of a bioactive temperature range (eg, 37 ± 20 ° C.).

  Another advantageous property of the anti-EGFR or anti-IGF-IR polypeptides disclosed herein as compared to conventional antibodies is, for example, binding to albumin or, for example, serum albumin, according to the present invention. Examples include regulation of circulating half-life, which can be regulated by binding to one or more Nanobodies directed to serum proteins such as: One aspect of the present invention is a bispecific anti-EGFR polypeptide having specificity for a serum protein such as serum albumin and specificity for a target disclosed in WO 04/041865, incorporated herein by reference. is there. As another means for increasing the half-life, coupling of the polypeptide of the present invention to a nanobody directed to Fc or other EGFR (creation of a multivalent nanobody such as bivalent or trivalent) or Examples include coupling with polyethylene glycol. The ability to control the half-life is desirable in order to adjust the dose with immediate effect.

  Conventional antibodies are not suitable for use in environments outside the normal physiological pH range. They are unstable at low and high pH and are therefore not suitable for oral administration. Camelized antibodies are resistant to harsh conditions such as extreme pH, denaturing agents, and high temperatures, so that the anti-EGFR or anti-IGF-IR polypeptides disclosed herein can be delivered by oral administration. Is preferred. Camelized antibodies are resistant to proteolytic enzymes, whereas conventional antibodies are less resistant.

  The expression yield of conventional antibodies is very low, and the production method is very laborious. Furthermore, the mammalian cell lines required to express a complete and functional antibody require a high level of assistance from a time and equipment standpoint, and the yield is very low, so that the production or Production on a scale is expensive. The anti-EGFR or anti-IGF-IR polypeptide of the present invention is inexpensively produced by fermentation in convenient recombinant host organisms such as Escherichia coli and yeast unlike conventional antibodies that require expensive mammalian cell culture facilities. The expression level that can be achieved is high. Examples of yields of the polypeptides of the invention are 1-10 mg / ml (E. coli) and up to 1 g / l (yeast).

  The anti-EGFR or anti-IGF-IR polypeptides of the present invention exhibit high binding affinity for a wide range of different antigen types, the ability to bind to epitopes not recognized by conventional antibodies, eg, they enter the cavity A possible long CDR3 loop is shown.

The anti-EGFR or anti-IGF-IR polypeptides of the present invention are readily bifunctional or multifunctional by the (head-to-tail) fusion disclosed in WO 96/34103 (incorporated herein by reference). A molecule can be generated.
Due to the small size, the anti-EGFR or anti-IGF-IR polypeptide of the present invention has better tissue penetration and ability to reach any part of the body than conventional antibodies. Can do.

  Lama single domain antibodies can cross the brain-blood barrier. In one embodiment of the invention, the anti-EGFR or anti-IGF-IR polypeptide can cross the blood brain barrier. In another embodiment of the invention, the anti-EGFR or anti-IGF-IR polypeptide is unable to cross the brain-blood barrier.

  The anti-EGFR or anti-IGF-IR polypeptides disclosed herein are less immunogenic than conventional antibodies. A subclass of camelid antibodies that have 95% amino acid homology to the human VH framework region has been discovered. This suggests that the immunogenicity can be expected to be low or absent at the time of administration to human patients. Alternatively, humanization of Nanobodies surprisingly requires only a few residue substitutions that need to be substituted when such a need arises.

  According to one preferred but non-limiting embodiment, the polypeptides of the invention have an isoelectric point of 4-11. Preferably, the polypeptide of the present invention has an isoelectric point of 5-10.

  According to one preferred but non-limiting embodiment, the polypeptides of the present invention comprise two covalently linked amino acid chains (referred to herein as “heavy chains”).

  The heavy chains of the present invention are preferably linked via disulfide bonds. More preferably, the heavy chain of the present invention is linked via a cysteine residue forming a disulfide bond.

  According to one preferred but non-limiting embodiment, the heavy chain of the present invention has a molecular weight of about 35 kDa to 50 kDa. Molecular weight is determined according to the description of Hamers-Casterman et al. (Nature 1993). Preferably, the heavy chain of the present invention has a molecular weight of 40 kDa to 50 kDa.

  More preferably, the heavy chain of the present invention has a molecular weight of 41 kDa to 49 kDa, 42 kDa to 48 kDa, 43 kDa to 47 kDa, or 44 kDa to 46 kDa. Most preferably, the heavy chain of the present invention has a molecular weight of 43 kDa to 46 kDa.

  According to one preferred but non-limiting embodiment, the heavy chain of the invention has a molecular weight of 43 kDa.

  According to another preferred but non-limiting embodiment, the heavy chain of the invention has a molecular weight of 46 kDa.

  A single domain antibody is an antibody whose complementarity determining regions are part of a single domain polypeptide. Examples include heavy or light chain antibodies, antibodies that naturally lack light chains, single domain antibodies derived from conventional four chain antibodies, VH domains derived from conventional antibodies, each of which encodes a portion thereof Human germline sequences, recombinant antibodies other than those derived from these antibodies, and single domain scaffolds, but are not limited to these. A single domain antibody may be any conventional or any future single domain antibody. Single domain antibodies can be derived from any species including but not limited to mice, humans, camels, llamas, sharks, puffers, goats, rabbits, cows and the like. According to one embodiment of the present invention, the single domain antibody used in the present invention is an immunoglobulin lacking a naturally occurring light chain. Such single domain antibodies are disclosed, for example, in WO 94/04678. For reasons of clarity, variable regions derived from heavy chain antibodies that naturally lack a light chain are used herein to distinguish them from VHH or or Known as nanobody. The VHH molecule can be derived, for example, from antibodies obtained from camelid species such as camels, dromedary, llama, vicugna, alpaca and guanaco. Other species other than camelids (for example, sharks and puffers) also produce functional antigen-binding heavy chain antibodies that naturally lack light chains, and VHH derived from these heavy chain antibodies Included within the scope of the invention.

  Nanobodies according to the present invention include heavy chain variable regions (hereinafter referred to as VHH domains or Nanobodies) derived from immunoglobulins that naturally lack light chains, such as those derived from camelids, as described in WO 94/04678. Call). VHH molecules are 10 times smaller than IgG molecules. These are single polypeptides that are very stable and resistant to extreme pH and temperature conditions. In addition, expression in the microbial host produces functional FHH that is properly folded in high yield. Thus, expression in a mammalian host can be avoided.

  Furthermore, they are resistant to proteolytic enzymes, whereas conventional antibodies are less resistant. The VHH of the present invention generally exhibits higher resistance to digestion by proteolytic enzymes in the digestive tract than conventional antibodies. This allows the VHH to be used therapeutically via uptake by the oral route. Furthermore, the VHH of the present invention has been shown to be stable over several months when incubated at 37 ° C. in human serum.

  Furthermore, antibodies produced in camelids recognize epitopes other than those recognized in vitro by the use of antibody libraries or by immunizing mammals other than camelids (WO No. WO 97/49805). Thus, anti-EGFR or anti-IGF-IRVHH can interact more efficiently than conventional antibodies or induce unique biological effects when binding to EGFR or IGF-IR, respectively. On each receptor, it can block the interaction with the EGFR ligand more efficiently or block the activity of the IGF-IR antigen. Since VHH is known to bind "unusual" epitopes such as cavities or grooves (WO 97/49805), the affinity of the VHH is more suitable for therapy.

  It has been reported that antibodies directed against EGFR, such as IMC-C225, ABX-EGF, Humax-EGFR, hR3 and EMD72000, show a growth inhibitory effect in human cancer cells. These biological mechanisms indicate that these EGFR-directed antibodies inhibit the binding of ligands to the extracellular region of the EGF receptor, and are therefore receptor-mediated downstream required for cell proliferation. Explained by its ability to block signal transduction. One commercially available EGFR antibody that exhibits cytostatic effects is Erbitux (chimerized mouse monoclonal antibody 225) recently approved for the treatment of colorectal cancer in combination therapy with irinotecan. One mechanism by which cells reduce signaling through the EGF receptor is the mechanism of receptor capture or downregulation. After binding of the ligand to the receptor, the cell can downregulate receptor signaling by internalizing the ligand-receptor complex and degrading the ligand-receptor complex in the lysosome. .

The inventors have found that certain anti-EGFR Nanobodies can surprisingly antagonize EGF, TGFα and / or erbitux. Novel anti-EGFR Nanobodies can be functionally classified into three types of ligand antagonist Nanobodies. Non-limiting examples of different types of Nanobodies are as follows:
-Type A Nanobodies (binding to the extracellular region of EGFR and antagonizing EGF and TGFα binding sites on EGFR but not erbitux binding sites. Examples of Nanobodies are 27-10-E8 (sequence No. 80: Family I) and PMP7A5 (SEQ ID No. 84; Family III)).
-Type B Nanobodies (binding to the extracellular region of EGFR and antagonizing EGF, erbitux, and TGFα binding sites on EGFR. Examples of Nanobodies are PMP7D12 and PMP7C12 (SEQ ID NO: 81-82: Family II) Is).
-Type C Nanobodies (binding to the extracellular region of EGFR and antagonizing TGFα binding sites but not EGF or erbitux binding sites in EGFR downregulation. Examples of Nanobodies are PMP8C7 (SEQ ID NO: 90: Family XIII)).

  Thus, one embodiment of the present invention is a single domain antibody that specifically binds to the extracellular region of EGFR and antagonizes EGF, TGFα binding sites on EGFR, but does not antagonize erbitux binding sites on EGFR. Or an anti-EGFR polypeptide comprising Nanobodies. Another embodiment of the invention is an anti-EGFR polypeptide comprising a single domain antibody or Nanobody that specifically binds to the extracellular region of EGFR and antagonizes EGF, erbitux, and TGFα binding sites on EGFR . In yet another embodiment of the invention, a single domain antibody that specifically binds to the extracellular region of EGFR and antagonizes a TGFα binding site on EGFR but does not antagonize an EGF or erbitux binding site on EGFR Or an anti-EGFR polypeptide comprising Nanobodies.

  One aspect of the invention is an anti-EGFR or anti-IGF-IR polypeptide comprising at least one anti-EGFR Nanobody. It is one embodiment of the present invention that the polypeptide contains other components. The component may be, for example, a polypeptide sequence such as one or more anti-EGFR Nanobodies, one or more antibody IGF-IR Nanobodies, one or more antiserum albumin Nanobodies. Other fusion proteins are encompassed within the scope of the invention and include, for example, fusion proteins with carrier proteins, signaling molecules, tags, and enzymes. Examples of other components include radioactive labels, organic dyes, and fluorescent compounds.

  One embodiment of the present invention is an anti-EGFR or anti-IGF-IR polypeptide consisting of a sequence corresponding to a camelid VHH directed against EGFR or IGF-IR, respectively, or a closely related family member .

  The single domain antibodies of the invention are directed against each of EGFR or IGF-IR, or closely related family members.

  Another embodiment of the invention is a multivalent anti-EGFR or anti-IGF-IR polypeptide disclosed herein comprising at least two single domain antibodies directed against EGFR. Another embodiment of the invention is a multivalent anti-IGF-IR polypeptide as disclosed herein comprising at least two single domain antibodies directed against IGF-IR. The multivalent anti-EGFR or anti-IGF-IR polypeptide has the advantage of exhibiting an unusually high functional affinity for the target, much higher inhibition than expected compared to a monovalent analog.

  The multivalent anti-EGFR or anti-IGF-IR polypeptide has a functional affinity several orders of magnitude greater than the parent monovalent anti-EGFR or anti-IGF-IR polypeptide, respectively. The present inventors have found that the functional affinity of these multivalent polypeptides is much higher than previously reported bivalent and multivalent antibodies. Surprisingly, anti-EGFR or anti-IGF-IR polypeptides of the invention comprising single domain antibodies linked together, either directly or via a short linker sequence, are theoretically predicted with conventional multivalent four chain antibodies. Show high functional affinity.

  The inventors have shown that such a large increase in functional activity is preferably in a direct binding assay or a functional assay such as a cytotoxicity assay, using an antigen consisting of multidomain and multimeric proteins. I also found that I can decide.

  The inventors have also found that multivalent polypeptides have increased residence time and / or affinity / binding for each of their target IGF-IR and / or EGFR. These multimeric polypeptides can be modified to be labeled with radioactive labels, enzyme labels, or magnetic resonance paramagnetic chelates, or encapsulated in microparticles, ultrasonic bubbles, microspheres, emulsions or liposomes, or binding sites as optical dyes. Can be used to provide an IGF-IR or EGFR specific contrast agent.

  As used herein, a multivalent anti-EGFR or anti-IGF-IR polypeptide refers to a polypeptide comprising two or more anti-EGFR single domain antibodies linked by a covalent bond. As used herein, a multivalent anti-IGF-IR polypeptide refers to a polypeptide comprising two or more anti-IGF-IR single domain antibodies linked by a covalent bond. Anti-EGFR or IGF-IR single domain antibodies may be identical in sequence or different in sequence, but directed against the same target or their same antigen or epitope Yes. Alternatively, the multivalent construct may be directed against different epitopes of the same target.

  Depending on the number of anti-EGFR or IGF-IR single domain antibodies bound, a multivalent anti-EGFR or anti-IGF-IR polypeptide may be bivalent (two anti-EGFR single domain antibodies or two anti-IGF-IR Single domain antibody), trivalent (three anti-EGFR or anti-IGF-IR single domain antibodies), tetravalent (four anti-EGFR or anti-IGF-IR single domain antibodies), or higher valent molecules It may be.

  According to another aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptide may comprise at least two anti-EGFR Nanobodies. It is one embodiment of the present invention that the polypeptide may contain the above-mentioned other components.

  Examples of anti-EGFR or anti-IGF-IR polypeptides of the invention comprising two anti-EGFR Nanobodies or two anti-IGF-IR Nanobodies are shown in Table 4 below (SEQ ID NOS 122-133, and 141-143), And polypeptides described in Table 6 (SEQ ID NOs: 134 to 135).

  According to a further aspect of the present invention, the anti-EGFR or anti-IGF-IR polypeptide of the present invention is directed to 1, 2, 3, 4, 5, 6, 7, 8 having directional properties to EGFR or IGF-IR, respectively. , 9, 10, 11, 12, 13, 14, 15 or more than 15 nanobodies.

  According to one aspect of the invention, an anti-EGFR or anti-IGF-IR polypeptide of the invention may comprise at least two identical or non-identical anti-EGFR Nanobody sequences. At least two of the sequences do not have equal affinity for EGFR, thus forming an anti-EGFR or anti-IGF-IR polypeptide that combines a low affinity binding sequence and a high binding sequence To do may be an aspect of the present invention.

  Examples of bivalent anti-EGFR or anti-IGF-IR polypeptides of the present invention comprising two identical Nanobodies directed to EGFR or IGF-IR, respectively, include the following Table 4 (SEQ ID NOs: 122-133), And polypeptides described in each of Table 6 (SEQ ID NOs: 134 to 135).

  As described below, the Nanobody may be bound with or without a linker sequence.

  Methods for constructing bivalent polypeptides are well known in the art (eg, US Patent Application Publication No. 2003/0088074) and are described below.

  In order to increase the therapeutic effect, it is desirable to modify the anti-EGFR or anti-IGF-IR polypeptides of the present invention with respect to effector function. For example, Nanobody fusions with certain Fc regions, particularly fusions with human-derived Fc regions, may be advantageous.

  The present invention also surprisingly provides an anti-EGFR or anti-IGF-IR polypeptide as disclosed herein further comprising one or more Nanobodies directed against a serum protein of the subject. In the circulation of the anti-EGFR or IGF-IR Nanobody when it is not part of the polypeptide has a significantly longer half-life. In addition, the anti-EGFR or anti-IGF-IR polypeptide may be, for example, highly stable to maintain integrity in mice, resistant to extreme pH, high temperature stability, and high target specificity and affinity. Have been found to have desirable properties similar to the Nanobodies.

  Thus, the anti-EGFR or anti-IGF- disclosed herein, comprising one or more Nanobodies directed against each of EGFR or IGF-IR, and one or more Nanobodies with specificity for serum proteins. IR polypeptides are much more efficient than polypeptides that target only EGFR or IGF-IR, respectively.

  For example, examples of bispecific anti-EGFR polypeptides comprising one Nanobody for EGFR and one Nanobody for serum albumin are essentially as described in WO 05/044858 and WO 04/041867. It may be a thing.

  The serum protein may be any suitable protein or fragment thereof found in the subject's serum. In one embodiment of the invention, the serum protein is any of serum albumin, serum immunoglobulin, thyroxine binding protein, transferrin or fibrinogen. The subject may be, for example, a rabbit, goat, mouse, rat, cow, calf, camel, llama, monkey, donkey, guinea pig, chicken, sheep, dog, cat, horse, and preferably a human. Depending on the intended use, such as the half-life required for effective therapy and / or localization of the target antigen, the paired Nanobodies are directed against one of the serum proteins described above. Good.

  According to one aspect of the invention, the number of Nanobodies directed to serum proteins in the anti-EGFR or anti-IGF-IR polypeptides of the invention is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 15 or more.

  Another aspect of the present invention provides an anti-EGFR or anti-IGF-IR further comprising at least one agent linked (coupled) or non-covalently to increase the in vivo half-life of the polypeptide. It is a polypeptide. Examples of substances that increase half-life are well known in the art and include, for example, polyethylene glycol and serum albumin.

  Methods for combining Nanobodies and other materials to form bispecific and multispecific polypeptides are known to those of skill in the art and are described below.

  In order to increase the half-life, the polypeptides of the present invention that have not been modified according to the present invention are characterized in that they are excreted more rapidly from the body. Conversely, a bispecific polypeptide comprising one or more Nanobodies directed to EGFR and one or more antiserum protein Nanobodies can circulate in a subject's body for several days, and the frequency of treatment , Increase the duration of functional activity in the body, reduce inconvenience to the subject, and thereby reduce the cost of treatment. Similar advantageous features are found for polypeptides of the invention that include other substances to increase half-life.

  Furthermore, it is an aspect of the present invention that the half-life of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein can be controlled by the number of antiserum protein Nanobodies present in the polypeptide. A controllable half-life is desirable in some situations, such as application to regular administration of therapeutic anti-EGFR polypeptides.

  Pharmacokinetic analysis and half-life determination are well known to those skilled in the art. Details are described in Kenneth, A, et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists, and Peters, et al., Pharmacokinete analysis: A Practical Approach (1996). See also "Pharmacokinetics", M Gibaldi and D Perron, Marcel Dekker, 2nd edition (1982).

  According to one aspect of the invention, the polypeptide can bind to one or more molecules that can increase the in vivo half-life of the polypeptide.

  Half-life is the time required for the serum concentration of a polypeptide to decrease to 50% in vivo, for example, due to degradation of the ligand and / or elimination or capture of the ligand by natural mechanisms. The polypeptides of the present invention are stabilized in vivo and their half-life is increased by binding to molecules that are resistant to degradation and / or excretion or capture of the ligand. Usually, the molecule is a naturally occurring protein that itself has a long half-life in vivo.

  In vivo, the half-life of a polypeptide of the invention is increased if its functional activity persists longer than a similar polypeptide that is not specific for a molecule that increases half-life. Thus, polypeptides of the invention specific for HSA and target molecules are compared to similar polypeptides that lack specificity for HSA and do not bind HSA but bind to other molecules. For example, it binds to the second epitope of the target molecule. Typically, the half-life is increased by 10%, 20%, 30%, 40%, 50% or more. An increase in half-life in the range of 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x or more is possible. Alternatively or in addition, half-life increases in the range of 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold are possible.

Typically, molecules that can increase the half-life of a polypeptide in vivo are polypeptides that are naturally occurring in vivo and are resistant to degradation or removal by endogenous mechanisms that remove unwanted substances from the body. is there. For example, molecules that increase the half-life in a living body can be selected from:
Extracellular matrix derived proteins; for example, collagen, laminin, integrin and fibronectin. Collagen is the major protein of the extracellular matrix. About 15 types of collagen found in different parts of the body, such as bone I, skin, tendons, ligaments, cornea, viscera, type I collagen (accounting for about 90% of body collagen), or cartilage, intervertebral disc Type II collagen found in the vitreous humor of the notochord and the eyeball is currently known;
Proteins found in blood: fibrin, α-2-macroglobulin, serum albumin, fibrinogen A, fibrinogen B, serum amyloid protein A, heptaglobin, profilin, ubiquitin, intrauterine globulin, β-2-macroglobulin, etc. Plasma proteins, etc .;
Enzymes and inhibitors such as plasminogen, lysozyme, cystatin C, α-1-antitrypsin and pancreatic trypsin inhibitor. Plasminogen is an inactive precursor of plasmin, a trypsin-like serine protease. This has been found to circulate normally through the bloodstream. When plasminogen is activated and converted to plasmin, it expands the active enzyme region that breaks down the fibrinogen fibers that aggregate blood cells and generate thrombi. This is called fibrinolysis.

  Immune system proteins such as IgE, IgG, and IgM.

  Transport proteins such as retinol binding protein and α-1 macroglobulin.

  Defensins such as β-defensin 1, anti-neutrophil defensins 1, 2, and 3.

  A protein present in the blood-brain barrier or nerve tissue of melanocortin receptor, myelin, ascorbate transporter

  Transferrin receptor specific ligand-neurotherapeutic fusion protein (see US Pat. No. 5,977,307);

Brain capillary epithelial cell receptor, transferrin, transferrin receptor, insulin, insulin-like growth factor 1 (IGF1 receptor), insulin-like growth factor 2 (IGF2 receptor), insulin receptor.

  Proteins localized in the kidney, such as polycystin, type IV collagen, organic anion transporter KI, Heymann antigen.

  Proteins localized in the liver such as alcohol dehydrogenase and G250.

  Blood aggregation element X, α-1 antitrypsin, HNF1α.

  Proteins localized to the lung, such as secretory components (coupled to IgA).

  A protein localized to the heart, such as HSP27. This is associated with dilated cardiomyopathy.

  Proteins localized to the skin, such as keratin.

Bone-specific proteins such as bone morphogenetic protein (BMP), which is a subset of the transforming growth factor β superfamily that exhibits osteogenic activity.
Examples include BMP-2, -4, -5, -6, -7 (also called bone morphogenetic protein (OP-1) and -8 (OP-2)).

  Tumor-specific proteins such as human trophoblast antigens, herceptin receptors, estrogen receptors, cathepsins such as cathepsin B (found in liver and spleen).

  Disease-specific proteins such as antigens expressed only in activated T cells: LAG-3 (lymphocyte activation gene), osteoprotegerin ligand (OPGL), OX40 (expressed on activated T cells, type I human The only costimulatory T cell molecule known to be specifically upregulated in T cell leukemia virus (HTLV-I) producing cells, a component of the TNF receptor family), CG6512 Drosophila, human parapregine, human Metalloproteases (related to arthritis / cancer) such as FtsH, human AFG3L2, mouse ftsH, acidic fibroblast growth factor (FGF-1), alkaline fibroblast growth factor (FGF-2), vascular epidermal growth factor / Vascular permeability factor (VEGF / VPF), transforming growth factor α (TGFα), tumor necrosis factor-α (TNF-α), a Diogenin, interleukin-3 (IL-3), interleukin-8 (IL-8), platelet derived epidermal growth factor (PD-ECGF), placental growth factor (PlGF), midkine platelet derived growth factor-BB (PDGF) ), Angiogenic growth factors such as fractalin.

  Stress protein (heat shock protein): HSP is commonly found in cells. When seen outside the cell, it indicates that the cell is dead and the contents are leaking. This unprogrammed cell death (necrosis) occurs in vivo only as a result of trauma, disease or injury, and extracellular HSPs elicit responses from the immune system that fight infection or disease. Bispecific binding to extracellular HSPs allows localization to disease sites.

  Protein involved in Fc transport: Brambell receptor (also known as FcRB). This Fc receptor has two functions, both of which are very useful for delivery. Function is the extension of IgG half-life in serum by transport of IgG from the mother to the fetus through the placenta and protection from degradation of IgG. This receptor is thought to recover IgG from endosomes.

  Polypeptides according to the present invention need not be increased in vivo half life or can be designed to be specific for the above target without increasing it. For example, a polypeptide according to the present invention may be specific for a target selected from the above targets that are tissue specific, so that, regardless of the increase in half-life, Tissue specific targeting of the polypeptide is possible. Furthermore, if the polypeptide is tropic to the kidney or liver, this allows the polypeptide to be transferred to another elimination pathway in vivo (e.g., the polypeptide from the kidney via the elimination pathway from the liver). Can be changed to the discharge route).

  Another embodiment of the invention is an anti-EGFR or anti-IGF-IR polypeptide as described herein, wherein one or more Nanobodies are humanized. The humanized Nanobody may be an anti-EGFR Nanobody, an anti-IGF-IR Nanobody, an antiserum albumin, another Nanobody useful according to the present invention, or these It may be a combination.

  One embodiment of the invention is an anti-EGFR or anti-IGF-IR polypeptide comprising one or more humanized anti-EGFR or IGF-IR Nanobodies and one or more humanized anti-human serum albumin Nanobodies.

  Humanized means that the subject has undergone a mutation that has little or no potential for immunogenicity when administered to a human patient. Humanizing a polypeptide according to the present invention means that one or more amino acids of a non-human immunoglobulin are converted into their normal form by their human homologues found in human consensus sequences or human germline gene sequences. Substitution may be included so as not to lose the properties, that is, so as not to significantly affect the antigen-binding ability of the resulting polypeptide.

  According to one aspect of the invention, a humanized Nanobody has at least 50% homology (eg, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%) with a human framework region. %, 95%, 98%, 100%) as defined above.

  The inventors have determined the amino acid sequence of Nanobodies that may have been modified to not lose their natural affinity in order to reduce immunogenicity against heterologous species.

  We have humanized Nanobody polypeptides by introducing only a few amino acids or mutagenesis in a single polypeptide chain without dramatically losing binding and / or inhibitory activity. I found that it is not necessary. This involves the humanization of scFv, Fab, (Fab) 2 and IgG, which requires the introduction of amino acid changes into the two molecular chains of the light and heavy chains, and the maintenance of the association of both molecular chains. In contrast.

  The inventors have surprisingly found that our Nanobodies comprising framework regions with high homology with human germline sequences such as DP29, DP47 and DP51 are very effective. . They are found naturally in several species, including species belonging to the camelid family. The Nanobody has an amino acid belonging to the group consisting of glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, methionine, serine, threonine, asparagine, or glutamine at position 45, for example L45. Features. Furthermore, they may have a human germline “J” tryptophan at position 103 according to Kabat numbering. This type of camelid VHH, or mutant Nanobody having one or more of this type of framework sequence is included within the scope of the present invention.

  Nanobodies belonging to the above types or having this type of mutation show high amino acid homology with the human VH framework region, and the polypeptides of the present invention comprising these may cause an undesirable immune response. And can be administered directly to humans without the need for further humanization. The present invention also relates to a nucleic acid capable of encoding the polypeptide.

  The humanization approach involves any Nanobody residue, alone or in combination, corresponding framework 1, 2, 3 (FR1, FR2, FR3) residues (DP47, DP29, And DP51).

  According to one aspect of the present invention, humanization of Nanobodies is carried out by substituting one or more amino acids at the positions described below with corresponding amino acids derived from the germline VH gene framework in said Nanobodies. Numbering is according to Kabat numbering.

  According to one aspect of the invention, the framework region of the unsubstituted Nanobody remains as the framework of the original Nanobody.

  According to one aspect of the invention, one or more residues of FR1, FR2, and FR3 are substituted according to the above scheme.

  According to one aspect of the present invention, at least 1, 2, 3, or all residues of FR1 are substituted according to the above scheme.

  According to one aspect of the present invention, at least 1, 2, 3, or all residues of FR2 are substituted according to the above scheme.

  According to one aspect of the present invention, at least one, two, three, four, five, six, or all residues of FR3 are substituted according to the above scheme.

  According to one aspect of the invention, at least 1, 2, 3, or all residues of FR4 are substituted according to the above scheme.

  In another embodiment of the invention, the humanized Nanobody is obtained by transplanting all or part of the CDR region of the Nanobody into the germline human VH framework region scaffold.

  According to one aspect of the present invention, humanization of Nanobodies is performed by replacing one or more of said Nanobodies CDR1, CDR2, and CDR3 on a germline human VH framework region scaffold. Examples of suitable framework skeletons include those of DP47, DP29, and DP51.

  The Nanobodies of the present invention obtained by the above humanization method are part of the present invention.

  Conventional four chain antibodies directed against EGFR or IGF-IR have camelization that removes the light chain and mutates to replace one or more amino acid residues with Camelidae-specific residues. May be made (see, eg, WO 94/04678, incorporated herein by reference). The position is preferentially found in the VH-VL interface, including positions 37, 44, 45, 47, 103, and 108, and so-called camelid feature residues. The camelized antibody is a Nanobody according to the present invention. A polypeptide in which at least one Nanobody is a VH in which one or more amino acid residues are partially substituted by a specific sequence of the Nanobody or an amino acid residue is a Nanobody according to the invention.

  To form any anti-EGFR or anti-IGF-IR polypeptide comprising two or more Nanobodies disclosed herein, the Nanobodies can be attached using known methods. For example, they can be fused by chemical cross-linking by reacting amino acid residues with organic derivatizing agents such as those reported by Blattler et al. (Biochemistry 24, 1517-1524, EP 294703). it can. Alternatively, the Nanobodies may comprise a complete anti-EGFR or IGF-IR polypeptide at the DNA level, i.e. comprising one or more anti-EGFR or anti-IGF-IR Nanobodies and optionally one or more antiserum protein Nanobodies. By forming the encoding polynucleotide, it can be genetically fused. A method for producing divalent or multivalent nanobodies is disclosed in WO 96/34103, a PCT international application.

  According to another aspect of the invention, the Nanobodies may be bound to each other directly or via a linker sequence. The construct is difficult to produce using conventional antibodies due to bulky subunit steric hindrance, and loses or significantly reduces function. Looking at the Nanobodies of the present invention, their binding greatly increases the function compared to a monovalent anti-EGFR or anti-IGF-IR polypeptide.

  According to one aspect of the invention, the Nanobodies are directly attached to each other without using a linker. In contrast to binding bulky conventional antibodies that require a linker sequence to maintain binding activity in the two subunits, the polypeptides of the present invention can bind directly, thereby The possibility of linker sequence problems such as immunogenicity when administered to a human subject, or instability of the linker sequence that causes subunit dissociation, can be avoided.

  According to another aspect of the invention, the Nanobodies are linked to each other via a peptide linker sequence. The linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. The linker sequence is expected not to be immunogenic in a subject to whom an anti-EGFR or anti-IGF-IR polypeptide is administered. The linker sequence may provide sufficient flexibility to the multivalent anti-EGFR or anti-IGF-IR polypeptide and at the same time be resistant to degradation by proteolytic enzymes. Non-limiting examples of linker sequences are those that can be derived from the hinge region of the Nanobody, as disclosed in WO 96/34103. Another example is the linker sequence 3a (Ala-Ala-Ala).

  Another linker sequence constructed by the present inventors for fusion of bispecific and bivalent anti-EGFR or anti-IGF-IR polypeptides is listed in the pending international application WO 06/040153. Has been. One linker sequence is the llama upper long hinge region. Other linkers are Gly / Ser linkers of various lengths. It will be apparent to those skilled in the art that the sequence linker can be used to join any two of the monovalent sequences of the invention.

According to one aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptide may be a homologous sequence of a full-length anti-EGFR or anti-IGF-IR polypeptide. According to another aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptide may be a functional part of a full-length anti-EGFR or anti-IGF-IR polypeptide. According to another aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptide may be a functional part of a homologous sequence of a full-length anti-EGFR or anti-IGF-IR polypeptide. According to one aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptide may comprise the sequence of an anti-EGFR or anti-IGF-IR polypeptide.

  According to one aspect of the invention, the Nanobody used to form the anti-EGFR or anti-IGF-IR polypeptide may be a full length Nanobody or a homologous sequence thereof. According to another aspect of the invention, the Nanobody used to form an anti-EGFR or anti-IGF-IR polypeptide may be a functional part of a full length Nanobody. According to another aspect of the invention, the Nanobody used to form the anti-EGFR or anti-IGF-IR polypeptide may be a homologous sequence of the full length Nanobody. According to another aspect of the invention, the Nanobody used to form an anti-EGFR or anti-IGF-IR polypeptide may be a functional part of a homologous sequence of a full-length Nanobody.

  As used in the present invention, a homologous sequence of the present invention may include one or more amino acid additions, deletions, or substitutions that do not substantially alter the functional characteristics of the polypeptide of the present invention. The number of amino acid deletions or additions is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 Up to 64, 65, 66, 67, 68, 69 or 70 amino acids.

  Homologous sequences according to the present invention are modified by amino acid additions, deletions or substitutions, said modification being an anti-EGFR or anti-antigen that essentially does not change functional characteristics compared to an unmodified polypeptide. It may be an IGF-IR polypeptide.

  Homologous sequences according to the present invention may be sequences present in other camelid species such as camels, dromedaries, llamas, alpaca, guanacos and the like.

  If a homologous sequence shows sequence identity, it is highly sequence identity (70%, 75%, 80%, 85%, 90%, greater than 95%, or 98% sequence identity with the parental sequence). ), And is preferably characterized by the same properties as the parent sequence, ie, binding to the same target.

  A homologous nucleotide sequence according to the present invention refers to a base sequence under stringent hybridization conditions (such as those described in Sambrook et al., Molecular Cloning, Laboratory Manuel, Cold Spring, Harbor Laboratory press, New York). It can refer to a nucleotide sequence of more than 50, 100, 200, 300, 400, 500, 600, 800 or 1000 nucleotides that can hybridize to a complementary sequence of nucleotide sequences that can be encoded.

  As used herein, a functional moiety refers to an array of Nanobodies of sufficient size such that the interaction of interest is maintained with an affinity of 1 × 10 −6 M or higher.

  Alternatively, the functional part contains a partial deletion of the complete amino acid sequence and still retains the binding site and the region of the protein necessary for binding and interaction with the target.

  Alternatively, the functional part of the Nanobody of the present invention contains a partial deletion of the complete amino acid sequence and still retains the binding site and the region of the protein necessary for binding and interaction with the target.

  Alternatively, the functional portion comprises a partial deletion of the complete amino acid sequence and is a polysite that still retains the binding site as well as the region of the protein necessary to inhibit EGFR binding to other EGFR. It is a peptide.

  Alternatively, the functional moiety is a polypeptide that contains a partial deletion of the complete amino acid sequence and still retains the binding site and region of the protein required for binding and interaction with EGFR.

  Alternatively, the functional moiety contains a partial deletion of the complete amino acid sequence and still retains the binding site and the region of the protein required for binding and interaction with the target antigen It is. It includes but is not limited to nanobodies.

  As used herein, a functional moiety is less than 100% of a complete sequence (eg, 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% 10%, 5%, 1%, etc.) but includes 5 or more amino acids or 15 or more nucleotides.

  The homologous sequence of the invention may comprise a humanized anti-EGFR or anti-IGF-IR polypeptide. Humanization of new types of Nanobody antibodies further reduces the potential for undesirable immune responses in human subjects upon administration.

  Yet another example of a Nanobody includes a “functional fragment” (described in WO 03/035694), meaning a fragment that has the function of binding to an antigen. The fragment contains an active antigen binding region. The fragment may be a functional Nanobody fragment, a molecular fragment that behaves like a functional Nanobody, a functionalized antibody fragment, or one or more amino acid residues as Camelidae-specific residues. It may be a nanobody fragment derived from a conventional four-chain antibody, modified by substitution.

  “Functional” for heavy chain antibodies, Nanobodies, VH domains or fragments thereof retains sufficient binding to the epitope (dissociation constants on the micromolar level or higher) when compared to binding in vivo. Show no aggregation or only a limited range of aggregation (soluble and not aggregated above 1 mg / ml), so that it is possible to use antibodies as conjugates means.

  “Functionalized”, with respect to heavy chain antibodies, Nanobodies, or fragments thereof, means to impart functionality to the heavy chain antibodies, Nanobodies, or fragments thereof. “Fragments thereof”, when used with respect to functional fragments, is greater than 95% of the sequence, greater than 90% of the sequence, greater than 85% of the sequence, greater than 80% of the sequence, greater than 75% of the sequence Means a portion corresponding to more than 70% of the sequence, more than 65% of the sequence, more than 60% of the sequence, more than 55% of the sequence, more than 50% of the sequence.

  According to the present invention, the target is either EGFR, IGF-IR, or serum protein. The target is a mammal and is derived from a species such as a rabbit, goat, mouse, rat, cow, calf, camel, llama, monkey, donkey, guinea pig, chicken, sheep, dog, cat, horse, and preferably human. The

  Targets such as EGFR, IGF-IR and serum proteins (eg, serum albumin, serum immunoglobulin, thyroxine binding protein, transferrin, fibrinogen) described herein may be fragments of the target. Thus, a target is also a fragment of said target that can elicit an immune response. The target is also a fragment of the target that can bind to the Nanobody generated against the full-length target.

  A Nanobody directed to a target preferably means a Nanobody that can bind to the target with an affinity of 1 × 10 −6 M or higher.

  EGFR is to be understood as full length EGFR or any fragment of EGFR. Also, the polypeptides and Nanobodies of the present invention generally bind to EGFR analogs, mutants, mutants, alleles, portions or fragments, all naturally occurring or synthesized, or EGFR At least one of the EGFR comprising more than one antigenic determinant or epitope essentially the same as the antigenic determinant or epitope to which the Nanobodies and polypeptides of the invention bind (eg, in wild-type EGFR). It is also expected to normally bind to their analogs, mutants, mutants, alleles, parts or fragments.

  IGF-IR (also referred to as IGF-1R) should be understood to be full length IGF-IR or any fragment of IGF-IR. In addition, the polypeptides and Nanobodies of the present invention generally generally bind to, or are all naturally occurring or synthesized, IGF-IR analogs, mutants, mutants, alleles, portions or fragments, or In IGF-IR (eg, in wild-type IGF-IR) an IGF comprising one or more antigenic determinants or epitopes that are essentially identical to the antigenic determinant or epitope to which the Nanobodies and polypeptides of the invention bind. It is also normally expected to bind to at least one of their analogs, variants, mutants, alleles, parts or fragments of -IR.

  As used herein, a fragment refers to less than 100% of a sequence (eg, 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, etc.). Pointing to 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25 or more amino acids. The fragment is preferably of sufficient length so that the interaction of interest is retained with an affinity of 1 × 10 −6 M or higher.

  As used herein, a fragment also refers to any insertion, deletion and substitution for one or more amino acids that does not substantially alter the ability of the target to bind to the Nanobody generated against the wild-type target. The number of amino acid insertions, deletions or substitutions is preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 amino acids or less.

One embodiment of the invention relates to a polypeptide comprising at least one Nanobody in which one or more amino acid residues are substituted without substantially altering antigen binding capacity.
Targets described herein such as EGFR, IGF-IR and serum proteins are present in any species including, but not limited to, mouse, human, camel, llama, shark, puffer, goat, rabbit, cow. It may be an array.

  The target may be a homologous sequence of the complete target. The target may be a fragment of the homologous sequence of the complete target.

  One skilled in the art will recognize that the anti-EGFR and anti-IGF-IR polypeptides of the present invention may be modified and that such modifications are encompassed within the scope of the present invention. For example, polypeptides can be used as drug carriers, in which case they may be fused to drugs with therapeutic activity, and their solubility is altered by fusion to ionic / polar groups. They can also be used for imaging by fusing to appropriate imaging markers, and they may contain modified amino acids and the like. They may be prepared as salts. Such modifications that essentially retain binding to EGFR and / or IGF-IR are included within the scope of the present invention.

  According to one aspect of the invention, an anti-EGFR or anti-IGF-IR polypeptide can be used for oral administration. Conventional antibody-based therapeutics are promising as drugs because of their superior specificity for their targets and low intrinsic toxicity, but they have one important drawback: It is relatively unstable and susceptible to degradation by proteolytic enzymes. This means that conventional oral, sublingual, topical, nasal, vaginal, rectal, or inhalation administration of conventional antibody drugs does not have low pH tolerance at these sites, Means that it is impossible due to the action of proteolytic enzymes in these sites or in the blood and / or their large size. In order to overcome some of these problems, they must be administered by injection (intravenous, subcutaneous, etc.). Administration by injection requires expert training to use the hypodermic syringe or needle accurately and safely. It further requires a sterile instrument, a liquid formulation of the therapeutic polypeptide, a sterile stable form of the outer polypeptide vial container, and a suitable subject site to pierce the needle. Moreover, in many cases, subjects typically experience physical and psychological stress before and upon receiving an injection. However, the polypeptides of the invention can be used for administration by injection.

  One aspect of the present invention overcomes these problems of the prior art by providing an anti-EGFR or anti-IGF-IR polypeptide of the present invention. The polypeptide is sufficiently small to be resistant to oral, sublingual, topical, nasal, vaginal, rectal or inhalation with virtually no loss of activity. And stable. In addition, the polypeptides of the present invention can avoid injections and not only save cost / time, but are also easier and more comfortable for the subject.

  One embodiment of the present invention controls and prevents symptom of a disease susceptible to regulation by a substance that controls EGFR or IGF-IR, respectively, and can pass through the gastric environment without being inactivated. And / or an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for use in alleviating.

  As known to those skilled in the art, once the polypeptide is obtained, the formulation technique can be applied to release the maximum amount of polypeptide to the correct location (in the stomach, large intestine, etc.). This delivery method is important for treating, preventing and / or alleviating the symptoms of a disease whose target is the digestive system.

  One aspect of the present invention is to control EGFR or IGF-IR, respectively, by orally administering to a subject an anti-EGFR or anti-IGF-IR polypeptide disclosed herein, and It is a method for treating, preventing and / or alleviating symptoms of diseases susceptible to regulation by substances that can pass through the environment.

  Another embodiment of the present invention treats the symptoms of a disease susceptible to modulation by a substance that controls EGFR or IGF-IR, respectively, and can pass through the gastric environment without being inactivated, Use of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for the preparation of a medicament for prevention and / or alleviation.

  According to one embodiment of the present invention, a substance that controls each of EGFR or IGF-IR is administered to a subject by oral administration of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein to the subject. It is a method for delivering to the digestive system without being converted.

  According to one embodiment of the present invention, a substance that controls each of EGFR or IGF-IR is administered to a subject by oral administration of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein to the subject. A method for delivering to the bloodstream of a subject without undergoing oxidization.

  Another embodiment of the invention is for use in treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by the Nanobodies or polypeptides of the invention delivered to the vagina and / or gastrointestinal tract. Of the Nanobodies or polypeptides disclosed herein.

  Examples of diseases include any that cause inflammation, including but not limited to rheumatoid arthritis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, and multiple sclerosis. In a non-limiting example, a formulation according to the present invention is in the form of a gel, cream, suppository, film, or sponge, or as a nanobody or poly disclosed herein as a vaginal ring that gradually releases the active ingredient over time. Peptides are included (the formulations are described in EP 707473, EP 684814, US Pat. No. 5,629,001).

  One aspect of the present invention is described herein wherein a Nanobody or polypeptide disclosed herein is delivered to the vagina and / or gastrointestinal tract by transvaginal and / or rectal administration to a subject. A method for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by Nanobodies or polypeptides.

  Another embodiment of the present invention is for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by the Nanobodies or polypeptides described herein delivered to the vagina and / or gastrointestinal tract. The use of the Nanobodies or polypeptides disclosed herein for the preparation of a medicament.

  One aspect of the present invention provides a Nanobody as described herein without administration of the Nanobodies or polypeptides disclosed herein into the vagina and / or rectum of a subject without inactivating the substance. Or a method for delivering the polypeptide into the vagina and / or rectum.

  One aspect of the present invention provides a Nanobody as described herein without administration of the Nanobodies or polypeptides disclosed herein into the vagina and / or rectum of a subject without inactivating the substance. Or a method for delivering a polypeptide to the bloodstream of a subject.

  Another embodiment of the present invention treats, prevents and / or alleviates the symptoms of diseases that are susceptible to modulation by substances that control EGFR or IGF-IR, respectively, delivered to the nose, upper respiratory tract and / or lung. An anti-EGFR or anti-IGF-IR polypeptide as disclosed herein for use.

  In a non-limiting example, a formulation according to the invention includes an anti-EGFR or anti-IGF-IR polypeptide disclosed herein in the form of a nasal spray (eg, aerosol) or inhaler. Because the polypeptide has a low molecular weight, it can reach the target much more efficiently than therapeutic IgG molecules.

  One aspect of the present invention relates to an EGFR delivered to the upper respiratory tract and lung, respectively, by administering an anti-EGFR or anti-IGF-IR polypeptide disclosed herein to a subject by inhalation through the mouth or nose, respectively. Alternatively, a method for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by substances that control IGF-IR.

  Another embodiment of the present invention is susceptible to regulation by substances that control EGFR or IGF-IR, respectively, delivered to the nose, upper respiratory tract and / or lung without being inactivated. Use of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for the preparation of a medicament for treating, preventing and / or alleviating the symptoms of a disease.

  One aspect of the present invention is that an anti-EGFR or anti-IGF-IR polypeptide disclosed herein is administered to a subject's nose, upper respiratory tract and / or lung so that the substance is not inactivated. A method for delivering substances that regulate EGFR or IGF-IR, respectively, to the nose, upper respiratory tract and lungs.

  One aspect of the present invention is that an anti-EGFR or anti-IGF-IR polypeptide disclosed herein is administered to a subject's nose, upper respiratory tract and / or lung so that the substance is not inactivated. A method for delivering a substance that regulates EGFR or IGF-IR, respectively, into the bloodstream of a subject.

  One embodiment of the present invention controls, prevents, and / or alleviates symptoms of diseases that are susceptible to modulation by substances that control EGFR or IGF-IR, respectively, and can efficiently penetrate sublingual tissue. An anti-EGFR or anti-IGF-IR polypeptide as disclosed herein for use in. Formulations of the polypeptide disclosed herein, such as tablets, sprays, drops, are administered sublingually and are adsorbed through the mucosa into the capillary network under the tongue.

One aspect of the present invention is to administer EGFR or IGF-IR, respectively, to a sublingual tissue efficiently by sublingual administration of the anti-EGFR or anti-IGF-IR polypeptide disclosed herein to a subject. It is a method for treating, preventing and / or alleviating symptoms of diseases that are susceptible to modulation by substances that can permeate.

  Another embodiment of the invention is for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by substances capable of controlling EGFR or IGF-IR respectively and having sublingual tissue. Use of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for the preparation of a medicament.

  In one embodiment of the present invention, a substance that regulates EGFR or IGF-IR, respectively, is not inactivated by sublingual administration of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein to a subject. For delivery to sublingual tissue.

  In one embodiment of the present invention, an anti-EGFR or anti-IGF-IR polypeptide disclosed in the present specification is orally administered to a subject without causing inactivation of a substance that regulates EGFR or IGF-IR, respectively. A method for delivery to the bloodstream of a subject.

  One embodiment of the present invention is for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by Nanobodies or polypeptides described herein that can efficiently penetrate the skin. Nanobodies or polypeptides disclosed herein for use.

  Examples of diseases include any that cause inflammation, including but not limited to rheumatoid arthritis, psoriasis, Crohn's disease, ulcerative colitis, inflammatory bowel syndrome, and multiple sclerosis. Formulations of the polypeptide construct, such as creams, films, sprays, drops, patches, are administered to the skin and penetrate.

  One aspect of the invention is directed to a Nanobody or polypeptide described herein that can efficiently penetrate the skin by transdermally administering the Nanobody or polypeptide disclosed herein to a subject. A method for treating, preventing and / or alleviating the symptoms of a disease that is susceptible to modulation.

  Another embodiment of the present invention is an agent for treating, preventing and / or alleviating symptoms of diseases susceptible to modulation by Nanobodies or polypeptides described herein, which can efficiently penetrate the skin. Is the use of the Nanobodies or polypeptides disclosed herein.

  One aspect of the present invention is to deliver a Nanobody or polypeptide described herein to the skin without inactivation by topically administering to the subject a Nanobody or polypeptide disclosed herein. It is a method.

  One aspect of the present invention is to locally administer a Nanobody or polypeptide disclosed herein to a subject so that the Nanobody or polypeptide described herein is not inactivated without subjecting the subject to blood flow. It is a method for delivering to.

  One embodiment of the present invention is to treat, prevent and / or treat symptoms of diseases that increase permeability of the intestinal mucosa that are susceptible to modulation by substances that control EGFR or IGF-IR, respectively, delivered to the intestinal mucosa. Or an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for alleviation. Due to its small size, the anti-EGFR or anti-IGF-IR polypeptides disclosed herein can more efficiently penetrate the intestinal mucosa to subjects suffering from diseases that cause increased permeability of the intestinal mucosa. Can reach the bloodstream.

  One aspect of the present invention is directed to a substance that regulates EGFR or IGF-IR, respectively, delivered to the intestinal mucosa by orally administering to a subject an anti-EGFR or anti-IGF-IR polypeptide disclosed herein. A method for treating, preventing and / or alleviating symptoms of diseases that are susceptible to regulation and that increase permeability of the intestinal mucosa.

  This method can be further facilitated by the use of an active transport carrier that is a further aspect of the present invention. In this aspect of the invention, the Nanobody is fused to a carrier that facilitates transport through the intestinal mucosa wall into the bloodstream. In a non-limiting example, the “carrier” is a second Nanobody fused to a therapeutic Nanobody. The fusion polypeptide is produced using methods well known in the art. “Carrier” Nanobodies specifically bind to receptors on the intestinal mucosal wall that induce active transport through the wall.

  Another embodiment of the invention is to treat, prevent, and treat symptoms of diseases that increase permeability of the intestinal mucosa that are susceptible to modulation by substances that control EGFR or IGF-IR, respectively, delivered to the intestinal mucosa. Use of an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for the preparation of an agent to / or alleviate.

  In one embodiment of the present invention, an anti-EGFR or anti-IGF-IR polypeptide of the present specification is orally administered to a subject, whereby a substance that regulates EGFR or IGF-IR is inactivated without inactivation. It is a method for delivering to.

  In one embodiment of the present invention, an anti-EGFR or anti-IGF-IR polypeptide of the present specification is orally administered to a subject, whereby a substance that regulates EGFR or IGF-IR, respectively, is inactivated without being inactivated. A method for delivering to the bloodstream. This method can be further facilitated by the use of an active transport carrier that is a further aspect of the present invention. In this aspect of the invention, the anti-EGFR or anti-IGF-IR polypeptides described herein are fused to a carrier that facilitates transport through the intestinal mucosal wall into the bloodstream. In a non-limiting example, the “carrier” is a Nanobody fused to the polypeptide. The fusion polypeptide can be produced using methods well known in the art. “Carrier” Nanobodies specifically bind to receptors on the intestinal mucosal wall that induce active transport through the wall.

  In another embodiment of the invention, an anti-EGFR or anti-IGF-IR polypeptide disclosed herein is a carrier Nanobody (acting as an active transport carrier for transporting said polypeptide to the blood through the lung lumen) For example, Nanobody) is further included.

The anti-EGFR or anti-IGF-IR polypeptide may further comprise a carrier that specifically binds to a receptor present on the mucosal surface (bronchial epithelial cells) and causes active transport from the lung lumen to the blood. . The carrier Nanobody may be fused to the polypeptide. The fusion polypeptide can be produced using known methods described herein. “Carrier” Nanobodies specifically bind to receptors on the mucosal surface that induce active transport through the surface.
Another aspect of the invention is a method of determining which Nanobodies (eg, Nanobodies) are actively transported into the bloodstream upon nasal administration. Similarly, nasal administration of immature or immune Nanobody phage libraries can be used to isolate blood or organs to recover phage that are actively transported into the bloodstream at multiple time points after administration. it can. A non-limiting example of a receptor for active transport from the lung lumen to the bloodstream is Fc receptor N (FcRn). One aspect of the invention includes Nanobodies identified by this method. The Nanobodies can then be used as carrier Nanobodies for delivering therapeutic Nanobodies from the bloodstream to the corresponding target upon nasal administration.

  The polypeptide of the present invention can be used as the following administration agents: as repeated administration in combination with conventional chemotherapy and radiotherapy; as intraluminal administration by stereotaxic surgery such as treatment of brain tumor; or EGFR As a diagnostic to identify patients with overexpressing tumors.

  One embodiment of the present invention provides an anti-EGFR or anti-IGF-IR polypeptide disclosed herein for use in treating, preventing and / or alleviating a symptom of a disease associated with an inflammatory process, or cancer, or A nucleic acid capable of encoding the polypeptide.

  Another embodiment of the invention encodes an anti-IGF-IR polypeptide disclosed herein or the polypeptide for use in the treatment, prevention and / or alleviation of symptoms of a disease associated with cancer. It is a nucleic acid that can.

  Another embodiment of the present invention encodes an anti-EGFR or anti-IGF-IR polypeptide disclosed herein or the polypeptide for preparing a medicament for treating a disease associated with an inflammatory process, or cancer The use of nucleic acids that can be made.

  EGFR is involved in the inflammatory process, and blocking the action of EGFR can have anti-inflammatory effects and is highly desirable in certain medical conditions such as inflammatory arthritis or psoriasis. Furthermore, EGFR and IGF-IR blockade can inhibit human tumor growth, so that the anti-EGFR or anti-IGF-IR polypeptides of the present invention have cytostatic or cytotoxic effects on tumors. Can have. Our examples demonstrate that VHH according to the present invention that binds to EGFR and blocks ligand binding to EGFR inhibits (hetero) dimerization of the receptor and / or induces apoptosis. ing.

  The polypeptides and methods of the present invention include lung cancer, liver cancer, central nervous system tumor, bone cancer, blood and lymphoid cancer, colon cancer, breast cancer, prostate cancer, rectal cancer, bladder cancer, head and neck cancer, ovarian cancer, It can be applied to the treatment and diagnosis of epithelial cancers such as testicular cancer, pancreatic cancer, testicular cancer, kidney cancer, and squamous cell carcinoma. This list of human tumors is intended to be illustrative rather than exhaustive.

  The anti-EGFR or anti-IGF-IR polypeptides and methods of the present invention can also be applied to other diseases associated with EGFR and / or IGF-IR (overexpression). Examples of such diseases and disorders will be apparent to those skilled in the art.

  The present invention provides an antitumor agent or chemotherapeutic agent alone or in combination with an antitumor agent or chemotherapeutic agent, whereby the VHH binds to human EGFR or IGF-IR of tumor cells, respectively, thereby A therapeutic composition comprising an anti-EGFR or anti-IGF-IR polypeptide that inhibits or kills is provided. Anti-tumor or chemotherapeutic agents such as doxorubicin or cisplatin are known in the art. Therapeutic compositions comprising both anti-EGFR and anti-IGF-IR single domain antibodies are also encompassed within the scope of the present invention.

  The present invention also provides diagnostic methods using the anti-EGFR or anti-IGF-IR polypeptides disclosed herein for detection of corresponding conditions. Diagnostic methods are well known in the art and include screening assays and imaging techniques (discussed in detail below).

Another embodiment of the present invention is a method for diagnosing a disease characterized by abnormal EGFR signaling comprising the following steps:
(A) contacting the sample with the anti-EGFR or anti-IGF-IR polypeptide,
(B) detecting the binding of the polypeptide to the sample, and (c) a standard, wherein the difference in binding compared to the sample is useful for the diagnosis of a disease characterized by abnormal EGFR signaling A step of comparing with the binding detected in (b).

Another embodiment of the present invention is a method for diagnosing a disease characterized by the presence of IGF-IR comprising the following steps:
(A) contacting the sample with the anti-IGF-IR polypeptide,
(B) detecting the binding of the polypeptide to the sample, and (c) a standard wherein the difference in binding compared to the sample is useful in diagnosing a disease characterized by the presence of IGF-IR; comparing the binding detected in b).

  In one aspect of the invention, an anti-EGFR or anti-IGF-IR polypeptide disclosed herein can be used to screen for agents that modulate binding of said polypeptide to EGFR or IGF-IR, respectively. it can. When identified in an assay that measures binding or said polypeptide substitution alone, the agent can be subjected to a functional test to determine whether the agent modulates the action of EGFR or IGF-IR in vivo. .

  In general, “therapeutically effective amount”, “therapeutically effective dose”, and “effective amount” refer to achieving the desired effect (modulation of EGFR or IGF-IR binding, treatment or prevention of cancer or inflammation). Means the amount needed to do. One skilled in the art will recognize that the efficacy, and thus the “effective amount”, can vary for various compounds that modulate the binding of EGFR or IGF-IR used in the present invention. One skilled in the art can readily assess the efficacy of the compound.

  As used herein, the term “compound” refers to an anti-EGFR or anti-IGF-IR Nanobody or polypeptide of the invention, or a nucleic acid that can encode the polypeptide, or a screening described herein. Means an agent identified by the method, or said polypeptide comprising more than one derivatized amino acid.

  “Pharmaceutically acceptable” means a substance that is not biologically or otherwise undesirable, that is, without causing or causing an undesirable biological effect. The substance can be administered to an individual along with other compounds without causing deleterious interactions with any of the other ingredients in the object.

  A VHH polypeptide of a type similar to a human sequence disclosed herein is useful for treating or preventing a physical condition in a subject and includes administering a therapeutically effective amount of a compound or composition. .

  The polypeptides herein are useful in the treatment or prevention of diseases associated with cancer, rheumatoid arthritis and psoriasis in a subject and therapeutically for compounds or compositions that bind to EGFR, or IGF-IR, or both. Administration of an effective amount.

  An anti-EGFR or anti-IGF-IR polypeptide disclosed herein is useful for treating or preventing a disease associated with cancer, rheumatoid arthritis and psoriasis in a subject, and comprising a therapeutically effective amount of a mixed compound, For example, administration with another such as doxorubicin.

  The present invention is not limited to the administration of formulations containing only a single compound of the invention. It is also within the scope of the present invention to provide a combination therapy in which a formulation comprising more than two compounds of the present invention is administered to a patient in need thereof.

  Diseases mediated by EGFR and / or IGF-IR include, but are not limited to, cancer, rheumatoid arthritis and psoriasis.

  The compounds useful in the present invention can be formulated as pharmaceutical compositions and are orally or parenterally, nasally, intravenously, intramuscularly, topically, or subcutaneously to human patients or mammalian hosts such as livestock. It can be administered in a variety of ways to suit the chosen route of administration, such as the route. The pharmaceutical composition of the present invention may contain the compounds listed below and a suitable excipient.

  The compounds of the present invention can also be administered using gene therapy delivery methods. See, for example, US Pat. No. 5,399,346, which is incorporated herein by reference in its entirety. Using gene therapy delivery methods, primary cells transfected with a gene for a compound of the invention can be driven by a tissue-specific promoter to target specific organs, tissues, grafts, tumors or cells. It can be transfected and can also be transfected with signals and stabilizing sequences for expression localized within the cell.

  Thus, the present compounds can be systemically administered, eg, orally, in combination with an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft gelatin capsules, compressed into tablets, or added directly to food in the patient's diet. For oral therapeutic administration, the active compound is combined with one or more excipients, such as ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. Can be used in form. The compositions and preparations should contain at least 0.1% of active compound. Composition and formulation proportions can, of course, vary and are conveniently from about 2 to about 60% of the weight of the given unit dosage form. The amount of active compound in the therapeutically useful composition is such that an effective dosage level will be obtained.

  Tablets, troches, pills, capsules and the like may contain the following components: binders such as gum tragacanth, gum arabic, corn starch or gelatin; disintegrants such as corn starch, potato starch and alginic acid; Lubricants such as magnesium stearate; and sweeteners such as sucrose, fructose, lactose or aspartame, or flavors such as peppermint, winter green oil, cherry flavors may be added. When the unit dosage form is a capsule, it may contain a liquid carrier such as vegetable oil or polyethylene glycol in addition to the above types of substances. Various other materials may be present as coatings or to change the physical form of the solid unit dosage form by other methods. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl or propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, all materials used in preparing unit dosage forms should be pharmaceutically acceptable and almost non-toxic in the amounts employed. In addition, the active compound may be encapsulated in sustained release agents and devices.

  The active compound can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof, and in oils. Under normal conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

  Suitable pharmaceutical dosage forms for injection or infusion are sterile aqueous solutions or dispersions adapted for the ready preparation of sterile injectable or injectable solutions or dispersions, optionally containing the active ingredient encapsulated in liposomes. Liquid or sterilized powder may be mentioned. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. Liquid carriers or solvents are solvents or liquid dispersion media containing, for example, water, ethanol, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glyceryl esters, and suitable mixtures thereof. It may be. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions, or by the use of surfactants. For example, the use of various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal can interfere with the action of microorganisms. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffering agents or sodium chloride. The use of agents that delay absorption, such as aluminum monostearate and gelatin, in the composition can result in prolonged absorption of the injectable composition.

  Sterile injectable solutions are prepared by mixing the desired amount of active compound, if necessary, in a suitable solvent containing the various other ingredients listed above and sterilizing with a filter. In the case of sterile powders for preparing sterile injectable solutions, the preferred method of preparation is the vacuum drying and lyophilization techniques, which are active ingredient powders, and any solution present in a filter sterilized solution. Other desirable ingredients are obtained.

  For topical administration, when the compound is a liquid, it can be applied in pure form. In general, however, they are desirably administered to the skin as a composition or formulation with a dermatologically acceptable carrier that may be solid or liquid.

  Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, hydroxyalkyl or glycols or water-alcohol / glycol mixtures, which can dissolve or disperse at effective concentrations, sometimes by the action of non-toxic surfactants. be able to. Adjuvants such as fragrances and other antimicrobial agents may be added to optimize properties for a given application. The resulting liquid composition can be applied from an absorbent pad for use in drug-impregnated bandages and other bandages, or can be sprayed onto the affected area using a pump-type or aerosol spray.

  Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified cellulose or inorganic substances to form application pastes, gels, ointments, soaps, etc. for direct application to the user's skin It can be used with a liquid carrier.

  Examples of useful dermatological compositions that can be used to deliver compounds to the skin are known, for example, Jacquet et al. (US Pat. No. 4,608,392), Geria (US Pat. 992, 478), Smith et al. (US Pat. No. 4,559,157) and Wortzman (US Pat. No. 4,820,508).

  Useful doses of the compounds can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective doses in mice and other animals to humans are known, see, eg, US Pat. No. 4,938,949.

  In general, the concentration of the compound in a liquid composition such as a lotion is about 0.1 to 25% by weight, preferably about 0.5 to 10% by weight. The concentration in a semi-solid or solid composition such as a gel or a powder is about 0.1-5% by weight, preferably about 0.5-2.5% by weight.

  The amount of compound, or salt or derivative thereof, required for use in therapy will vary not only depending on the particular salt selected, but also on the route of administration, the nature of the physical condition being treated, and the age and condition of the patient, and ultimately Is left to the judgment of the doctor or clinician in charge. The dose of the compound will also vary depending on the target cell, tumor, tissue, graft or organ.

  Desirable doses may be present in a single dose or in divided doses at appropriate intervals, for example, as a partial dose twice, three times, four times or more per day. The partial dose itself may be further divided into a number of separate, roughly spaced doses, such as multiple inhalations from an inhaler or multiple drops of eye drops.

  The dosing regimen may include a long-term daily administration. “Long term” means a period of at least 2 weeks, preferably weeks, months or years. Necessary modifications in this dosage range can be determined by one of ordinary skill in the art using only routine experimentation described herein. See Remington's Pharmaceutical Sciences (Martin, E.W., 4th edition), Mack Publishing Co., Easton, PA. The dose can also be adjusted by the individual physician if any complications occur.

  The pharmaceutical compounds of the present invention can be administered in combination with conventional chemotherapy and radiation therapy.

  The high-throughput screening kits of the invention preferably interact with EGFR / ligand or fragments thereof by interacting with EGFR or IGF-IR, respectively, or fragments thereof, in the presence of a concentration of polypeptide in the range of 1 μM to 1 mM. It includes all the means and media necessary to carry out the detection of agents that modulate the IGF-IR / ligand interaction.

  The kit includes the following: In particular, nucleotides that grow according to the kit and encode EGFR or a fragment thereof on a solid support such as a microtiter plate, more preferably a 96-well microtiter plate, by methods well known to those skilled in the art described in WO 00/02045 A recombinant cell of the invention comprising and expressing a sequence. Alternatively, EGFR or a fragment thereof is supplied in purified form by those skilled in the art, for example, so that it is immobilized on a 96-well microtiter plate. Alternatively, in the kit, for example, EGFR or fragment thereof previously immobilized on a 96 well microtiter plate is supplied. The EGFR may be complete EGFR or a fragment thereof. Similar kits for IGF-IR can also be made.

  The modulator according to the present invention is an anti-EGFR or anti-IGF-IR polypeptide at a concentration in the range of about 1 μM to 1 mM or above, at a suitable concentration, preferably in the range of 1 μM to 1 mM, their In the presence of homologous sequences, their functional parts or functional parts of their homologous sequences are added to a given well. The kit may include one or more anti-EGFR or anti-IGF-IR polypeptides as described herein.

  The inventors have found that the anti-EGFR or anti-IGF-IR polypeptides of the invention provide an excellent marker for in vivo imaging when labeled with a suitable contrast agent. Excellent specificity was obtained without compromising the high anti-IGF-IR affinity of IGF-IR Nanobodies with excellent localization to tumors in vivo. As a labeling agent with a short half-life of 6 hours, the radionuclide 99mTc is used in conjunction with the rapidly excreted Nanobodies of the present invention, resulting in a low background compared to currently available alternatives to conventional antibodies. Radiation and hence excellent imaging results were obtained.

  One aspect of the invention is an anti-EGFR or anti-IGF-IR polypeptide disclosed herein further comprising one or more contrast agents. The contrast agent is suitable for any in vivo use, including but not limited to 99mTc, 111 indium, 123 iodine. Other contrast agents suitable for magnetic resonance imaging include paramagnetic compounds and magnetic resonance paramagnetic chelates. Another contrast agent includes an optical dye.

  Another aspect of the invention is the use of an anti-EGFR or anti-IGF-IR polypeptide further comprising one or more contrast agents for in vivo imaging. The anti-EGFR or anti-IGF-IR polypeptide can be labeled with a contrast agent using known methods.

  It is an embodiment of the present invention that the labeled polypeptide is encapsulated in microparticles, ultrasonic bubbles, microspheres, emulsions or liposomes. The formulation is capable of more efficient delivery.

  The anti-EGFR or anti-IGF-IR polypeptides of the present invention can be used to direct radiation therapy doses directly to the tumor. The polypeptide is labeled with one or more radioisotopes that cause damage or destruction of the tumor. Examples of suitable radioisotopes include, but are not limited to, 188Re, 131I, and 211At. These isotopes can be attached to the polypeptide using conventional techniques or using a tag (eg, His tag) fused to the polypeptide of the invention.

  The polypeptide of the present invention is conjugated to one or more anticancer agents that cause tumor damage or destruction when the anti-EGFR or anti-IGF-IR polypeptide binds to EGFR or IGF-IR, respectively. Also good. Suitable chemotherapeutic agents are known to those skilled in the art and include anthracyclines, methotrexate, vindesine, cisplatin, lysine and calicheamicin.

  Further, the anticancer drug bound to the polypeptide of the present invention may be an enzyme that activates a prodrug. This activates the inactive prodrug into an active cytotoxic form. Further, the anticancer agent bound to the polypeptide of the present invention may be a cytokine such as interleukin-2, interleukin-4, or tumor necrosis factor α.

  It is an aspect of the invention that the polypeptide labeled for treatment is encapsulated in microparticles, ultrasound bubbles, microspheres, emulsions, or liposomes. The formulation allows for more efficient delivery of the labeled polypeptide.

  In a further aspect, the present invention provides one or more nucleic acid molecules encoding Nanobodies as defined herein.

  Multivalent or multispecific Nanobodies may be encoded on a single nucleic acid molecule, or each Nanobody may be encoded on a separate nucleic acid molecule. If a multivalent or multispecific Nanobody is encoded by a single nucleic acid molecule, the Nanobody that forms part of it may be expressed as a fusion polypeptide, such as an scFv molecule, and separately after expression For example, by using a chemical binder. Multivalent or multispecific Nanobodies expressed from separate nucleic acid molecules will be bound by suitable means.

  The nucleic acid may further encode a signal sequence for releasing the polypeptide from the host cell upon expression, and may be fused to the surface component of the filamentous bacteriophage particle (or other component of the selective display system) upon expression. Good.

  In a further aspect, the present invention provides a vector comprising a nucleic acid encoding a polypeptide according to the present invention.

  In yet another aspect, the present invention provides a host cell transfected with a vector encoding a polypeptide according to the present invention.

  Expression from the vector can be set, for example, to produce selected Nanobodies on the surface of bacteriophage particles. This makes it possible to select the presented Nanobodies and thus select polypeptides using the method of the invention.

  The present invention further provides a kit comprising at least a polypeptide according to the present invention.

  Cells useful according to the invention include, for example, any bacterial cell such as E. coli, for example, yeast cells such as S. budding yeast and Pichia pastoris, insect cells, mammalian cells, or those belonging to the genus Aspergillus or Trichoderma. It is mold.

  As defined herein, a useful cell according to the present invention is a nucleic acid encoding a Nanobody or polypeptide according to the present invention, so that the polypeptide is expressed at or above the natural level. It can be any cell into which the sequence can be introduced. Preferably, the polypeptide of the invention expressed in a cell exhibits a natural or near-natural pharmacological action, as defined herein.

  According to a preferred embodiment of the present invention, the cells are a group consisting of COS7 cells, CHO cells, LM (TK-) cells, NIH-3T3 cells, HEK-293 cells, K-562 cells, or 1321N1 astrocytoma. From another transfectable cell line.

  Finally, the use of the Nanobodies of the invention and the polypeptides of the invention (as defined herein) is much preferred, but based on the description herein, those skilled in the art will be able to Other (single) domain antibodies to each of EGFR or IGF-IR, and polypeptides comprising said (single) domain antibodies (the terms “domain antibody” and “single domain antibody” are used in the art. It will be clear that it has the usual meaning and can be designed and / or generated (see, for example, the prior art documents described herein).

  Thus, a further aspect of the invention comprises and / or essentially consists of a domain or single domain antibody against each of EGFR or IGF-IR and at least one (single) domain antibody. ) It relates to a polypeptide comprising a domain antibody.

  Specifically, the (single) domain antibody against each of the EGFR or IGF-IR may comprise three CDRs, such CDRs are as defined above for the Nanobodies of the present invention. . For example, the (single) domain antibody may be a single domain antibody known as “dAb”, eg, as described in Ward et al., Supra, but above for Nanobodies of the present invention. Has a CDR as defined. However, as mentioned above, the use of the “dAb” usually has several drawbacks compared to the corresponding use of the Nanobodies of the present invention. Thus, any (single) domain antibody against each of EGFR or IGF-IR according to this aspect of the present invention preferably has an EGFR or IGF-IR, respectively, whose properties are approximately equivalent to the Nanobodies of the present invention. It has a framework region that provides these (single) domain antibodies against.

  In addition, this aspect of the present invention provides a nucleic acid encoding the (single) domain antibody and / or polypeptide, a composition comprising the (single) domain antibody, polypeptide or nucleic acid, the (single) domain antibody Or a host cell that expresses the polypeptide, and a method for preparing the (single) domain antibody, polypeptide or nucleic acid, which comprises a polypeptide, nucleic acid, composition, as described above for the Nanobodies of the invention, Almost similar to host cells, methods and uses.

  Furthermore, it is possible that one or more of the CDRs described above for the Nanobodies of the present invention can be “transplanted” onto other “skeletons” including, but not limited to, human scaffolds or non-immunoglobulin scaffolds. It will be obvious to those skilled in the art. Suitable scaffolds and procedures for implantation of the CDRs will be apparent and well known to those skilled in the art, for example, US Pat. No. 6,180,370, WO 01/27160, European Patent 0 605 522. EP 0 460 167, US Pat. No. 6,054,297, Nicaise et al., Protein Science (2004), 13: 1882-1891, Ewert et al., Methods, October 2004, 34 (2): 184. -199, Kettleborough et al., Protein Eng. October 1991, 4 (7): 773-783, O'Brien and Jones, Methods Mol. Biol. 2003: 207: 81-100, and Skerra, J. Mol. Recognit. 2000: 13: 167-187, and Saerens et al., J. Mol. Biol. September 2005, 23; 352 (3): 597-607, and other references cited therein. For example, methods known per se for transplanting mouse or rat CDRs onto human frameworks and scaffolds include one or more Nanobody CDRs of the present invention and one or more human framework regions or sequences. Can be used as well to provide chimeric proteins comprising

  Accordingly, in another embodiment, the invention provides a chimeric polypeptide comprising at least one CDR sequence selected from the group consisting of the CDR1 sequence, CDR2 sequence, and CDR3 sequence described herein for the Nanobody of the invention. Contains. Preferably, the chimeric polypeptide comprises at least one CDR sequence selected from the group consisting of the CDR3 sequences described herein for the Nanobodies of the present invention, and also described herein for the Nanobodies of the present invention. Optionally, at least one CDR sequence selected from the group consisting of CDR1 and CDR2 sequences is included. For example, the chimeric polypeptide comprises one CDR sequence selected from the group consisting of the CDR3 sequences described herein for the Nanobodies of the invention and the group consisting of the CDR1 sequences described herein for the Nanobodies of the invention. And a CDR sequence selected from the group consisting of the CDR2 sequences described herein for the Nanobodies of the invention. The CDR combinations described herein as preferred for the Nanobodies of the present invention are usually also preferred for these chimeric polypeptides.

  In the chimeric polypeptide, the CDRs may be linked to sequences of other amino acid sequences and / or may be linked to each other via amino acid sequences, the amino acid sequences are preferably framework sequences or frames It is an amino acid sequence that acts as a work sequence or together forms a backbone for presenting CDRs. Again, please refer to the prior art documents listed in the above paragraph. According to a preferred embodiment of the invention, the amino acid sequence is a human framework sequence, eg a VH3 framework sequence. However, non-human synthetic, semi-synthetic, or non-immunoglobulin framework sequences can also be used. Preferably, the framework sequence is (1) at least 1%, preferably at least 5%, more preferably at least 10% of the corresponding Nanobody of the invention for each of the EGFR or IGF-IR chimeric polypeptides. For example, can bind with an affinity of at least 25% and not more than 50%, or greater than 90%, (2) the chimeric polypeptide is suitable for pharmaceutical use, and (3) the chimeric polypeptide is preferably Mostly under conditions (indications, dosage forms, administration and treatment regimens) intended for their pharmaceutical use (which may be almost similar to the conditions described herein for the use of Nanobodies of the invention) It is like having no immunogenicity.

  According to one non-limiting embodiment, the chimeric polypeptide comprises at least two CDR sequences (described above) linked to at least one framework sequence, preferably at least one of the two CDR sequences. One is a CDR3 sequence and the other CDR sequence is a CDR1 or CDR2 sequence. According to a preferred but non-limiting embodiment, the chimeric polypeptide comprises at least three CDR sequences (described above) linked to at least two framework sequences, preferably of the three CDR sequences At least one is a CDR3 sequence and the other two CDR sequences are CDR1 or CDR2 sequences, preferably one CDR1 and one CDR2 sequence. According to one particularly preferred but non-limiting embodiment, the chimeric polypeptide has the structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4 ′, wherein CDR1, CDR2, and CDR3 are The Nanobody CDRs of the invention are as defined herein, where FR1 ′, FR2 ′, FR3 ′ and FR4 ′ are framework sequences. Specifically, FR1 ′, FR2 ′, FR3 ′ and FR4 ′ are respectively framework region 1, framework region 2, framework region 3, framework region 4 and / or human antibody (VH3 sequence and the like). Alternatively, it may be a part or a fragment of the framework sequence. A portion or fragment of a chimeric polypeptide having the structure FR1′-CDR1-FR2′-CDR2-FR3′-CDR3-FR4 ′ can also be used. Preferably, the portion or fragment is such that it meets the criteria as set forth in the above paragraph.

  The present invention also includes proteins and polypeptides comprising and / or consisting essentially of the chimeric polypeptides, nucleic acids encoding the proteins or polypeptides, methods for preparing the proteins and polypeptides, the proteins or A host cell that expresses or is capable of expressing a polypeptide, the protein or polypeptide, a nucleic acid or a composition comprising the host cell, in particular a pharmaceutical composition, and the protein or polypeptide, the nucleic acid, the host cell, and It also relates to the use of the composition for prophylactic, therapeutic or diagnostic purposes, in particular for the prophylactic, therapeutic or diagnostic purposes described herein. For example, the protein, polypeptide, nucleic acid, method, host cell, composition, and use are the same as the protein, polypeptide, nucleic acid, method, host cell, composition, and use described herein for the Nanobody of the invention. It may be the same.

  Where the Nanobodies of the present invention comprise one or more other CDR sequences that differ from the preferred CDR sequences described above, these CDR sequences can be, for example, (preferably) Nanobodies, conventional antibodies (specifically Is a human antibody) derived VH domain, heavy chain antibody, conventional 4-chain antibody (specifically human 4-chain antibody), or another immunoglobulin sequence directed to EGFR or IGF-IR, respectively. Note that it can be obtained by any method known per se. The immunoglobulin sequences directed against each of EGFR or IGF-IR can be determined by any method known per se, immunization by EGFR or IGF-IR, EGFR or IGF-IR, respectively, which will be apparent to those skilled in the art. Can be used to screen a suitable library of immunoglobulin sequences, or any suitable combination of these. In some cases, techniques such as random or position-specific mutagenesis and / or other techniques for affinity maturation known per se may then be performed. Suitable techniques for generating the immunoglobulin sequences will be apparent to those skilled in the art and include, for example, the screening techniques described in Nature Biotechnology, 23, 9, 1105-1116 (2005), a review by Hoogenboom. Other techniques for generating immunoglobulins for specific targets include, for example, the nanoclone approach (eg, described in the unpublished international application PCT / EP2005 / 011819), the so-called SLAM approach (eg, European patents) (See Application 0 542 810), the use of transgenic mice expressing human immunoglobulins, or well-known hybridoma techniques (see, eg, Larrick et al., Biotechnology, Vol. 7, 1989, p. 934). )). All of these approaches can be used to generate immunoglobulins for each of EGFR or IGF-IR, and the CDRs of the immunoglobulins can be used in the Nanobodies of the invention outlined above. For example, the CDRs can all be sequenced, synthesized and / or isolated using techniques known per se, such as those described herein (eg, to replace the corresponding native CDRs). A) a Nanobody of the invention (or a nucleic acid encoding it) that can be inserted into the sequence of a Nanobody of the invention, or that comprises the CDR, is also de novo using the techniques described herein It can also be synthesized.

Example 1: Induction of αEGFR heavy chain-dependent immune response in llamas Intact human squamous cell carcinoma cells (A431; ATCC CRL 1555, Giard DJ, Aaronson SA, Todaro GJ, Arnstein P, Kersey JH, Dosik H, Parks WP 1973. In vitro cultivation of human tumors: establishment of cell lines derived from a series of solid tumors. J. Natl. Cancer Inst. 51: 1417-1423), membrane vesicles derived from A431 (Cohen S, Ushiro H , Stoscheck C, Chinkers M, 1982. A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J. Biol. Chem. 257: 1523-31) or purified EGFR (Sigma) injection The heavy chain-dependent immune response in the four llamas is described in WO 05/044858. EGFR (Sigma) is prepared from EGFR overexpressing tumor cells (A431) using an immobilized antibody affinity resin by Panayotou et al. (Receptor Purification 1990; 1: 289).

Example 2: Expression and Purification of EGFR-Fcγ1 A genetic fusion sequence (EGFR-Fcγ1) of the N-terminal 618 amino acid residue of the extracellular region of EGFR and a sequence encoding human IgG1 hinge-CH2-CH3 was used. . The plasmid expresses a 140 kDa EGFR-Fc fusion protein and transports it to the culture supernatant. Chinese hamster ovary cells (CHO) were transiently transfected with the EGFR-Fcγ1 construct, and the culture supernatant was collected for further processing. The EGFR-Fcγ1 fusion was purified from the supernatant by protein G affinity chromatography. In the ELISA, the function of EGFR-Fcγ1 was evaluated. EGFR-specific mAb528 and biotinylated EGF bind well to the EGFR-Fcγ1 fusion captured by rabbit anti-human IgG in an ELISA, indicating that the EGFR ligand and mAb528 binding site on the extracellular region of EGFR The conformation shows that it is not dramatically affected.

Example 3 Generation of Monovalent Fab Fragment of Erbitux Erbitux is a chimeric antibody for anti-EGFR therapy derived from mouse monoclonal mAb 225 (Sato JD, Kawamoto T, Le AD, Mendelsohn J, Polikoff J, Polikoff J, Sato GH 1983. Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Mol. Biol. Med. 1: 511-529). The Erbitux-derived Fab fragment was prepared by the method of Papain digestion by Fan et al. (Fan Z, Mendelsohn J, Masui H, Kumar R, 1993. Regulation of Epidermal Growth Factor Receptor in NIH3T3 / HER14 Cells by Antireceptor Monoclonal Antibodies. J. Biol. Chem 268: 21073-21079). Papain digested Fab fragments were purified from undigested IgG and Fc fragments by affinity chromatography on a protein A column followed by size exclusion chromatography.

Example 4: Biotinylation of EGFR extracellular region and receptor binding protein Recombinant encoding amino acid residues 1-614 of EGFR produced in baculovirus and purified by immobilized metal affinity chromatography (IMAC) Human EGFR extracellular domain (EGFR-ECD) was kindly provided by Dr. KM Ferguson (Ferguson KM, Darling PJ, Mohan MJ, Macatee TL, Lemmon MA, 2000. Extracellular domains drive homo-but not hetero-dimerization of erbB receptors. EMBO J. 19: 4632-4643). Fab Erbitux and EGFR-ECD are biotinamidohexanoic acid 3-sulfo-N-hydroxysuccinimide ester (sodium salt; Sigma), an active biotin derivative, according to manufacturer's recommended conditions, 5 or Biotinylation was performed by applying a 3-fold molar excess of biotin reagent. Unreacted biotin was removed by dialysis. Both the bivalent antibody erbitux and its monovalent derivative Fab erbitux can bind to biotinylated EGFR-ECD entrapped in neutravidin in an ELISA, indicating that by biotin incorporation, It shows that the site of interaction with ERBITUX or Fab ERBITUX on EGFR-ECD is not dramatically affected. Biotinylated EGF and TGFα were obtained from Peprotech or Invitrogen.

Example 5 Nanobodies Can Inhibit Ligand Binding to EGFR Cloning of the heavy chain antibody fragment (VHH) repertoire and subsequent isolation of EGFR-specific Nanobodies is almost as described in WO 05/044858. It was performed as follows. Individual Nanobody periplasmic extracts were screened for EGFR specificity by ELISA on solid phase coated EGFR (Sigma). As a result of the sequence analysis, a panel of 14 EGFR Nanobodies (SEQ ID NO: 80-93: Table 1) belonging to a family of 13 distinct Nanobodies (Table 2) is identified. A Nanobody family is defined as a panel of Nanobodies that have the same CDR3 or have only a limited number of mutations in the CDR3.

Several assays have been performed to identify Nanobodies that can antagonize proteins that interact with EGFR (ligand EGF, TGFα or anti-EGFR neutralizing antibodies). For the first assay based on ELISA, A431-derived vesicles were prepared as described in Example 1 and total protein content was spectroscopically analyzed using the BCA protein assay kit from Pierce according to the manufacturer's protocol. Decided. A431 vesicles were immobilized overnight in Maxisorp microtiter plates (Nunc) at 4 ° C. in PBS at a concentration of 5 μg / ml. The plate was washed with 0.05% PBS-Tween (registered trademark) and then blocked for 2 hours at room temperature using 1% PBS-casein. After washing, the same volume of 1.6 nM biotinylated EGF (EGFbiot) was added in 1% PBS-casein in the presence of various dilutions of soluble Nanobodies (final concentration 160-0.009 nM) purified by IMAC. Simultaneously added and incubated for 1 hour at room temperature (rt). EGFbiot bound to the receptor was detected by color reaction using extraavidin-alkaline phosphatase complex (Sigma) followed by p-nitrophenyl phosphate (Sigma). As a result, eight families of Nanobodies were identified that antagonize EGF binding to EGFR-expressing A431 endoplasmic reticulum (Families I-VI and XI-XII summarized in Table 2). As an example, the EGF inhibitory ability of Nanobodies PMP7A5, PMP7C12, PMP7D12 and 27-10-E8 is shown in FIG. However, the two types of Nanobodies (PMP9C1 and PMP8C7 belonging to Nanobody families X and XIII, respectively) were unable to antagonize the EGF epitope on EGFR in this assay (FIG. 1).
To evaluate EGF inhibition ability, another antagonistic ELISA was performed. Polyclonal anti-human IgG (Dako, catalog number A0424) was diluted 1: 1000 in PBS and coated overnight at 4 ° C. The next day, the plate was washed 3 times with 200 μl PBS and blocked with 200 μl 2% BSA in PBS. The EGFR-Fcγ1 fusion (Example 2) was captured in 3 μg / ml 1% BSA in PBS for 1 hour at room temperature. After washing the plate again, serially diluted various Nanobodies were made in individual plates in 50 μl of 1% BSA in PBS. 50 μl of 20 ng / ml EGFbiot was then added to each well (final concentration 10 ng / ml) and the Nanobody and EGFbiot mixture was added to the immobilized EGFR-Fcγ1. After incubation for 1.5 hours at room temperature, the plates were washed 4 times and the bound EGF was used with streptavidin conjugated with peroxidase (Jackson Immuno-research Laboratories, diluted 1: 5000 in 1% BSA in PBS). And staining with o-phenylenediamine (OPD) / H 2 O 2 was performed. Absorbance was measured at 490 nm. This screening method and subsequent sequence analysis identified Nanobody family IX (typical example 27-1-H7) as having EGFR inhibitory capacity (Table 2).

  To screen for Nanobodies that can inhibit the interaction between TGFα and EGFR, a similar antagonistic ELISA was performed. In this competitive ELISA, the binding of 4 nM biotinylated TGFα to A431 endoplasmic reticulum was evaluated in the presence of various dilutions of Nanobodies (periplasmic extracts or purified by IMAC). Surprisingly, Nanobody families X and XIII, which have already been identified and failed to inhibit EGF binding to the receptor, inhibit TGFα binding to EGFR (Table 2, FIG. 2).

  Yet another assay for screening for inhibition of EGFR, called the so-called alpha screen, was performed. The Amplified Luminescent Proximity Homogeneous Assay screen is a proximity bead-based homogeneous assay that allows quantitative detection of two biologically interacting molecules. Envision (Perkin Elmer at 520-620 nm, due to fluorescence chromophores, due to the migration of singlet oxygen molecules generated by excitation at 680 nm as these beads approach due to interaction partners bound to the acceptor and donor beads, respectively. ) Causes the acceptor beads to be detected to emit light. This automated high throughput screening method allows screening for inhibition of interaction pairs in a 384 well format. Donor beads that bind to streptavidin and capture EGFbiot, or biotinylated Fab erbitux in another screening method, with acceptor beads conjugated to EGFR-Fcγ1 prepared according to the manufacturer's instructions (Perkin Elmer) Were used in two alpha screen assays to identify inhibition of EGF or erbitux, respectively. EGF or Fab erbitux was added at 1 and 0.4 nM, respectively. In the first screen, the inhibition of EGF-EGFR-Fcγ1 interaction by periplasmic nanobodies was measured for a total of 3000 Nanobodies. About 10 percent of the tested Nanobodies showed inhibition of EGF binding to the EGFR-Fcγ1 fusion. This screening method and subsequent sequence analysis identified two additional EGF antagonist Nanobody families (VII and VIII) (Table 2). As an example of the obtained inhibition curve, the concentration-dependent ability of EGF inhibition (to Erbitux and Fab Erbitux) of Nanobody PMP9G8 is shown in FIG. In a subsequent alpha screen, all EGF antagonist Nanobodies were screened for their ability to inhibit the interaction of Fab erbitux with EGFR-Fc, resulting in inhibition of interaction with EGFR for both EGF and Fab erbitux Only one Nanobody family (family II, typical PMP7D12) was identified (Figure 4).

  In summary, as a result of detailed screening and sequence analysis, compared to the EGFR Nanobody published in WO 05/044858, it was possible to inhibit the binding of a ligand to EGFR, which was previously unidentified 13 An EGFR-specific Nanobody family (Family I-XIII) has been identified. Of these 13 Nanobody families, 11 can inhibit the binding of EGF to EGFR. Surprisingly, the remaining two Nanobody families (X and XIII) inhibit only TGFα but not the interaction of EGF with EGFR. An overview of all 13 αEGFR Nanobody families is shown in Table 2.

The novel anti-EGFR Nanobodies are functionally classified into three types of ligand antagonistic Nanobodies. Non-limiting examples of different classes of Nanobodies are as follows:
Type A Nanobodies (binding to the extracellular region of EGFR and antagonizing EGF and TGFα binding sites on EGFR but not erbitux binding sites. Examples of Nanobodies are 27-10-E8 (Family I) And PMP7A5 (family III).
Type B Nanobodies (binding to the extracellular region of EGFR and antagonizing EGF, erbitux and TGFα binding sites on EGFR. Examples of Nanobodies are PMP7D12 and PMP7C12 (family II).
Type C Nanobodies (binding to the extracellular region of EGFR and antagonizing the TGFα binding site on EGFR, but not EGFR downregulation with EGF or erbitux binding sites. Examples of Nanobodies are PMP8C7 (family XIII).

Example 6: Screening of dissociation rate constant by surface plasmon resonance (BIAcore) A surface plasmon resonance sensor chip (CM5) was coated with purified EGFR (Sigma) at a density of 5500 RU or less according to the manufacturer's instructions. The chip surface was regenerated by flowing 50 mM NaOH for 3 seconds. First, periplasmic nanobody extracts were screened to determine the dissociation rate. The best nanobody families (I-III and VI-IX) that inhibit EGF binding and show the highest IC50 in the antagonistic endoplasmic reticulum ELISA are screened for representatives of multiple Nanobodies belonging to the same family, the lowest Nanobodies showing dissociation rates are displayed as typical examples of the nanobody family. The dissociation rates of typical examples of related families were confirmed by Nanobodies purified by IMAC and are shown in Table 3.

  Of the seven Nanobody families tested, 27-10-E8, PMP7A5, PMP7E12, PMP9G8 and PMP3867 (Table 3), which are typical examples of the five Nanobody families, were Fab Erbitux (kd = 1.7 × 10 × 10). -3s-1) showed an equal or lower dissociation rate.

Example 7: Recognition of EGFR epitopes expressed on the cell surface of cells of different origin by Nanobodies Flow cytometric analysis allows detection of EGFR expressed on the cell surface. The EGFR cell line used to evaluate Nanobody binding is the mouse fibroblast line HER14 transfected with EGFR (estimated receptor density of about 1 × 10 5), the untransfected maternal mouse fibroblast The cell line NIH3T3 and the human squamous cell carcinoma cell line A431 tumor cells (estimated receptor density approximately 1.2 × 10 6). 1 × 10 5 cells (100 μl) were mixed with 1 μg Nanobody and detection was performed using polyclonal rabbit anti-nanobody phycoerythrin (PE) labeled antiserum (R23-PE). All experiments were performed on a FACSarray (BD Biosciences). Type A, B and C Nanobodies (described in Example 5), known to inhibit binding of ligand to EGFR and tested in flow cytometry, are 27-10-E8 (type A), PMP7D12 (type B), PMP7A5 (type A) and PMP9C1 (type C) (represented as Nanobody families I, II, III, and X, respectively). Without exception, all antagonistic Nanobodies tested showed a shift for A431 and HER14 compared to background fluorescence (detected using the R23-PE complex), as shown in FIG. There was no shift for NIH3T3 that did not express, indicating that these Nanobodies specifically recognize EGFR expressed on the surface.

Example 8 Nanobodies Inhibit EGF-Mediated Receptor Signaling HER14 cells were placed in DMEM (Gibco Life Technologies) with 10% (v / v) serum in 12-well tissue culture plates. 100,000 cells were seeded per well and cultured at 37 ° C. in 5% CO 2. After 8 hours, the cells were washed once with DMEM containing 0.5% (v / v) FCS and subjected to serum starvation culture overnight in the same medium. On the day of the assay, the medium was replaced with medium supplemented with 2% skim milk. Ligand (8 nM) or excess Nanobody (1 μM) was added at 37 ° C. After 15 minutes, the cells were rapidly cooled with ice and washed twice with iced PBS. Whole cell lysates were prepared by scraping cells from plates in 50 μl of Laemmli protein sample buffer. Proteins were separated by size on a 6% (w / v) polyacrylamide gel (packed with 20 μl lysate for each gel on two parallel gels) and then blotted onto a PVDF membrane (Roche) did. For total amount of EGFR, use rabbit polyclonal antiserum to the receptor (Santa Cruz Biotechnology), for phosphorylated receptor use mouse monoclonal anti-phosphotyrosine antibody TyrlO68 (Cell signaling) and then bind to peroxidase. Blots were stained with secondary antibodies (donkey anti-rabbit and donkey anti-mouse antibodies) against each of the above. As a loading control, parallel blots were stained with a monoclonal anti-actin antibody (ICN Biomedicals Inc) for the presence of actin. Bound antibody was visualized by amplified chemiluminescence using Western Lightning ™ substrate (Perkin Elmer Life Sciences). The presence of exogenous EGF ligand activates unambiguous EGFR tyrosine phosphorylation and thereby the receptor (FIG. 6, lane 1), but the residual level of receptor phosphorylation depends on the exogenous ligand It was detected in the absence (FIG. 6, lane 2). Nanobodies PMP7C12 or PMP7D12 (Family II) do not activate EGFR expressed on HER14 in the absence of EGF, as indicated by the absence of tyrosine phosphorylation of the receptor (FIG. 6, lane 3 and 4). However, compared to the level of background receptor phosphorylation, Nanobody PMP9C1 (family X) (FIG. 6, lane 5) showed an increase in the phosphorylation state of EGFR under the applied conditions.

  Receptor-mediated activation downstream of EGF-induced receptor phosphorylation occurs. One of the downstream effector proteins involved in signal transduction mediated by the EGF receptor is mitogen-activated protein kinase (MAPK). After phosphorylation, MAPK triggers an event in which quiescent cells undergo cell division (Well A, 1999. Molecules in focus: EGF receptor. Int. J. Biochem. Cell Biol. 31: 637-643). Phosphorylation by downstream MAPK was assessed by staining the blot with a mouse monoclonal anti-phosphate MAPK antibody (Cell Signaling). Induction with exogenous EGF (FIG. 6, lane 1), to a lesser extent, with PMP9C1 (FIG. 6, lane 5), a clear induction of MAPK phosphorylation in HER14 is detected. It was not detected in the presence or in the presence of PMP7C12, PMP7D12 (FIG. 6, lanes 2, 3, and 4 respectively).

  Next, the ability of Nanobodies to inhibit receptor activation in the presence of exogenous ligands was evaluated under experimental conditions similar to those described above. In this experiment, diluted Nanobodies (1, 10, 100, or 1000 nM) were added to HER14 cells in the presence of 8 nM EGF ligand. Receptor phosphorylation was assessed by Western blot using a mouse monoclonal antibody (Cell Signaling) that binds to phosphorylated EGFR Tyr1068. When exogenous EGF ligand was added, only clear phosphorylation of Tyr1068 occurred (FIG. 7A, lane EGF), but residual levels of receptor phosphorylation were observed even when no exogenous ligand was added. (FIG. 7A, lane nc). In this cellular EGF antagonism assay, Nanobody families I (27-10-E8), II (PMP7D12), III (PMP7A5), VII (PMP9G8), VIII (PMP38G7) and X (PMP9C1) were tested. Concentration-dependent inhibition of EGF-mediated receptor phosphorylation is induced by Nanobody Family I, II, III, VII, VIII but not by Nanobody Family X (PMP9C1, not shown) This demonstrates the EGF inhibitory ability of Nanobody Family I, II, III, VII, VIII described in Example 5 (Table 2). FIG. 7 shows the concentration-dependent inhibition of EGF-mediated receptor phosphorylation by Nanobodies PMP9G8, 27-10-E8 (Panel B) and PMP7D12 (Panel A).

Example 9 Nanobodies Inhibit Cell Growth of EGFR-expressing Tumor Cells In Vitro 1000 μl in carbonate buffered DMEM (Gibco) + 10% FCS with 100 μl penicillin and streptomycin in culture treated 96 well plates. A431 cells / well were seeded and incubated for 48 hours at 37 ° C., 5% CO 2. To determine growth that is uninhibited (100%) at t = 0 after incubation, one complete 96-well A431 seeded plate was obtained from Sken et al. (Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd MR, 1990. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl Cancer Inst 82: 1107-1112) Sulforhodamine B staining (SRB) ) And kept as a control plate. A series of diluted Nanobodies or growth regulatory control components (0-1000 nM) were then added to the medium containing 1% FCS without adding exogenous ligand. The plates were then incubated at 37 ° C., 5% CO2. After 72 hours of incubation, cell number was assessed by a colorimetric assay. Cell proteins were fixed with trichloroacetic acid and then stained with SRB. Unbound dye was removed by washing and the dye bound to the protein was extracted with 10 mM unbuffered Tris base solution. The optical density was measured at 564 nm. The relative activity of the tested Nanobodies was verified by comparison with dose-response curves for various Nanobody erbitaxes (complete or Fab fragments).

  Family I, II, III, VII, VIII and XIII Nanobodies were evaluated for their ability to inhibit (compared to monovalent or divalent erbitux) and the results are illustrated in FIG. There is a strong positive correlation between Nanobodies (see Examples 5 and 8) showing significant inhibition of ligand binding to the receptor and the activity of A431 to inhibit cell growth and proliferation in vitro. To do.

Example 10: Nanobodies induce receptor internalization from the cell surface To detect internalization (down-regulation) of EGFR from the cell surface, Hela cells were treated with 10% (v / v) 10,000 cells / well were seeded in DMEM supplemented with FCS and 2 mM L-glutamine. After 24 hours, the cells were incubated at 37 ° C. in the presence of either recombinant human EGF (8 nM) or excess antibody or Nanobody (both 1 μM for both) for various times at intervals of 120 minutes or less. After a predetermined time interval, the cells were rapidly cooled with ice, and a whole cell lysate was prepared as in Example 8, separated by size and blotted. Subsequently, to measure the total amount of EGFR, staining was performed with a polyclonal rabbit EGFR antiserum (Santa Cruz Biotechnologies). As a loading control, the same blot was stained for actin measurement. Visualization of bound antibody was performed by amplified chemiluminescence (Example 8).

  As expected, stimulation of Hela cells with 8 nM exogenous EGF resulted in nearly complete EGFR degradation after 2 hours (FIG. 9, panel A), but erbitux and tested Nanobody families I, II, For III and III (labeled 27-10-E8, PMP7D12, and PMP7A5, respectively), no receptor degradation was detected in Hela cells. For Nanobody family XIII (PMP8C7) only, slight receptor internalization was detected after 120 min in Hela cells (FIG. 9). Previously, Fan et al. (Fan Z, Mendelsohn J, Masui H, Kumar R 1993. Regulation of epidermal growth factor receptor in NIH3T3 / HER14 cells by antireceptor monoclonal antibodies. J. Biol. Chem. 268: 21073-21079, Fan Z, Lu Y, Wu X, Mendelsohn J 1994. Antibody-induced epidermal growth factor receptor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J. Biol. Chem. 269: 27595-27602), slightly under their experimental conditions Could demonstrate mAb225-dependent internalization of EGFR. Our experiments were performed repeatedly, but with a 2 hour incubation with EGF inhibitory mouse mAb225 (data not shown), or its chimeric derivative erbitux (FIG. 9), the detectable EGF receptor Control effects did not occur under our experimental conditions.

Example 11: Optimization of nanobody morphology to increase serum half-life and inhibitory activity The constructed multivalent / multispecific Nanobodies are shown in Table 4 (SEQ ID NOs: 110-133 and 141-143). To test whether selected Nanobodies have potential as anti-cancer agents in animal models, the serum half-life of monovalent or divalent Nanobodies (approximately 15 or 30 kDa, respectively) Strategies for increasing serum half-life are preferred (eg as described in WO 04/041865) because they are not optimal for application. Here, trivalent, bispecific, consisting of two anti-EGFR molecules located at the N-terminus and fused to a third Nanobody with serum albumin specificity, all separated by a 9 (GS) amino acid linker peptide The construction and evaluation of nanobodies will be described. Human serum albumin-specific Nanobody ALB8 (Table A-9: SEQ ID NO: 33) having cross-reactivity with mouse serum albumin was selected. Constructs 7D12-GS-7D12-GS-ALB8 and 7A5-GS-7A5- in which DNA segments encoding Nanobodies PMP7D12 or PMP7A5 and ALB8 are head-to-tail fused and the two αEGFR Nanobodies are separated by a 9 (GS) amino acid linker GS-ALB8 (Table 4; SEQ ID NOs 126 and 124, respectively) was obtained. Two Nanobodies were expressed in E. coli and purified by affinity chromatography (IMAC) on a NiNTA matrix to a purity level of about 95%.

  In order to verify whether the bispecific construct can bind to EGFR and MSA simultaneously, a sandwich ELISA was performed. Immobilization of A431 endoplasmic reticulum (5 μg / ml) and blocking of the plate was performed as described in Example 5. After adding a series of diluted Nanobodies, detect the Nanobody bound to the endoplasmic reticulum by biotinylated MSA followed by extraavidin-alkaline phosphatase complex (Sigma) and subsequent color reaction as described in Example 5. did. As expected, only the bispecific trivalent construct (FIG. 10) bound to A431 endoplasmic reticulum and serum albumin at the same time, while the bivalent construct without albumin specific Nanobody (data not shown) did not bind simultaneously. .

  Subsequently, Nanobodies 7D12-GS-7D12-GS-ALB8 and 7A5-GS-7A5-GS-ALB8 were evaluated for the ability to inhibit the ligand by EGF-A431 endoplasmic reticulum antagonism ELISA (Example 9). As illustrated in FIG. 11, the IC50 values for 7D12-GS-7D12-GS-ALB8 and 7A5-GS-7A5-GS-ALB8 are 1/5 and 1 respectively compared to the respective monovalent anti-EGFR Nanobodies. / 10.

  To clearly show that our Nanobodies can inhibit the growth of tumor cells expressing EGFR in vitro, as described in Example 9, Nanobodies 7D12-GS-7D12-GS-ALB8 and 7A5-GS-7A5 -GS-ALB8 was added to proliferating A431 tumor cells. As illustrated in FIG. 12, the IC50 values of 7D12-GS-7D12-GS-ALB8 and 7A5-GS-7A5-GS-ALB8 are clearly lower than those of monovalent EGFR Nanobodies PMP7A5 and PMP7D12 (FIG. 8) .

  Nanobodies directed to individual paratopes on EGFR (double paratope Nanobodies) can be produced in the absence or presence of compounds to increase serum half-life. By way of example, Nanobodies comprising Nanobody ALB8 (EGFRPMP7D12-GS-EGFRPMP7A5-GS-ALB8; SEQ ID NO: 142 and EGFRPMP7D12-) combining EGFR Nanobodies PMP7D12 and PMP7A5 (belonging to Type B and Type A, respectively) and increasing serum half-life GS-ALB8-GS-EGFRPMP7A5; SEQ ID NO: 143, Table 4), or Nanobodies (EGFRPMP7D12-GS-EGFRPMP7A5; SEQ ID NO: 141, Table 4) that do not contain groups that increase serum half-life are produced. The anti-EGFR dual paratope target Nanobody inhibits ligand binding in an inhibition ELISA (described in Example 5) and inhibits the growth of tumor cells expressing EGFR. The group for increasing half-life may be an albumin specific Nanobody or an Fc receptor specific Nanobody. In the same construct, for example, to supplement immune effector functions (eg, Nanobodies that bind to anti-CD3 Nanobodies or complement proteins), serum half-life functions and binding proteins with other biological functions Can be replaced.

Example 12: Nanobody format is involved in conditional binding to EGFR A single αEGFR Nanobody (PMP7D12 or PMP7A5) and an α-albumin Nanobody (ALB8) are bound, 3 (3A), 5 (GGGGGS), A panel of 12 bispecific Nanobody constructs separated by 9 (GS) or 25 (GS5) amino acid linkers was constructed and shown in Table 4 (SEQ ID NOs: 110-121).

  Three parallel flow cytometry experiments assessing binding to A431 tumor cells were performed on 14 bispecific Nanobodies using separate detection methods, adding equimolar amounts of Nanobodies. In the first experiment, Nanobodies were detected with a polyclonal rabbit α-Nanobody-PE complex (R23-PE). In a second experiment, Nanobodies were incubated with A431 cells and then detected with mouse albumin (mSA-647) conjugated with Alexa-647. By this detection method, it is possible to evaluate whether albumin can also interact with the Nanobody bound to A431. In a third experiment, Nanobodies and mSA-647 were pre-incubated for 30 minutes (to bind to albumin before interaction with EGFR), after which bispecific Nanobody bound to albumin was expressed on the surface To verify whether it can also bind to EGFR, add to A431 cells. The obtained FACS results are shown in FIG.

When ALB8 is located at the C-terminus in a bispecific construct, maximal binding to the surface of A431 cells is detected by R23-PE, regardless of αEGFR Nanobody (mean fluorescence intensity> 5 × 10 4). . When similar detection with R23-PE is performed, ALB8 located at the N-terminus is less pronounced with increasing linker length, but negatively affects binding to A431 (compared to ALB8-EGFR construct). As indicated by a decrease in the average fluorescence intensity). For both Nanobodies tested in the ALB8-EGFR format, clear binding to A431 was detected, but the fusion of ALB8 to the N-terminus appears to have a greater effect on EGFR binding for PMP7D12 than for PMP7A5 It is. This means
The ALB8-PMP7D12 bispecific Nanobody is supported by surface plasmon resonance experiments showing that binding to EGFR is significantly reduced (data not shown) compared to the PMP7D12-ALB8 format. When detection of binding to A431 by mSA-647 was performed without preincubation, the average fluorescence intensity of the bispecific construct was low (regardless of EGFR type and ALB8 position), indicating that A431 This suggests that it has a negative effect on the binding to the. Applying this detection method, for ALB8-PMP7D12, the length of the linker appears to have a positive effect on the binding of the bispecific construct to A431 (FIG. 13).

  Surprisingly, however, preincubation with mSA-647 dramatically increased binding to A431, regardless of the EGFR Nanobody, ALB8 position or linker length tested, indicating that albumin Suggests a conditional interaction with EGFR after binding to. In fact, the interaction with albumin greatly increases the average fluorescence intensity of the bispecific Nanobody construct compared to without preincubation with mSA-647 (FIG. 13).

Example 13 Nanobodies Can Inhibit IGF-I Ligand Binding to IGF-IR A cloned VHH repertoire derived from a llama immunized with A431 cells described in Example 5 -Used for identification of IR-specific Nanobodies. Recombinant human IGF-IR (rhIGFIR) and IGF-I were obtained from Peprotech. Isolation of IGF-IR specific Nanobodies is described generally in WO 05/044858 using IGF-I ligands to select Nanobodies that bind to IGF-IR on at least an epitope that overlaps the ligand binding site. It was executed as follows. Both A431 endoplasmic reticulum encapsulating IGF-IR and rhIGF-IR were used for selection as antigen formats. Following selection, periplasmic extracts of individual Nanobodies were screened by ELISA on solid phase coated receptors for IGF-IR selectivity. Sequence analysis identified a panel of four individual Nanobodies PMP4B11, PMP3G7, PMP2C7, and PMP1C7 (SEQ ID NOs: 106-109, respectively, Table 5) belonging to four different families.

  To screen for Nanobodies capable of inhibiting the interaction of IGF-I with IGF-IR, an antagonistic ELISA similar to that described in Example 5 was performed. As described in Example 4, IGF-I was biotinylated by applying a 5-fold molar excess of biotin reagent. Excess unreacted biotin was removed by dialysis. rhIGFIR was immobilized on a solid phase (1 μg / ml) and the binding of biotinylated IGF-I (50 ng / ml) was detected by extraavidin-alkaline phosphatase complex in the absence or presence of inhibitor. As shown in FIG. 14, only PMP1C7 can significantly inhibit the interaction of IGF-I with rhIGFIR. Subsequent surface plasmon resonance analysis (BIAcore) showed that the dissociation constant of PMP1C7 on immobilized IGF-IR was 1.9 × 10 −3 s −1.

  Bispecific anti-IGF-IR Nanobodies separated by 9 (GS) or 25 (GS5) amino acid linkers were constructed (SEQ ID NOs: 134-135, Table 6) and these Nanobodies were compared to monovalent IGF-IR Nanobodies Thus, the inhibition of IGF-I (inhibition ELISA) and the growth of tumor cells expressing IGF-IR was dramatically increased.

Example 14: Bispecific αEGFR-αIGF-IR Nanobodies Can Inhibit Signaling Cascade by IGF-IR and EGFR EGFR and IGF-IR in the absence or presence of compounds that increase serum half-life Nanobodies can be produced that simultaneously target the above epitopes. As an example, nanobodies (SEQ ID NOs: 136-140, Table 7) are produced that combine EGFR Nanobody PMP7D12 and IGF-IR Nanobody PMP1C7 with or without Nanobody ALB8 to increase serum half-life. Nanobodies directed against EGFR-IGF-IR inhibit the binding of ligands to their respective receptors in an inhibition ELISA (described in Example 5) and inhibit the growth of tumor cells expressing EGFR / IGF-IR To do. The group for increasing half-life may be an albumin specific Nanobody or an Fc receptor specific Nanobody. In the same construct, for example, to supplement immune effector functions (eg, Nanobodies that bind to anti-CD3 Nanobodies or complement proteins), serum half-life functions can be combined with binding proteins with other biological functions. Can be replaced.

Table 1: Nanobodies against preferred EGFR

Table 2: Anti-EGFR Nanobody Family

Table 3: Dissociation rates of EGFR-specific Nanobodies that inhibit EGF binding

Table 4: Arrays of multivalent / multispecific EGFR Nanobodies

Table 5: Nanobodies against preferred IGF-IR

Table 6: Array of multivalent / multispecific IGF-IR Nanobodies

Table 7: Nanobodies against preferred EGFR and IGF-IR

Table 8: SEQ ID NO: 1-41

Table 9: Preferred CDRs of Nanobodies for EGFR

Table 10: Preferred CDRs of Nanobodies against IGF-IR

Inhibition of interaction between EGF and A431 endoplasmic reticulum encapsulating EGFR by Nanobodies of the present invention. Inhibition of interaction between TGFα and A431 endoplasmic reticulum encapsulating EGFR by Nanobodies of the present invention. Inhibition of EGF binding to EGFR-Fcγ1. Inhibition of Fab erbitux binding to EGFR-Fcγ1. Binding of EGFR Nanobodies to A431 tumor cells expressing EGFR. After regulation of the phosphorylation state of EGFR in HER14, incubation with EGF (lane 1), PMP7C12 (lane 3), PMP7D12 (lane 4), and PMP9C1 (lane 5). Background EGFR phosphorylation in the absence of exogenous ligand is shown in lane 2. References relating to the detection of antibodies for Western blot development are described in the text. Inhibition of signal transduction by EGFR-mediated EGFR in HER14 cells. Inhibition of tumor cell growth by Nanobodies. The concentration-dependent inhibition of cell proliferation is expressed as the relative number of cells compared to the number of cells in the absence of Nanobodies or antibodies. Degradation mediated by Nanobodies of EGFR expressed on Hela cells. A sandwich ELISA showing that Nanobodies simultaneously bind to EGFR encapsulated A431 endoplasmic reticulum and serum albumin. Inhibition of EGF binding to EGFR encapsulated A431 endoplasmic reticulum by Nanobodies or polypeptides of the invention. Inhibition of A431 tumor cell growth by trivalent bispecific Nanobodies. The concentration-dependent inhibition of cell proliferation is expressed as the relative number of cells compared to the number of cells in the absence of Nanobodies or antibodies. Effect of albumin interaction on binding at A431. Inhibition of IGF-I binding to rhIGF-IR by Nanobodies of the invention.

Claims (37)

Nanobodies against EGFR comprising or consisting of four framework regions (respectively FR1-FR4) and three complementarity determining regions (respectively CDR1-CDR3):
CDR1 is an amino acid sequence selected from the group consisting of:
TYTMA [SEQ ID NO: 42]
SYGMG [SEQ ID NO: 43]
GFAMG [SEQ ID NO: 44]
SNNMG [SEQ ID NO: 45]
GDVMG [SEQ ID NO: 46]
SYVVG [SEQ ID NO: 47]
DYNMA [SEQ ID NO: 48]
TYTMA [SEQ ID NO: 49]
NNAMA [SEQ ID NO: 50]
SYVMG [SEQ ID NO: 51]
SYAMG [SEQ ID NO: 52]
A [SEQ ID NO: 53]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having two or only one “amino acid difference” (as defined herein):
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
CDR2 is an amino acid sequence selected from the group consisting of:
GISRSDGGTYDADSVKG [SEQ ID NO: 54]
GISWRGDSTGYADSVKG [SEQ ID NO: 55]
AISWSGGSLLYVDSVKG [SEQ ID NO: 56]
AIGWGLETHYSDSVGKG [SEQ ID NO: 57]
GFSRSTSTTHYADSVKG [SEQ ID NO: 58]
GIAWGDGITYYADSVKG [SEQ ID NO: 59]
HISWLGGRTYYRDSVKG [SEQ ID NO: 60]
GFSGSGGATYYAHSVEG [SEQ ID NO: 61]
AISWRGGSTYYADSVKG [SEQ ID NO: 62]
AINWSSGSTYADSVKG [SEQ ID NO: 63]
TIAWDGSTYYADSVKG [SEQ ID NO: 64]
GLSWSADSTYYADSVKG [SEQ ID NO: 65]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
CDR3 is an amino acid sequence selected from the group consisting of:
ASVKLVYVNPRNRYSY [SEQ ID NO: 66]
AAGSAWYGTLYEYDY [SEQ ID NO: 67]
AAGSTWYGTLYEYDY [SEQ ID NO: 68]
MVGPPPRSLDYGLGNNHYEYDY [SEQ ID NO: 69]
SSTRVIVITLPRMYNY [SEQ ID NO: 70]
NSRSSWVIFTIKGQYDR [SEQ ID NO: 71]
RPGMIITTIQATYGF [SEQ ID NO: 72]
GSPYGTELLPYTRIEQYAY [SEQ ID NO: 73]
ANEYWVYVNPRNRYTY [SEQ ID NO: 74]
RRKSGEVVFTIPARYDY [SEQ ID NO: 75]
VYRVGAISEYSGTDYYTDEYDY [SEQ ID NO: 76]
GYQINSGNYNFKDYEYDY [SEQ ID NO: 77]
SYNVYYNNYYYPISRDEYDY [SEQ ID NO: 78]
HRRPFASVFTTRMYDY [SEQ ID NO: 79]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions,
Nanobody.   A polypeptide having a molecular weight of less than 15 kDa and directed against IGF-IR.   The polypeptide according to claim 2, which is capable of inhibiting the interaction between IGF-I and IGF-IR.   4. A polypeptide according to any of claims 2 or 3, which binds to IGF-IR with a binding affinity of at least 107M-1.   5. A polypeptide according to any of claims 2 to 4, selected from a single domain antibody, a domain antibody, "dAb", VH, VHH, or Nanobody.   Nanobody against IGF-IR. 7. Nanobody according to claim 6, comprising or consisting of four framework regions (respectively FR1-FR4) and three complementarity determining regions (respectively CDR1-CDR3):
CDR1 is an amino acid sequence selected from the group consisting of:
FNAMG [SEQ ID NO: 94]
INVMA [SEQ ID NO: 95]
NYAMG [SEQ ID NO: 96]
RTAMA [SEQ ID NO: 97]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions, no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having two or only one “amino acid difference” (as defined herein):
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
CDR2 is an amino acid sequence selected from the group consisting of:
VIISGGSTHYVDSVKG [SEQ ID NO: 98]
EITRSGRTNYVDSVKG [SEQ ID NO: 99]
AINWNSRSTYYADSVKG [SEQ ID NO: 100]
TITWNSGTTRYADSVKG [SEQ ID NO: 101]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions;
And / or:
CDR3 is an amino acid sequence selected from the group consisting of:
KKFGDY [SEQ ID NO: 102]
IDGSWREY [SEQ ID NO: 103]
SHDSDYGGTNANLYDY [SEQ ID NO: 104]
TAAAAVITPTRGYNY [SEQ ID NO: 105]
Or from an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99% sequence identity (as defined herein) with one of the above amino acid sequences An amino acid sequence selected from the group consisting of:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) said amino acid sequence is preferably an amino acid when compared to the amino acid sequence described above. Contains only substitutions and no amino acid deletions or insertions;
And / or an amino acid sequence selected from the group consisting of one of the above amino acid sequences and an amino acid sequence having only 3, 2, or 1 "amino acid difference" (as defined herein) Yes:
(I) any amino acid substitution is preferably a conservative amino acid substitution (as defined herein); and / or (ii) when said amino acid sequence is compared to the above amino acid sequence, preferably only amino acid substitutions Including no amino acid deletions or insertions,
Nanobody. The method of obtaining the Nanobody of Claim 6 including the following processes:
a) (i) immunizing a mammal belonging to the family Camelidae with IGF-IR or a part or fragment thereof, and improving an immune response against IGF-IR and / or an antibody (more specifically, a heavy chain antibody) (Ii) obtaining a biological sample containing a heavy chain antibody sequence and / or a VHH sequence directed against IGF-IR from the immunized mammal as described above; and (iii) from the biological sample; Obtaining (eg, isolating) a heavy chain antibody sequence and / or a VHH sequence directed against IGF-IR, generally by a method and / or (i) IGF-IR or at least a portion or fragment thereof Screening a library comprising heavy chain antibody sequences and / or VHH sequences having directivity to (ii) said library Providing at least one VHH domain directed against IGF-IR by a method generally comprising obtaining (eg, isolating) a heavy chain antibody sequence and / or VHH sequence directed against IGF-IR The step of:
b) In order to increase the affinity and / or specificity of the heavy chain antibody sequence and / or VHH sequence for IGF-IR, the heavy chain antibody sequence and / or VHH sequence thus obtained for IGF-IR Optionally subjecting to affinity maturation, mutagenesis (eg, random mutagenesis or site-directed mutagenesis), and / or any other technique;
c) determining the sequence of the CDRs of the heavy chain antibody sequence and / or VHH sequence obtained in this way against IGF-IR; and d) at least one, preferably at least two, more preferably three Providing a Nanobody, wherein all of the CDRs (CDR1, CDR2 and CDR3, specifically at least CDR3) have the sequence determined in step c).   8. At least one Nanobody according to any of claims 1 or 6-7, or at least one polypeptide according to any of claims 2-5, or an EGFR or IGF-IR binding portion thereof or A polypeptide comprising a fragment.   A polypeptide comprising at least two binding sites, each of said at least two binding sites being directed against a tumor associated antigen or epitope.   11. The polypeptide of claim 10, wherein each of the at least two binding sites is directed against a different tumor associated antigen.   11. The polypeptide of claim 10, wherein each of the at least two binding sites is directed to a different epitope on the same tumor associated antigen.   13. A polypeptide according to any of claims 10 to 12, wherein at least one of the binding sites is selected from VH, VHH, domain antibody, single domain antibody, "dAb" or Nanobody.   The polypeptide according to any one of claims 10 to 13, wherein each of the binding sites is a Nanobody.   11. The polypeptide of claim 10, comprising at least one Nanobody for EGFR and at least one Nanobody for an EGFR family member selected from the group consisting of HER2, HER3, and HER4.   11. The polypeptide of claim 10, comprising at least one Nanobody for EGFR and at least one Nanobody for IGF-IR.   A polypeptide comprising at least two Nanobodies, wherein one binding of said at least two Nanobodies modulates said binding by a second of said at least two Nanobodies.   18. The polypeptide of claim 17, wherein binding by a second of the at least two Nanobodies is increased.   18. The polypeptide of claim 17, wherein binding by a second of the at least two Nanobodies is reduced.   18. The polypeptide of claim 17, wherein binding by a second of the at least two Nanobodies is inhibited.   A nucleic acid encoding the Nanobody according to any one of claims 1 or 6 to 7, or the polypeptide according to any one of claims 2 to 5 or 9 to 20.   Expressing the Nanobody according to any one of claims 1 or 6-7, or expressing the polypeptide according to any one of claims 2-5 or 9-20, or comprising the nucleic acid according to claim 21; Host cell. A method for preparing the Nanobody according to any one of claims 1 or 6-7, or the polypeptide according to any one of claims 2-5 or 9-20, comprising the following steps:
Expressing in a suitable host cell, or host organism, or in another suitable nucleic acid expression system according to claim 21, and then optionally;
Isolating and / or purifying the Nanobody or polypeptide thus obtained.   22. At least one Nanobody according to any of claims 1 or 6-7, at least one polypeptide according to any of claims 2-5 or 9-20, and / or at least according to claim 21. A composition comprising one nucleic acid.   22. At least one Nanobody according to any of claims 1 or 6-7, at least one polypeptide according to any of claims 2-5 or 9-20, and / or at least according to claim 21. A pharmaceutical composition comprising one nucleic acid and optionally at least one pharmaceutically acceptable carrier.   22. At least one Nanobody according to any of claims 1 or 6-7, at least one polypeptide according to any of claims 2-5 or 9-20, and / or at least according to claim 21. A diagnostic composition comprising one nucleic acid and optionally at least one contrast agent.   23. Nanobody according to any of claims 1 or 6-7, polypeptide according to claims 2-5 or 9-20, nucleic acid according to claim 21 for use in prevention and / or treatment. 26. A pharmaceutical composition according to claim 25.   A therapeutically effective amount of the Nanobody according to any one of claims 1 or 6-7, the polypeptide according to any one of claims 2-5 or 9-20, and / or the pharmaceutical composition according to claim 25. A method for preventing and / or treating a disease or disorder associated with EGFR and / or IGF-IR comprising the step of administering.   29. The method of claim 28, wherein the disease or disorder is associated with or characterized by EGFR and / or IGF-IR overexpression.   30. The method of claim 29, wherein the disease or disorder is cancer or a tumor.   30. The method of claim 28, wherein the disease or disorder is associated with an inflammatory process.   32. The method of claim 31, wherein the disease or disorder is rheumatoid arthritis, psoriasis, or mucus hypersecretion in the lung.   A method of inhibiting an interaction between EGF and EGFR comprising administering the Nanobody of claim 1, or the polypeptide of any of claims 9-20, or the pharmaceutical composition of claim 25. .   An anti-EGFR therapy comprising the step of administering a Nanobody according to any of claims 6-7, a polypeptide according to any of claims 2-5 or 9-20, or a pharmaceutical composition according to claim 25. How to strengthen. A method of diagnosing a disease or disorder associated with EGFR and / or IGF-IR, comprising the following steps:
(A) contacting the sample with the Nanobody according to any one of claims 1 or 6-7, or the polypeptide according to any one of claims 2-5 or 9-20,
(B) detecting the binding of the Nanobody or polypeptide to the sample, and (c) a difference in binding compared to the sample of a disease or disorder associated with each of EGFR and / or IGF-IR. Comparing the standard useful for diagnosis with the binding detected in step (b).   Administering a Nanobody according to any of claims 1 or 6-7, a polypeptide according to any of claims 2-5 or 9-20, or a diagnostic composition according to claim 26, A method of imaging an EGFR or IGF-IR target.   Nanobody according to any of claims 1 or 6-7 for the preparation of a medicament for the treatment of cancer, tumors, diseases associated with inflammatory processes, rheumatoid arthritis, psoriasis or mucus hypersecretion in the lung, The use of the polypeptide according to any one of Items 2 to 5 or 9 to 20.