Cysteine-linked nanobody dimers

Abstract

The present invention relates to a dimer comprising a first polypeptide and a second polypeptide, wherein each of said first and second polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine moiety, preferably at the C-terminus, wherein said first polypeptide and said second polypeptide are covalently linked by a disulfide bond between the cysteine moiety of said first polypeptide and the cysteine moiety of said second polypeptide, wherein the dimer outperforms benchmark constructs, such as homomultivalent and multispecific constructs, in a variety of assays. The present invention provides methods for making the dimer of the invention.

Description

Invention Field

[0001] This invention relates to a dimer comprising a first polypeptide and a second polypeptide, wherein each of the first and second polypeptides comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​moiety (preferably at the C-terminus), wherein the first polypeptide and the second polypeptide are covalently linked by a disulfide bond between the cysteine ​​moieties of the first polypeptide and the cysteine ​​moieties of the second polypeptide, wherein the dimer outperforms a baseline construct in various assays, such as homologous multivalent and multispecific constructs. The invention provides a method for preparing the dimer of the invention. The invention also provides variable domains (as defined herein) and polypeptides comprising one or more variable domains obtainable by the methods of the invention (also referred to as "polypeptides of the invention"), and compounds comprising such variable domains and / or polypeptides coupled to one or more groups, residues, or portions (also referred to as "compounds of the invention").

[0002] The present invention also relates to nucleic acids encoding such variable domains and / or polypeptides; to host cells comprising such nucleic acids and / or expressing or capable of expressing such variable domains and / or polypeptides; to compositions comprising such variable domains and / or polypeptides, compounds, nucleic acids and / or host cells, and particularly to pharmaceutical compositions; and to the use of such variable domains, polypeptides, nucleic acids, host cells and / or compositions, especially for preventive, therapeutic or diagnostic purposes.

[0003] Other aspects, embodiments, advantages, and applications of the invention will become clear from the further description herein.

[0004] background

[0005] More than 20 monoclonal antibodies (mAbs) have been approved for treatment, with many more in clinical development. These molecules have become established therapeutic agents for a variety of diseases (Reichert (2011) MAbs 3:76-99; Nelson et al., (2010) Nat Rev Drug Discov 9:767-74). However, complex diseases such as cancer or inflammatory conditions are often multifactorial in nature, involving numerous disease-mediating ligands and receptors, as well as crosstalk between signaling cascades. Blocking multiple targets or multiple sites on a single target should result in enhanced therapeutic efficacy. The limited ability of conventional monoclonal antibody therapy to induce significant antitumor activity has led to the development of bispecific substances (which can simultaneously bind two different antigens / antibodies) (Kontermann (2012) mAbs 4:182-197). Over the past decade, dual targeting using bispecific antibodies has emerged as an alternative to combination therapy or mixtures. The concept of dual targeting using bispecific antibodies is based on targeting multiple disease-modulating molecules with a single drug. From a technical and regulatory perspective, this makes development less complex because manufacturing, preclinical, and clinical testing are simplified to a single bispecific molecule (Kontermann (2012) above). Treatment using a single, dual-targeting drug, rather than a combination, should also be less complex for patients.

[0006] Bispecific antibodies can be produced by biochemical or genetic means. Recombinant technology has produced a wide range of bispecific antibodies, generating 45 forms in the last two decades (Byrne et al. (2013) Trends Biotechnol. 31, 621-32). Despite the large number of topologies, this approach is not suitable for every protein combination. Fusion of proteins through their N or C terminals can lead to a reduction or loss of biological activity, and variations in expression yield can be observed due to complexities in folding and processing (Schmidt (2009) Curr. Opin. Drug Discovery Dev. 12, 284-295; Baggio et al. (2004) Diabetes 53, 2492-2500; Chames and Baty (2009) mAbs 1, 539-47).

[0007] An alternative method for generating bispecific therapeutic agents is homo- or hetero-bifunctional conjugates (Doppalapudi et al. (2010) Proc Natl Acad Sci USA 107:22611-6). To date, this has been a less successful method for preparing such conjugates. A fundamental drawback of the chemical techniques employed in this region is their dependence on modified lysine residues. Each conventional antibody contains an average of 100 lysine residues, and their distribution is uniform throughout the surface topology of the antibody or its fragments such as Fab, Fc, and immunoglobulin single variable domain (ISVD) regions. Therefore, conjugation techniques utilizing lysine residues will randomly crosslink with virtually all regions of the antibody molecule, resulting in a highly heterogeneous mixture of products with unpredictable properties.

[0008] A strategy to overcome this problem is provided by inserting non-natural amino acids that allow for site-specific introduction of chemical linkers. However, the substitution of non-natural amino acids is often incomplete, and expression yields are typically low due to the cytotoxicity of artificial amino acids at the required high concentrations.

[0009] Site-directed mutagenesis, which introduces a single nucleophilic cysteine ​​residue at a desired site in the antibody, offers an alternative approach to overcoming the problem of random crosslinking. Cysteine ​​residues are found in low native abundance in proteins, but are typically found bound in intramolecular disulfide bonds, providing structural and functional integrity, hence the lack of free cysteine ​​residues in antibodies and antibody fragments (Fodje and Al-Karadaghi (2002) Protein Eng. Des. Sel. 15, 353-358). However, controlling intramolecular crosslinking relative to intermolecular crosslinking using these reagents is very difficult. Some control can be achieved through appropriate selection of reaction parameters such as protein / reagent ratio, pH, ionic strength, etc., but the results remain unsatisfactory.

[0010] WO2004 / 03019 hypothesizes that variable domains can be linked together to form multivalent ligands by, for example, providing dAbs (where cysteine ​​is a bound disulfide) each having a cysteine ​​residue at the C-terminus of a domain, using a chemical coupling process with 2,2'-dithiodipyridine (2,2'-DTDP) and a reduced monomer. However, 2,2'-DTDP is irritating, limiting its practical application. Furthermore, because 2,2'-DTDP also makes Ca... 2+The application of reactive disulfides that migrate from cells is further limited. WO2004 / 03019 not only fails to address whether this method is practically feasible, especially without disrupting and rearranging intramolecular thiol bonds, but also, given the nature of 2,2'-DTDP, requires laborious measurements to completely remove this reagent.

[0011] Baker et al. (2014, Bioconjugate Chem., DOI:10.1021 / bc5002467) described bispecific antibody constructs using a synthetic bis-dibromomaleimide crosslinking agent, which are constructed by the reduction and bridging of antibody fragment disulfide bonds.

[0012] Carlsson et al. (1978, Biochem J. 173:723-737) proposed a thiolation process for proteins using succinimide-3-(2-pyridyldithio)propionate to yield reversible protein-protein conjugates. However, this process requires extensive purification. Furthermore, reduced activity has been reported when following the protocol (Carlsson et al., 1978). Carlsson et al. (1978) did not mention whether this process could be used for antibodies or their fragments.

[0013] Typically, intermolecular crosslinking by introducing cysteine ​​residues is restricted because cysteine ​​mutagenesis usually leads to reduced expression yields and undesirable properties such as susceptibility to unwanted dimerization, mixed disulfide formation, or disulfide scrambling (Schmiedl et al. (2000) J. Immunol. Methods 242, 101-14; Junutula et al. (2008) Nat. Biotechnol. 26, 925-32; Albrecht et al. (2004) Bioconjugate Chem. 15, 16-26).

[0014] Graziano and Guptill discussed methods for constructing bispecific substances with Fab'×Fab' chemical linkages using free thiol groups generated during the reduction of disulfide bonds within the heavy chain of the F(ab')2 segment. However, conditions must be selected to achieve efficient reduction of inter-chain disulfides without extensive reduction of heavy-light chain disulfide bonds. It should be noted that bispecific substances formed using the o-phenylene dimaleimide (o-PDM) method can be more stable than those produced by Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB), but it is more difficult to purify the o-PDM-generated bispecific substances to a biochemically homogeneous state. Another significant drawback of the o-PDM method is the requirement for an odd number of heavy chain disulfide bonds in the antibody molecule to be maleiminated (Graziano and Guptill (2004), Chapter 5, Chemical Production of Bispecific Antibodies, pp. 71-85, from: Methods in Molecular Biology, Vol. 283: Bioconjugation Protocols: Strategies and Methods; edited by: CMNiemeyer) Humana Press Inc. (Totowa, NJ). This prevented its application in the construction of human-to-human bispecific substances.

[0015] Another strategy for improving treatment, especially cancer treatment, is the use of antibody-drug conjugates (ADCs). Although more than 50 different ADCs are currently in clinical trials, many of which are active, a wide range of problems remain in the development, purification, and prevention of their toxicity. First, there is little control over the physicochemical properties of ADCs, such as the heterogeneity of the amount of drug conjugated to each antibody, PK / biodistribution, payload, and delivery vehicle. For example, many drugs are conjugated to antibodies via lysine. As mentioned above, this results in an uncontrollable drug-to-antibody ratio because lysine is distributed throughout the antibody. Furthermore, this conjugation interferes with the concept of bispecificity utilizing lysine conjugation. In addition, most drugs used in cancer treatment are highly hydrophobic, causing unpredictable and mostly unfavorable aggregation, PK, and biodistribution characteristics of the ADC moiety. This is especially true for small antibody fragments. Conventional antibodies have a size of approximately 150 kDa, while drugs have an average size of approximately 1 kDa. Therefore, the antibody:drug size ratio is approximately 150:1. In stark contrast to conventional antibodies, antibody fragments, such as ISVDs, have a size of only about 15 kDa. Therefore, the ISVD:drug size ratio is only 15:1, which is 10 times smaller than that of conventional antibodies. Consequently, the hydrophobic characteristics of the drug have a disproportionately large influence on the physicochemical properties of the conjugated antibody fragment. In fact, the main problem with conjugated antibody fragments is aggregation (Feng et al., 2014 Biomedicines 2:1-13). Further analysis shows that large molecular constructs of IgG size exhibit a favorable balance between systemic clearance and extravasation, resulting in maximum tumor uptake (Dane Wittrup et al., 2012 Methods Enzym. 503 Chapter 10, pp. 255-268). These difficulties effectively limit the use of conjugated drugs to smaller antibody fragments.

[0016] Along with HER-2, HER-3, and HER-4, epidermal growth factor receptor (EGFR; also known as HER-1) is a member of the HER kinase family. EGFR is overexpressed in a variety of human tumors, including non-small cell lung cancer, breast cancer, head and neck cancer, gastric cancer, colorectal cancer, esophageal cancer, prostate cancer, bladder cancer, kidney cancer, pancreatic cancer, and ovarian cancer. EGFR activation triggers signaling pathways that can lead to cell division, increased motility, angiogenesis, and decreased apoptosis. These effects are mediated through a complex series of signaling mechanisms, such as the binding of mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 kinase (PI3K) pathways.

[0017] EGFR is also involved in a number of other diseases, such as inflammatory arthritis and excessive mucus secretion in the lungs.

[0018] Many EGFR-targeting antibodies, such as IMC-C225 (Erbitux, Imclone), EMD72000 (Merck Darmstadt), ABX-EGF (Abgenix), h-R3 (theraCIM, YM Biosciences), and Humax-EGFR (Genmab), act as antibodies that prevent the binding of the ligand to the receptor. However, these antibodies, or existing available drugs, are not entirely effective for cancer treatment, and most are limited by severe toxicity. Invention Overview

[0020] The purpose of this invention is to overcome or improve at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0021] Given their modularity, immunoglobulin single variable domains (ISVDs), and especially nanobodies, are particularly suitable for inclusion in multivalent constructs. A convenient and preferred method for generating multivalent constructs is through genetic fusion of individual nucleic acids encoding the ISVD via amide bonds, wherein the nucleotide sequence encoding the ISVD is coupled via its 3' terminal nucleic acid to the 5' terminal nucleic acid of another nucleotide sequence encoding the ISVD, or, if desired, via a (nucleic acid) linker of multiple lengths. Thus, the ISVD is coupled via amide bonds and possibly via peptide linkers.

[0022] ISVDs contain intramolecular disulfide bonds between cysteine ​​residues, thus maintaining the integrity and functionality of the part. It has been widely confirmed that, following genetic fusion of the nucleotide sequence encoding the ISVD, the inherent property of forming typical (also known as intramolecular) disulfide bonds in an individual ISVD remains unaffected during translation.

[0023] Given the ease of genetic fusion and versatility, chemical conjugation of ISVDs is not a preferred method, especially because it requires selectively coupling ISVDs at predetermined sites without impeding intramolecular disulfide bonds and / or using laborious methods involving non-self, potentially harmful components.

[0024] This invention provides a convenient method in which intermolecular dimerization via disulfide bonds between two polypeptides is facilitated, with substantially no aberrant disturbances or intramolecular disulfide bonds involving ISVD. The method utilizes a cysteine ​​residue introduced into the C-terminal extension of a polypeptide that also contains ISVD.

[0025] The present invention also provides a method for preparing the dimer of the present invention. In particular, the present invention relates to a method for preparing (peptide-)dimers, the method comprising at least the following steps:

[0026] (i) providing a first polypeptide, wherein the first polypeptide comprises

[0027] - At least one immunoglobulin single variable domain (ISVD) and

[0028] - Includes a C-terminal extension, preferably a cysteine ​​moiety at the C-terminus;

[0029] (ii) providing a second polypeptide, wherein the second polypeptide comprises

[0030] - At least one immunoglobulin single variable domain (ISVD) and

[0031] - Includes a C-terminal extension, preferably at the C-terminus of a cysteine ​​residue; and

[0032] (iii) Optionally, copper ions (Cu) are added. 2+ The dimer is prepared by oxidizing the thiol portion of the cysteine ​​moiety extending from the C-terminus of the first polypeptide and the thiol portion of the cysteine ​​moiety extending from the C-terminus of the second polypeptide to the disulfide derivative cystine at pH 6.5 to pH 7.5; and the cystine is the only intermolecular disulfide bond present in the dimer.

[0033] Preferably, the step of reducing the [C-terminal] cystine of the dimer is carried out under conditions where the intramolecular disulfide bonds of the first polypeptide and / or the second polypeptide remain oxidized. In other words, the integrity of the ISVD is maintained. The method optionally further includes the step of reducing the (C-terminal) cystine of the dimer.

[0034] Further, it was unexpectedly discovered that the dimer of the present invention is superior to baseline constructs, such as homologous multivalent and multispecific constructs. The baseline constructs consist of the same polypeptides as the dimer of the present invention, but are generated through genetic fusion of nucleic acids encoding these polypeptides, thus the first polypeptide is coupled to the second polypeptide via an amide bond in the N-terminal to C-terminal direction. In particular, the dimer of the present invention can exhibit a better affinity (suitably as K) than baseline, such as homologous divalent constructs. D Value (actual or apparent), K A Value (actual or apparent), k on rate and / or k offThe target binding rate is high. On the other hand, even when containing albumin with two ISVD-binding proteins, the dimer of the present invention exhibits similar biodistribution characteristics to the baseline containing only one ISVD-binding albumin. Furthermore, the dimer of the present invention unexpectedly shows improved internalization compared to the baseline construct, particularly on cells with low target expression. Since internalization is crucial for good efficacy, improved internalization may result in better efficacy. Moreover, improved internalization can reduce side effects, such as toxicity, because less drug is required and less drug will detach. Internalization of the dimer on cells with low target expression can expand the range of treatable tumors and reduce the chance of drug resistance development. On the other hand, the dimer of the present invention exhibits favorable biodistribution characteristics similar to the baseline construct.

[0035] Therefore, the present invention relates to a dimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​moiety (preferably at the C-terminus); wherein the second polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​moiety (preferably at the C-terminus); and wherein the first polypeptide and the second polypeptide are covalently linked by a disulfide bond between the cysteine ​​moiety of the first polypeptide and the cysteine ​​moiety of the second polypeptide.

[0036] Therefore, the present invention relates to a (peptide) dimer comprising a first polypeptide and a second polypeptide, wherein the first polypeptide and the second polypeptide are covalently linked by a disulfide bond located at the C-terminus.

[0037] The inventors further observed that the dimer of the present invention possesses unexpectedly advantageous binding and functional characteristics. These characteristics are also maintained for extended periods without any apparent or substantial loss of effectiveness. This makes the dimer suitable for storage and transport. Therefore, the present invention further relates to a method for storing polypeptides comprising a reactive cysteine ​​moiety, the method comprising at least the step of temporarily inactivating the reactive cysteine ​​moiety by oxidizing the thiol portion of the reactive cysteine ​​moiety to a disulfide derivative cystine, wherein the polypeptide further comprises an (internal) cystine bond.

[0038] The inventors of this invention hypothesize that dimers may be particularly suitable as pools for transient use, such as by coupling C-terminal cysteine, for example, via functional groups of maleimide chemistry. A scheme using mild reducing conditions has been developed in which the intermolecular disulfide bridges of the dimer are reduced to activate the thiol groups of the constituent polypeptide. Optimized conditions induce the reduction of the disulfide, forming the dimer without reducing the internal typical ISVD disulfide bonds. Therefore, this invention relates to a method for generating a polypeptide comprising a reactive cysteine ​​moiety, the method comprising at least the following steps:

[0039] (i) Providing a polypeptide dimerized via cystine bonds;

[0040] (ii) Reduce the cystine bond;

[0041] This generates a polypeptide containing a reactive cysteine ​​moiety. Preferably, the cysteine ​​bond is located at the C-terminus of the polypeptide. Preferably, the reduction conditions of step (ii) are selected such that the internal cysteine ​​bond is not reduced.

[0042] Furthermore, the present invention provides a method for conjugating the effective load with the peptides of the present invention at a highly controllable drug-to-antibody ratio (DAR) and a purity exceeding 95%. Completely unexpectedly, conjugating the peptide with the effective load (DAR = 1) has no effect on biodistribution characteristics. Moreover, these conjugated peptides have demonstrated in vitro cytotoxicity and in vivo inhibition of tumor growth. Therefore, the present invention provides a method for treating subjects using the peptides of the present invention. Attached Figure Description

[0043] Figure 1 shows a schematic diagram of the construct used.

[0044] Figure 2 Competition combined with FACS.

[0045] Figure 3 Blockage of EGF-mediated EGFR phosphorylation in HER14 cells (0.5 mM EGF).

[0046] Figure 4 A schematic diagram of the reduction of disulfide dimers of C-terminal GGC-extended peptides.

[0047] Figure 5 SEC characteristics of reduced cysteine-extended peptides.

[0048] Figure 6 Maleimide-val-cit-MMAE.

[0049] Figure 7SDS-PAGE analysis of T0238-00001-mc-val-cit-PAB-MMAE (ABL100-NC003-1). 1) Novex label; 2) T0238-00001 dimer; 3) Reduced T0238-00001 (10 mM DTT, 2-8℃, O / N); 4) Crude conjugated mixture of ABL100-NC003-1; 5) ABL100-NC003-1.

[0050] Figure 8 Chromatogram of superimposed hydrophobic interactions of reduced T023800001-A, oxidized T023800001-A, and T0238-00001-mc-val-cit-PAB-MMAE.

[0051] Figure 9 In vitro cell killing by peptide-MMAE conjugates: Impedimetric monitoring of the effect of different concentrations of unconjugated and conjugated nanobodies on the proliferation of MDA-MB-468 cells as a measure of fluctuation in the normalized cell index (CI). For data analysis, arrows indicate the time point of nanobody application (i.e., 20 h post-inoculation) and dashed lines indicate the endpoint (i.e., 116 h post-inoculation). The cell index obtained from cell growth in the absence of nanobodies was selected as a control.

[0052] Figure 10. Dose-dependent effects of unconjugated and MMAE-conjugated peptides.

[0053] Figure 11 In vivo efficacy of peptide-MMAE conjugates.

[0054] Figure 12 uses NCS-Bz-Df and 89 Nb modification and radiolabeling of Zr.

[0055] Figure 13 Average %ID / g of the three peptides.

[0056] Figure 14 Dose-response profiles of internalized peptides and constructs. Invention Details

[0058] Any discussion of prior art throughout the specification should never be considered an admission that such prior art is widely known or constitutes part of common knowledge in the art.

[0059] Unless otherwise indicated or limited, all terms used have their usual meanings that will be clear to a person skilled in the art. For example, references were made to standard manuals such as Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2nd ed.), Volumes 1–3, Cold Spring Harbor Laboratory Press (1989); F. Ausubel et al., ed., *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 ed., University of California Press, Berkeley, CA (1981); Roitt et al., *Immunology* (6th ed.), Mosby / Elsevier, Edinburgh (2001); and Roitt et al., *Roitt's Essential Immunology*. Immunology, 10th edition, Blackwell Publishing, UK (2001); and Janeway et al., Immunology (6th edition), Garland Science Publishing / Churchill Livingstone, New York (2005), and the general background techniques cited herein.

[0060] Unless otherwise stated, all methods, steps, techniques and operations not specifically described in detail can and have been performed in a manner known to the art, which will be clear to a person skilled in the art. For example, reference is made again to the standard manual and the general background techniques mentioned herein, and to other references cited therein; and to, for example, to the following reviews: Presta 2006 (Adv. Drug Deliv. Rev. 58(5-6): 640-56), Levin and Weiss 2006 (Mol. Biosyst. 2(1): 49-57), Irving et al., 2001 (J. Immunol. Methods 248(1-2): 31-45), Schmitz et al., 2000 (Placenta 21 Suppl. A: S106-12), Gonzales et al., 2005 (Tumour Biol. 26(1): 31-43), which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins.

[0061] A nucleic acid or amino acid sequence is considered to be "substantially separated"—for example, compared to the reaction or culture medium from which it is obtained—meaning it has been separated from at least one other component (such as another nucleic acid, another protein / peptide, another biological component or macromolecule, or at least one contaminant, impurity, or trace component) that it normally associates with in the said source or medium. In particular, a nucleic acid or amino acid sequence is considered "substantially separated" when it has been purified at least 2-fold, especially at least 10-fold, more especially at least 100-fold, and up to 1000-fold or higher. As determined using suitable techniques, such as suitable chromatographic techniques like polyacrylamide gel electrophoresis, a nucleic acid or amino acid sequence "in a substantially separated form" is preferably substantially homogeneous.

[0062] Unless the context clearly requires otherwise, throughout the description and claims, the words “comprising,” “including,” etc., shall be interpreted as inclusive, as opposed to exclusive or exhaustive; that is, “including but not limited to.”

[0063] For example, when a nucleotide sequence, amino acid sequence, or polypeptide is described as "comprising" another nucleotide sequence, amino acid sequence, or polypeptide, or is described as "consistently composed of" another nucleotide sequence, amino acid sequence, or polypeptide, this may mean that the subsequent nucleotide sequence, amino acid sequence, or polypeptide has been incorporated into the first-mentioned nucleotide sequence, amino acid sequence, or polypeptide, respectively. However, more generally, this usually means that the first-mentioned nucleotide sequence, amino acid sequence, or polypeptide contains within its sequence a segment of nucleotide or amino acid residues having the same nucleotide or amino acid sequence as the subsequent sequence, regardless of how the first-mentioned sequence was actually generated or obtained (e.g., it may be generated by any suitable method described herein). By way of non-limiting example, when the polypeptide of the present invention is described as containing an immunoglobulin monovariable domain, this may mean that the immunoglobulin monovariable domain sequence has been incorporated into the sequence of the polypeptide of the present invention. However, more generally, this usually means that the polypeptide of the present invention contains the sequence of an immunoglobulin monovariable domain within its sequence, regardless of how the polypeptide of the present invention was generated or obtained. Furthermore, when a nucleic acid or nucleotide sequence is described as containing another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that when expressed into an expression product (e.g., a polypeptide), the amino acid sequence encoded by the subsequent nucleotide sequence constitutes part of said expression product (in other words, the subsequent nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence).

[0064] The phrases “consistently composed of” or “consistently composed of” mean that the polypeptide used herein is identical to or corresponds to the polypeptide of the present invention having a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues, and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5, or 6 amino acid residues, added to the N-terminus, C-terminus, or both of the immunoglobulin monovariable domain.

[0065] For specific antigenic determinants, epitopes, antigens, or proteins (or for at least a portion, fragment, or epitope thereof) that can (specifically) bind, have affinity, and / or have specific amino acid sequences (such as the immunoglobulin monovariable domain, antibody, polypeptide, or generally antigen-binding protein or polypeptide or fragment thereof of the present invention) are described as “against” or “directed against” the said antigenic determinant, epitope, antigen, or protein.

[0066] Affinity represents the strength or stability of molecular interactions. Affinity is usually expressed in terms of K0. DAlternatively, the dissociation constant can be given, which has units of moles per liter (or M). Affinity can also be expressed as the binding constant K. A It is equal to 1 / K D And it has (moles / liter) -1 (or M) -1 The unit is ). In this specification, the stability of the interaction between two molecules will be primarily determined by the K0 of their interaction. D Value representation; what is clear to technicians is that, considering relation K A =1 / K D The relationship, through its K D The strength of the molecular interaction specified by the value can also be used to calculate the corresponding K. A Value. K D The value characterizes the strength of molecular interactions in a thermodynamic sense, because it is determined by DG = RT.ln(K). D (Equivalent to DG = -RT.ln(K)) A It relates to the change in the free energy of combination (DG), where R is equal to the gas constant, T is equal to the absolute temperature, and ln represents the natural logarithm.

[0067] K of biological interactions considered meaningful (e.g., specific) D Usually in 10 -10 M(0.1nM) to 10 -5 Within the range of M (10000nM). The stronger the interaction, the greater its K. D The lower.

[0068] K D It can also be represented as k off The dissociation rate constant of the complex is expressed as k on The ratio of their binding rates (therefore K) represents the ratio of their binding rates. D =k off / k on And K A =k on / k off Dissociation rate k off With unit s -1 (Where s is the SI unit symbol for seconds). Combination rate k on With unit M -1 s -1 The binding rate can be 10. 2 M -1 s -1 To about 10 7 M -1 s -1 The variation between these values ​​approaches the diffusion-limited binding rate constant of biomolecular interactions. The dissociation rate depends on the relationship t... 1 / 2=ln(2) / k off It is related to the given half-life of the molecular interaction. The dissociation rate can be as high as 10. -6 s -1 (approximately t with several days) 1 / 2 Irreversible complexes) up to 1s -1 (t 1 / 2 It varies between 0.69s.

[0069] The specific binding of antigen-binding proteins such as ISVD to antigens or antigenic determinants can be determined in any suitable manner known in itself, including, for example, Scatchard analysis and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA) and sandwich competitive assays, and various variants thereof known in the art; as well as other techniques mentioned herein.

[0070] The affinity of molecular interactions between two molecules can be measured using various techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see, for example, Ober et al., 2001, Intern. Immunology 13:1551-1559), in which one molecule is immobilized on a biosensor chip and the other molecule flows through the immobilized molecule to obtain k. on k off The measured value and therefore K D (or K) A ) value. For example, this can be done using well-known values. The assay was performed using an instrument (Pharmacia Biosensor AB, Uppsala, Sweden). Kinetic exclusion assay. (Drake et al., 2004, Analytical Biochemistry 328:35-43) measured binding events in solution without labeling the binding partner and based on kinetic exclusion of the dissociation of the complex.

[0071] Immunoassay systems provide a platform for automated bioanalysis and rapid sample transfer (Fraley et al., 2013, Bioanalysis 5:1765-74).

[0072] It will also be clear to technicians that if the measurement process somehow affects the inherent binding affinity of the implied molecule (e.g., through artificial actions related to coating a molecule onto a biosensor), then the measured K... D It can be equivalent to apparent K DFurthermore, if a molecule contains more than one recognition site for other molecules, the epigenetic K can be measured. D In such cases, the measured affinity may be affected by the antibody affinity of the interaction between the two molecules.

[0073] The term "specificity" has the meaning assigned to it in paragraph n) on pages 53-56 of WO 08 / 020079; and as mentioned therein, it refers to the number of different types of antigens or antigenic determinants that a particular antigen-binding molecule or antigen-binding protein (such as the dimer or polypeptide of the present invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and / or antibody affinity, as described on pages 53-56 of WO 08 / 020079 (by reference incorporated herein), which also describes some preferred techniques for measuring the binding between an antigen-binding molecule (such as the polypeptide or ISVD of the present invention) and the associated antigen. Typically, antigen-binding proteins (such as the immunoglobulin monovariable domain and / or polypeptide of the present invention) will be measured at 10... -5 Up to 10 -12 Below 10 mol / L, and preferably 10 -7 Up to 10 12 Below 10 mol / L and more preferably 10 -8 Up to 10 -12 dissociation constant (K) per mole / liter D (i.e., 10) 5 Up to 10 12 10 liters / molar or higher, and preferably 10 liters / molar. 7 Up to 10 12 liters / molar or higher, and more preferably 10 8 Up to 10 12 Binding constant (K) per liter / molar A )) bind to their antigens. It is generally believed that any antigen greater than 10 -4 moles / liter of K D Value (or anything below 10) 4 K in liters / moles AThe value indicates nonspecific binding. Preferably, the monovalent immunoglobulin single variable domain of the present invention will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. The specific binding of the antigen-binding protein to the antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays, and various variants thereof known per se in the art; as well as other techniques mentioned herein. As will be apparent to those skilled in the art, and as described on pages 53-56 of WO 08 / 020079, the dissociation constant can be an actual or apparent dissociation constant. The methods used to determine the dissociation constant will be apparent to those skilled in the art, and include, for example, the techniques mentioned on pages 53-56 of WO 08 / 020079.

[0074] Another method that can be used to assess affinity is the two-step ELISA (enzyme-linked immunosorbent assay) procedure described by Friguet et al., 1985 (J. Immunol. Methods 77:305-19). This method establishes a solution-bound equilibrium measurement and avoids possible artificial behavior associated with the adsorption of a molecule on a carrier such as plastic.

[0075] However, K D Accurate measurement can be very laborious, and as a result, the apparent K is usually determined. D The value is used to assess the binding strength of two molecules. It should be noted that the apparent K value can be used as long as all measurements are performed in a consistent manner (e.g., keeping the measurement conditions constant). D Measurement as the true K D The approximation of K is therefore given to K in this paper with equal importance or relevance. D and apparent K D .

[0076] Finally, it should be noted that in many cases, experienced scientists can determine whether it is convenient to measure the binding affinity relative to some reference molecule. For example, to assess the binding strength between molecule A and molecule B, a reference molecule C, known to bind to B, can be used, for example, and appropriately labeled with a fluorophore or chromophore group or other chemical moiety, such as biotin or other forms readily detectable in ELISA or FACS (fluorescence-activated cell sorting) (fluorophores for fluorescence detection, chromophores for light absorption detection, and biotin for streptavidin-mediated ELISA detection). Typically, reference molecule C is maintained at a fixed concentration, while the concentration of A varies with a given concentration or amount of B. The result is the IC50 corresponding to the concentration of A. 50 The value of C measured under conditions lacking A is halved under the concentration conditions of A. If K D ref K of the reference molecule D It is known, and the total concentration of the reference molecule, c, is... ref Since this is also known, the apparent K of the interaction AB can be obtained according to the following formula. D :K D =IC 50 / (1+c ref / K D ref Note that if c ref < <K D ref Then K D ≈IC 50 If, for the conjugates being compared, IC 50 Measurements are taken in a consistent manner (e.g., maintaining c) ref If it is fixed, it can be done through IC. 50 The strength or stability of molecular interactions is evaluated, and this measurement is determined to be equivalent to K throughout this paper. D or apparent K D .

[0077] Half-maximum inhibition concentration (IC50) 50 A half-maximum inhibitory concentration (IC50) is a measure of a compound’s effectiveness (e.g., pharmacological action) in inhibiting biological or biochemical functions. This quantitative measure indicates how many ISVs or nanobodies (inhibitors) are needed to inhibit a given biological process (or its components, such as enzymes, cells, cell receptors, chemotaxis, anaplasia, metastasis, invasion, etc.) by half. In other words, it is the half-maximum (50%) inhibitory concentration (IC50) of a substance. 50 The IC50 of the drug can be determined by constructing dose-response curves and examining the effect of different concentrations of antagonists, such as the ISV or nanobody of this invention, on the activity of the reversal agonist. 50The IC50 of a given antagonist, such as the ISV or nanobody of this invention, can be calculated by determining the concentration required to inhibit half of the maximum biological response of the agonist. 50 value.

[0078] The term half-maximal effect concentration (EC50) 50 EC50 (Effective Coefficient of Motion) refers to the concentration of a compound that induces a response at half the range between baseline and maximum after a specified exposure time. In the context of this paper, it is used as a measure of the potency of peptides, ISVs, or nanobodies. EC50 of graded dose-response curves. 50 This indicates the concentration of the compound in which the maximum effect was observed in 50% of cases. Concentration is preferably expressed in moles.

[0079] In biological systems, small changes in ligand concentration typically induce rapid changes in the response, following a sigmoid function. The inflection point at which the increase in response begins to slow down with increasing ligand concentration is EC50. 50 This can be determined mathematically by differentiating the best-fit line. In most cases, relying on graphs for estimation is convenient. EC is provided in the Examples section. 50 In this case, experiments are designed to reflect KD as accurately as possible. In other words, EC can then be considered... 50 The value is the KD value. The term "average KD" refers to the average KD value obtained from at least one, but preferably more than one, such as at least two experiments. The term "average" refers to the mathematical term "average" (the sum of the data divided by the number of terms in the data).

[0080] It is also related to the IC50, which is a measure of the compound's inhibition (50% inhibition). 50 Relevant. For competitive binding assays and functional antagonist assays, IC50 is... 50 It is the most commonly used summary measure of dose-response curves. For agonist / stimulant assays, the most commonly used summary measure is EC. 50 .

[0081] As used herein, the term "genetic fusion" refers to the coupling of individual nucleic acids encoding ISVD via amide bonds, wherein the nucleotide sequence encoding ISVD is coupled via its 3' terminal nucleic acid to the 5' terminal nucleic acid of another nucleotide sequence encoding ISVD via a phosphodiester bond, and, if suitable, via a (nucleic acid) linker of multiple lengths, for example, the nucleotide sequence encoding ISVD is coupled via its 3' terminal nucleic acid to the 5' terminal nucleic acid of a linker sequence via a phosphodiester bond, and the linker sequence is coupled via its 3' terminal nucleic acid to the 5' terminal nucleic acid of another nucleotide sequence encoding ISVD via a phosphodiester bond (i.e., ISVD and optional linker are genetically fused). Genetic fusion can be performed according to standard recombinant DNA protocols (see above) or according to the examples described, for example, in the Examples section of Garaicoechea et al. (2008, J Virol. 82: 9753-9764).

[0082] Based on the context, an amino acid sequence is interpreted as an unbranched sequence of a single amino acid or two or more amino acids. A nucleotide sequence is interpreted as an unbranched sequence of three or more nucleotides.

[0083] Amino acids are those L-amino acids commonly found in naturally occurring proteins and are listed in Table 1 below. This definition is not intended to include amino acid sequences containing D-amino acids. Any amino acid sequence containing post-translational modifications can be described as an amino acid sequence first translated using the symbols shown in the table below and having the modified position; for example, hydroxylation or glycosylation, but these modifications should not be explicitly shown in the amino acid sequence. This definition includes any peptide or protein that can be represented as a sequence modified by bonds, crosslinks and terminal caps, non-peptide bonds, etc.

[0084] Table 1: Common Amino Acids

[0085]

[0086] The terms “protein,” “peptide,” “protein / peptide,” and “polypeptide” are used interchangeably throughout this disclosure and each has the same meaning for the purposes of this disclosure. Each term refers to an organic compound consisting of a straight chain of two or more amino acids. Compounds may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids; two hundred or more amino acids; and even three hundred or more amino acids. Those skilled in the art will understand that although there is no art-recognized cutoff point for the number of amino acids that distinguishes polypeptides from proteins, polypeptides generally contain fewer amino acids than proteins; polypeptides can be prepared by chemical synthesis or recombinant methods; and proteins are generally prepared in vitro or in vivo by recombinant methods as known in the art.

[0087] To facilitate understanding of the invention, a brief discussion of the terminology used in conjunction with the invention will be provided. Conventionally, the amide bonds in the primary structure of a polypeptide are written in the order of the amino acids, where the amine terminus (N-terminus) of the polypeptide is always on the left and the acid terminus (C-terminus) is on the right.

[0088] The polypeptide of the present invention comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​residue preferably at the C-terminus. In its simplest form, the polypeptide of the present invention consists of an ISVD and subsequent (bound to or conjugated thereto) cysteine ​​residues.

[0089] The C-terminal extension is located at the C-terminus of the last amino acid residue (usually a serine residue) of the last (most C-terminal) ISVD, and contains a cysteine ​​residue. Preferably, the cysteine ​​portion of the present invention is present at or located at the C-terminus of the C-terminal extension.

[0090] In the context of this invention, the C-terminal extension consists of at least one amino acid, i.e., a cysteine ​​portion, or consists of an amino acid sequence of at least two amino acid residues to a maximum of 50 amino acid residues (preferably between 2 and 40 amino acid residues, such as between 2 and 30 amino acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 amino acid residues) containing at least one cysteine ​​residue present in or located at the C-terminus of the C-terminal extension. For example, the C-terminal extension can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid residues, wherein the amino acid at the C-terminus is a cysteine ​​moiety, such as, for example, the C-terminal extension consists only of cysteine ​​residues; for example, the C-terminal extension can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 amino acid residues followed by a cysteine ​​moiety; for example, the C-terminal extension can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 glycine residues followed by a cysteine ​​moiety; for example, the C-terminal extension can consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 alanine residues followed by a cysteine ​​moiety.

[0091] In another aspect, cysteine ​​residues are present or located at sites in the C-terminal extension that are different from the C-terminal end (C-terminus). For example, cysteine ​​residues are present or located at an amino acid residue preceding (upstream) the last amino acid residue of the C-terminal extension (i.e., the penultimate amino acid residue of the polypeptide of the present invention) or preceding (upstream) the last two amino acid residues of the C-terminal extension (i.e., the penultimate amino acid residue of the polypeptide of the present invention). For example, the C-terminal extension may consist of 2, 3, 4, 5, 6, 7, or 8 amino acid residues of cysteine ​​(e.g., glycine or alanine) of its first, second, third, fourth, fifth, sixth, or seventh amino acid residue (i.e., the penultimate amino acid residue of the polypeptide of the present invention); or the C-terminal extension may consist of 3, 4, 5, 6, 7, or 8 amino acid residues of cysteine ​​(e.g., glycine or alanine) of its first, second, third, fourth, fifth, or sixth amino acid residue (i.e., the penultimate amino acid residue of the polypeptide of the present invention).

[0092] Preferred examples of C-end extensions are given in Table 2.

[0093] Table 2: C-end Extension

[0094] 1 C 2 GC 3 GGC 4 GGGC 5 GGGGC 6 AC 7 AAC 8 AAAC 9 AAAAC 10 CG 11 GCG 12 GGCG 13 GGGCG 14 GGGGCG 15 GGGGCGGGG

[0095] In one embodiment, the present invention relates to a dimer as described herein, wherein the first polypeptide and / or the second polypeptide comprises a C-terminal extension of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue having a cysteine ​​motif preferably at the C-terminus. In one embodiment, the present invention relates to a dimer as described herein, wherein the C-terminal extension comprises GlyGlyGlyCys (SEQ ID NO:4), GlyGlyCys (SEQ ID NO:3), GlyCys (SEQ ID NO:2), or Cys (SEQ ID NO:1).

[0096] In one embodiment, the present invention relates to dimers as described herein, wherein the polypeptide comprises a C-terminal extension selected from the group consisting of SEQ ID NO:1-15.

[0097] C-terminal extensions can be coupled to ISVDs using any suitable technique known to those skilled in the art, such as, for example, the recombinant DNA technique described above for genetic fusion.

[0098] The polypeptides of the present invention may contain more than one ISVD, such as two, three, four, or even more ISVDs. Therefore, the present invention relates to polypeptides of the present invention containing at least two ISVDs. Furthermore, the present invention relates to dimers comprising a first polypeptide and a second polypeptide as described herein, wherein the first polypeptide contains at least two ISVDs and / or the second polypeptide contains at least two ISVDs.

[0099] The ISVDs contained in the polypeptides of the present invention may be the same or different. In one embodiment, the ISVDs may bind to the same target, regardless of whether the ISVDs are the same or different. Therefore, the present invention relates to polypeptides of the present invention that respectively contain the same ISVD, ISVDs binding to the same target, and / or ISVDs containing the same CDR1, CDR2, and CDR3. In one embodiment, the ISVDs may bind to different targets. In one embodiment, the present invention relates to a dimer comprising a first polypeptide and a second polypeptide as described herein, wherein the at least two ISVDs of the first polypeptide are the same and / or the at least two ISVDs of the second polypeptide are the same.

[0100] In the polypeptides of the present invention, whether or not they are included in the dimers of the present invention, they can be directly linked or linked to ISVDs via a connector.

[0101] Relative affinity can depend on the position of the ISVD in the peptide. It should be understood that the order (orientation) of the ISVDs in the peptides of this invention can be selected according to the needs of those skilled in the art. The order of individual ISVDs and whether the peptide contains a linker are design choices. Some orientations (with or without a linker) can provide preferred binding characteristics compared to others. Additionally, the order of the first ISVD (e.g., ISVD 1) and the second ISVD (e.g., ISVD 2) in the peptides of this invention can be (from N-terminus to C-terminus): (i) ISVD 1 (e.g., nanobody 1) - [linker] - ISVD 2 (e.g., nanobody 2); or (ii) ISVD 2 (e.g., nanobody 2) - [linker] - ISVD 1 (e.g., nanobody 1); (wherein the linker is optional). All orientations are included in this invention. Peptides containing ISVD orientations that provide the desired binding characteristics can be readily identified by conventional screening, for example, as illustrated in the Examples section.

[0102] In the polypeptides of the present invention, two or more ISVDs, such as nanobodies, may be directly linked to each other (e.g., as described in WO99 / 23221) and / or may be linked to each other via one or more suitable adapters, or any combination thereof. Suitable adapters for the polypeptides of the present invention will be apparent to those skilled in the art and may generally be any adapter used in the art for linking amino acid sequences. Preferably, the adapters are suitable for use in constituting proteins or polypeptides intended for pharmaceutical use.

[0103] Some particularly preferred adapters include those used in the art for linking antibody fragments or antibody domains. These include adapters mentioned in the publications cited above, as well as adapters used in the art, for example, for constructing biantibodies or ScFv fragments (however, for this purpose, it should be noted that, although the adapter sequence used in biantibodies and ScFv fragments should have a permissible V...). H and V L The length, flexibility, and other properties of the domains together to form a complete antigen-binding site are not particularly limited to the length or flexibility of the linkers used in the peptides of this invention, since each ISVD, such as a nanobody, itself forms a complete antigen-binding site.

[0104] For example, the linker can be a suitable amino acid or amino acid sequence, and especially between 1 and 50, preferably between 1 and 30, such as an amino acid sequence of 1 to 10 amino acid residues. Some preferred examples of such amino acid sequences include gly-ser linkers, such as type (gly x ser y ) z Examples of preferred linkers include (e.g., (gly4ser)3 or (gly3ser2)3, as described in WO 99 / 42077, and the GS30, GS15, GS9, and GS7 linkers described in the Ablynx applications mentioned herein (see, for example, WO 06 / 040153 and WO 06 / 122825), as well as hinge-like regions, such as naturally occurring heavy chain antibodies or hinge regions of similar sequences (as described in WO 94 / 04678). Preferred linkers are described in Table 3.

[0105] Some other particularly preferred connectors are polyalanine (such as AAA), as well as connectors GS30 (SEQ ID NO:85 in WO 06 / 122825) and GS9 (SEQ ID NO:84 in WO 06 / 122825).

[0106] Including within the scope of this invention, the length, flexibility, and / or other properties of one or more linkers used (though not critical, as they are generally specific to linkers used in ScFv fragments) may have some influence on the properties of the final peptides and / or dimers of this invention, including but not limited to affinity, specificity, or antibody affinity for chemokines or one or more other antigens. Based on the disclosure herein, those skilled in the art will be able to determine one or more optimal linkers for use in the specific peptides and / or dimers of this invention, optionally after some limited routine experiments.

[0107] When more than two linkers are used in the polypeptides of the present invention, these linkers may be the same or different. Similarly, based on the disclosure herein, those skilled in the art will be able to determine the optimal linker for use in the specific polypeptides of the present invention after some optional limited routine experiments.

[0108] In the polypeptides of the present invention, the ISVD may precede the N-terminal extension. In the context of the present invention, the N-terminal extension consists of an amino acid sequence of at least one amino acid residue to a maximum of 40 amino acid residues (preferably between 2 and 30 amino acid residues, such as between 2 and 20 amino acid residues, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues). The N-terminal extension is present at the N-terminus of the first (i.e., the N-terminal, typically designated as amino acid 1 according to Kabat numbering) amino acid residue of the first (i.e., the N-terminal) ISVD in the polypeptides of the present invention. Therefore, the present invention relates to a first polypeptide comprising an N-terminal extension and / or a second polypeptide.

[0109] In one embodiment, the present invention relates to dimers as described herein, wherein at least two ISVDs of the first polypeptide are identical and / or at least two ISVDs of the second polypeptide are identical.

[0110] In one embodiment, the dimer of the first polypeptide of the present invention and the second polypeptide of the present invention are different.

[0111] In one embodiment, the first polypeptide and the second polypeptide of the present invention used to prepare the dimer are identical. Therefore, the first polypeptide and the second polypeptide of the present invention are identical.

[0112] Table 3: Some connector sequences of the present invention

[0113] A3 16 AAA GS5 (5GS) 17 GGGGS GS7 (7GS) 18 SGGSGGS GS9 (9GS) 19 GGGGSGGGS GS10 (10GS) 20 GGGGSGGGGS GS15 (15GS) 21 GGGGSGGGGSGGGGS GS18 (18GS) 22 GGGGSGGGGSGGGGGGGGS GS20 (20GS) 23 GGGGSGGGGSGGGGSGGGGS GS25 (25GS) 24 GGGGSGGGGSGGGGSGGGGSGGGGS GS30 (30GS) 25 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS GS35 (35GS) 26 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS

[0114] As further explained below, ISVD can originate from V HH V H or VL Domains, however, are selected such that they do not form V in the peptides or dimers of the present invention. H and V L Complementary pairs of structural domains. The nanobody, V HH and humanized V HH What's unusual is that they originate from antibodies from natural camelids that lack light chains, and in fact, these domains cannot bind to camelid light chains to form complementary V. HH and V L Yes. Therefore, the dimers and peptides of the present invention do not contain complementary ISVDs and / or form complementary ISVD pairs, such as, for example, complementary V H / V L right.

[0115] In one embodiment, the present invention relates to dimers as described herein, wherein the connector is selected from the group consisting of SEQ ID NO:16-26.

[0116] Monovalent peptides contain only one binding unit (e.g., an immunoglobulin monovariable domain) or are substantially composed of it. Peptides containing two or more binding units (e.g., immunoglobulin monovariable domains) will also be referred to herein as “multivalent” peptides, and the binding units / immunoglobulin monovariable domains present in such peptides will also be referred to herein as “multivalent forms.” For example, a “bivalent” peptide may contain two immunoglobulin monovariable domains optionally linked by one adapter sequence, while a “trivalent” peptide may contain three immunoglobulin monovariable domains optionally linked by two adapter sequences; and a “tetravalent” peptide may contain four immunoglobulin monovariable domains optionally linked by three adapter sequences, and so on.

[0117] In a multivalent polypeptide, two or more immunoglobulin monovariable domains may be the same or different, and may target the same antigen or antigenic determinant (e.g., targeting the same one or more parts or one or more epitopes or targeting different parts or epitopes) or alternatively target different antigens or antigenic determinants; or any suitable combination thereof. A polypeptide containing at least two binding units (e.g., immunoglobulin monovariable domains) of a first antigen targeting a first target and at least one binding unit targeting a second target (e.g., different from the first target) will also be referred to as a “multispecific” polypeptide, and the binding units present in such a polypeptide (e.g., immunoglobulin monovariable domains) will also be referred to herein as “multispecific forms.” Thus, for example, the “bispecific” polypeptide of the present invention is a polypeptide comprising at least one immunoglobulin monovariable domain targeting a first antigen targeting a first target and at least one additional immunoglobulin monovariable domain targeting a second antigen (i.e., different from the first target).

[0118] A "multi-complementary-site polypeptide," such as, for example, a "bi-complementary-site polypeptide" or a "triple-complementary-site polypeptide," comprises or is substantially composed of two or more binding units, each having a different complementary site. In another aspect, the polypeptide of the present invention is a multi-complementary-site polypeptide (also referred to herein as "one or more multi-complementary-site polypeptides of the present invention"), such as, for example, "one or more bi-complementary-site polypeptides of the present invention" or "one or more triple-complementary-site polypeptides of the present invention." As used herein, the terms "multi-complementary-site (antigen) binding molecule" or "multi-complementary-site" polypeptide shall mean a polypeptide comprising at least two (i.e., more than two) immunoglobulin monovariable domains, wherein a "first" immunoglobulin monovariable domain is directed to a first target and a "second" immunoglobulin monovariable domain is directed to the same first target, wherein the "first" and "second" immunoglobulin monovariable domains have different complementary sites. Therefore, the multi-complementary polypeptide comprises or is composed of two or more immunoglobulin monovariable domains targeting a first target, wherein at least one "first" immunoglobulin monovariable domain targets a first epitope on the first target and at least one "second" immunoglobulin monovariable domain targets a second epitope on the first target that is different from the first epitope on the first target.

[0119] In one embodiment, the present invention relates to dimers as described herein, wherein the first polypeptide and / or the second polypeptide is selected from the group consisting of monovalent polypeptides, divalent polypeptides, multivalent polypeptides, single-specific polypeptides, bispecific polypeptides, and multispecific polypeptides.

[0120] As used herein, the “target” of the present invention is any suitable antigen (e.g., any target) to which the ISVD can bind. For example, the ISVD of the present invention can bind to or target antigenic determinants, epitopes, portions, domains, subunits, or conformations (if applicable) of a target (e.g., receptor tyrosine kinase (RTK) or G protein-coupled receptor (GPCR) (e.g., involved in malignancy). The targets of the present invention can be any targets, preferably on the cell surface, such as those known to be involved in malignancy, such as cellular receptors. For example, receptor tyrosine kinases (RTKs) and RTK-mediated signaling pathways are involved in tumorigenesis, maintenance, angiogenesis, and angiogenesis. Approximately 20 different RTK types have been identified, among which the most extensively studied are: 1. Type I RTK (EGF receptor family) (ErbB family), 2. Type II RTK (insulin receptor family), 3. Type III RTK (PDGF receptor family), 4. Type IV RTK (FGF receptor family), 5. Type V RTK (VEGF receptor family), 6. Type VI RTK (HGF receptor family), 7. Type VII RTK (Trk receptor family), 8. Type VIII RTK (Eph receptor family), 9. Type IX RTK (AXL receptor family), 10. Type X RTK (LTK receptor family), 11. Type XI RTK (TIE receptor family), 12. Type XII RTK (ROR receptor family), 13. Type XIII RTK (DDR receptor family), 14. Type XIV RTK (RET receptor family), 15. Type XV RTK (KLG receptor family), 16. Type XVI RTK (RYK receptor family), and 17. Type XVII RTK (MuSK receptor family). In particular, targets such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), c-Met, HER3, plexin, integrin, CD44, RON, and receptors of mammalian targets involved in pathways such as the Ras / Raf / mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI3K) / Akt / mammalian target pathway.

[0121] Therefore, the present invention relates to dimers as described herein, wherein the first target and the second target are independently selected from the group consisting of: GPCRs, receptor tyrosine kinases, DDR1, cyclophosphamide I (CD167a antigen), DDR2, ErbB-1, C-erbB-2, FGFR-1, FGFR-3, CD135 antigen, CD 117 antigen, protein tyrosine kinase-1, c-Met, CD148 antigen, C-ret, ROR1, ROR2, Tie-1, Tie-2, CD202b antigen, Trk-A, Trk-B, Trk-C, VEGFR-1, VEGFR-2, VEGFR-3, Notch receptors 1-4, FAS receptor, DR5, DR4, CD47, CX3CR1, CXCR-3, CXCR-4, CXCR-7, chemokine-binding protein 2, and CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, and CCR11; MART-1, carcinoembryonic antigen (“CEA”), gp100, MAGE-1, HER-2, and Lewis Y Antigens, CD123, CD44, CLL-1, CD96, CD47, CD32, CXCR4, Tim-3, CD25, TAG-72, Ep-CAM, PSMA, PSA, GD2, GD3, CD4, CD5, CD19, CD20, CD22, CD33, CD36, CD45, CD52, and CD147; growth factor receptors, including ErbB3 and ErbB4; and cytokine receptors, including interleukin-2 receptor γ chain (CD132 antigen); interleukin-10 receptor α chain (IL-10R-A); interleukin-10 receptor β chain (IL-10R-B); interleukin-12 receptor β-1 chain (IL-12R-β1); and interleukin-12 receptor β-2 chain (IL-12 receptor β-2). Interleukin-13 receptor α-1 chain (IL-13R-α-1) (CD213a1 antigen); Interleukin-13 receptor α-2 chain (interleukin-13 binding protein); Interleukin-17 receptor (IL-17 receptor); Interleukin-17B receptor (IL-17B receptor); Interleukin-21 receptor precursor (IL-21R); Interleukin-1 receptor, type I (IL-1R-1) (CD121a); ​​Interleukin-1 receptor, type II (IL-1R-β) (CDw121b); Interleukin-1 receptor antagonist protein (IL-1ra); Interleukin-2 receptor α chain (CD25 antigen); Interleukin-2 receptor β chain (CD122 antigen); Interleukin-3 receptor α chain (IL-3R-α) (CD123 antigen).

[0122] Exemplary molecular targets (e.g., antigens) include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, and CD52; ErbB receptor family members such as EGF receptors (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3), or HER4 (ErbB4) receptors; macrophage receptors such as CRIg; tumor necrosis factors such as TNFα or TRAIL / Apo-2; and cell adhesion molecules such as LFA-1, Mad, p150, p95, VLA-4, ICAM-1, VCAM, and ανβ3 integrin (including its α or β). β subunit; growth factors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE; cytokines such as IL1; cytokine receptors such as IL2 receptor; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptors; mpl receptors; CTLA-4; protein C; neutropilin; ephrin and receptors; netrin and receptors; slit and receptors; chemokines and chemokine receptors such as CCL5, CCR4, CCR5; amyloid β; complement factors such as complement factor D; lipoproteins such as oxidized LDL (oxLDL); lymphotoxins such as lymphotoxin α (LTa). Other molecular targets include Tweak, B7RP-1, proprotein convertase subtilisin / kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

[0123] It is also anticipated that the immunoglobulin single variable domains, peptides and / or dimers of the present invention will generally bind to all naturally occurring or synthetic analogs, variants, mutants, alleles, portions and fragments of their targets.

[0124] Therefore, the present invention relates to dimers as described herein, wherein the ISVD of the first polypeptide binds to a first target and / or the ISVD of the second polypeptide binds to a second target.

[0125] Therefore, the present invention relates to dimers as described herein, wherein the first polypeptide binds to a first target:

[0126] - ICs with a maximum 100nM, such as 50nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, preferably even up to 2nM, such as 1nM. 50 Measured by competitive FACS;

[0127] -with 10 -5 Up to 10 -12 Below mol / L, and preferably 10 -7 Up to 10 -12 Below 10 mol / L and more preferably 10 -8 Up to 10 -12 dissociation constant (K) per mole / liter D );

[0128] -has in 10 2 M -1 s -1 To about 10 7 M -1 s -1 Between, 10 are preferred 3 M -1 s -1 Up to 10 7 M -1 s -1 Between, preferably 10 4 M -1 s -1 Up to 10 7 M -1 s -1 Between, such as 10 5 M -1 s -1 Up to 10 7 M -1 s -1 The binding rate between them (k) on (rate); and / or

[0129] -has in 1s -1 Up to 10 -6 s -1 Between, 10 are preferred -2 s -1 Up to 10 -6 s -1 Between, preferably 10 -3 s -1 Up to 10 -6 s -1 Between, such as 10 -4 s -1 Up to 10 -6 s -1 Dissociation rate between (k) off rate).

[0130] Therefore, the present invention relates to dimers as described herein, wherein the second polypeptide binds to a second target:

[0131] - ICs with a maximum 100nM, such as 50nM, 20nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, preferably even up to 2nM, such as 1nM. 50 Measured by competitive FACS;

[0132] -with 10 -5 Up to 10 -12 Below mol / L, and preferably 10 -7 Up to 10 -12 Below 10 mol / L and more preferably 10 -8 Up to 10 -12 dissociation constant (K) per mole / liter D );

[0133] -has in 10 2 M -1 s -1 To about 10 7 M -1 s -1 Between, 10 are preferred 3 M -1 s -1 Up to 10 7 M -1 s -1 Between, preferably 10 4 M -1 s -1 Up to 10 7 M -1 s -1 Between, such as 10 5 M -1 s -1 Up to 10 7 M -1 s -1 The binding rate between them (k) on (rate); and / or

[0134] -has in 1s -1 Up to 10 -6 s -1 Between, 10 are preferred -2 s -1 Up to 10 -6 s -1 Between, preferably 10 -3 s -1 Up to 10 -6 s -1 Between, such as 10 -4 s -1 Up to 10 -6 s -1 Dissociation rate between (k) off rate).

[0135] In one embodiment, the present invention relates to dimers as described herein, wherein the first target and the second target are different.

[0136] In one embodiment, the present invention relates to dimers as described herein, wherein the first target and the second target are identical.

[0137] Unless otherwise indicated, the term "immunoglobulin sequence" is used as a general term, whether referring to heavy chain antibodies or conventional 4-chain antibodies in this document, to include full-size antibodies, their individual chains, and all their portions, domains, or fragments (including, but not limited to, antigen-binding domains or fragments such as V...). HH Domain or V H / V L Domain). Furthermore, the term "sequence" as used herein (e.g., in phrases such as "immunoglobulin sequence," "antibody sequence," "variable domain sequence," "V...") HH In the term “sequence” or “protein sequence”, it should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence that encodes it, unless the context requires a more restrictive interpretation.

[0138] Immunoglobulin monovariable domains can be used as “binding units,” “binding domains,” or “building units” (these terms are used interchangeably) for preparing peptides containing one or more additional immunoglobulin monovariable domains that can act as binding units (i.e., the same or different epitopes for the same target and / or for one or more different targets).

[0139] The term "immunoglobulin single variable domain" ("ISVD"), used interchangeably with "single variable domain" ("SVD"), defines a molecule in which the antigen-binding site is located on and formed by a single immunoglobulin domain. This distinguishes an immunoglobulin ISVD from "conventional" immunoglobulins or fragments thereof, where two immunoglobulin domains, specifically two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy chain variable domain (VH) and the light chain variable domain (VL) interact to form the antigen-binding site. In this case, the complementarity-determining regions (CDRs) of both the VH and VL will favor the antigen-binding site, meaning a total of six CDRs will be involved in the formation of the antigen-binding site.

[0140] In contrast, the binding site of the immunoglobulin monovariable domain is changed from a single V... H or V LDomain formation. Therefore, the antigen-binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0141] The terms "immunoglobulin single variable domain" and "single variable domain" therefore do not include conventional immunoglobulins or fragments thereof that require at least two variable domains to interact to form an antigen-binding site. However, these terms do include fragments of conventional immunoglobulins in which an antigen-binding site is formed via a single variable domain.

[0142] Typically, a single variable domain will be an amino acid sequence consisting of approximately four framework regions (FR1 to FR4) and three complementarity-determining regions (CDR1 to CDR3). Such single variable domains and fragments are most preferred, as they contain immunoglobulin folds or are capable of forming immunoglobulin folds under suitable conditions. Therefore, a single variable domain may, for example, contain a light chain variable domain sequence (e.g., V...). L Sequences) or suitable fragments thereof; or heavy-chain variable domain sequences (e.g., V H Sequence or V HH (sequence) or a suitable fragment thereof; provided that it can form a single antigen-binding unit (i.e., a functional antigen-binding unit consisting essentially of a single variable domain, such that the single antigen-binding unit does not need to interact with another variable domain to form a functional antigen-binding unit, for example, for those present in, for example, conventional antibodies and scFv fragments, requiring, for example, via V H / V L This is the case for a variable domain that interacts with another variable domain to form a functional antigen-binding domain.

[0143] In one embodiment of the invention, the immunoglobulin monovariable domain is a light chain variable domain sequence (e.g., V). L Sequence) or heavy-chain variable structural domain sequence (e.g., V) H (Sequence); More specifically, the immunoglobulin single variable domain can be a heavy chain variable domain sequence derived from a conventional four-chain antibody or a heavy chain variable domain sequence derived from a heavy chain antibody.

[0144] For example, a single variable domain or immunoglobulin single variable domain (or an amino acid suitable for use as an immunoglobulin single variable domain) can be a (single) domain antibody (or an amino acid suitable for use as a (single) domain antibody), a "dAb" or dAb (or an amino acid suitable for use as a dAb), or a nanobody (as defined herein, and including but not limited to V). HH ); other single variable structural domains, or any suitable fragment of any of them.

[0145] For a general description of (single)domain antibodies, reference is also made to the prior art cited herein and to EP0368684. The term “dAb” is used, for example, in Ward et al., 1989 (Nature 341:544-546), Holt et al., 2003 (Trends Biotechnol. 21:484-490); and to other published patent applications such as WO 04 / 068820, WO 06 / 030220, WO06 / 003388, WO 06 / 059108, WO 07 / 049017, WO 07 / 085815 and Domantis Ltd. It should also be noted that, although less preferred in the context of this invention because they are not of mammalian origin, single variable domains can be derived from certain species of sharks (e.g., the so-called “IgNAR domain,” see, for example, WO 05 / 18629).

[0146] In particular, immunoglobulin single variable domains can be (as defined in this document) or its appropriate fragment. [Note:] and [This is a registered trademark of Ablynx NV.] A general description of nanobodies is given by reference to the following further description, as well as to prior art referenced herein, such as, for example, that described in WO 08 / 020079 (page 16).

[0147] For V HHFor a further description of nanobodies, refer to the review article in Muyldermans 2001 (Reviews in Molecular Biotechnology) 74:277-302, and refer to the following patent applications mentioned as general background art: Vrije Universiteit Brussel WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103; Unilever WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193; Vlaams Instituutvoor Biotechnologie (VIB) WO 97 / 49805, WO WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527; WO 03 / 050531 of Algonomics NV and Ebolings Inc.; WO 01 / 90190 of the National Research Council of Canada; WO 03 / 025020 of the Institute of Antibodies; and WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551, WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO The patent applications 06 / 122825 and Ebolings Inc. are also referenced. Further prior art mentioned in these applications is also referenced, and in particular the list of references mentioned on pages 41-43 of International Application WO 06 / 040153, which, together with the references, is incorporated herein by reference. As described in these references, nanobodies (especially VHH sequences and partially humanized nanobodies) may be characterized, in particular, by the presence of one or more “marker residues” in one or more framework sequences.Further descriptions of nanobodies can be found, for example, in WO 08 / 101985 and WO 08 / 142164, including humanization and / or camelification of nanobodies, as well as other modifications, portions or fragments, derivatives or “nanobody fusions,” multivalent constructs (including some non-limiting examples of linker sequences), and various modifications to increase the half-life of nanobodies and their preparation.

[0148] Therefore, in the sense of this invention, the term "immunoglobulin single variable domain" or "single variable domain" includes polypeptides derived from non-human, preferably camelid, and more preferably camelid heavy chain antibodies. As previously described, they may be humanized. Furthermore, the term includes polypeptides derived from non-camelid sources such as mice or humans that have been "camelized," as described in Davies and Riechmann 1994 (FEBS 339:285-290), 1995 (Biotechnol. 13:475-479), 1996 (Prot. Eng. 9:531-537) and Riechmann and Muyldermans 1999 (J. Immunol. Methods 231:25-38).

[0149] The term "immunoglobulin single variable domain" encompasses immunoglobulin sequences from various sources, including mouse, rat, rabbit, donkey, human, and cameloid immunoglobulin sequences. It also includes fully human, humanized, or chimeric immunoglobulin sequences. For example, it includes cameloid immunoglobulin sequences and humanized cameloid immunoglobulin sequences, or camel-derived immunoglobulin single variable domains, such as camel-derived dAb (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995, and 1996) and camel-derived VH, as described by Ward et al., 1989 (see, for example, WO 94 / 04678 and Davies and Riechmann 1994, 1995, and 1996).

[0150] Similarly, such immunoglobulin single variable domains can be derived from any suitable source in any suitable manner, and can, for example, be naturally occurring V. HH Sequences (i.e., derived from suitable camel species) or synthetic or semi-synthetic amino acid sequences, including but not limited to partially or fully "humanized" V. HH "Camel-derived" immunoglobulin sequences (especially camel-derived V) H ), and nanobodies and / or V obtained through the following techniques HH Such as affinity maturation (e.g., from synthetic, random, or naturally occurring immunoglobulin sequences like V). HH(sequence start), CDR transplantation, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques known to the art for modifying immunoglobulin sequences; or any suitable combination of any of the foregoing.

[0151] The amino acid sequence and structure of an immunoglobulin single variable domain can be considered—however, not limited to—to consist of four framework regions or “FRs,” referred in the art and herein as “framework region 1” or “FR1”; “framework region 2” or “FR2”; “framework region 3” or “FR3”; and “framework region 4” or “FR4”, respectively; said framework regions are separated by three complementarity-determining regions or “CDRs,” referred in the art as “complementarity-determining region 1” or “CDR1”; “complementarity-determining region 2” or “CDR2”; and “complementarity-determining region 3” or “CDR3”, respectively.

[0152] The total number of amino acid residues in the immunoglobulin single variable domain can be in the range of 110-120, preferably 112-115, and most preferably 113.

[0153] As further described in paragraphs q) on pages 58 and 59 of WO 08 / 020079 (incorporated herein by reference), the amino acid residues of the immunoglobulin monovariable domain are numbered according to the general numbering for the VH domain given by Kabat et al. (“Kabat Numbering”) (“Sequence of proteins of immunological interest”, US Public Health Services, NIH Bethesda, MD, Publication No. 91), as in Riechmann and Muyldermans 2000 (J. Immunol. Methods 240:185-195; see, for example, that publication). Figure 2 The article applies to the VHH domain of camels, and accordingly, the FR1 of the immunoglobulin monovariable domain contains amino acid residues at positions 1-30, the CDR1 of the immunoglobulin monovariable domain contains amino acid residues at positions 31-35, the FR2 of the immunoglobulin monovariable domain contains amino acid residues at positions 36-49, the CDR2 of the immunoglobulin monovariable domain contains amino acid residues at positions 50-65, the FR3 of the immunoglobulin monovariable domain contains amino acid residues at positions 66-94, the CDR3 of the immunoglobulin monovariable domain contains amino acid residues at positions 95-102, and the FR4 of the immunoglobulin monovariable domain contains amino acid residues at positions 103-113.

[0154] Based on the examples of immunoglobulin monovariable domain (CDR) sequences given herein and in WO 08 / 020079, WO 06 / 040153, and in other references cited therein concerning immunoglobulin monovariable domains, it will be clear that the precise number of amino acid residues will also depend on the length of the specific CDR present in the immunoglobulin monovariable domain. Regarding CDRs, as is well known in the art, there are various definitions and conventions for describing CDRs of VH or VHH fragments, such as the Kabat definition (which is based on sequence variability and is the most commonly used) and the Chothia definition (which is based on the position of the structural loop region). See, for example, http: / / www.bioinf.org.uk / abs / . For the purposes of this specification and claims, although reference may be made to CDRs according to Kabat, it is most preferably to define CDRs based on the Abm definition (which is based on the AbM antibody modeling software of OxfordMolecular), as this is considered the best compromise between the Kabat and Chothia definitions. See also the website http: / / www.bioinf.org.uk / abs / .

[0155] In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS, i.e., each of positions 109, 110, 111, 112, and 113. The invention also includes an ISVD terminated at position 109, 110, 111, or 112. In one aspect of the invention, FR4 terminates with the C-terminal amino acid sequence VTVS (positions 109-112), FR4 terminates with the C-terminal amino acid sequence VTV (positions 109-111), FR4 terminates with the C-terminal amino acid sequence VT (positions 109-110), or FR4 terminates with the C-terminal amino acid V (position 109). The C-terminal extension may be present at the C-terminus of the last amino acid residue of FR4 in the last (most C-terminal) ISVD, such as V109, T110, V111, S112, or S113, wherein the cysteine ​​moiety of the invention is preferably present or located at the C-terminus of the C-terminal extension. In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVSS and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVSSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTVS and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVSC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VTV and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTVC). In one embodiment, FR4 comprises the C-terminal amino acid sequence VT and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VTC). In one embodiment, FR4 comprises the C-terminal amino acid V and the C-terminal extension is a cysteine ​​residue (e.g., the polypeptide of the present invention terminates with VC).

[0156] In one embodiment, the present invention relates to dimers as described herein, wherein ISVD is a light chain variable domain sequence (VL) and a heavy chain variable domain sequence (VH), derived from conventional tetrachain antibodies or derived from heavy chain antibodies.

[0157] In one embodiment, the present invention relates to dimers as described herein, wherein the ISVD is selected from the group consisting of: single-domain antibodies, domain antibodies, amino acid sequences suitable for use as single-domain antibodies, amino acid sequences suitable for use as domain antibodies, dAbs, amino acid sequences suitable for use as dAbs, nanobodies, VHHs, humanized VHHs, and camel-derived VHs. Preferably, the ISVD comprises between 100 and 140 amino acids, such as between 110 and 130 amino acids.

[0158] In one embodiment, the present invention relates to a dimer as described herein, wherein the ISVD is selected from the group consisting of nanobodies, VHH, humanized VHH, and camel-derived VH, and comprises between 105 and 125 amino acids, preferably between 110 and 120 amino acids, such as 110, 111, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119 or 120 amino acids, most preferably 113 amino acids.

[0159] This invention relates to dimers as described herein, wherein the ISVD selected from the group consisting of nanobodies, VHH, humanized VHH, and camel-derived VH terminates at amino acid positions 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 according to the Kabat number, preferably at amino acid position 113.

[0160] This invention relates to dimers as described herein, wherein the ISVD is selected from the group consisting of: single-domain antibodies, domain antibodies, amino acid sequences suitable for use as single-domain antibodies, amino acid sequences suitable for use as domain antibodies, dAbs, amino acid sequences suitable for use as dAbs, and camel-derived VHs, wherein the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, amino acid sequence suitable for use as a dAb, and camel-derived VHs are derived from VHs.

[0161] This invention relates to dimers as described herein, wherein the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, amino acid sequence suitable for use as a dAb, and camel-derived VH contain 110-130 amino acids, preferably 115-127 amino acids, such as 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, or 127 amino acids, most preferably 123 amino acids. Preferably, the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, amino acid sequence suitable for use as a dAb, and camel-derived VH terminate at amino acid 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, or 130 according to Kabat numbering, preferably at amino acid 123.

[0162] Therefore, the present invention relates to dimers as described herein, wherein the ISVD is selected from the group consisting of: single-domain antibodies, domain antibodies, amino acid sequences suitable for use as single-domain antibodies, amino acid sequences suitable for use as domain antibodies, dAbs, and amino acid sequences suitable for use as dAbs, wherein the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, or amino acid sequence suitable for use as a dAb is derived from VL. Preferably, the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, and amino acid sequence suitable for use as dAb contain 100-120 amino acids, preferably 105-115 amino acids, such as 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 amino acids, most preferably 108 amino acids. Preferably, the single-domain antibody, domain antibody, amino acid sequence suitable for use as a single-domain antibody, amino acid sequence suitable for use as a domain antibody, dAb, and amino acid sequence suitable for use as dAb terminate at amino acid 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, or 120 according to Kabat numbering, preferably at amino acid 108.

[0163] In specific aspects of the invention, the polypeptides or dimers of the invention may have an increased half-life compared to the corresponding polypeptides or dimers of the invention. Based on the further disclosure herein, some preferred, but non-limiting, examples of such polypeptides or dimers will become apparent to those skilled in the art, including, for example, polypeptides of the invention that have been chemically modified to increase their half-life (e.g., by means of polyethylene glycolation); polypeptides of the invention that contain at least one additional binding site for binding to serum proteins (such as serum albumin); or polypeptides of the invention that contain at least one portion of a polypeptide of the invention linked to increase the half-life of the polypeptide of the invention.

[0164] According to a specific, preferred, but non-limiting aspect of the invention, in addition to one or more immunoglobulin monovariable domains targeting epitopes on target cells, the polypeptides of the invention may also contain at least one immunoglobulin monovariable domain targeting human serum albumin. These immunoglobulin monovariable domains targeting human serum albumin may be those commonly described in applications of Eberlinx Inc. cited herein (see, for example, WO04 / 062551). Some particularly preferred ISVDs, such as nanobodies, that provide increased half-life and can be used in the peptides of the present invention include the ISVDs disclosed in WO 06 / 122787 (see Tables II and III), such as nanobodies ALB-1 to ALB-10 (of which ALB-8 (SEQ ID NO: 62 in WO06 / 122787) is particularly preferred), as well as the ISVDs disclosed in WO 2012 / 175400, such as nanobodies (SEQ ID NO: 1-11 in WO 2012 / 175400), and the ISVD with SEQ ID NO: 109 disclosed in co-pending U.S. Provisional Application No. 62 / 047,560 entitled “Improved immunoglobulin singlevariable domains” (filed: September 8, 2014; assigned to: Eberlinx, Inc.), such as nanobodies.

[0165] In another aspect, the present invention relates to dimers as described herein, wherein the first polypeptide and / or the second polypeptide further comprises one or more other groups, residues, portions, or binding units (as further defined herein), wherein the one or more other groups, residues, portions, or binding units increase the half-life of the dimer (compared to a dimer lacking the one or more other groups, residues, portions, or binding units). Preferably, the one or more other groups, residues, portions, or binding units that increase the half-life of the dimer are ISVDs that increase the half-life of the dimer.

[0166] In one embodiment, the present invention relates to a dimer as described herein, wherein the ISVD that increases the half-life of the dimer binds to serum albumin, preferably human serum albumin, or serum immunoglobulin, preferably human IgG.

[0167] In one embodiment, the present invention relates to a dimer as described herein, having a serum half-life at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times greater than the corresponding dimer of the ISVD without the increased half-life of the dimer.

[0168] In one embodiment, the present invention relates to a dimer as described herein, having a serum half-life that is increased by more than 1 hour, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding ISVD for increasing the half-life of the dimer.

[0169] In one embodiment, the present invention relates to a dimer as described herein, having a serum half-life in humans of at least about 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 72 hours or more; for example, at least 5 days (e.g., about 5 to 10 days), preferably at least 9 days (e.g., about 9 to 14 days), more preferably at least about 10 days (e.g., about 10 to 15 days), or at least about 11 days (e.g., about 11 to 16 days), more preferably at least about 12 days (e.g., about 12 to 18 days or more), or more than 14 days (e.g., about 14 to 19 days).

[0170] In a particularly preferred but non-limiting aspect of the invention, the invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD); and further comprising one or more (preferably one) serum albumin-binding immunoglobulin single variable domains as described herein, such as those selected from SEQ ID NO:32-44, such as Alb11, Alb23, Alb129, Alb132, Alb8, Alb11(S112K)-A, Alb82, Alb82-A, Alb82-AA, Alb82-AAA, Alb82-G, Alb82-GG, Alb82-GGG (see Table 10).

[0171] Table 10: Serum albumin bound to ISVD sequences (“ID” refers to SEQ ID NO as used herein)

[0172]

[0173] ISVDs, such as nanobodies, contain highly conserved internal (also known as canonical or intramolecular) disulfide bonds. Removal of these specific internal disulfide bonds impairs the activity of ISVDs.

[0174] The inventors of this invention unexpectedly observed that oxidizing the thiol moiety (-SH) of an unpaired cysteine ​​residue (abbreviated as Cys, cys, or C; 2-amino-3-mercaptopropionic acid; an α-amino acid having the chemical formula HO2CCH(NH2)CH2SH) located in the C-terminal extension of the first polypeptide of this invention, preferably at the C-terminus, and the thiol moiety of an unpaired cysteine ​​residue located in the C-terminal extension of the second polypeptide of this invention, preferably at the C-terminus, yields a disulfide derivative cysteine, thereby preparing a dimer, but in which the intramolecular thiol moiety does not react. In other words, as demonstrated in the Examples section, without any abnormalities or re-oxidation of the intramolecular thiol group, the thiol group of the C-terminal cysteine ​​is specifically oxidized to form an intermolecular bond, thereby maintaining the integrity of the ISVD. Coupling of the polypeptide into the dimer is achieved by chemical conjugation, wherein the thiol moiety of the cysteine ​​in the C-terminal extension of each of the two polypeptides is oxidized to the disulfide derivative cysteine. Preferably, the cystine (e.g., a disulfide bridge) is the only interchain disulfide bond present in the dimer, for example...

[0175]

[0176] in

[0177]

[0178] -[Cys-S] and -[AA] x ]-[Cys-S]-[AA y ] indicates the C-terminal extension of the cysteine ​​residue containing the polypeptide;

[0179] “AA” means any amino acid as defined in this document;

[0180] The prefix "N" indicates the N-terminus of the polypeptide;

[0181] The suffix "C" indicates the C-terminus of the polypeptide;

[0182] The subscripts “x”, “y”, “p”, and “q” indicate quantities, which are independently selected from the range of 0-50, such as from the range of 1-40, or integers from the range of 2-30, such as, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. For example, if “x”, “y”, “p”, and “q” are all 0, then the C-terminal extension is only cysteine; and if both “y” and “q” are 0 but “x” and “p” are not 0, then the C-terminus of the C-terminal extension is cysteine.

[0183] This invention relates to a method for preparing a (peptide-)dimer, the method comprising at least the following steps: (i) providing a first polypeptide, wherein the first polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​moiety preferably at the C-terminus; (ii) providing a second polypeptide, wherein the second polypeptide comprises at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension comprising a cysteine ​​moiety preferably at the C-terminus; and (iii) oxidizing the thiol portion of the cysteine ​​moiety preferably at the C-terminus of the first polypeptide and the thiol portion of the cysteine ​​moiety preferably at the C-terminus of the second polypeptide to a disulfide derivative cystine; thereby preparing the dimer; and wherein the disulfide derivative cystine is the only intermolecular disulfide bond present in the dimer.

[0184] The present invention further relates to a method as described herein, wherein the first polypeptide comprises at least two ISVDs and / or the second polypeptide comprises at least two ISVDs.

[0185] The present invention further relates to a method as described herein, wherein the at least two ISVDs of the first polypeptide are identical and / or the at least two ISVDs of the second polypeptide are identical.

[0186] The present invention further relates to a method as described herein, wherein the first polypeptide and the second polypeptide are the same or different.

[0187] As used herein, the term "bispecific dimer" refers to a dimer in which the first polypeptide, independent of the valence (e.g., monovalent, divalent, or polyvalent) or specificity (e.g., monospecific, bispecific, or multispecific) of the first and second polypeptides, is different from the second polypeptide in the dimer. It should be understood that a dimer may comprise two identical but bispecific polypeptides.

[0188] Methods for generating bispecific dimers are provided, for example, where the first polypeptide of the dimer differs from the second polypeptide. In a first embodiment, a host strain, such as Pichia pastoris, is transformed with two different vectors, wherein the first vector encodes the first polypeptide and the second vector encodes the second polypeptide. Alternatively, a single vector is used, but the vector contains a first gene encoding the first polypeptide and a second gene encoding the second polypeptide. Alternatively, two host cells, each expressing one or the other polypeptide, are used, for example, a first vector encoding the first polypeptide is expressed in a first host cell (e.g., Pichia pastoris), and a second vector encoding the second polypeptide is expressed in a second host cell (e.g., also Pichia pastoris). For example, in a culture medium in which Pichia pastoris has been grown, the coupling of peptides to bispecific dimers is carried out by chemical conjugation, wherein at near-neutral pH, such as, for example, between pH 6.5 and pH 7.5, for example, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, to pH 7.5, the cysteine ​​(preferably located at the C-terminus) in the C-terminus of each of the two peptides is oxidized to the disulfide derivative cystine via their sulfhydryl groups.

[0189] Therefore, the present invention relates to a method for preparing a bispecific dimer, the method comprising at least the following steps:

[0190] (i) providing a first polypeptide, wherein the first polypeptide comprises

[0191] - At least one immunoglobulin single variable domain (ISVD) and

[0192] - Includes a C-terminal extension, preferably a cysteine ​​moiety at the C-terminus;

[0193] (ii) providing a second polypeptide, wherein the second polypeptide comprises

[0194] - At least one immunoglobulin single variable domain (ISVD) and

[0195] - Includes a C-terminal extension, preferably a cysteine ​​moiety at the C-terminus;

[0196] The first polypeptide is different from the second polypeptide; and

[0197] (iii) By optionally adding copper ions (Cu) 2+ The dimer is prepared by oxidizing the thiol portion of the cysteine ​​moiety at the C-terminus of the first polypeptide and the thiol portion of the cysteine ​​moiety at the C-terminus of the second polypeptide to the disulfide derivative cystine at pH 6.5 to pH 7.5.

[0198] Preferably, the integrity of the ISVD is maintained and the cystine is the only intermolecular disulfide bond present in the dimer.

[0199] As used herein, the term “integrity” means maintaining the structure, stability, and / or function of the ISVD, such as, for example, maintaining the proper intramolecular disulfide bonds that connect the two antiparallel β-sheet structures of the immunoglobulin domains and bind their homologous antigens.

[0200] This invention relates to a method as described herein, wherein a gene encoding a first polypeptide and a gene encoding a second polypeptide are present on two different vectors. Preferably, the vectors are present in a single host cell, such as Pichia pastoris. Alternatively, the polypeptides are encoded by different genes located on a single vector.

[0201] The vector of the present invention can be any suitable vector, such as, for example, plasmids, granules, YAC, viral vectors, or transposons. In particular, the vector can be an expression vector, i.e., a vector that can provide in vitro and / or in vivo expression (e.g., in suitable host cells, host organisms, and / or expression systems).

[0202] The vector of the present invention can be used to transform host cells or host organisms, i.e., for expressing and / or preparing the polypeptides of the present invention. Suitable hosts or host cells will be apparent to those skilled in the art, and can be, for example, any suitable fungus, prokaryotic or eukaryotic cell or cell line, or any suitable fungus, prokaryotic or (non-human) eukaryotic organism, such as:

[0203] - Bacterial strains, including but not limited to Gram-negative strains such as *Escherichia coli*; *Proteus* strains such as *Proteus mirabilis*; *Pseudomonas* strains such as *Pseudomonas fluorescens*; and Gram-positive strains such as *Bacillus* strains such as *Bacillus subtilis* or *Bacillus brevis*; *Streptomyces* strains such as *Streptomyces lividans*; *Staphylococcus* strains such as *Staphylococcus carnosus*; and *Lactococcus* strains such as *Lactococcus lactis*.

[0204] - Fungal cells, including but not limited to cells from the following species: Trichoderma, such as Trichoderma reesei; Neurospora, such as Neurosporacrassa; Sordaria, such as Sordaria macrospora; Aspergillus, such as Aspergillus niger or Aspergillus sojae; or other filamentous fungi;

[0205] - Yeast cells, including but not limited to cells from the following species: *Saccharomyces*, such as *Saccharomyces cerevisiae*; *Schizosaccharomyces*, such as *Schizosaccharomyces pombe*; *Pichia*, such as *Pichia pastoris* or *Pichia methanolica*; *Hansenula*, such as *Hansenula polymorpha*; *Kluyveromyces*, such as *Kluyveromyces lactis*; *Arxula*, such as *Arxula adeninivorans*; *Yarrowia*, such as *Yarrowia lipolytica*.

[0206] -Amphibian cells or cell lines, such as Xenopus oocytes;

[0207] - Cells or cell lines derived from insects, such as cells / cell lines derived from Lepidoptera, including but not limited to Spodoptera SF9 and Sf21 cells, or cells / cell lines derived from Drosophila, such as Schneider and Kc cells;

[0208] - Plants or plant cells, such as in tobacco plants; and / or

[0209] - Mammalian cells or cell lines, such as human cells or cell lines, mammalian cells or cell lines, including but not limited to CHO-cells (e.g., CHO-K1 cells), BHK-cells, and human cells or cell lines, such as HeLa, COS, Caki and HEK293H cells;

[0210] And all host cells or (non-human) hosts known per se for the expression and preparation of antibodies and antibody fragments (including, but not limited to, (single)-domain antibodies and ScFv fragments), will be clear to those skilled in the art. Also refer to the general background art cited above, and to reference, for example, WO 94 / 29457; WO 96 / 34103; WO 99 / 42077; Frenken et al. (Res Immunol. 149:589-99, 1998); Riechmann and Muyldermans (1999), see above; van der Linden (J. Biotechnol. 80:261-70, 2000); Joosten et al. (Microb. Cell Fact. 2:1, 2003); Joosten et al. (Appl. Microbiol. Biotechnol. 66:384-92, 2005); and other references cited herein.

[0211] In this specification, a gene is defined as the complete nucleic acid sequence required to synthesize a functional polypeptide. Therefore, a gene includes more nucleotides (coding regions) than the amino acid sequence encoding a polypeptide, and also includes the complete DNA sequence required to synthesize a specific RNA transcript. Preferably, step (iii) is performed in a culture medium previously used to grow Pichia pastoris.

[0212] Methods for processing nucleic acids, such as adding, inserting, mutating, substituting, or deleting nucleic acids relative to the nucleic acid encoding ISVD, are well known to those skilled in the art. See the above for reference in the standards manual.

[0213] The inventors of this invention provide a further optimized method for preparing bispecific dimers, wherein the efficiency exceeds 50%, such as 60%, 70%, 80%, or even greater than 90%, such as ≥95%. This is achieved by binding a first reactive polypeptide to a (solid) carrier and allowing a second reactive polypeptide to flow through the first polypeptide bound to the carrier. Any unreacted second polypeptide can be regenerated (reduced) and flowed again through the first polypeptide bound to the carrier. This step can be repeated until all the first and / or second polypeptides have reacted.

[0214] In step 1, the first polypeptide is reduced to obtain monomeric material, preferably 100% monomeric material. General conditions for reducing typical polypeptide solutions are given herein.

[0215] In step 2, the first polypeptide in the buffer binds to the carrier under reducing conditions. The carrier is preferably a chromatographic resin. Preferably, the carrier binds only the first polypeptide and not the second polypeptide. To avoid the possible formation of homodimers of the first polypeptide, the first polypeptide can be immobilized to the carrier at a low density during immobilization. Such spatial separation of individual first polypeptides can be achieved by using the carrier under suboptimal binding conditions (e.g., excessively high flow rates for typical affinity resins) or by loading via expanded bed chromatography. The methods and conditions for spatially separating individual polypeptides on the carrier are common knowledge or can be achieved by those skilled in the art using routine experiments. In a preferred embodiment, the carrier binds only the first polypeptide and not the second polypeptide. For example, if the first polypeptide (and preferably not the second polypeptide) binds to protein A, a carrier such as protein A can be used. Alternatively, in the case where both the first and second polypeptides bind to the carrier, after immobilization of the first polypeptide and before application of the second polypeptide, the carrier is saturated with a dummy polypeptide, such as a non-cysteine-extended nanobody.

[0216] In step 3, an excess of the second polypeptide, also in its reduced form (see above), is applied to a buffer solution and cycled through a column (optionally under slightly oxidizing conditions). The second polypeptide is passed through a support until the immobilized first polypeptide is fully conjugated (conjugated) to the second polypeptide via disulfide bonds. Preferably, the concentration of the second polypeptide is then measured to match the saturation of the first polypeptide group. As is known to those skilled in the art, if desired, conditions for this step are optimized to limit the amount of single-specific dimer formation of the second polypeptide. The second polypeptide group that does not bind to the support can be recovered and used in future coupling reactions, e.g., by being reduced again and applied to a column containing the first polypeptide until the first polypeptide is saturated.

[0217] In step 4, as is known to those skilled in the art, the bispecific dimer is recovered from the vector by typical elution conditions for the vector used (e.g., acidic conditions for protein A).

[0218] In the context of this document, the term "fixation" refers to a molecule whose movement in space is completely restricted or confined to a finite small region by being attached to a solid structure, such as a carrier. Generally, the term fixation refers to an action that restricts or prevents movement, such as slowing down movement. The dimers of the present invention can be fixed by any suitable method, such as, for example, by adsorption, covalent bonding, entrapment, encapsulation, and (reversible) crosslinking, preferably covalent bonding, more preferably by affinity. Any suitable carrier for fixation can be used. Those skilled in the art will understand that the suitability of the carrier depends on the method of fixation. For example, carriers used for covalent bonding are agarose, cellulose, cross-linked dextran, polystyrene, polyacrylamide gel, and porous silica gel. A preferred carrier is protein A resin.

[0219] Suitable buffers may include, but are not limited to, acetate buffer, phosphate buffer, citrate buffer, sulfate buffer, glycine buffer, carboxylate buffer and / or Tris buffer.

[0220] Reduction and oxidation conditions are well known in the art. Refer to the Examples section, the specification, and, for example, standard chemistry handbooks such as Principles of Modern Chemistry (2011, Oxtoby, Gillis and Campion, 7th edition). Preferred reduction conditions are carried out at a concentration of up to 10 mg / ml of peptide in 1-15 mM, such as 2-12 mM, 4-11 mM, 5-10 mM, preferably 10 mM DTT, at room temperature for at least 1 h (up to 8 h) or overnight at 4 °C, thereby maintaining the typical -SS- still oxidized. Preferred oxidation conditions are carried out in 0.1-10 mM, 0.5-5 mM, preferably 1 mM CuSO4 for 1-4 h, preferably 2 h at room temperature, or by using a convenient redox couple, which can be readily determined by those skilled in the art.

[0221] Therefore, the present invention relates to a method for preparing a bispecific dimer, the method comprising at least the following steps:

[0222] 1. Providing a first polypeptide, wherein the first polypeptide comprises

[0223] - At least one immunoglobulin single variable domain (ISVD) and

[0224] - Includes a C-terminal extension, preferably a cysteine ​​moiety at the C-terminus;

[0225] 2. Reduce the first polypeptide;

[0226] 3. The first polypeptide reduced in step 2 is bound to the carrier under reducing conditions;

[0227] 4. Providing a second polypeptide, wherein the second polypeptide comprises

[0228] - At least one immunoglobulin single variable domain (ISVD);

[0229] - Includes a C-terminal extension, preferably a cysteine ​​moiety at the C-terminus;

[0230] The first polypeptide is different from the second polypeptide;

[0231] 5. Reduce the second polypeptide;

[0232] 6. Optionally, under slightly oxidizing conditions, the reduced second polypeptide from step 5 is applied to the reduced first polypeptide bound to the carrier from step 3, oxidizing the thiol portion of the cysteine ​​moiety preferably at the C-terminus of the first polypeptide and the thiol portion of the cysteine ​​moiety preferably at the C-terminus of the second polypeptide to the disulfide derivative cystine; thereby preparing the bispecific dimer; optionally until the entire first polypeptide is completely conjugated to the second polypeptide via disulfide bonds;

[0233] 7. Optionally, recover, reduce, and reapply the unconjugated second polypeptide according to steps 5 and 6;

[0234] 8. Elute the bispecific dimer from the carrier.

[0235] The present invention further relates to any method as described herein, wherein the first polypeptide and / or the second polypeptide comprises an N-terminal extension.

[0236] The present invention further relates to a method as described herein, wherein the first polypeptide and / or the second polypeptide comprises a C-terminal extension of 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residue having a cysteine ​​motif preferably at the C-terminus.

[0237] The present invention further relates to a method as described herein, wherein the C-terminal extension is selected from the group consisting of SEQ ID NO:1-15, and preferably, the C-terminal extension is composed of GlyGlyGlyCys (SEQ ID NO:4), GlyGlyCys (SEQ ID NO:3), GlyCys (SEQ ID NO:2) or Cys (SEQ ID NO:1).

[0238] The present invention further relates to a method as described herein, wherein the C-terminal extension is genetically fused with the C-terminal end of the ISVD located at the C-terminus of the polypeptide.

[0239] In one embodiment, copper ions (CuSO4) are added, for example, in the form of oxidizing copper ions (CuSO4). 2+ The oxidation process is optimized by [method name missing]. It has been observed that almost 100% of the C-terminal thiol moiety is oxidized during copper treatment. Therefore, the present invention relates to a method as described herein, wherein at least 80%, such as 85%, 90%, 95%, 99%, or even more than 99%, such as 100%, of the first and / or second polypeptide is dimerized. The degree of oxidation can be determined by any suitable method, but is preferably determined by mass spectrometry.

[0240] In another embodiment, the dimer is purified to homogeneity. Purification can be accomplished by any suitable technique known in the art, such as chromatography, preferably size exclusion chromatography, as is fully known to those skilled in the art. Therefore, the present invention relates to a method as described herein, which further includes the optional step of purifying the dimer by size exclusion chromatography. Thus, the present invention relates to a method as described herein, wherein the dimer is purified to a purity of at least 90%, for example, at least 95%, such as 97%, 98%, 99%, or even 100%. Purity can be determined by any suitable method known in the art, and preferably by mass spectrometry.

[0241] In one embodiment, the present invention relates to dimers that can be prepared by the method described above.

[0242] The inventors of this invention unexpectedly observed that not only was the binding of the polypeptide in the dimer and other functional characteristics, such as potency, maintained, but it was even improved compared to the corresponding benchmark.

[0243] Without any theoretical constraints, it is assumed that the complementary sites of ISVDs can be in more "favorable" positions for antigen recognition of such dimer assemblies compared to the corresponding baseline assemblies.

[0244] As used herein, a “benchmark” is used as a reference point for evaluating the performance of a molecule, such as one or more functional characteristics (e.g., affinity, efficacy, and potency as described herein). A particular dimer will determine the appropriateness of a benchmark, which can be readily assessed by those skilled in the art. Preferably, the benchmark will consist of the same number and / or the same ISVD as the dimer. Preferably, the benchmark comprises the same polypeptides that make up the dimer, but in this benchmark, as described herein, these polypeptides are formed by genetic fusion rather than chemical conjugation (see, for example, the Examples section). Comparisons between the dimer and the one or two polypeptides that individually constitute the dimer have provided a wealth of information about the properties of the dimer.

[0245] The dimer of the present invention comprises a first polypeptide containing at least one ISVD and a second polypeptide containing at least one ISVD. The affinity of the dimer can be determined by treating the two polypeptides together as a whole, or by determining the affinity of each polypeptide that individually constitutes the dimer. In other words, in the latter case, the affinity of the polypeptides is determined independently of the antibody affinity attributed to the other polypeptides.

[0246] As used herein, the term "potency" is a measure of the biological activity of a reagent such as a dimer, benchmark, peptide, ISVD, or nanobody. The potency of a reagent can be determined by any suitable method known in the art, such as those described, for example, in the Examples section. Cell culture-based potency assays are generally the preferred form for determining biological activity because they measure the physiological response induced by the reagent and can produce results within a relatively short timeframe. Based on the mechanism of action of the product, assays based on various cell types can be used, including but not limited to proliferation assays, cytotoxicity assays, reporter gene assays, cell surface receptor binding assays, and assays measuring the induction / inhibition of functionally essential proteins or other signaling molecules (such as phosphorylated proteins, enzymes, cytokines, cAMP, etc.), all of which are well known in the art. Cell-based potency results from cell-based assays can be expressed as a "relative potency" as determined by comparing the dimer of the present invention with the response obtained against a corresponding benchmark (see Examples section).

[0247] When the response obtained for a compound, such as the dimer of the present invention, is at least 1.5 times better (e.g., functionally better) than the response obtained by a reference compound, such as a corresponding benchmark in a given assay, such as a reference compound, such as an construct containing the corresponding polypeptide, the compound is described as more potent than a benchmark, such as a reference compound.

[0248] Depending on the specific disease or condition involved, any suitable in vitro assay, cell-based assay, in vivo assay, and / or animal model known per se, or any combination thereof, may be used to test the efficacy or potency of the dimers, immunoglobulin monovariable domains, and peptides of the present invention, as well as compositions comprising them. Suitable assays and animal models will be clear to the technicians and include, for example, ligand substitution assays (e.g., Burgess et al., Cancer Res 2006 66:1721-9), dimerization assays (e.g., WO 2009 / 007427A2, Goetsch, 2009), signal transduction assays (e.g., Burgess et al., Mol Cancer Ther 9:400-9), proliferation / survival assays (e.g., Pacchiana et al., J Biol Chem 2010 Sep M110.134031), cell adhesion assays (e.g., Holt et al., Haematologica 2005 90:479-88) and migration assays (e.g., Kong-Beltran et al., Cancer Cell 6:75-84), endothelial cell budding assays (e.g., Wang et al., J Immunol. 2009; 183:3204-11), and in vivo xenograft models (e.g., Jin et al., Cancer Res. 2008 68:4360-8), as well as assays and animal models used in the experimental section below and in the prior art cited herein. In vitro inhibition of the first target is represented by IC50 assays. 50 .

[0249] In particular, the dimer of the present invention exhibits a better affinity than the reference (suitably as K) D Value (actual or apparent), K A Value (actual or apparent), k on rate and / or k off (Rate measurement) combined with target.

[0250] In one embodiment, the present invention relates to a polypeptide-containing dimer as described herein, wherein the dimer is expressed as a fraction of a reference IC 50 Good or at least 10% higher, such as 20%, 30%, 50%, 80%, 90%, or even 100% of IC. 50 Binding to targets, for example, in ligand competition assays, competitive FACS, and functional cell assays, such as ligand-induced inhibition of chemotaxis. As determined by methods such as measurement, competitive FACS is preferred.

[0251] In one embodiment, the present invention relates to a polypeptide-containing dimer as described herein, wherein the dimer is expressed as a fraction of a reference IC 50 It's better to have at least 1.5 times, such as 2, 3, or 4 times, and even 5 or 10 times the IC. 50 Binding to targets, for example, in ligand competition assays, competitive FACS, and functional cell assays, such as ligand-induced inhibition of chemotaxis. As determined by methods such as measurement, competitive FACS is preferred.

[0252] In one embodiment, the present invention relates to a polypeptide dimer as described herein, having an IC concentration between 200 nM and 0.01 nM. 50 For example, concentrations of 0.01, 0.05, 0.1, 0.15, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nM, are used in ligand competition assays, competitive FACS, and functional cell assays, such as ligand-induced inhibition of chemotaxis. Determined through measurements, etc.

[0253] Transportation, manufacturing, storage, and delivery processes can subject peptides to various stresses, including chemical and physical stresses. During storage, chemical modifications may occur, such as deamidation, racemization, hydrolysis, oxidation, isomerization, β-elimination, or disulfide exchange. Physical stresses can lead to denaturation and unfolding, aggregation, particle formation, precipitation, opalescence, or adsorption. These stresses are known to affect the physicochemical integrity of protein therapeutics, such as antibody therapeutics.

[0254] As noted above, the inventors have observed that the dimers of the present invention possess unexpectedly advantageous binding and functional characteristics. These characteristics are also maintained over long periods without any apparent or substantial loss of effectiveness. This makes the dimers suitable for storage and transport. The present invention provides stable dimers of the present invention. “Stable” generally means that after long periods of storage, such as 1 month to 36 months, the dimer does not suffer significant physical or chemical changes, especially oxidation, even when exposed to one or more chemical or physical stresses such as high temperatures (equal to or above +25°C) or physical stresses such as shaking or stirring. More specifically, “stable” means that after long periods of storage under various conditions (as defined), one or more degradation products are formed only to a limited extent, such as low molecular weight (LMW) derivatives (e.g., peptides) of the dimers of the present invention; and / or, for example, high molecular weight (HMW) derivatives (oligomers or polymers) formed by the aggregation of the dimers.

[0255] Therefore, the present invention relates to dimers as described herein, wherein the dimer is stable for at least 2 months, such as 4 months, 6 months, 12 months or even longer, such as 18 months, 24 months or 36 months, at -20°C, +4°C, room temperature, such as +20°C or even +25°C, wherein the stability is characterized by limited formation or non-formation of LMW and / or HMW, such as less than 10%, such as less than 5%, less than 2% or even undetectable LMW and / or HMW.

[0256] Common techniques that can be used to assess protein stability include static light scattering, tangential flow filtration, Fourier transform infrared spectroscopy, circular dichroism, urea-induced protein unfolding, intrinsic tryptophan fluorescence, and / or 1-aniline-β8-naphthalenesulfonic acid protein binding. These techniques are also applicable to the dimers of the present invention. Furthermore, the dimers of the present invention exhibit little or no loss of potency / biological activity during storage and / or under the influence of one or more stresses as defined herein.

[0257] Therefore, the present invention relates to a method for storing a polypeptide comprising a reactive cysteine ​​moiety, the method comprising at least the step of oxidizing the thiol portion of the reactive cysteine ​​moiety to a disulfide derivative cystine, thereby temporarily inactivating the reactive cysteine ​​moiety, wherein the polypeptide further comprises an (internal) cystine bond.

[0258] Although the dimer of the present invention possesses advantageous functional properties, the inventors hypothesize that the dimer may be particularly suitable as an aggregate for transient use, for example, by coupling with a functional group of maleimide chemistry, such as a C-terminal cysteine. A scheme using mild reducing conditions was developed in which the intermolecular disulfide bridges of the dimer are reduced to activate the thiol groups of the constituent polypeptide. The optimized conditions induce the reduction of the disulfide, forming the dimer without reducing the internal typical ISVD disulfide bonds.

[0259] Preferred reducing agents are acid-based reducing agents such as oxalic acid (C2H2O4), formic acid (HCOOH), ascorbic acid (C6H8O6), phosphorous acid, or β-mercaptoethanol, lithium aluminum hydride (LiAlH4), nascent (atomic) hydrogen, sodium amalgam, diborane, sodium borohydride (NaBH4), and those containing Sn. 2+ Ionic compounds such as tin(II) chloride, sulfite compounds, hydrazine, zinc amalgam (Zn(Hg)), diisobutylaluminum hydride (DIBAL-H), Lindlar catalysts, phosphites, hypophosphites, and compounds containing Fe 2+ Compounds such as ferric sulfate (II), carbon monoxide (CO), carbon (C), dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine HCl (TCEP), with DTT and TCEP being preferred.

[0260] The preferred final concentration of the reducing agent is between 50 mM and 1 mM, such as between 40 mM and 2 mM, 30 mM and 5 mM, and between 20 mM and 7.5 mM, preferably 10 mM.

[0261] It has been determined that overnight treatment with 10 mM DTT at 4 °C (or for at least 2 hours at room temperature) is highly suitable for reducing intermolecular disulfide bonds in ISVD at concentrations up to 10 mg / ml without affecting the typical internal disulfide bonds. DTT or TCEP can be preferred for reduction. Unlike TCEP, DTT is preferably removed to create optimal coupling conditions. Size exclusion chromatography (SEC) can separate the monomeric peptide from the unreduced dimer and DTT.

[0262] The extent of reduction can be monitored by any means known in the art, such as, for example, by SEC or SDS-PAGE under non-reduction conditions.

[0263] Therefore, the present invention relates to a method as described herein, the method further comprising the step of reducing the (C-terminal) cystine of the dimer, preferably under conditions in which the internal disulfide bonds of the first polypeptide and / or the second polypeptide remain oxidized.

[0264] Therefore, the present invention relates to a method for generating a polypeptide comprising a reactive cysteine ​​moiety, the method comprising at least the following steps:

[0265] (i) Provides a polypeptide according to the invention, which is obtained by cystine dimerization (a disulfide bond between two cysteine ​​residues; an SS-bond or a disulfide bridge);

[0266] (ii) Reduce the cystine;

[0267] This generates a polypeptide containing a reactive cysteine ​​moiety; preferably, the cysteine ​​bond is located at the C-terminus of the polypeptide. Preferably, the reduction conditions of step (ii) are selected such that the internal cysteine ​​bond is not reduced.

[0268] Following dimer reduction, the reduced monomeric peptide is preferably used immediately, for example within 0.5 h, but preferably within 10 minutes, for conjugation, or frozen to prevent re-oxidation, although freezing cannot completely prevent re-oxidation. Experimental evidence shows that the reduced monomeric peptide of the present invention is stable in D-PBS at 4 °C for up to 24 h.

[0269] In one embodiment, the dimer and constituent polypeptide of the present invention comprise one or more functional groups, residues, or portions. In one embodiment, the present invention relates to a dimer as described herein, which further comprises one or more other groups, residues, portions, or binding units. In one embodiment, the present invention relates to a dimer as described herein, wherein the first polypeptide and / or the second polypeptide further comprises one or more other groups, residues, portions, or binding units. For example, functional groups, residues, or portions may be coupled or linked to the (reactive) thiol portion of a cysteine ​​residue at the C-terminus of the polypeptide and / or functional groups, residues, or portions may be coupled or linked to the N-terminus of the polypeptide of the present invention. In one embodiment, one or both N-termini of the dimer of the present invention comprise functional groups, residues, or portions.

[0270] Examples of such groups, residues, or portions, and methods and techniques for linking such groups, residues, or portions, as well as the potential uses and advantages of such groups, residues, or portions, will be clear to those skilled in the art. Without limitation, thiol-reactive groups used for antibody modification include maleimide groups, vinyl sulfone groups, acetyl halide groups, or dithiopyridine groups. Although reactive with amine groups at higher pH values, maleimides selectively react with cysteine ​​at neutral pH to form stable thioether bonds.

[0271] One or more functional groups, residues, or portions may be linked to the dimers and / or peptides of the present invention, thereby conferring one or more desired properties or functions to the dimers and / or peptides of the present invention. Examples of such functional groups, residues, or portions will be apparent to those skilled in the art. For example, one or more such functional groups, residues, or portions may increase the half-life, solubility, and / or absorption of the dimers and / or peptides of the present invention; one or more such functional groups, residues, or portions may reduce the immunogenicity and / or toxicity of the dimers and / or peptides of the present invention; one or more such functional groups, residues, or portions may eliminate or attenuate any undesirable side effects of the dimers and / or peptides of the present invention; and / or one or more such functional groups, residues, or portions may confer other advantageous properties and / or reduce undesirable properties of the dimers and / or peptides of the present invention; or any combination of two or more of the foregoing. Examples of such functional groups, residues, or portions and the techniques used to introduce them will be clear to those skilled in the art, and generally include all functional groups, residues, or portions and techniques mentioned in the general background art cited herein, as well as functional groups, residues, or portions and techniques known per se for the modification of pharmaceutical proteins, and especially for the modification of antibodies or antibody fragments (including ScFv and single-domain antibodies), for which reference is made, for example, Remington's Pharmaceutical Sciences, 16th edition, Mack Publishing Co., Easton, PA (1980).

[0272] Due to their specificity, the dimers and / or peptides of the present invention are also well-suited for conjugation with imaging agents. Suitable imaging agents for conjugation with antibodies are well known in the art, and similarly, can be used for conjugation with the dimers and / or peptides of the present invention. Suitable imaging agents include, but are not limited to, molecules preferably selected from the group consisting of: organic molecules, enzyme markers, radiolabels, colored markers, fluorescent markers, chromogenic markers, luminescent markers, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold, fluorescent markers, metal markers, biotin, chemiluminescent substances, bioluminescent substances, chromophores, and mixtures thereof.

[0273] Therefore, the present invention relates to dimers and / or polypeptides according to the invention that also contain imaging agents, said imaging agents including, but not limited to, molecules preferably selected from the group consisting of: organic molecules, enzyme markers, radiolabels, colored markers, fluorescent markers, chromogenic markers, luminescent markers, haptens, digoxigenin, biotin, metal complexes, metals, colloidal gold, fluorescent markers, metal markers, biotin, chemiluminescent substances, bioluminescent substances, chromophores, and mixtures thereof.

[0274] Depending on the intended use of the labeled polypeptide, one or more detectable markers or other signal-generating groups, residues, or portions may be conjugated to the dimers and / or polypeptides of the present invention. Suitable markers and techniques for their conjugation, use, and detection will be apparent to those skilled in the art, and include, for example, but not limited to, fluorescent markers, phosphorescent markers, chemiluminescent markers, bioluminescent markers, radioactive isotopes, metals, metal chelates, metal cations, chromophores, and enzymes, as mentioned on page 109 of WO 08 / 020079. Radioactive isotopes and radionuclides known in the art for their use as detection agents include, but are not limited to, 3 H 14 C 15 N 18 F 35 S 64 Cu 67 Cu 75 Br 76 Br 77 Br 89 Zr 90 Y 97 Ru 99 Tc 105 Rh 109 Pd 111 ln 123 l 124 l 125 l 131 l 149 Pm 153 Sm 166 Ho 177 Lu 186 Re 188 Re 198 Au 199 Au 203 Pb 211 At 212 Pb 212 Bi 213 Bi 223 Ra 225 Ac. Indium 111It is particularly preferred as a diagnostic radionuclide because: it can be safely administered between about 1 and about 10 mCi without detectable toxicity; and imaging data are generally predictive of subsequent PDC distribution (see below). See, for example, Murray JL, 26 J.Nuc.Med.3328 (1985) and Carraguillo, JA et al., 26 J.Nuc.Med.67 (1985).

[0275] Other suitable markers will be apparent to those skilled in the art, and include, for example, those detectable by NMR or ESR spectroscopy. For instance, as illustrated in the Examples section, markers can be used... 89 Zr is used to radiolabel the polypeptides of the present invention. Depending on the choice of specific marker, the thus-labeled polypeptides of the present invention can be used, for example, for in vitro, in vivo, or in situ assays (including immunoassays known per se such as ELISA, RIA, EIA, and other “sandwich assays”) and for in vivo diagnostic and imaging purposes. In a preferred embodiment, the radiolabeled polypeptides and / or dimers of the present invention are detected by microPET imaging. Images can be reconstructed using AMIDE Medical Image Data Examiner software (version 1.0.4, Stanford University).

[0276] Functional groups, residues, or portions may be linked to form a specific binding pair, such as the biotin-(streptavidin)binding pair. Such functional groups can be used to link the dimers and / or peptides of the present invention to another protein, peptide, or compound that binds to the other half of the binding pair, i.e., by forming a binding pair. For example, the dimers and / or peptides of the present invention may be conjugated to biotin and linked to another protein, peptide, compound, or carrier conjugated to avidin or streptavidin. For example, such conjugated dimers and / or peptides can be used as reporter proteins, for example, in diagnostic systems where a reagent that generates a detectable signal is conjugated to avidin or streptavidin. Such binding pairs can also be used, for example, to bind the dimers and / or peptides of the present invention to a carrier, including carriers suitable for pharmaceutical purposes. A non-limiting example is the liposomal formulation described in Cao and Suresh 2000 (Journal of Drug Targeting 8(4):257). Such binding pairs can also be used to link therapeutic agents to the peptides of the present invention.

[0277] Other potential chemical and enzymatic modifications will be apparent to those skilled in the art. Such modifications may also be introduced for research purposes (e.g., to study function-activity relationships). See, for example, Lundblad and Bradshaw 1997 (Biotechnol. Appl. Biochem. 26:143-151).

[0278] In some embodiments, the dimers and / or peptides of the present invention are conjugated with a drug to form a dimer / peptide-drug conjugate (collectively abbreviated herein as "PDC"). The use of simultaneous antibody-drug conjugates (ADCs) in oncology applications, where the ADC is intended for the local delivery of a drug such as a cytotoxic agent or cell growth inhibitor, toxin, or toxin moiety, allows for targeted delivery of the drug moiety to the tumor, which can allow for higher efficacy, lower toxicity, etc. These ADCs have three components: (1) a monoclonal antibody conjugated to (3) a drug moiety such as a toxin moiety or toxin via (2) a linker. An overview of this technique is provided in Ducry et al., Bioconjugate Chem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008), and Senter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which are incorporated herein by reference in their entirety. The PDC of the present invention also has three components: (1) a dimer or polypeptide conjugated to (3) a drug such as a toxin moiety or toxin via (2) a linker. As noted above, although the conjugation of the linker and the drug has a greater and more adverse effect on the aggregation, biodistribution and PK characteristics of antibody fragments such as the polypeptides of the present invention compared to larger-sized antibodies, those skilled in the art will understand that the techniques, methods, and approaches of ADCs are generally equivalent to those of PDCs (see Feng et al., see above).

[0279] This invention provides polypeptides of the invention comprising a drug such as a toxin or a toxin moiety (whether or not contained in the dimer of the invention). For completeness, this invention provides dimers of the invention comprising a drug such as a toxin or a toxin moiety.

[0280] Drugs, such as toxin moieties or toxins, can be linked or conjugated to dimers and / or peptides using any suitable method. Typically, as is known in the art, conjugation is accomplished by covalent linking to dimers and / or peptides and generally depends on a linker, which is typically a peptide bond. For example, drugs such as toxin moieties or toxins can be covalently bound to peptides directly or via suitable linkers. Suitable linkers can include non-cleavable or cleavable linkers, such as pH-cleavable linkers containing cleavage sites for cellular enzymes (e.g., cellular esterases, cellular proteases such as cathepsin B, see, for example, the Examples section). Such cleavable linkers can be used to prepare ligands that can release drug moieties or toxins after peptide internalization. As those skilled in the art will understand, the amount of drug moieties in each dimer and / or peptide can be varied depending on the reaction conditions and can range from 1:1 to 20:1 drug:peptide variations (also expressed as drug-antibody ratios or DAR). As those skilled in the art will also understand, the actual amounts are averages when the reaction and / or purification are not strictly controlled. Preferably, the dimer of the present invention further comprises a drug, wherein the drug-to-dimer ratio (DAR) is 1. Various methods can be used to link or conjugate a drug, such as a toxin moiety or toxin, to a dimer and / or polypeptide. The specific method chosen will depend on the drug, such as the toxin moiety or toxin, and the dimer and / or polypeptide to be linked or conjugated. If desired, a connector containing terminal functional groups can be used to link the dimer and / or polypeptide and the drug, such as the toxin moiety or toxin. Typically, conjugation is accomplished by reacting a drug, such as a toxin moiety or toxin, containing reactive functional groups (or modified to contain reactive functional groups) with a connector or directly with the dimer and / or polypeptide. Covalent bonds are formed by reacting a drug, such as a toxin moiety or toxin, containing (or modified to contain) a chemical moiety or functional group that can react with a second chemical group under suitable conditions to form a covalent bond. If desired, suitable reactive chemical groups can be added to peptides or linkers using any suitable method (see, for example, Hermanson, GT, Bioconjugate Techniques, Academic Press: San Diego, CA (1996)). Many suitable combinations of reactive chemical groups are known in the art; for example, amine groups can react with electrophilic groups such as toluenesulfonate ions, methanesulfonate ions, halogens (chlorine, bromine, fluorine, iodine), N-hydroxysuccinimide esters (NHS), etc. Thiol groups can react with maleimide, iodoacetyl, acryloyl, pyridyl disulfide, 5-mercapto-2-nitrobenzoic acid thiol (TNB-thiol), etc. Aldehyde functional groups can be coupled to amine- or hydrazide-containing molecules, and azide groups can react with trivalent phosphorus groups to form aminophosphate esters or phosphorimide linkages.Suitable methods for introducing activating groups into molecules are known in the art (see, for example, Hermanson, above).

[0281] As shown in the examples, it was unexpectedly discovered that the peptides of the present invention, comprising a C-terminal extension containing a cysteine ​​moiety at the C-terminus, are remarkably suitable for conjugating specific amounts / peptides of drugs, such as DAR=1, in a highly controlled manner. This results in better controlled efficacy and safety compared to prior art molecules. Therefore, the present invention relates to peptides as described herein, such as DAR=1, comprising a single conjugated drug. The process of the present invention thus allows for the preparation of peptides and dimers with improved uniformity.

[0282] As described below, the pharmaceutical agent of PDC can be any number of reagents, including but not limited to cell growth inhibitors, cytotoxic agents such as chemotherapeutic agents, growth inhibitors, toxins (e.g., enzyme-active toxins or fragments thereof of bacterial, fungal, plant, or animal origin), toxic fractions, or radioactive isotopes (i.e., radioactive conjugates). In other embodiments, the invention also provides methods of using PDC. The invention also relates to radioimmunotherapy (RIT), wherein the polypeptides or dimers of the invention are labeled with radioactive isotopes to deliver cytotoxic radiation to target cells.

[0283] The pharmaceutical agents used in this invention include cytotoxic agents, particularly those used for cancer treatment. Such agents typically include DNA damaging agents, antimetabolites, natural products, and analogues thereof. Exemplary types of cytotoxic agents include enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidine synthase inhibitors, DNA intercalating agents, DNA breaking agents (cleaver), topoisomerase inhibitors, anthracycline antibiotics, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, pteridine family drugs, diynene, podophyllotoxin, dolastatin, maytansinoids, differentiation inducers, and taxol.

[0284] These types of members include, for example, methotrexate, methotrexate, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine arbinoside, melphalan, leurosine, leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C, and mitomycin A. A) Caminomycin, aminopterin, tallysomycin, podophyllotoxin and podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxanes including paclitaxel, taxotere, retinoic acid, butyric acid, N8-acetylsemine, camptothecin, calicheamicin, esperamicin, enediyne derivatives, duocarmycin A, duocarmycin SA, calicheamicin, camptothecin, maytansinoids (including DM1), monomethyl-auristatin E E, MMAE), monomethylauristatin F (MMAF), and maytansine compounds (DM4) and their analogues, preferably MMAE. Preferably, the polypeptide conjugated with the toxin is selected from the group consisting of: ABL 100-NC003-1, ABL 100-NC003-3, ABL 100-NC003-5, ABL 100-NC003-6 and ABL 100-BF012-1, most preferably ABL 100-BF012-1.

[0285] Drugs such as toxins can be used as peptide-toxin conjugates and / or dimer-toxin conjugates, including bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al. (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansine compounds (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and cazithromycin (Lode et al. (1998) ...) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19) Cancer Inst. 92(19 Res. 58:2928; Hinman et al. (1993) Cancer Res. 53:3336-3342). Toxins can exert their cytotoxic and cell growth-inhibiting effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

[0286] The present invention is contemplated into polypeptides and / or dimers and one or more small molecule toxins such as maytansinoids, dolastatin, auristatin, trichothecene, calicheamicin, and CC1065, as well as conjugates of derivatives of these toxins having toxic activity.

[0287] Other drugs that can be conjugated with the dimers and / or peptides of the present invention, such as antitumor agents including BCNU, streptozotocin, vincristine and 5-fluorouracil, the family of agents collectively referred to as the LL-E33288 complex as described in U.S. Patent Nos. 5,053,394 and 5,770,710, and esperamicin (U.S. Patent No. 5,877,296).

[0288] Available medications, such as enzyme-active toxins and their fragments, include diphtheria A chain, non-conjugated active fragments of diphtheria toxin, exotoxin A chain (from *Pseudomonas aeruginosa*), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, *Aleurites fordii* protein, dianthin protein, *Phytolacca amaranth* protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitors, curcin, crotin, and sapaonaria. Inhibitors of *Acinetoxin*, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecene. See, for example, WO 93 / 21232, published October 28, 1993.

[0289] The present invention further considers the formation of PDCs between the dimers and / or polypeptides of the present invention and compounds with nucleolytic activity (e.g., ribonucleases or DNA endonucleases such as deoxyribonuclease; DNase).

[0290] To selectively destroy tumors, the dimers and / or peptides of the present invention may contain highly radioactive atoms. Various radioactive isotopes can be used to prepare radioconjugated PDCs. Examples include At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 And radioactive isotopes of Lu.

[0291] Radiolabeled or other markers can be incorporated into conjugates in known ways. For example, peptides can be biosynthesized or synthesized via chemical amino acid synthesis using suitable amino acid precursors (e.g., including replacing hydrogen with fluorine-19). Markers such as Tc... 99m or I 123 Re 186 Re 188 and In 111iodine-90 can be linked via cysteine ​​residues in the peptide. Iodogen-90 can be linked via lysine residues. The Iodogen method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80:49-57) can be used to bind iodine-123. Iodine-125 can be radiolabeled using the iodobead method as described in Valentine, MA et al. (1989) J. Biol. Chem. 264:11282. Other methods are described in detail in "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989).

[0292] Those skilled in the art can establish an effective single therapeutic dose (e.g., a therapeutically effective dose) for radioconjugated PDCs, which depends particularly on the specific radiolabel, the half-life of the PDC, its toxicity, the target, etc. Preferably, the effective single therapeutic dose range is between about 5 and about 75 mCi, more preferably between about 10 and about 40 mCi.

[0293] The generation of PDC compounds can be accomplished using any technique known to those skilled in the art of ADCs. In short, PDC compounds may comprise the dimers and / or peptides of the present invention as antibody units, pharmaceuticals, and optionally linkers connecting pharmaceuticals and binders.

[0294] Methods for determining whether a drug or antibody-drug conjugate exerts an effect, such as cell growth inhibition and / or cytotoxicity, are known. Typically, the effects of antibody-drug conjugates, such as cytotoxic or cell growth-inhibiting activity, can be measured by exposing mammalian cells expressing the target protein to the antibody-drug conjugate in cell culture medium; culturing the cells for a period of approximately 6 hours to approximately 5 days; and measuring cell viability. Cell-based in vitro assays can be used to measure the viability (proliferation), cytotoxicity, and apoptosis induction (cysteine ​​activation) of antibody-drug conjugates. These methods are also applicable to PDCs (Proton Drug Conjugates).

[0295] Therefore, this invention relates to polypeptides of the invention (whether or not contained in the dimer of the invention) that also contain a drug such as a toxin or a toxin portion. For clarity, this invention relates to dimers (containing polypeptides of the invention) that also contain a drug such as a toxin or a toxin portion.

[0296] Therefore, this invention relates to polypeptides according to the invention (whether or not contained in the dimers of the invention) conjugated to drugs such as toxins or toxins. For clarity, this invention relates to dimers conjugated to drugs such as toxins or toxins (containing the polypeptides of the invention).

[0297] PDCs combine the selectivity of a highly selective targeting moiety with the cytotoxic efficacy of a drug. For a successful PDC component to function as a peptide according to the invention (whether or not contained in the dimer of the invention), the peptide needs to bind to a target antigen on the surface of target cells, such as tumor cells. For most drugs, PDCs become effective through cellular internalization (Trail 2013 Antibodies 2:113-129 review). After internalization, the PDC is delivered to lysosomes, where subsequent intracellular processing of the PDC will release the bioactive drug to exert its (toxic) effect on target cells, such as tumor cells. Not only should the bioactive drug be internalized, but it is also preferable to internalize the radioactive isotope used in radioimmunotherapy (RIT), thereby highly localizing the toxic effects of the radioactive payload. Precise targeting with the radiolabeled peptides of the invention (whether or not contained in the dimer of the invention) results in selective and highly effective cytotoxicity to target cells (e.g., tumor cells) at lower radioactive doses, minimizing side effects.

[0298] The inventors have demonstrated that the overall internalization of the dimer of this invention appears to be more efficient and effective compared to the corresponding monomers and divalent benchmarks, especially in cells with low target quantities. This difference in internalization is less pronounced, however, it remains evident in cells expressing the target at extremely high levels.

[0299] Therefore, the present invention relates to a dimer for the treatment of cancer, wherein the dimer is internalized. Preferably, the dimer is conjugated with a (cytotoxic) drug.

[0300] Therefore, this invention relates to the use of the dimer of the present invention in the manufacture of a medicament for the treatment of cancer, wherein the dimer is internalized. Preferably, the dimer is conjugated with a (cytotoxic) drug.

[0301] In one embodiment, the dimer of the present invention can be used to target cells expressing low numbers of binding sites for the corresponding ISVD, such as less than 10*102 5 There are 5*10 binding sites. 5 One binding site, or even fewer than 10*10 4 One binding site, 5*10 4 One binding site, 1*10 4 1000 binding sites, or less than 5000 binding sites, such as less than 4000, 3000 or even less than 2000 binding sites, such as 1000 binding sites or even less.

[0302] In one embodiment, the present invention relates to a dimer as described herein for the treatment of cancer, wherein the dimer is internalized. Preferably, the dimer is conjugated with a (cytotoxic) drug.

[0303] In one embodiment, the present invention relates to the use of dimers as described herein for the manufacture of medicaments for the treatment of cancer, wherein the dimer is internalized. Preferably, the dimer is conjugated with a (cytotoxic) drug.

[0304] Most drugs used in cancer treatment are hydrophobic. This is advantageous because these hydrophobic drugs can penetrate cell membranes. However, these drugs can also penetrate membranes from non-cancer cells. These drugs remain effective because cancer cells divide much faster than “normal” cells. It should be understood that the use of these drugs results in serious side effects. To obtain a more targeted pathway, many of these drugs have been conjugated with conventional antibodies, which are used as carriers for preferential targeting of cancer cells. These conventional antibodies have a size of approximately 150 kDa, while the drugs have an average size of approximately 1 kDa. Therefore, the antibody:drug size ratio is approximately 150:1. This ratio is one of the reasons why the hydrophobicity of the drug has little effect on the antibody-drug conjugate (ADC) overall.

[0305] It has been confirmed that the physicochemical properties of ISVDs depend particularly on the amino acids exposed on their surface when exposed to solvents. This is reflected in a large number of different formulations used for ISVDs. In stark contrast to conventional antibodies, ISVDs have a size of only about 15 kDa. Therefore, the ISV:drug size ratio is only 15:1, which is 10 times smaller than that of conventional antibodies. Consequently, the hydrophobic characteristics of the drug have a disproportionately large influence on the properties of PDCs. In fact, the main problem with PDCs is aggregation. However, it has been unexpectedly observed that the PDCs of the present invention are stable, easy to administer in vivo, and capable of reducing tumor growth in vivo.

[0306] In one embodiment, the present invention provides a polypeptide or a toxin-conjugated dimer for treating a subject in need of it.

[0307] This invention relates to the dimer described above for therapeutic purposes, preferably for cancer treatment. Furthermore, this invention relates to the use of the dimer described above in the manufacture of medicaments for cancer treatment.

[0308] The term "cancer" refers to any cancer caused by the proliferation of malignant tumor cells, such as tumors, growths, carcinomas, sarcomas, leukemia, and lymphomas. Target cancers for treatment include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, leukemia, malignant lymphomas, breast cancer, ovarian cancer, testicular cancer, lung cancer, colon cancer, rectal cancer, pancreatic cancer, liver cancer, central nervous system cancers, head and neck cancer, kidney cancer, bone cancer, blood cancers, or lymphatic system cancers. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, stomach cancer or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, oral cancer, liver cancer, bladder cancer, urethral cancer, hepatocellular carcinoma, breast cancer (including, for example, HER2-positive breast cancer), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer or kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, multiple myeloma and B-cell lymphoma, brain cancer, head and neck cancer, and related metastases.

[0309] "Solid tumor cancer" is cancer that contains abnormal masses of tissue. In some implementations, cancer is solid tumor cancer (e.g., carcinoma, lymphoma, breast cancer, non-small cell lung cancer, prostate cancer, pancreatic cancer, head and neck cancer, colon cancer, sarcoma, or adrenocortical carcinoma).

[0310] The present invention provides methods for treating and / or preventing and / or alleviating symptoms associated with cancer (e.g., as defined above).

[0311] As used herein, and as fully understood in the art, “treatment” of a condition or illness (e.g., cancer as described herein) is a means of achieving a beneficial or desired outcome, such as a clinical result. A beneficial or desired outcome may include, but is not limited to, relief or improvement of one or more symptoms or conditions; reduction in the severity of the disease, symptom, or condition; a stable (i.e., non-deterioration) state of the disease, symptom, or condition; prevention of the spread of the disease, symptom, or condition; delay or postponement of the development of the disease, symptom, or condition; improvement or mitigation of the disease, symptom, or condition; and detectable or undetectable reduction (partial or complete). To “mitigate” a disease, symptom, or condition means to reduce the severity and / or undesirable clinical manifestations and / or delay or prolong the time course of development of the disease, symptom, or condition compared to its severity or time course without treatment.

[0312] As used herein, the term “effective amount” refers to an amount of a pharmaceutical agent (e.g., any of the aforementioned conjugates) that is sufficient to produce a beneficial or desired result, such as a clinical outcome, and therefore, “effective amount” depends on the context in which it is applied.

[0313] "Subject" refers to a human or a non-human animal (e.g., a mammal).

[0314] This invention relates to a method for treating cancer, the method comprising administering the dimer of the invention to a patient.

[0315] The present invention also provides a method for treating and / or preventing and / or alleviating conditions associated with rheumatoid arthritis, psoriasis, or excessive mucus secretion in the lungs, the method comprising administering (an effective amount) of a toxin-conjugated polypeptide as described herein to a subject requiring such treatment.

[0316] This invention provides methods for delivering a toxin-conjugated prophylactic or therapeutic peptide or dimer to a specific site, tissue, or cell type in the body, the methods comprising administering to a subject a toxin-conjugated peptide or a toxin-conjugated dimer as described herein. This invention also provides methods for treating a subject in need of it, the methods comprising administering a toxin-conjugated peptide as described herein.

[0317] In one embodiment, the present invention relates to a pharmaceutical composition comprising a toxin-conjugated polypeptide as described above or a toxin-conjugated dimer as described herein. The invention provides dimers of the invention in conjunction with a pharmaceutical carrier; optionally in conjunction with additional pharmaceutical agents.

[0318] Therefore, the present invention provides polypeptides of the invention (whether or not contained in the dimer of the invention) conjugated to a drug such as a toxin or a toxin portion as described herein. Preferably, the polypeptide comprises an ISV targeting EGFR, and may further comprise an ISVD targeting serum albumin.

[0319] In one embodiment, the present invention provides a toxin-conjugated polypeptide as described herein, wherein at least one ISVD inhibits and / or prevents the interaction between epidermal growth factor (EGF) and EGFR.

[0320] As used herein, the term "pharmaceutical composition" means a composition comprising a compound described herein, formulated together with a pharmaceutical excipient. In some embodiments, the pharmaceutical composition is manufactured or marketed as part of a treatment regimen for treating diseases in mammals, subject to approval by a government regulatory authority. For example, the pharmaceutical composition may be formulated for oral administration in unit dosage forms (e.g., tablets, capsules, caplets, gelcapsules, or syrups); for topical application (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution without microemboli and in a solvent system suitable for intravenous use); or for any other formulation described herein.

[0321] In one embodiment, the present invention relates to compositions comprising the dimer of the invention, preferably pharmaceutical compositions that optionally further comprise at least one pharmaceutical carrier, diluent or excipient and / or adjuvant, and optionally one or more additional pharmaceutically active peptides and / or compounds. An effective amount of the composition may be administered for radiation therapy programs, diagnostic treatments, or therapeutic treatments. When administered for radiation therapy programs or diagnostic purposes, the conjugate is administered to the subject at a diagnostically effective dose and / or an amount that effectively determines a therapeutically effective dose. In therapeutic applications, the composition is administered to a subject (e.g., a person) already suffering from a condition (e.g., cancer) at an amount sufficient to cure or at least partially prevent symptoms of the disease and its complications. An amount sufficient to achieve this purpose is defined as a “therapeutically effective amount,” that is, an amount of compound sufficient to substantially improve at least one symptom associated with the disease or medical condition. For example, in the treatment of cancer, an agent or compound that reduces, prevents, delays, inhibits, or prevents any symptoms of the disease or condition would be therapeutically effective. A therapeutically effective amount of a drug or compound does not necessarily cure the disease or condition, but rather provides treatment to the disease or condition, thereby delaying, stopping, or preventing the onset of the disease or condition, or improving the symptoms of the disease or condition, or altering the severity of the disease or condition (e.g., making it less severe), or accelerating recovery in an individual. The dimer of the present invention can be used to treat cancer by administering a first dose of any of the aforementioned dimer or composition to a subject in an amount effective for a radiation therapy program, followed by administering a second dose of any of the aforementioned dimer or composition in a therapeutically effective amount.

[0322] The effective amount for these uses can depend on the severity of the disease or condition and the subject's weight and overall condition. The therapeutically effective amount of the dimers and compositions of the present invention and used in the methods of the present invention, administered to mammals (e.g., humans), can be determined by a person skilled in the art, taking into account individual differences in the age, weight, and condition of the mammal. Because certain PDCs of the present invention exhibit enhanced targeting of cancer cells and are residual, the dosage of the compounds of the present invention can be lower than the equivalent dose required for the therapeutic effect of the unconjugated agent (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%). The agents of the present invention are administered to subjects (e.g., mammals, such as humans) in an effective amount that produces desirable results in the treated subject. The therapeutically effective amount can also be determined empirically by a person skilled in the art. The composition of the invention, comprising an effective amount, can be administered once or multiple times at a dosage level and in a manner selected by the treating physician. The dosage and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which can be monitored throughout treatment according to methods typically performed by a clinician or those described herein.

[0323] The dimer of the present invention can be used in combination with conventional treatment methods or therapies or can be used separately from conventional treatment methods or therapies.

[0324] When the dimer of the present invention is administered in combination therapy with other pharmaceutical agents, they may be administered sequentially or simultaneously to an individual. Alternatively, the pharmaceutical composition according to the present invention may consist of a combination of the compound of the present invention together with a pharmaceutical excipient as described herein and another therapeutic or prophylactic agent known in the art.

[0325] Typically, for pharmaceutical use, the dimers and / or polypeptides of the present invention can be formulated into pharmaceutical preparations or compositions comprising at least one dimer and / or polypeptide of the present invention and at least one pharmaceutical carrier, diluent or excipient and / or adjuvant, and optionally comprising one or more other pharmaceutically active polypeptides and / or compounds. By way of non-limiting examples, such formulations may be suitable for oral administration, parenteral administration (e.g., by intravenous, intramuscular or subcutaneous injection or intravenous infusion), topical administration, administration by inhalation, administration by skin patch, administration by implant, administration by suppository, etc., wherein parenteral administration is preferred. Such suitable forms of administration—which may be solid, semi-solid or liquid depending on the method of administration—and the methods and carriers used to prepare them will be clear to those skilled in the art and are further described herein. Such pharmaceutical preparations or compositions will generally be referred to herein as “pharmaceutical compositions.” Pharmaceutical preparations or compositions intended for use in non-human organisms will generally be referred to herein as “veterinary compositions.”

[0326] Therefore, in another aspect, the present invention relates to a pharmaceutical composition comprising at least one polypeptide or at least one dimer of the present invention and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use) and optionally one or more other active substances.

[0327] Generally, the polypeptides and / or dimers of the present invention can be formulated and applied in any suitable manner known per se. For example, refer to the general background art cited above (and especially to WO 04 / 041862, WO 04 / 041863, WO 04 / 041865, WO 04 / 041867 and WO 08 / 020079) and to standard manuals such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, USA (1990), Remington, the Science and Practice of Pharmacy, 21st edition, Lippincott Williams and Wilkins (2005); or Handbook of Therapeutic Antibodies (edited by S. Dubel), Wiley, Weinheim, 2007 (see, for example, pp. 252-255).

[0328] The polypeptides and / or dimers of the present invention can be formulated and administered in any manner known per se for use with conventional antibodies and antibody fragments (including ScFv and biantibodies) and other pharmaceutically active proteins. Such formulations and methods for their preparation will be apparent to those skilled in the art, and include, for example, formulations suitable for parenteral (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intracavitary, intra-arterial, or intrathecal) or topical (i.e., percutaneous or intradermal) administration.

[0329] For example, formulations intended for parenteral administration may be sterile solutions, suspensions, dispersions, or emulsions suitable for infusion or injection. Suitable carriers or diluents for such formulations include, but are not limited to, those mentioned on page 143 of WO 08 / 020079. Aqueous solutions or suspensions are generally preferred.

[0330] Therefore, the polypeptides and / or dimers of the present invention can be combined with pharmaceutical carriers such as inert diluents or assimilated edible carriers for systemic administration, e.g., orally. They can be encapsulated in hard-shell or soft-shell gelatin capsules, compressed into tablets, or directly combined with food in a patient's diet. For oral therapeutic administration, the polypeptides and / or dimers of the present invention can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, trochetes, capsules, elixirs, suspensions, syrups, rice paper wafers, etc. Such compositions and formulations should contain at least 0.1% of the polypeptides and / or dimers of the present invention. Of course, their percentage in the composition and formulation can vary and can conveniently be between about 2% and about 60% by weight of a given unit dosage form. The amount of the polypeptides and / or dimers of the present invention in such therapeutically useful compositions results in an effective dose level.

[0331] Tablets, lozenges, pills, capsules, etc., may also contain conjugates, excipients, disintegrants, lubricants, and sweeteners or flavorings, such as those mentioned on pages 143-144 of WO 08 / 020079. 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 materials of the types mentioned above. A variety of other materials may be present as coatings or additionally used to alter the shape of the unit dosage form. For example, tablets, pills, or capsules may be coated with gelatin, wax, shellac, or sugar. Syrups or elixirs may contain the polypeptides, compounds, and / or constructs of the present invention, sucrose or fructose as sweeteners, methylparaben and propylparaben as preservatives, dyes, and flavorings such as cherry or orange flavorings. Of course, any materials used in the preparation of any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amount used. Furthermore, the polypeptides and / or dimers of the present invention can be incorporated into sustained-release formulations and devices.

[0332] Enteric coatings can also be provided for formulations and preparations intended for oral administration, allowing the constructs of the invention to resist the gastric environment and enter the intestine. More generally, formulations and preparations for oral administration can be suitably formulated for delivery in any desired portion of the gastrointestinal tract. Furthermore, suitable suppositories can be used for delivery into the gastrointestinal tract.

[0333] The polypeptides and / or dimers of the present invention can also be administered intravenously or intraperitoneally by infusion or injection. Specific examples are further described on pages 144 and 145 of WO 08 / 020079 or in PCT / EP2010 / 062975 (all documents).

[0334] For topical application, the polypeptides and / or dimers of the present invention can be applied in their pure form, i.e., when they are liquids. However, it would generally be desirable to apply them to the skin as compositions or formulations in combination with dermatologically acceptable carriers, which may be solid or liquid. Specific examples are further described on page 145 of WO 08 / 020079.

[0335] The effective dose of the peptides, compounds, and / or constructs of the present invention 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 in the art; see, for example, US4,938,949.

[0336] Typically, the concentration of the polypeptides and / or dimers of the present invention in liquid compositions such as detergents will be about 0.1-25% by weight, preferably about 0.5-10% by weight. The concentration in semi-solid or solid compositions such as gels or powders will be about 0.1-5% by weight, preferably about 0.5-2.5% by weight.

[0337] The amount of the polypeptides and / or dimers of the present invention required for use in treatment will vary not only with the specific polypeptide and / or dimer selected, but also with the route of administration, the nature of the condition being treated, and the patient's age and condition, and will ultimately depend on the judgment of the assistant physician or clinician. Furthermore, the dosage of the polypeptides and / or dimers of the present invention varies depending on the target cells, tumor, tissue, graft, or organ.

[0338] The ideal dosage can be conveniently given as a single dose or as a fractional dose administered at appropriate intervals (e.g., as two, three, four or more sub-dose per day). The sub-dose itself can be further divided into, for example, discrete, loosely spaced administration intervals.

[0339] Administration regimens may include long-term, routine treatment. "Long-term" means at least two weeks, and preferably several weeks, months, or years. Necessary modifications to this dosage range can be determined by a person skilled in the art using only the routine experiments given in the teachings herein. In the event of any complications, the dosage may also be adjusted by an individual physician. Example

[0340] 1. Generation of building blocks

[0341] Various constructs were generated in Pichia pastoris, starting with EGFR-binding nanobodies 7D12 and 9G08, and albumin-binding nanobodies ALB11, as described in Table 4.

[0342] Table 4: Constructs

[0343] T023800001-A 7D12-20GS-ALB11-GGC-A T023800003-A 7D12-20GS-7D12-GGC-A T023800005-A 7D12-20GS-9G08-GGC-A T023800006-A 7D12-20GS-7D12-20GS-ALB11-GGC-A T023800008-A 7D12-20GS-9G08-20GS-ALB11-GGC-A T023800001-Dimer <![CDATA[ N [7D12-ALB11-GGC] C -S-S- C [CGG-ALB11-7D12] N ]]>

[0344] 1.1 Gene Fusion

[0345] According to standard protocols, such as those described by Garaicoechea et al. (Garaicoechea et al. (2008) JVirol. 82:9753–9764), gene fusion is used to couple the building blocks 7D12 and Alb11 with the linker in multiple transposable sequences in T023800001, T023800003, T023800005, and T023800006. This produces peptides containing various linker lengths and compositions (sequences and individual ISVDs). C-terminal extensions containing GGCs are also constructed via gene fusion. The sequences T023800001 (SEQ ID NO:27), T023800003 (SEQ ID NO:28), T023800005 (SEQ ID NO:29), T023800006 (SEQ ID NO:30), and T023800008 (SEQ ID NO:31) are provided in Table 6.

[0346] The feasibility of constructing different C-terminal extensions containing a cysteine ​​moiety at the C-terminus was demonstrated by manufacturing a variety of peptides with different C-terminal extensions: -C (SEQ ID NO:1), -GC (SEQ ID NO:2), -GGC (SEQ ID NO:3), -GGGC (SEQ ID NO:4), -CG (SEQ ID NO:10), -GCG (SEQ ID NO:11), -GGGCG (SEQ ID NO:13), -GGGGCGGGG (SEQ ID NO:15), and -AAAC (SEQ ID NO:8) (data not shown).

[0347] 1.2 Alanine extension

[0348] According to a widely accepted protocol (see below), the alanine moiety (N-maleimino-β-alanine; Sigma-Aldrich) is conjugated to the thiol group (-SH) of the C-terminal cysteine ​​via maleimide chemistry under near-neutral conditions (pH 6.5–7.5) to form a stable thioether bond. Briefly, the concentration of the peptide under consideration is first determined. 2–5 moles of excess N-maleimino-β-alanine are added to the peptide to block all available cysteine. The mixture is incubated at RT for 1 h, followed by overnight incubation at 4 °C. The conjugation efficiency is determined by LC-MS the following day. The peptide containing the Ala extension is purified to homogeneity by SEC chromatography to remove excess N-maleimino-β-alanine.

[0349] The resulting constructs are designated as T023800001-A, T023800003-A, T023800005-A and T023800006-A (Figure 1; Table 4).

[0350] Further evidence confirms that alanine can also be conjugated to constructs with a C-terminal extension containing cysteine ​​that differs from GGC (see also 1.1 above; data not shown).

[0351] 1.3 Dimerization

[0352] In a culture medium in which Pichia pastoris has been grown, peptides are coupled into dimers via chemical conjugation, wherein the C-terminal cysteine ​​in the C-terminal extension of each of the two peptides is oxidized to the disulfide derivative cysteine ​​at near-neutral pH via their sulfhydryl moiety. Essentially as given in WO2010 / 125187, to optimize the oxidation process, copper ions (CuSO4 in the form of Cu) are added. 2+ The dimer was purified to homogeneity and subsequently analyzed by size exclusion chromatography. The sample was also confirmed by LC-MS. The obtained data confirmed that almost 100% of the thiol moiety was oxidized after treatment with 1 mM CuSO4 at room temperature for 2 h. No obvious (undesirable) peak formation was observed in any of the chromatograms. Furthermore, no evidence of obvious peak formation was seen in any of the chromatograms, suggesting that copper treatment did not appear to oxidize methionine in the protein, and no +16 Da mass increase was detected in the total mass analysis, which would be consistent with single oxidation at, for example, methionine.

[0353] Dimers were prepared from T023800001, T023800003, T023800005 and T023800006. The dimer of T023800001 (denoted as T023800001-dimer) is shown in Figure 1.

[0354] 1.4 Stability

[0355] After storage under severe stress conditions, the stability of different constructs of the invention, such as dimers, peptides, and benchmarks, was tested. These conditions were essentially as given in WO2014 / 184352, and consisted of incubating the peptides of the invention at different temperatures (25°C and 40°C) for extended periods (3 weeks and 6 weeks).

[0356] The peptides (e.g., having a C-terminal extension comprising a cysteine ​​moiety) and dimers of the present invention have been shown to possess properties similar to those of their parent molecules and to be stable over extended periods at 4°C, 25°C, and 40°C without chemical degradation or modification (data not shown). Furthermore, as shown in the functional assays (refer to below), stability remains unchanged after multiple freeze-thaw cycles and extended storage at 4°C for extended periods (i.e., >4 days).

[0357] Characterization of peptides that bind to MDA-MB-468 cells

[0358] The peptide was characterized in a binding competition assay to assess EGFR binding affinity.

[0359] The MDA-MB-468 breast cell carcinoma line (breast / mammary gland; derived from metastatic site: pleural effusion; ABL216) was used.

[0360] To detect the binding of the peptide to EGFR-expressing cells, FLAG-labeled 7D12 was used as a competitor. To set up the assay, a titration series of FLAG-labeled 7D12 was first performed on MDA-MB-468 cells. In the competitive setup for titrating unlabeled peptides, FLAG-labeled 7D12 was selected for ECG. 90 (41nM).

[0361] In short, 100,000 cells were transferred to plates. The plates were washed twice by centrifugation at 200g for 3 minutes at 4°C. The supernatant was removed, and 50 μl of purified peptide along with 50 μl of FLAG-labeled 7D12 (final concentration 41 nM) was added to each well, totaling 100 μl. After incubation at 4°C for 90 minutes, the plates were washed three times by centrifugation at 4°C for 30 minutes. The supernatant was removed, and 100 μl / well of 0.5 μg mouse anti-flagmAb (Sigma-Aldrich, catalog number F1804) or FACS buffer was added, followed by incubation at 4°C for 30 minutes. The cells were washed three times by centrifugation at 200g for 3 minutes at 4°C. After removing the supernatant, 110 μl / well of goat anti-mouse PE or goat anti-human IgG PE was added to the cells, and the plates were incubated at 4°C for 30 minutes. The plates were then centrifuged at 200g for 30 minutes at 4°C. The supernatant was removed, and 100 μl / well of FACS buffer was added. The plates were then washed three times sequentially by centrifugation at 200g for 3 minutes at 4°C. Next, dead cells were stained with 100 μl of TOPRO (Molecular Probes, T3605) per well, and the cells were sequentially measured on a FACS Canto (Becton Dickinson) spectrometer. First, gates were set on intact cells as determined based on scattering characteristics. Then, dead cells were selected by gating them using fluorescence characteristics derived from TOPRO staining (5 nM, Molecular Probes, T3605). As a control, conditions were selected where no peptides were present or where known unrelated peptides were present (data not shown).

[0362] The unit price of T023800001-A (also referred to as T023800001 in this paper), the extended half-life (HLE) T023800003-A (also referred to as T023800003 in this paper), T23800005-A (also referred to as T023800005 in this paper), T023800006-A (also referred to as T023800006 in this paper) and the unreduced T023800001-dimer were evaluated.

[0363] The result is Figure 2 As described in the text.

[0364] Based on these results, it can be concluded that T023800001, T023800003, T023800005, and T023800006 are related to EC. 90The competition for 7D12-FLAG (i.e., 41nM) yielded K values ​​of 15nM, 0.63nM, 0.25nM, and 1.16nM for T023800001, T023800003, T023800005, and T023800006, respectively. i The absolute inhibition constant K was calculated using the Cheng-Prusoff equation. i .

[0365]

[0366] Surprisingly, the T023800001-dimer showed a K0.12 nM. i This is twice as good as the best-performing gene fusion construct T023800005-A, and even more than nine times better than the direct comparison of the T023800001-dimer, T023800006-A.

[0367] 3. Quantification of EGFR phosphorylation in the HER14 cell line

[0368] To confirm whether the increased potency observed in competitive FACS (see Example 2 above) also translates into regulation of EGFR-mediated signal transduction, the inventors provided an experiment to block EGF-mediated EGFR phosphorylation in NHI3T3 / HER14 cells using nanobodies. The constructs used were T023800001-A, T023800006-A, and T023800001-dimer. Dose-dependent inhibition of EGFR phosphorylation was evaluated in EGFR-only HER14 cells.

[0369] In short, HER14 cells were seeded in duplicate into 96-well plates coated with 0.1% gelatin and grown for 24 h in DMEM medium containing 10% FBS / BS. The following day, the cells were serum-starved for 24 h in medium supplemented with 0.1% FCS and then incubated with the construct, followed by stimulation with 0.5 nM recombinant human EGF (R&D Systems, catalog number 236-EG) for 10 min. The EGF concentration was based on the EC50 (EC50) obtained in HER14 cells. 50=3.5 ng / ml). An unrelated control peptide was included as a reference in each plate (data not shown). The monolayer was washed twice with ice-cold D-PBS and subsequently lysed in ice-cold RIPA buffer replaced with 1 mM PMSF. EGF-dependent receptor activation in the cell lysates was measured using the Phospho (Tyr1173) / Total EGFR Whole Cell Lysate Kit (Meso Scale Discovery-K15104D). Plates were loaded with 30 μl of lysate, incubated at RT for 1 h with shaking, and processed according to the manufacturer's protocol. Plate readings were taken on a Sector Imager 2400 (Meso Scale Discovery). The percentage of phosphoprotein relative to total protein was calculated using the following formula: (2 × p-protein) / (p-protein + total protein) × 100.

[0370] The result is Figure 3 As described in the text.

[0371] Dose-dependent inhibition of EGFR phosphorylation was observed only in Her-14 cells expressing EGFR. Since functional phosphorylation is mediated solely by EGFR signaling, the increase in antibody affinity caused by multivalent formatting is expected to translate into an increase in EGFR phosphorylation in a cell-specific manner.

[0372] Even more pronounced than results from competing FACS studies, the T023800001-dimer (4.4 nM) showed a 5-6 fold increase in potency compared to the formed divalent nanobody T023800006-A (26.6 nM). The monovalent T023800001-A achieved a potency of 10.5 nM.

[0373] 4. Preparation of monomeric nanobodies extended by SEC using cysteine

[0374] 4.1 Background Information

[0375] It is recognized that the polypeptides of the present invention, comprising at least one immunoglobulin single variable domain (ISVD) and a C-terminal extension containing a cysteine ​​moiety at the C-terminus, are suitable for maleimide chemistry based on coupling reaction (refer to Example 1.2 above).

[0376] To convert the dimer into a monomeric polypeptide and make the C-terminal cysteine ​​available for coupling, reduction is required. However, careful design of optimized conditions is necessary to induce the reduction of the disulfide dimer without reducing the internal typical ISVD disulfide bond. DTT or TCEP is preferred for reduction. Unlike TCEP, DTT needs to be removed to create optimal coupling conditions. The monomeric nanobody is separated from the unreduced dimer nanobody and DTT by size exclusion chromatography (SEC).

[0377] 4.2 Restoration Scheme

[0378] The reduction protocol consisted of treatment with 10 mM DTT in D-PBS overnight at 4°C (or for a minimum of 2 hours at room temperature). ISVD concentrations ranged from 2 to 10 mg / ml. These conditions confirmed that they did not affect the typical internal disulfide bonds (see below). Figure 4 Similar results were obtained using TCEP, where we used fixed TCEP (Pierce, fixed TCEP disulfide reduction gel, #77712) according to the manufacturer's protocol. Alternatively, short exposures to 10 mM TCEP were used over a period of 30 minutes at 4°C.

[0379] 4.3 Size Exclusion Chromatography (SEC)

[0380] For purification, a Superdex 75 column (GE Healthcare) (separation range 5-100 kDa) is preferably used for peptides containing up to three ISVDs to generate monomer reduction products. For analytical purposes, an HPLC column such as the Agilent SEC-3 is used. Equilibration and flow buffers are D-PBS.

[0381] exist Figure 5 The document provides an example of a completely restored, clean product after SEC.

[0382] Following SEC, the reduced monomeric peptides were immediately used for conjugation or frozen to prevent re-oxidation to dimers. Experimental evidence suggests that the reduced monomer fractions of GGC-extended peptides are stable in D-PBS at 4°C for up to 24 hours (data not shown).

[0383] The peptide containing two ISVDs was reduced at ambient temperature with 10 mM DTT over 2 h and its size was determined on a Superdex 75XK 16 / 60 column (GE Healthcare) equilibrated in D-PBS. Only a small amount of dimer was detected; the remainder of the material was reduced to monomer and thus prepared for conjugation. The molecular weight of the gel filtration standard (Biorad) is shown above the corresponding peak, as dashed lines.

[0384] 5. Peptides conjugated with MMAE

[0385] Monomethyl auristatin E (MMAE), a hydrophobic antimitotic agent, is a synthetic analogue of the natural product dolastatin 10. MMAE is a potent inhibitor of microtubule polymerization in dividing cells.

[0386] In this embodiment, we proceed to conjugate MMAE to the released C-terminal cysteine ​​residue of the peptide of the present invention. In short, for drug targeting purposes, MMAE is conjugated to the peptide via a valine-citrulline linker. The valine-citrulline linker is highly stable in serum, but is cleaved by lysosomal enzymes such as cathepsin B after the conjugate is internalized by the target cell. The following linker abbreviations are used herein and have the indicated definitions: ValCit is the valine-citrulline dipeptide site in the protease-cleavable linker; PAB is a para-aminobenzoyl group; and mc is the conjugated maleimide.

[0387] The nanobody was reduced overnight with 10 mM DTT at 4 °C, followed by buffer exchange to remove excess DTT. The reaction was performed at 22 °C using mc-val-cit-PAB-MMAE (MW approximately 0.7 kDa). Figure 6 The conjugation of N-acetylcysteine ​​was performed. After 1 hour, the reaction was quenched at 20 equivalents of N-acetylcysteine / free drug. The resulting product was purified by centrifugation and the buffer was exchanged for the final buffer. For larger-scale purification, a non-centrifugal percolation method is more suitable. The product solution was sterile filtered (0.2 mm).

[0388] Peptides conjugated with MMAE:

[0389] T023800001=>T023800001-mc-val-cit-PAB-MMAE(ABL 100-NC003-1)

[0390] T023800003 => T023800003-mc-val-cit-PAB-MMAE(ABL 100-NC003-3)

[0391] T023800005=>T023800005-mc-val-cit-PAB-MMAE(ABL 100-NC003-5)

[0392] T023800006=>T023800006-mc-val-cit-PAB-MMAE(ABL 100-NC003-6)

[0393] T023800008=>T023800008-mc-val-cit-PAB-MMAE(ABL 100-BF012-1)

[0394] HIC-HPLC analysis was performed to determine the drug-to-peptide ratio (DAR). Briefly, analytical HIC of the conjugates was performed using a TOSOH, TSKgel butyl-NPR column (35 × 4.6 mm) connected to a Dionex Ultimate 3000RS HPLC system. A linear gradient was applied over 30 min at a flow rate of 0.8 mL / min from 100% buffer A (1.5 M ammonium sulfate in 50 mM sodium phosphate, pH 7.0) to 100% buffer B (20% isopropanol (v / v) in 50 mM sodium phosphate). The column temperature was maintained at 30 °C and UV detection was performed at 280 nm throughout the analysis. 10 μg of sample was injected for each analysis. The drug-to-peptide ratio (DAR) was assigned to each peak based on the shift to higher retention times and the A248 / A280 ratio. The average DAR was calculated by summing the individual DAR values ​​and multiplying by the species fraction (expressed as a decimal). The peptides used are ABL100-NC003-1, ABL 100-NC003-3, ABL 100-NC003-5 and ABL 100-NC003-6.

[0395] The results are provided in Table 5.

[0396]

[0397] SDS-PAGE analysis was performed to determine the oxidative state of the peptide. In short, using... 4-12% Bis-Tris gel (Invitrogen, catalog number NP0321BOX) was used for SDS-PAGE analysis with MES buffer under non-reducing conditions. For analysis, 1 μg of sample (protein-based) was loaded onto each lane of the gel. Electrophoresis was performed at 200V for 35 min. The gel was then rinsed with INSTANTBLUE. TM (Expedeon, catalog number ISB1LUK) staining for protein detection and using Analysis using imaging equipment (GE Healthcare).

[0398] A summary of the results is also provided in Table 5. Exemplary results are provided as follows: Figure 7 supply.

[0399] To further confirm and elucidate the SDS-PAGE results, SE-HPLC analysis was performed to determine percentage purity and aggregation. Briefly, a Waters system connected to an Agilent Infinity 1260 Bioinert system was used. SE-HPLC was performed using a UPLC BEH200SEC column (4.6 mm x 30 cm, 1.7 μm). The mobile phase was 0.1 M sodium phosphate buffer, pH 6.8, containing 15% (v / v) isopropanol. The flow rate was kept constant at 0.15 mL / min. The column was maintained at 25 °C throughout the analysis. Analysis was performed using UV detection at 280 nm after 30 min of isocratic elution. For each analysis, 10 μg of sample was injected. The percentage purity and percentage aggregation were calculated by comparing the peak areas of the main peak and the early elution peak with the total peak area, respectively.

[0400] The results are summarized in Table 5. An exemplary HIC analysis of the coupling results is presented in... Figure 8 As described in the text.

[0401] This means that, for all peptides, the reaction yields an efficiency of over 98% for conjugation with the ADC. Furthermore, the reaction yields a DAR of 1, indicating that the ISVD is intact, for example, without the use of internal thiol groups, and that the amount of drug per peptide is highly controllable. This results in better safety compared to the Gaussian distribution of drugs conjugated with conventional antibodies.

[0402] Table 6

[0403]

[0404]

[0405] 5.1 In vitro cytotoxicity of peptides conjugated with MMAE

[0406] use The instrument (analyzer model W380; SN: 281081212038, Roche) was used to test the effects of MMAE-conjugated peptides on cell proliferation and / or cytotoxicity. The instrument quantifies changes in electrical impedance as cells attach and spread in a culture dish, displaying them as a dimensionless parameter known as the cell index, which is a direct proportion to the total area of ​​the tissue culture wells covered by cells (Duchateau et al., 2013. Phys. Status Solidi 10:882-888 and Giaever and Keese 1991. Proc. Natl. Acad. Sci. USA 88:7896-7900). The instrument (analyzer model W380; SN: 281081212038) used E-plate 96 (ACEABiosciences; catalog number 05 232 368 001; batch number 20140138; plate 1: ID number 079605; plate 2: ID number 079606) as tissue culture well plates for cell seeding. The constructs used were T023800001-A and T023800001-MMAE, T023800003-A and T023800003-MMAE, T023800005-A and T023800005-MMAE, and T023800006-A and T023800006-MMAE. The following protocol was used... Instrumental evaluation was conducted to assess the dose-dependent inhibitory effect of unconjugated and MMAE-conjugated nanobodies on the proliferation of MDA-MB-468 (mammary gland / breast; derived from metastatic site: pleural effusion; ABL216) cells.

[0407] In short, in the presence of 5% CO2, The plates were placed in an incubator at 37°C. MDA-MB-468 cells were grown in T175 flasks containing RPMI (Gibco, catalog number: 72400-021) supplemented with 1% P / S (Gibco, catalog number: 15140-122); 1% Na pyruvate (Gibco, catalog number: 11360-039); and 10% FBS (Sigma-Aldrich, catalog number: F7524). Cells were harvested by trypsinization, centrifuged, and resuspended to the specified cell density. 50 μl of cell culture medium was added to each well of an E-plate 96 and... The system was used to perform blank readings to measure background impedance in the absence of cells. 6000 cells (50 μl) were transferred to each well of an E-plate 96 and incubated for 20 h to allow cell adhesion. Starting at 500 nM (total volume / well 200 μl), 100 μL of each peptide was serially administered at a 1:3 dilution. Impedance readings were programmed at 15-minute intervals. The experiment was stopped at time point 162 h (± 7 d). Cell indexes measured at time point 116 h after seeding for all test concentrations were used for dose-response analysis.

[0408] The proliferation curve and dose-response curve were respectively at Figure 9 As described in Figure 10. The obtained IC 50 The values ​​are given in Table 7.

[0409] Table 7. IC50 values ​​observed for unconjugated and MMAE-conjugated peptides 50 and % inhibition

[0410]

[0411]

[0412] Apart from the bicomplementary site T023800005-A, which exhibited a slight inhibitory effect on cell proliferation, the unconjugated peptides T023800001-A, T023800003-A, T023800005-A, and T023800006-A did not show a significant effect on the proliferative properties of MDA-MB-468 cells. In contrast, as in Figure 9 As shown, the MMAE-conjugated peptides T023800001-MMAE, T023800003-MMAE, T023800005-MMAE and T023800006-MMAE clearly exhibit a dose-dependent inhibitory effect on cell proliferation, with almost complete inhibition at the highest dose.

[0413] 5.2 In vivo efficacy of peptides conjugated with MMAE

[0414] In vivo efficacy study of anti-EGFR peptide drug conjugates in a subcutaneous xenograft mouse model.

[0415] 1x10 g of the drug was injected subcutaneously into the right ventral midline of healthy 6-8 week old SWISS nude female mice. 7 FaDu cells were used to induce tumor growth. The FaDu cell line was a head and neck cancer cell line established from a punch biopsy of a hypopharyngeal tumor removed from a 56-year-old Caucasian / Indian male patient. Tumors were induced when they reached 100-200 mm in size. 3 Treatment begins when the average volume between the two values ​​is reached.

[0416] Animals received daily injections of 5 mg / kg T023800008-MMAE every 4 days for a total of 6 injections (Q4Dx6). The first control group received daily injections of a non-EGFR-binding, but equimolar dose of the MMAE-conjugated peptide every 4 days for a total of 6 days. The second control group received daily injections of the vector every 4 days for a total of 6 injections. Each group consisted of 12 animals.

[0417] Measure the length and width of the tumor twice a week using a caliper, and estimate the tumor volume using the following formula:

[0418] Compared with the two control groups, the peptide-MMAE conjugate T023800008-MMAE showed significant inhibition of tumor growth. Figure 11 ).

[0419] 6. Formation of bispecific dimers

[0420] In this embodiment, a scheme is provided to generate bispecific dimers, i.e., dimers in which polypeptide 1 and polypeptide 2 are dissimilar.

[0421] 6.1 Standard Scheme

[0422] In the first case, the scheme is similar to that in Example 1.3 above, but one of the Pichia pastoris strains produces two dissimilar polypeptides. In the culture medium where Pichia pastoris has grown, the polypeptides are also coupled to the dimer via chemical conjugation, wherein the C-terminal cysteine ​​in the C-terminal extension of each of the two polypeptides is oxidized to the disulfide derivative cysteine ​​at near-neutral pH via their sulfhydryl moiety. Essentially as given in WO2010 / 125187, to optimize the oxidation process, copper ions (CuSO4 in the form of Cu) are added. 2+ The dimer was purified to homogeneity (ion-exchange chromatography) and subsequently analyzed by size exclusion chromatography. The sample was also confirmed by LC-MS. The standard protocol will produce the expected bispecific NB1-NB2 dimer. However, it is anticipated that the fraction will also contain monospecific dimers, such as NB1-NB1 and NB2-NB2.

[0423] 6.2 Alternative Solutions

[0424] Nanobody heterodimers (bispecific dimers) can be generated without the use of cross-linking agents by using two different C-terminal cysteine-extended Nb.

[0425] This can be achieved by non-covalently immobilizing the first nanobody (=NbA) while simultaneously forming C-terminal heterodimer disulfide bonds with the free thiol groups available to the second nanobody (=NbB).

[0426] In the first step, NbA is reduced to obtain 100% monomeric material. General conditions for reducing a typical nanobody solution [5-10 mg / ml] are 10 mM DTT in D-PBS overnight at 4°C or over 1-2 hours at room temperature (RT). Preferably, optimal conditions are determined for each individual nanobody so that its typical disulfide bonds remain intact.

[0427] In step 2, the NbA monomer fraction binds to the support under reducing conditions. Such a support can be a chromatographic resin that preferably binds only NbA and not NbB. NbA is immobilized at a low density to avoid the formation of NbA-NbA dimers during immobilization. Spatial separation of such individual NbA fractions is achieved by using suboptimal binding conditions on the column (e.g., excessively high flow rates for typical affinity resins) or by means of loading via expanded bed chromatography. Preferably, the support binds only NbA. Therefore, if NbA (and preferably not NbB) is a protein A binder, protein A can be used. If both nanobodies A and B bind to the support, the support should be saturated with non-cysteine-extended nanobodies after NbA immobilization and before the application of NbB.

[0428] In step 3, an excess of the second nanobody (NbB) (see above), also in its reduced form, is applied and cycled through a column (optionally under slightly oxidizing conditions). The NbB is passed through the support until the immobilized NbA is fully coordinated with the NbB via disulfide bonds. The decrease in NbB concentration can then be measured to match the saturated NbA population. For this step, conditions are optimized to limit the amount of NbB-NbB dimer formation. This population will not bind to the support and can be recovered and used in future coupling reactions.

[0429] In step 4, the NbA-NbB dimer formulation is recovered from the resin under typical elution conditions for the column (i.e., acidic conditions for protein A) and further processed / formulated.

[0430] Research on 7-dimers and drug-conjugated peptides

[0431] As noted above, due to size differences, the dimerization and conjugation of the drug with the peptides of this invention have a much greater effect than with conventional antibodies. Therefore, the inventors set out to evaluate the effect of the effective payload conjugated with nanobodies having DAR=1 on PK properties.

[0432] Furthermore, the inventors set out to evaluate the PK properties of the dimers of the present invention containing two human serum albumin-binding domains (“Alb”). As will be apparent from the above examples, dimers containing two identical parts (i.e., polypeptide 1 = polypeptide 2) are easier and more cost-effective to generate and purify than dimers having two dissimilar parts. In several instances used to extend the half-life of the construct, the human serum albumin-binding domain is required. However, having an additional human serum albumin-binding domain should not have any negative impact on the PK characteristics of the construct.

[0433] 7.1 Radiolabeling of peptides

[0434] PK properties are tested using radiolabeled peptides. In short, using...89 Zr and NCS-Bz-Df were randomly conjugated to free -NH2 to radiolabel the peptide (see Figure 12). 22 nmol of the peptide (1.0 mg) was mixed with 0.9% NaCl until a final volume of 500 μL (final concentration 2 mg / mL) was reached. Next, the pH was adjusted to 8.9–9.1 by adding 0.1 M Na2CO3. Finally, 66 nmol of NCS-Bz-Df in DMSO (3 eq, 10 μL) was added and the reaction was carried out at 37 °C for 30 min. After 30 min, the reaction mixture was purified using a PD10 column prewashed with 50 mM NaOAc / 200 mM sucrose. The product was collected in a 1.0 mL fraction. The reaction mixture was then... 89 Zr radiolabeled Df-PK-peptide at room temperature for 60 min at pH ~7 (the reaction mixture contained: 100 μL 1M oxalic acid, containing...) 89 Zr, 45 μL 2M Na2CO3, 500 μL 0.5M Hepes buffer (pH 7.2), and 355 μL NCS-Df-peptide (~0.4 mg) were added. Next, the pre-washed PD10 column was used to purify the reaction mixture with 50 mM NaOAc / 200 mM sucrose, and the product was collected in 1.5 mL.

[0435] Table 8 Labeling Results of Peptides

[0436]

[0437] The radiolabeling results are summarized in Table 8. The radiolabeling yield varied between 9.6% and 37.6% (a yield of 70% is typically expected). This low labeling yield is likely related to the low amount of peptide used during modification. HPLC and spin-filter analyses showed that the amount used... 89 Zr-T023800006A and 89 For Zr-T023800006-MMAE, the radiochemical purity is satisfactory (>96%). For rotary filter analysis, 89 Zr-T023800001 showed a purity of <90.0%, and HPLC showed 8.6% free content. 89 Zr. Typically, the construct should have a radiolabeling purity >90.0%. In this case, it was decided to use [Zr]. 89 Zr-T023800001 is used for PK research because 89The rotary filter purity of Zr-T023800001 is close to 90%, and HPLC analysis shows a product purity >90%. For all peptides, Lindmo binding is >90% (data not shown).

[0438] Then, 89 The Zr-PK radiolabeled peptide was prepared at an activity of 0.22 MBq / mouse and a concentration of 50 μg / mL, with an injection volume of 130 μL.

[0439] In summary, the radiolabeling yield was not as effective as expected. 89 Zr-T023800006A and 89 Zr-T023800006-MMAE exhibits good radiochemical purity (>97% according to rotary filter and >96% according to HPLC). Lindmo binding results are high, >90%. 89 Zr-T023800001 was not as pure as required for rotary filter analysis. Ultimately, it was decided to still use... 89 Zr-T023800001. All peptides were successfully formulated and injected.

[0440] 7.2 In vivo PK study

[0441] Radiolabeled ( 89 The Zr peptide was injected into three mice, and cpm (counts per minute) values ​​were measured at nine time points: 5 min, 1 h, 3 h, 24 h, 48 h, 72 h, 140 h, 168 h, and 192 h. These values ​​were then used to calculate the %injection dose / g mice (%ID / g) of the peptide. The results from the three mice were averaged for each peptide.

[0442] The results are summarized in Figure 13 middle.

[0443] The results unexpectedly showed that the biodistribution characteristics of the divalent peptide (T-023800006-A) were similar to those of the drug-conjugated peptide (T-023800006-MMAE). Conjugation of the peptide of the present invention to the effective payload had no effect on the biodistribution characteristics. Without any theoretical constraints, the assumption of a tightly controlled conjugation process with DAR=1 predicted the PK properties (in which case there was no change compared to the unconjugated peptide).

[0444] Surprisingly, the biodistribution characteristics of the cys-linked dimer (T-023800001) of the present invention are similar to those of the corresponding divalent polypeptide (T-023800006-A). The presence of two serum albumin-binding units in the cys-linked dimer of the present invention does not affect the distribution characteristics.

[0445] 8. Internalization improved through dimerization

[0446] The objective of this experiment was to evaluate whether the Cys-linked dimer DIM T023800001 (i.e., functionally divalent) exhibited increased internalization compared to its monomeric counterpart (T023800001-A) and the conventionally linked bivalent form of a gene fusion (T023800006-A). For this purpose, internalization experiments were performed on NCI-H292 cells moderately expressing the EGF receptor. Flow CytoMetry (FCM) was performed using pHrodo... TM Labeled albumin (50 μg / ml) was used as a detection tool to measure the accumulation of internalized nanobodies in living cells. The dye is a pH-sensitive molecular probe and is almost non-fluorescent at neutral pH. In acidic environments, such as endosomes and lysosomes, it emits bright fluorescence. Cells were transferred in flat-bottomed 96-well plates (30,000 cells / well) and subjected to different concentrations of specific peptides and constructs along with pHrodo at 37°C. TM The cells were incubated with labeled albumin (50 μg / ml) for 5 hours. Afterwards, the cells were washed, harvested, and analyzed using FCM.

[0447] The obtained dose-response curves are in Figure 14 The relevant EC is given in the middle. 50 The values ​​and top MFI top levels are listed in Table 9.

[0448] Table 9. Estimated EC 50 Values ​​and MFI top level

[0449]

[0450] Clearly, the overall internalization of DIM T023800001 appears to be more potent and efficient in NCI-H292 cells compared to the monomeric T023800001-A and the conventionally linked bivalent nanobody T023800006-A. Furthermore, this difference in internalization is less pronounced, however, it remains evident in cells expressing EGFR at extremely high levels, such as MDA-MB-468 (data not shown).

[0451] All references cited throughout this application (including bibliographic references, granted patents, published patent applications, and co-pending patent applications) are expressly linked by reference, especially for the teachings cited above.

[0452] The accompanying drawings and experimental / exemplary sections are provided only to further illustrate the invention and should not be construed or understood in any way as limiting the scope of the invention and / or the scope of the appended claims, unless expressly stated otherwise herein.

Claims

1. A method for preparing a dimer, the method comprising at least the following steps: (i) providing a first polypeptide, wherein the first polypeptide comprises -At least one V HH domain and - Including the C-terminal extension of the cysteine ​​moiety; (ii) providing a second polypeptide, wherein the second polypeptide comprises -At least one V HH domain and - Including the C-terminal extension of the cysteine ​​moiety; and (iii) The thiol portion of the cysteine ​​moiety at the C-terminus of the first polypeptide and the thiol portion of the cysteine ​​moiety at the C-terminus of the second polypeptide are oxidized to the disulfide derivative cystine. Its characteristic is that it maintains the V HH The integrity of the structural domains and the fact that the cysteine ​​is the only intermolecular disulfide bond present in the dimer; thereby preparing the dimer, The C-terminal extension is selected from the group consisting of SEQ ID NO: 1-5.

2. The method of claim 1, wherein at least 80% of the first polypeptide and the second polypeptide are dimerized.

3. The method according to claim 1, further comprising the step of purifying the dimer.

4. The method according to claim 1, wherein the first polypeptide and the second polypeptide are identical.

5. The method according to claim 1, wherein the first polypeptide and the second polypeptide are different.

6. The method according to any one of claims 1 to 5, wherein the C-terminal extension is connected to the V-terminus located at the C-most end of the polypeptide. HH Genetic fusion at the C-terminus of the domain.

7. Use of the dimer in the preparation of a medicament for the treatment of cancer, wherein the dimer is internalized. The dimer comprises a first polypeptide and a second polypeptide. Wherein the first polypeptide comprises -At least one V HH domain and - Including the C-terminal extension of the cysteine ​​moiety; The second polypeptide includes -At least one V HH domain and - Including the C-terminal extension of the cysteine ​​moiety; and The first polypeptide and the second polypeptide are covalently linked by a disulfide bond between the cysteine ​​moieties extending from the C-terminus of the first polypeptide and the cysteine ​​moieties extending from the C-terminus of the second polypeptide; and the disulfide bond between the cysteine ​​moieties extending from the C-terminus of the first polypeptide and the cysteine ​​moieties extending from the C-terminus of the second polypeptide is the only intermolecular disulfide bond present in the dimer. The C-terminal extension is selected from the group consisting of SEQ ID NO: 1-5.

8. The use according to claim 7, wherein the first polypeptide and the second polypeptide are identical.

9. The use according to claim 7, wherein the first polypeptide and the second polypeptide are different.

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