RU2567006C2 - Humanisation of rabbit antibodies using universal antibody scaffold - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: acceptor scaffold of variable region of heavy chain of human antibody or humanised antibody for transplantation of CDR from Lagomorpha antibody is presented where the scaffold contains threonine (T) in the position 24; alanine (A) or glycine (G) in the position 56; threonine (T) or asparagine (N) in the position 84 and leucine (L) or valine (V) in the position 89 (AHo numbering). The named acceptor antibody scaffold for transplantation of Lagomorpha CDR is offered. Besides, the present invention describes the released nucleinic acid coding the acceptor antibody scaffold; the expression vector containing the named nucleinic acid; and the host cell containing the named expression vector for production of the acceptor antibody scaffold.

EFFECT: invention allows to obtain the acceptor scaffold of heavy chain of antibody suitable for change of hyper variable regions of Lagomorpha antibodies.

10 cl, 6 dwg, 5 tbl, 5 ex

Description

Related Information

This application claims the priority of US 61/075697, filed June 25, 2008; additionally claims priority US 61/155041, filed February 24, 2009 and US61 / 155105 of February 24, 2009.

BACKGROUND OF THE INVENTION

Monoclonal antibodies, their conjugates and derivatives are very commercially important as therapeutic and diagnostic agents. Non-human antibodies elicit a strong immune response in patients, usually after a single injection at a low dose (Schroff, 1985 Cancer Res. 45: 879-85, Shawler. J. Immunol. 1985 135: 1530-5; Dillman, Cancer Biother 1994 9: 17-28). Therefore, several methods have been developed to reduce the immunogenicity of antibodies of mice and other rodents, as well as technologies for producing fully human antibodies using, for example, transgenic mice or phage display. Chimeric antibodies were constructed in which rodent variable regions and human constant regions (e.g., Boulianne Nature 1984 312: 643-6) were combined, significantly reducing the immunogenicity problem (e.g., LoBuglio, Proc. Natl. Acad. Sci. 1989 86: 4220- 4; Clark, Immunol. Today 2000 21: 397-402). Humanized antibodies have also been designed in which the actual sequence of the variable region of the rodent is designed to match the human sequence as much as possible while maintaining at least the original CDRs, or where the CDRs of the rodent antibodies are transplanted into a human antibody framework (e.g., Riechmann, Nature 1988 332: 323- 7; US5693761). Rabbit polyclonal antibodies are widely used for biological assays, such as ELISA or Western blotting. Rabbit polyclonal antibodies are generally preferred among rodent polyclonal antibodies, usually because of their much higher affinity. In addition, in a rabbit it is often possible to obtain a good antibody response to antigens that elicit a weak immune response in mice and / or are not a source of satisfactory binding molecules when used in phage display. Due to these well-known advantages, rabbit antibodies may be ideal for use in the research and development of therapeutic antibodies. The reason they are not widely used is mainly due to technical difficulties in obtaining rabbit monoclonal antibodies. Since tumors like myelomas are not known in rabbits, traditional hybridoma technology cannot be used to produce rabbit monoclonal antibodies. For the first time, a study of a method for producing a partner cell line for fusion with rabbit antibody expressing cells was conducted by Knight et al. (Spieker-Polet et al., PNAS 1995, 92: 9348-52), and an improved fusion partner cell line has been described by Pytela et al. in 2005 (see, for example, US Pat. No. 7,429,487). However, this technology is not widely used, since the know-how is mainly controlled by one research group. To obtain monoclonal antibodies, alternative methods are described in the literature, including cloning antibodies from selected cells expressing antibodies by RT-PCR, but there have never been any reports of their successful use for rabbit antibodies.

It is believed that rabbit antibodies, like mouse antibodies, elicit a strong immune response when used to treat humans, so rabbit antibodies must be humanized before they can be used clinically. However, the methods that are used to obtain humanized rodent antibodies cannot simply be extrapolated for rabbit antibodies due to structural differences between rabbit and mouse antibodies and, accordingly, between rabbit and human antibodies. For example, light chain CDR3 (CDR L3) is often much longer than previously known human or mouse CDR L3 antibodies.

At the present stage of development in this area, several approaches for the humanization of rabbit antibodies have been described, which, however, are not a classic transplantation approach in which a CDR of a non-human donor antibody is transplanted onto a human acceptor antibody. WO 04/016740 describes the so-called "surface change" approach. The purpose of this “surface change” approach is to modify non-human framework residues available to the solvent so that they become more like human residues. Methods for humanizing rabbit antibodies similar to the methods described in WO 04/016740 are known in the art. WO08 / 144757 and WO05 / 016950 describe methods for humanizing a rabbit monoclonal antibody, comprising comparing the amino acid sequences of the original rabbit antibody with the amino acid sequences of a similar human antibody. The amino acid sequence of the original rabbit antibody is then changed so that its frame regions are more similar in sequence to the equivalent frame regions of such a human antibody. To obtain good binding abilities for each immuno-binding molecule separately, it is necessary to carry out laborious experimental design work.

A possible problem of the above approaches is that they do not use a human frame, but design a rabbit frame so that it looks more like a human frame. This approach has the risk that the amino acid regions located in the core of the protein may still contain immunogenic T-cell epitopes.

To date, applicants have not found rabbit antibodies that have been humanized using transplantation methods that are current in the art. This can be explained by the fact that the rabbit CDR can be completely different from the human or rodent CDR. As is known in the art, most rabbit V _H chains relative to mouse and human variants contain additional paired cysteines. In addition to the conservative disulfide bridge formed between cys22 and cys92, there is also the cys21-cys79 bridge, as well as the SS bridge between CDRs, which forms in some rabbit chains between the last CDR H1 residue and the first CDR H2 residue. In addition, pairs of cysteine residues are often found in CDR L3. In addition, many rabbit CDRs are not suitable for any of the previously known canonical structures. In particular, L3 CDRs are often much longer than human or mouse CDR L3 variants.

Thus, the transplantation of CDRs from non-human antibodies into the human framework is an important task of protein engineering. The transfer of antigen-binding loops from a naturally-evolved scaffold to another artificially selected human scaffold must be carried out so that the natural conformations of the loops are maintained for antigen binding. Often after transplantation of loops, the antigen binding affinity significantly decreases or disappears. The use of carefully selected human scaffolds when transplanting antigen-binding loops maximizes the likelihood of maintaining binding affinity in a humanized molecule (Roguzka et al 1996). Although there are approximate guidelines for CDR transplantation for the many transplant experiments available in the literature, this principle cannot be generalized. Typical problems after transplantation of CDR loops are associated with a loss of specificity, stability, or availability.

Thus, there is an urgent need for improved methods for reliable and rapid humanization of rabbit antibodies for use as therapeutic and diagnostic tools. In addition, there is a need for human acceptor scaffolds for the reliable humanization of rabbit antibodies, providing functional antibodies and / or antibody fragments with biophysical properties similar to drugs.

SUMMARY OF THE INVENTION

Surprisingly, it was found that the framework of a highly soluble and stable human antibody identified by “quality control” (QC) analysis (as described in WO 0148017 and Auf der Maur et al., (2001), FEBS Lett. 508, p. 407-412 ) is particularly suitable for embedding CDRs from other non-human animal species, for example, rabbit CDRs. Thus, in a first aspect, the invention relates to the variable regions of the light and heavy chains of a particular human antibody (the so-called "FW1.4" antibody), which are particularly convenient as a universal acceptor for CDRs from a number of antibodies, in particular from rabbit antibodies, with different binding specificity, regardless of the presence or absence of a disulfide bridge in the CDR. In addition, the present invention relates to two mutant scaffold sequences of a specific human antibody, namely rFW1.4 and rFW1.4 (V2), which are both scaffolds, particularly suitable as universal acceptor scaffolds for rabbit CDR transplantation. In another aspect, the invention relates to a repeat of framework residues, which makes a human framework suitable for incorporation of CDRs from other non-human animal species, in particular a rabbit CDR.

The humanized immunobinding molecules obtained by transplanting rabbit CDRs into these highly compatible frameworks of the variable regions of the heavy and light chains constantly and reliably maintain the spatial orientation of rabbit antibodies from which donor CDRs are derived. Thus, there is no need to introduce into the acceptor skeleton any structurally significant positions of the donor immunobinding molecule. Due to these advantages, high-performance humanization of rabbit antibodies can be achieved without or with little optimization of binding ability.

Thus, in another aspect, the invention relates to methods for transferring CDRs of rabbit and other non-human CDRs into the framework sequences of the light chain and / or heavy chain of a soluble and stable human antibody described herein, thereby obtaining humanized antibodies with excellent biophysical properties. In particular, the immunobinding molecules obtained by the methods of the invention exhibit excellent functional properties such as solubility and stability.

Brief Description of Drawings

The figure 1 shows the definition of CDR H1 used in this document for transplantation of antigen-binding sites from rabbit monoclonal antibodies into the frameworks of highly soluble and stable human antibodies.

Figure 2 schematically shows a B-cell 1 labeled with a fluorescent antibody 2, interacting with a target-expressing cell 3, stained with intracellular dye 4. Selected target: 5; BCR: 6.

Figure 3: a method for selecting FACS rabbit B cells that bind to a soluble target of ESBA903. FIG. 3A: lymphocytes are taken for direct and lateral scattering. FIG. 3B: Among them, IgG + IgM- cells (probably memory B cells) are selected (red window). FIG. 3C: ESBA903-PE and ESBA903-PerCP double-stained cells (green window) are believed to encode high affinity anti-ESBA903 IgG. Cells showing the highest fluorescence brightness (pink window) were sorted into 96-well plates.

Figure 4. Granules coated with anti-TNF-alpha antibodies (labeled with PE) bind to CHO cells transfected with TNF-alpha (top panel). Control granules coated with anti-CD19 antibodies (labeled APC) do not bind to TNF alpha transfected CHO cells (middle panel). Pellets coated with anti-TNF-alpha antibodies (labeled with PE) do not bind to wild-type (wt) CHO cells (bottom panel). Scatter plots on the left show forward and side scatter, which indicates, respectively, the size and degree of detail of the events. A group of single granules (≈3 μm) is selected in P2. CHO cells finally bound to the granules (≈30 μm) were selected in P1. Scatter plots in the middle show P1 events (CHO cells) regarding their PE or APC staining. Thus, if cells interact with granules with anti-TNF-alpha antibodies, they should appear in window P3, and if they interact with granules with anti-CD19 antibodies, they should appear in window P4. On the right, statistics are detailed for each of the samples.

Figure 5. Granules coated with anti-TNF-alpha-PE antibodies and granules coated with anti-CD19-APC antibodies are mixed together with TNF-alpha transfected CHO cells. CHO cells were selected (P1) and from them cells binding to granules coated with anti-TNF-alpha-PE antibodies or granules coated with anti-CD19-APC antibodies are shown in windows P3 and P4, respectively. Unbound granules are visible in the P2 window.

Figure 6. Sequence analysis of rabbit antibodies extracted from the Kabat database confirms that CDR3 of the heavy chain variable region is typically three amino acids longer than its mouse variant.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

For ease of understanding of the present invention, certain terms are defined below. Throughout the detailed description, additional definitions are indicated.

The term “antibody” refers to full-length antibodies and to any antigen-binding fragment. The terms “antigen binding polypeptide” and “immunoconjugating molecule” are used interchangeably herein. "Antibody" refers to a protein, optionally glycosylated, containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or to its antigen-binding part. Each heavy chain consists of a variable region of the heavy chain (abbreviated herein as V _H ) and a constant region of the heavy chain. The constant region of the heavy chain consists of three domains, C _H 1, C _H 2 and C _H 3. Each light chain consists of a variable region of the light chain (abbreviated herein as V _L ) and a constant region of the light chain. The constant region of the light chain consists of one domain, C _L. The V _H and V _L regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) located between more conservative regions called framework regions (FR). Each V _H and V _L consists of three CDRs and four FRs, located from the N-end to the C-end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. Antibody constant regions can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system.

The term “antigen-binding part” of an antibody (or simply “part of an antibody”) refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (eg, TNF). It was shown that antigen-binding function of an antibody can be performed by fragments of a full-sized antibody. Examples of binding fragments encompassed by the term "antigen binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and C _H 1 domains; (ii) an F (ab ') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the V _H and C _H 1 domains; (iv) an Fv fragment consisting of the V _L and V _H domains of one arm of an antibody; (v) a single dAb domain or fragment (Ward et al., (1989) Nature 341: 544-546) consisting of a V _H domain; and (vi) a selected complementarity determining region (CDR) or (vii) a combination of two or more selected CDRs, which optionally can connect a synthetic linker. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, using recombinant methods, they can be joined by a synthetic linker, which allows them to be obtained as a single protein chain in which the V _L and V _H regions are paired with the formation of monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al., (1988) Science 242: 423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single chain antibodies should also be included in the term “antigen binding portion” of an antibody. These antibody fragments are prepared by conventional methods known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding parts can be obtained by recombinant DNA methods, or by enzymatic or chemical cleavage of intact immunoglobulins. Antibodies can be of a different isotype, for example, an IgG antibody (for example, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM.

The term “immunobinding molecule” refers to a molecule containing all or part of an antigen binding portion of an antibody, for example, the entire heavy and / or light chain variable domain, or a portion thereof such that the immunobinding molecule specifically recognizes a target antigen. Non-limiting examples of immuno-binding molecules include full-length immunoglobulin and scFv molecules, as well as antibody fragments, including but not limited to (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , C _L and C _H 1 domains; (ii) an F (ab ') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a Fab ′ fragment, which is essentially a Fab with a part of the hinge region (see Fundamental Immunology (Paul ed., 3.sup.rd ed. 1993); (iv) an Fd fragment consisting of V _H domains and C _H 1; (v) an Fv fragment consisting of the V _L and V _H domains of one arm of an antibody, (vi) a single domain antibody, such as a dAb fragment (Ward et al., (1989) Nature 341: 544-546) consisting of the V _H or V _L domain, camelid antibodies (see Hamers-Casterman, et al., Nature 363: 446-448 (1993), and Dumoulin, et al., Protein Science 11: 500-515 (2002) ) or sharks (e.g., Nanobody® Ig-NAR sharks; and (vii) Nanobody, heavy chain variable region containing one variable domain and two constants antenna domain.

The term “single chain antibody”, “single chain Fv” or “scFv” refers to a molecule comprising an antibody heavy chain variable domain (or region; V _H ) and an antibody light chain variable domain (or region; V _L ) linked by a linker. Such scFv molecules can have the general structure: NH ₂ -V _L -linker-V _H -COOH or NH ₂ -V _H -linker-V _L -COOH. Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof. In a preferred embodiment of the present invention, a linker (GGGGS) ₄ with the amino acid sequence of SEQ ID NO: 8 is used, but 1-3 repetitions are also possible (Holliger et al., (1993), Proc. Natl. Acad. Sci. USA 90 : 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al., (1995), Protein Eng. 8: 725-731, Choi et al., (2001), Eur. J. Immunol. 31: 94-106, Hu et al., (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al., (1999), J. Mol. Biol. 293: 41-56 and Roovers et al., (2001), Cancer Immunol.

As used herein, the term “functional property” is a property of a polypeptide (eg, an immuno-binding molecule), the improvement of which (eg, relative to a conventional polypeptide) is desirable and / or preferred by one skilled in the art, for example, to improve technological properties or therapeutic efficacy of the polypeptide. In one embodiment, the functional property is stability (e.g., thermal stability). In another embodiment, the functional property is solubility (for example, under cell conditions). In yet another embodiment, the functional property is an aggregation pattern. In another embodiment, the functional property is protein expression (for example, in a prokaryotic cell). In yet another embodiment, the functional property is the nature of refolding after dissolution of inclusion bodies in the manufacturing process. In certain embodiments, the functional property does not represent an improvement in antigen binding affinity. In another preferred embodiment, the improvement of one or more functional properties does not have a significant effect on the binding affinity of the immunobinding molecule.

The term "CDR" refers to one of the six hypervariable regions in the variable domains of an antibody, which mainly contribute to antigen binding. One of the most commonly used definitions for six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, Kabat CDR determination is used only for CDR1, CDR2 and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or LI, L2, L3), as well as for CDR2 and CDR3 of the heavy chain variable domain ( CDR H2, CDR H3 or H2, H3). However, as used herein, the heavy chain variable domain CDR1 (CDR H1 or H1) is determined by the positions of the residues (Kabat numbering), starting from position 26 and ending at position 36. This definition is essentially a combination of CDR H1, different defined by Kabat and Chotia (also see figure 1 for illustration).

As used herein, the term “antibody framework” refers to a portion of the variable domain, V _L or V _H , which serves as support for the antigen binding loops (CDRs) of this variable domain. Essentially, it is a variable domain without CDR.

The term “epitope” or “antigenic determinant” refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a TNF molecule). Typically, an epitope includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-consecutive amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms “specific binding”, “selective binding”, “selectively binding” and “specifically binding” refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, an antibody binds with an affinity (K _D ) of approximately less than 10 ⁻⁷ M, such as approximately less than 10 ⁻⁸ M, 10 ⁻⁹ M, or 10 ⁻¹⁰ M or even less.

The term "K _D " or "K _d " refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. Typically, antibodies of the invention bind to TNF with an equilibrium dissociation constant (K _D ) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M or 10 ^-10 M or even less, for example as determined using surface plasmon resonance (SPR) technology on a BIACORE device.

As used herein, the term "nucleic acid molecule" refers to DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. A nucleic acid is “operably linked” if it is in functional association with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is connected. In one embodiment, the vector is a "plasmid" which means a closed circular double-stranded DNA into which additional DNA sections can be ligated. In another embodiment, the vector is a viral vector, where additional sections of DNA can be ligated into the viral genome. The vectors described herein may be capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with a bacterial replication origin and episomal mammalian vectors), or may integrate into the genome of the host cell after introduction into the cell -host and, thus, replicate together with the host genome (for example, non-episomal mammalian vectors).

The term "host cell" refers to a cell into which an expression vector is introduced. Host cells include bacterial, microbial, plant or animal cells, preferably Escherichia coli , Bacillus subtilis ; Saccharomyces cerevisiae , Pichia pastoris , CHO (Chinese hamster line) or NSO.

The term "rabbit-like" refers to representatives of the taxonomic order of Lagomorpha , including the families Leporidae (for example, hares and rabbits), and Ochotonidae (pikas). In a most preferred embodiment, the rabbit is a rabbit. As used herein, the term "rabbit" refers to an animal belonging to the Leporidae family.

As used herein, "identity" refers to the coincidence of the sequences of two polypeptides, molecules or two nucleic acids. When the position in the two compared sequences is occupied by the same base or the same amino acid monomeric subunit (for example, if the position in both of the two polypeptides is occupied by lysine), then the corresponding molecules are identical in this position. The “percent identity” of two sequences is a function of the number of identical positions detected in the sequences, taking into account the number of gaps and the length of each gap that must be entered for optimal alignment of the two sequences. Typically, comparisons are made when two sequences are aligned to maximize identity. Such alignment can be carried out, for example, using the algorithm Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)), on which the GAP program in the GCG software package is based, using the Blossum matrix 62 or the PAM250 matrix , and the weight of the pass 16, 14, 12, 10, 8, 6 or 4 and the length of the pass 1, 2, 3, 4, 5 or 6.

Unless otherwise specified, all technical and scientific terms used in this document have the same meaning, as usually understood by a specialist in the field to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of doubt, follow this description. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the sections below, various aspects of the invention are described in more detail. It should be understood that various embodiments, the most preferred embodiments and ranges can be arbitrarily combined. Furthermore, depending on the particular embodiment, the selected definitions, embodiments, or ranges may not be used.

Unless otherwise specified, amino acid positions are indicated in accordance with the AHo numbering scheme. The AHo numbering system is further described in Honegger, A. and Pluckthun, A. (2001) J. Mol. Biol. 309: 657-670). Alternatively, Kabat numbering can be used as further described in Kabat et al., (Kabat, EA, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No 91-3242). Conversion tables for two different numbering systems used to identify the positions of amino acid residues in the variable regions of the heavy and light chains of antibodies are provided by A. Honegger, J. Mol. Biol. 309 (2001) 657-670.

In a first aspect, the present invention relates to a universal acceptor framework for transplanting CDRs of other animal species, for example, from a rabbit. It has been previously described that antibodies or antibody derivatives containing human scaffolds identified in the so-called “quality control” screening (WO0148017) are generally characterized by high stability and / or solubility. Although the single-stranded human framework FW1.4 (a combination of SEQ ID NO: 1 (referred to in WO03 / 097697 a43) and SEQ ID NO: 2 (referred to in WO03 / 097697 KI27)) is clearly inferior to the “quality control” analysis in characteristic parameters, unexpectedly it was found that it has high intrinsic thermodynamic stability and is well-produced, moreover, in combination with a number of different CDRs. The stability of this molecule can mainly be considered a property of its frame regions. Furthermore, FW1.4 has been shown to be substantially highly compatible with antigen binding sites in rabbit antibodies. Thus, FW1.4 is a suitable support molecule for the construction of stable humanized fragments of scFv antibodies obtained by transplanting rabbit loops. Thus, in one aspect, the invention relates to an acceptor scaffold of an immunobinding molecule comprising a V _H sequence at least 90% identical to SEQ ID NO: 1 and / or a V _L sequence at least 85% identical to SEQ ID NO: 2 , more preferably, containing the sequence FW1.4 (SEQ ID NO: 3) for transplanting a rabbit CDR, or a sequence of at least 60%, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95% identical to SEQ ID NO: 3.

In addition, it was found that FW1.4 can be optimized by replacing several residues at positions in the heavy chain of FW1.4 and / or by replacing the residue at 1 position in the light chain of FW1.4. Thus, it was unexpectedly found that the conformation of the loops of a large number of rabbit CDRs in V _H can be fully preserved, as a rule, regardless of the sequence of the donor scaffold. These residues in the heavy chain, as well as at position 1 in the light chain of FW1.4, are conserved in rabbit antibodies. The consensus residue for the positions in the heavy chain, as well as one position in the light chain is established on the basis of the rabbit's repertoire and introduced into the sequence of the human acceptor frame.

As a result, the modified framework 1.4 (hereinafter referred to as rFW1.4) is compatible with almost any rabbit CDR. In addition, rFW1.4, containing various rabbit CDRs, is well expressed and well-produced unlike the wild-type rabbit single chains and almost completely retains the affinity of the original rabbit donor antibody.

Thus, the present invention relates to the framework of the variable region of the heavy chain of SEQ ID NO: 1, additionally containing one or more amino acid residues, which, as a rule, support the conformation of CDRs derived from rabbit immunocompatible molecules. In particular, these residues are in one or more amino acid positions selected from the group consisting of 24H, 25H, 56H, 82H, 84H, 89H, and 108H (AHo numbering). It has been proven that these provisions influence the conformation of CDRs and are thus intended to mutate to embed donor CDRs. Preferably, said one or more residues are selected from the group consisting of: threonine (T) at position 24, valine (V) at position 25, glycine or alanine (G or A) at position 56, lysine (K) at position 82, threonine (T) at position 84, valine (V) at position 89 and arginine (R) at position 108 (AHo numbering). Preferably, at least three, more preferably four, five, six, and most preferably all seven residues are present. It has been unexpectedly revealed that the presence of these residues enhances the stability of the immunobinding molecule.

In a preferred embodiment, the invention relates to an acceptor scaffold of an immunobinding molecule containing at least 50% V _H , more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and even more preferably 100% identical to SEQ ID NO: 4, provided that at least one, more preferably at least three, more preferably four, five, six, and most preferably seven residues from the group consisting of threonine (T) at position 24, valine (V) at position 25, alanine (A) or glycine ( G) at position 56, threonine (T) at position 84, lysine (K) at position 82, valine (V) at position 89 and arginine (R) at position 108 (AHo numbering). In a preferred embodiment, the acceptor scaffold of an immunobinding molecule is an acceptor framework of an immunobinding molecule for a rabbit CDR.

In a preferred embodiment, said heavy chain variable region framework is or comprises SEQ ID NO: 4 or SEQ ID NO: 6. Both of these frameworks of the variable regions of the heavy chains can, for example, be combined with any suitable light chain framework.

Thus, the present invention relates to an acceptor scaffold of an immunobinding molecule comprising

(i) the framework of the variable region of the heavy chain is at least 70% identical, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 4; and / or

(ii) the framework of the variable region of the light chain is at least 70% identical, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 2.

In a more preferred embodiment, the heavy chain variable region framework comprises threonine (T) at position 24, glycine (G) at position 56, threonine (T) at position 84, valine (V) at position 89, and arginine (R) at position 108 (AHo numbering).

In a preferred embodiment, the light chain variable region comprises threonine (T) at position 87 (AHo numbering).

In a preferred embodiment, said acceptor scaffold of an immunobinding molecule comprises

(i) the framework of the variable region of the heavy chain selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6; and / or

(ii) the framework of the variable region of the light chain of SEQ ID NO: 2 or SEQ ID NO: 9.

In a preferred embodiment, the framework of the variable region of the heavy chain is connected to the framework of the variable region of the light chain via a linker. The linker may be any suitable linker, for example, a linker containing 1 to 4 repeats of the GGGGS sequence, preferably a peptide (GGGGS) ₄ (SEQ ID NO: 8), or a linker as described in Alfthan et al. (1995) Protein Eng. 8: 725-731.

In another preferred embodiment, the acceptor scaffold of the immunobinding molecule is a sequence of at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 5, with the condition that the sequence is preferably not SEQ ID NO: 3. More preferably, the acceptor scaffold of the immunobinding molecule contains or is SEQ ID NO: 5.

In another preferred embodiment, the acceptor scaffold of the immunobinding molecule is a sequence of at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 7, with the condition that the sequence is preferably not SEQ ID NO: 3. More preferably, the acceptor scaffold of the immunobinding molecule contains or is SEQ ID NO: 7.

In addition, it was unexpectedly discovered that the presence of the amino acid repeat described above makes the scaffold, preferably the human scaffold, particularly suitable for introducing CDRs of other non-human animal species, particularly rabbit CDRs. Said repetition does not adversely affect the stability of the immunobinding molecule. CDRs are presented in a conformation similar to their natural spatial orientation in the rabbit immunobinding molecule; Thus, there is no need for transplantation into an acceptor framework of structurally significant provisions. Thus, the acceptor skeleton of an immuno-binding molecule of a person or a humanized acceptor framework of an immuno-binding molecule contains at least three amino acids, preferably four, five, six, and more preferably seven amino acids from the group consisting of threonine (T) at position 24, valine ( V) at position 25, alanine (A) or glycine (G) at position 56, lysine (K) at position 82, threonine (T) at position 84, valine (V) at position 89 and arginine (R) at position 108 (AHo numbering).

As described herein, the acceptor scaffolds of the immunobinding molecules may contain a solubility enhancing substitution in the heavy chain scaffold, preferably at positions 12, 103 and 144 (AHo numbering). Preferably, the hydrophobic amino acid is replaced with a more hydrophilic amino acid. Hydrophilic amino acids are, for example, arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glycine (G), histidine (H), lysine (K), serine (S) and threonine (T) . More preferably, the heavy chain framework comprises (a) serine (S) at position 12; (b) serine (S) or threonine (T) at position 103 and / or (c) serine (S) or threonine (T) at position 144.

In addition, in one or more positions from 1, 3, 4, 10, 47, 57, 91, and 103 of the framework of the variable region of the light chain (AHo numbering), stability-enhancing amino acids may be present. More preferably, the framework of the variable region of the light chain contains glutamic acid (E) at position 1, valine (V) at position 3, leucine (L) at position 4, serine (S) at position 10; arginine (R) at position 47, serine (S) at position 57, phenylalanine (F) at position 91 and / or valine (V) at position 103.

Since glutamine (Q) is subject to deamination, in another preferred embodiment, V _H contains at position 141 glycine (G). This replacement may increase the shelf life of the protein.

For example, the acceptor scaffolds described herein can be used to produce a human antibody or a humanized antibody that retains the binding properties of a non-human antibody from which non-human CDRs are derived. Thus, in a preferred embodiment, the present invention relates to an acceptor scaffold of an immunobinding molecule, as described herein, further comprising a heavy chain CDR1, CDR2 and CDR3 and / or a light chain CDR1, CDR2 and CDR3 from a donor immunobinding molecule, preferably from an immunobinding molecule a mammalian molecule, more preferably, from an immunobinding molecule of a rabbit, and most preferably a rabbit. Thus, in one embodiment, the invention relates to an immunobinding molecule specific for a desired antigen comprising

(i) CDR of the variable region of the rabbit light chain; and

(ii) the framework of the variable region of the human heavy chain of at least 50%, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and most preferably, 100% identical to SEQ ID NO: 4.

In one preferred embodiment, there is a condition that at least one amino acid from the group consisting of threonine (T) at position 24, valine (V) at position 25, alanine (A) is present in the indicated sequence of the framework of the variable region of the human heavy chain ) or glycine (G) at position 56, threonine (T) at position 84, lysine (K) at position 82, valine (V) at position 89, and arginine (R) at position 108 (AHo numbering).

Preferably, the rabbit is a rabbit. More preferably, the immunobinding molecule contains a heavy chain CDR1, CDR2 and CDR3 and a light chain CDR1, CDR2 and CDR3 from a donor immunobinding molecule.

As is known in the art, many rabbit V _H chains, in comparison with mouse and human variants, contain additional paired cysteines. In addition to the conservative disulfide bridge formed between cys22 and cys92, there is also the cys21-cys79 bridge, as well as the SS-bridge between CDRs formed in some rabbit chains between the last CDR H1 residue and the first CDR H2 residue. In addition, pairs of cysteine residues are often found in CDR L3. In addition, many rabbit CDRs are not suitable for any of the previously known canonical structures. In particular, L3 CDRs are often much longer than human or mouse CDR L3 variants.

As indicated previously, transplanting non-human CDRs into the scaffolds described herein provides a molecule where the CDRs are presented in the correct conformation. If necessary, the affinity of the immunobinding molecule can be improved by transplanting antigen-interacting framework residues of a non-human donor immunobinding molecule. These provisions can be identified, for example, by

(i) identifying the corresponding germline precursor sequence or, alternatively, in the case of highly homologous framework sequences, using consensus sequences;

(ii) aligning the sequence of the donor variable domain with the germline precursor sequence or the consensus sequence from step (i); and

(iii) identification of differing residues.

Differing residues on the surface of the molecule in many cases mutate during the formation of affinity in vivo , presumably for the formation of affinity for the antigen.

In another aspect, the present invention relates to an immunobinding molecule, which comprises an acceptor framework for an immunobinding molecule described herein. For example, said immunocompatible molecule may be an scFv antibody, a full-length immunoglobulin, Fab fragment, Dab, or Nanobody.

In a preferred embodiment, the immunobinding molecule is attached to one or more molecules, for example, a therapeutic agent, such as a cytotoxic agent, cytokine, chemokine, growth factor or other signaling molecule, an imaging agent or a second protein, such as a transcription activator or DNA binding domain.

As described herein, an immunobinding molecule can be used, for example, for diagnostic purposes, in therapy, to confirm a target or gene therapy.

The invention further relates to an isolated nucleic acid encoding an acceptor scaffold of an immunobinding molecule described herein, or an immunobinding molecule (s) as described herein.

In another embodiment, the invention relates to a vector comprising the nucleic acid described herein.

A nucleic acid or vector, as described herein, can be used, for example, in gene therapy.

The invention further relates to a host cell comprising the vector and / or nucleic acid described herein.

In addition, a composition is provided comprising an acceptor scaffold of an immunobinding molecule as described herein, an immunobinding molecule as described herein, an isolated nucleic acid as described herein, or a vector as described herein.

The sequences described herein are as follows (X residues are portions of the CDR inserts):

SEQ ID NO: 1: framework of the variable region of the heavy chain FW1.4 (a43)

EVQLVESGGGLVQPGGSLRLSCAAS (X) _{n = 1-50} WVRQAPGKGLEWVS (X) _{n = 1-50} RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (X) _{n = 1-50} WGQGTLVTVSS

SEQ ID NO: 2: framework of the variable region of the light chain FW1.4 (KI27)

EIVMTQSPSTLSASVGDRVIITC (X) _{n = 1-50} WYQQKPGKAPKLLIY (X) _{n = 1-50} GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC (X) _{n = 1-50} FGQGTKLTVLG

SEQ ID NO: 3: Frame FW1.4

_{EIVMTQSPSTLSASVGDRVIITC (X) n = 1-50 WYQQKPGKAPKLLIY} (X) n = 1-50 GVPSRFSGSGSGAEFTLTISSLQPDDFATYYC (X) n = 1-50 FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAAS (X) n = 1-50 WVRQAPGKGLEWVS (X) n = 1-50 RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK (X) n _{= 1-50} WGQGTLVTVSS

SEQ ID NO: 4: framework of the variable region of the heavy chain rFW1.4

EVQLVESGGGLVQPGGSLRLSCTAS (X) _{n = 1-50} WVRQAPGKGLEWVG (X) _{n = 1-50} RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR (X) _{n = 1-50} WGQGTLVTVSS

SEQ ID NO: 5: Frame rFW1.4

_{EIVMTQSPSTLSASVGDRVIITC (X) n = 1-50 WYQQKPGKAPKLLIY} (X) n = 1-50 GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (X) n = 1-50 FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTAS (X) n = 1-50 WVRQAPGKGLEWVG (X) n = 1-50 RFTISRDTSKNTVYLQMNSLRAEDTAVYYCAR (X) n _{= 1-50} WGQGTLVTVSS

SEQ ID NO: 6: framework of the variable region of the heavy chain rFW1.4 (V2)

EVQLVESGGGLVQPGGSLRLSCTVS (X) _n = _1-50 WVRQAPGKGLEWVG (X) _{n = 1-50} RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR (X) _{n = 1-50} WGQGTLVTVSS

SEQ ID NO: 7: Frame rFW1.4 (V2)

_{EIVMTQSPSTLSASVGDRVIITC (X) n = 1-50 WYQQKPGKAPKLLIY} (X) n = 1-50 GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (X) n = 1-50 FGQGTKLTVLGGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTVS (X) n = 1-50 WVRQAPGKGLEWVG (X) n = 1-50 RFTISKDTSKNTVYLQMNSLRAEDTAVYYCAR (X) n _{= 1-50} WGQGTLVTVSS

SEQ ID NO: 8: linker

GGGGSGGGGSGGGGSGGGGS

SEQ ID NO: 9: substituted framework of the variable region of the light chain FW1.4

EIVMTQSPSTLSASVGDRVIITC (X) _{n = 1-50} WYQQKPGKAPKLLIY (X) _{n = 1-50} GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (X) _{n = 1-50} FGQGTKLTVLG

In another aspect, the invention relates to methods for humanizing non-human antibodies by transplanting CDRs of non-human donor antibodies into scaffolds of stable and soluble antibodies. In a particularly preferred embodiment, the CDRs derived from rabbit antibodies and scaffolds are CDRs and scaffolds described above.

A general method for transplanting CDRs into human acceptor scaffolds is described by Winter in US Pat. No. 5,225,539 and Queen et al. in WO9007861 A1, which are hereby fully incorporated by reference. The general strategy for transplanting CDRs from rabbit monoclonal antibodies into selected scaffolds is similar to that of Winter et al. and Queen et al., but diverges in some key respects. In particular, the methods of the invention differ from the conventional Winter and Queen methodology known in the art in that human antibody scaffolds, as described herein, are particularly suitable as universal acceptors for human donor antibodies or non-human donor antibodies. Thus, in contrast to the general Winter and Queen method, the framework sequence used for the humanization methods of the invention is not necessarily a framework sequence showing the highest sequence similarity to the sequence of a non-human antibody (e.g., rabbit) from which donor CDRs are derived. In addition, to support CDR conformation, transplantation of the framework residues from the donor sequence is not required. To a greater extent, antigen-binding amino acids or other mutations that occur during somatic hypermutation can be located in the skeleton.

The following describes specific options for transplantation methods to obtain humanized antibodies with high solubility and stability derived from rabbit antibodies.

Thus, the invention relates to a method for humanizing an immunobinding rabbit CDR donor molecule that comprises heavy chain CDR1, CDR2 and CDR3 sequences and / or light chain CDR1, CDR2 and CDR3 sequences. The method includes the steps of:

(i) transplantation into the heavy chain of at least one, preferably two, more preferably three CDRs from the group consisting of sequences of CDR1, CDR2 and CDR3 into the acceptor framework of the human heavy chain by at least 50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 1; and / or

(ii) transplantation into the light chain of at least one, preferably two, more preferably three CDRs from the group consisting of the sequence CDR1, CDR2 and CDR3 into the acceptor framework of the human light chain, where the framework of the human light chain is at least 50% preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 2.

In a preferred embodiment, the variable chain acceptor framework comprises (i) a human heavy chain framework comprising an amino acid framework selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, and (ii) a framework human light chain containing the frame amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9.

In a more preferred embodiment, the method comprises the step of (i) transferring the heavy chain CDR1, CDR2, and CDR3 sequences to the heavy chain, and (ii) transferring the light chain CDR1, CDR2, and CDR3 sequences to the light chain of the immunobinding molecule by at least 75%, 80% , 85%, 90%, more preferably at least 95% identical to SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7. More preferably, the immunobinding molecule is or contains SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

In another embodiment, to improve antigen binding, the method may further include the step of replacing the scaffold residues of the acceptor with donor residues that are involved in antigen binding.

In illustrative embodiments of the methods of the invention, the amino acid sequence of the CDR donor antibody is first identified and the sequences are aligned by conventional sequence alignment methods (e.g., the Needleman-Wunsch algorithm and Blossum matrices). The introduction of gaps and the designation of the positions of the residues can be carried out using the traditional numbering system in antibodies. For example, you can use the AHo numbering system for the variable domains of immunoglobulins. You can also use the Kabat numbering scheme, as it is the most widely accepted standard for numbering residues in an antibody. For example, Kabat numbering can be determined using the SUBIM program. This program analyzes the variable regions of an antibody sequence and numbers the sequence according to a system developed by Kabat and colleagues (Deret et al 1995). The determination of framework regions and CDRs is generally carried out in accordance with the Kabat definition, which is based on sequence variability and is most often used. However, for CDR H1, the designation is preferably a combination of Kabat definitions, average contact data obtained by contact analysis of the antibody and antigen of a subset of three-dimensional complex structures (MacCallum et al., 1996), and Chotia, which is based on determining the position of regions of structural loops ( also see Fig. 1). Conversion tables for various numbering systems used to identify the positions of amino acid residues in the variable regions of the heavy and light chains of antibodies are provided by A. Honegger, J. Mol. Biol. 309 (2001) 657-670. Kabat numbering is further described in Kabat et al. (Kabat, E. A., et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The AHo numbering system is further described in Honegger, A. and Pluckthun, A. (2001) J. Mol. Biol. 309: 657-670.

For example, the variable domains of rabbit monoclonal antibodies can be classified into appropriate human subgroups using, for example, implementation of EXCEL sequence analysis algorithms and classification methods based on analysis of the repertoire of human antibodies (Knappik et al., 2000, J. Mol. Biol. Feb 11; 296 (1): 57-86).

In donor antigen-binding regions, the conformation of CDRs can be determined, then the positions of the residues necessary to maintain various canonical structures can also be determined. The canonical CDR structures for five of the six hypervariable regions of antibodies in rabbit antibodies (L1, L2, L3, H1 and H2) are determined using the definition of Chothia (1989).

In a preferred embodiment, CDRs are obtained, identified and isolated in the following manner: B cells, preferably rabbit B cells, are incubated with (i) target antigens (preferably purified) or (ii) with cells expressing antigen on their surface target.

In case (ii), said cells expressing the target antigen can be, for example, mammalian cells, preferably CHO or HEK293 cells, yeast cells, preferably yeast spheroblasts, or bacterial cells that naturally express the selected target or are transformed so to express a target protein on its surface. When expressed, the target antigen can be expressed on the cell surface either by being integrated into or attached to the cell membrane. Cells can be cultured, for example, in the form of isolated lines in cell culture or isolated from their natural environment, for example, from a tissue, organ or organism.

Providing a target antigen expressed on a cell surface, i.e. case (ii) is particularly preferred for transmembrane whites, even more preferably for transmembrane proteins with multiple transmembrane domains, such as GPCR (G-protein coupled receptors), or ion channels or any other protein which, after recombinant expression and purification is difficult to maintain the natural conformation. Conventional immunization with a recombinant protein in these cases is impractical or impossible due to the loss of integral membrane proteins / complexes during the purification of the natural conformation or due to insufficient quantities of pure protein. In a preferred embodiment of the invention, a mammal, more preferably a rabbit, instead of a recombinant protein is immunized with DNA, for example, according to the DNA vaccination protocol, as described in WO / 2004/087216. DNA vaccination induces a rapid immune response to a natural antigen. Since there is no need for a recombinant protein, this technology is on the one hand very cost-effective, on the other hand, and more importantly, this method provides the natural expression of integral membrane complexes and / or transmembrane proteins with several transmembrane domains. B cells can be isolated from said immunized mammal, preferably said rabbit, or, alternatively, be naive B cells.

In the next step of this method, B cells, preferably memory B cells, are isolated from the lymph organs (such as the spleen or lymph nodes) of the immunized animals, preferably the immunized rabbits. B cells are incubated in admixture with cells expressing antigen on their surface, or with fluorescently labeled soluble antigen. B cells are isolated that express target-specific antibodies on their surface and thus bind to the target antigen or to the target antigen expressed on the cell surface. In a more preferred embodiment, B cells and / or target cells are stained to allow isolation by flow cytometry based on sorting of B-cell / target-cell or B-cell / antigen complexes. Typically, flow cytometry measures the fluorescence emitted by individual cells at the intersection of a laser beam. However, some researchers are already using cytometers to study cell-cell interactions, such as cadherin-mediated adhesion (Panorchan et al., 2006, J. Cell Science, 119, 66-74; Leong et Hibma, 2005, J. Immunol. Methods, 302 116-124) or integrins (Gawaz et al, 1991, J. Clin. Invest, 88, 1128-1134). Thus, in case (ii), cells expressing the selected target are stained with an intracellular fluorescent dye (e.g., calcein). B cells are stained with fluorescent antibodies that bind to cell surface specific markers. Thus, two-color “events” can be selected, consisting of two cells connecting to each other through B-cell receptor-target interactions (see FIG. 2).

Since IgG, as a rule, have a higher affinity than IgM, it is preferable to select positive B cells expressing IgG on their surface rather than IgM (which is typical for memory B cells). For this purpose, preferably multicolor staining is used, where antibodies specific for IgG and IgM are labeled in different ways, for example, APC and FITC, respectively.

In one particular embodiment, data for sorting B cells can also be obtained by the ability of this interaction to functionally block / activate receptor signaling. For example, B cells can be incubated with cells that are functionally expressing GPCR (G protein conjugated receptor). An agonist that transmits a signal via GPCR can be added to the mixture to induce GPCR-mediated Ca ²⁺ release from the endoplasmic reticulum. In the event that the antibody, presented not to the B-cell, functionally blocks the signal transmission by the agonist, thus, the Ca ²⁺ output will also be blocked by this cell-cell interaction. The yield of Ca ²⁺ can be quantified by flow cytometry. Thus, only B-cell / target cell conglomerates that exhibit either an increase or decrease in Ca ²⁺ yield can be sorted.

The selection step is followed by culturing B cells under suitable conditions so that antibodies secrete into the culture medium. The antibodies produced are monoclonal antibodies. Cultivation may include the use of an auxiliary cell line, such as an auxiliary thymoma cell line (e.g., EL4-B5, see Zubler et al, 1985, J. Immunol. 134 (6): 3662-3668). Preferably, the confirmation step is carried out by testing the obtained antibodies for specific binding to the target, for example, to exclude antibodies that are directed to a protein expressed on the cell surface other than the target protein. For example, CELISA, i.e. Enzyme-linked immunosorbent assay (ELISA), where the coating step is carried out using whole cells. The specified method allows to evaluate the selectivity and ability of antibodies to compete with the ligand.

The antibodies obtained in the above step are then analyzed to identify the CDRs of these antibodies. This may include steps such as purification of antibodies, determination of their amino acid sequence and / or nucleic acid sequence.

As a result, the CDRs can then be transplanted into acceptor scaffolds, for example, by genetic synthesis, by the method of elongation of oligonucleotides, preferably into acceptor scaffolds described above. In one embodiment, a region-recognized CDR transplantation method can be used to transfer donor CDRs to acceptor scaffolds. In most cases, all three heavy chain CDRs are transplanted from a donor antibody to one acceptor framework, and all three light chain CDRs are transplanted to another acceptor framework. It is believed that transplantation of all three CDRs is not always necessary, since some CDRs may not participate in antigen binding, and CDRs with other sequences (of the same length) may have the same folding (and thus, despite different sequences, it is possible keep contacts of antigen with contacts of the main chain). In fact, separate domains (Ward et al, 1989, Nature 341, pp. 544-546) or even separate CDRs (R. Taub et al, 1989, J. Biol Chem 264, pp. 259-265) can possess antigen binding activity. However, regardless of whether all or only some of the CDRs are transplanted, the purpose of the CDR transplant is to transplant into the human antibody the same or almost the same antigen-binding portion of the animal (see, for example, US Pat. No. 5,225,539 (Winter)).

In another embodiment, the CDRs of the donor antibody can be changed before or after their introduction into the acceptor framework.

Alternatively, antibody characterization can only be carried out in their final format of the immunobinding molecule. In this approach, the CDR sequences of antibodies expressed on sorted B cells are restored by RT-PCR or from cultured sorted B cells, or directly from single sorted B cells. For this purpose, the propagation of B cells and / or the confirmation step described above and / or the analysis step as described above may be optional. The combination of two groups of partially overlapping oligonucleotides, in which one group of oligonucleotides encodes a CDR and the second group encodes a frame region of a suitable immunocompatible support molecule, makes it possible to obtain humanized immunocompatible molecules by one-stage PCR. High-throughput sequencing, cloning, and production allows the selection of clones based on the characteristics of purified humanized immunobonding molecules, rather than the characteristics of IgG secreted into the supernatant of cell culture. In a preferred embodiment, the immuno-binding molecule is scFv.

However, a CDR transplant can lead to a deterioration in the affinity of the resulting immunobinding molecule for the antigen due to scaffold residues that come in contact with the antigen. Such interactions may result from somatic hypermutation. Thus, transplantation of such amino acids from a donor framework to a humanized antibody framework may still be required. Amino acid residues from a non-human immuno-binding molecule involved in antigen binding can be identified by examining the sequences and structures of the variable regions of rabbit monoclonal antibodies. Each residue in the framework of the CDR donor that is different from the germ line can be considered significant. If the closest germ line cannot be established, the sequence can be compared with the consensus of the subgroup or the consensus of rabbit sequences with a high percentage of similarity. Rare framework residues are considered as a possible result of somatic hypermutation and, thus, play a role in binding.

The antibodies of the invention can be further optimized so that they exhibit improved functional properties, for example, increased solubility and / or stability. In certain embodiments, the antibodies of the invention are optimized in accordance with the “functional consensus” methodology described in PCT Application Serial No. PCT / EP2008 / 001958 entitled "Sequence Based Engineering and Optimization of Single Chain Antibodies" filed March 12, 2008, incorporated in this document by reference.

For example, the immunobinding molecules of the invention can be compared to a base of functionally selected scFvs used to identify positions of amino acid residues that are either more or less resistant to change than the corresponding positions in the immunobinding molecule, which thus means that such identified positions Residues may be suitable for engineering with improved functionality, such as stability and / or solubility.

The characteristic positions of the carcass residues for replacement and the characteristic replacements in the carcass are described in PCT Application No. PCT / CH2008 / 000285, entitled "Methods of Modifying Antibodies, and Modified Antibodies with Improved Functional Properties", filed June 25, 2008, and PCT Application No. PCT / CH2008 / 000284, entitled "Sequence Based Engineering and Optimization of Single Chain Antibodies," filed June 25, 2008. For example, at amino acid positions (for each of the amino acid positions listed below, AHo numbering is indicated), one or more of the following substitutions can be made in the variable region of the heavy chain of the immunocontaining molecule of the invention:

(a) Q or E at amino acid position 1;

(b) Q or E at amino acid position 6;

(c) T, S or A at amino acid position 7, more preferably T or A, even more preferably T;

(d) A, T, P, V or D, more preferably T, P, V or D, at amino acid position 10,

(e) L or V, more preferably L, at amino acid position 12,

(f) V, R, Q, M or K, more preferably V, R, Q or M at amino acid position 13;

(g) R, M, E, Q or K, more preferably R, M, E or Q, even more preferably R or E, at amino acid position 14;

(h) L or V, more preferably L, at amino acid position 19;

(i) R, T, K or N, more preferably R, T or N, even more preferably N, at amino acid position 20;

(j) I, F, L or V, more preferably I, F or L, even more preferably I or L, at amino acid position 21;

(k) R or K, more preferably K, at amino acid position 45;

(l) T, P, V, A or R, more preferably T, P, V or R, even more preferably R, at amino acid position 47;

(m) K, Q, H or E, more preferably K, H or E, even more preferably K, at amino acid position 50;

(n) M or I, more preferably I, at amino acid position 55;

(o) K or R, more preferably K, at amino acid position 77;

(p) A, V, L or I, more preferably A, L or I, even more preferably A, at amino acid position 78;

(q) E, R, T or A, more preferably E, T or A, even more preferably E, at amino acid position 82;

(r) T, S, I or L, more preferably T, S or L, even more preferably T, at amino acid position 86;

(s) D, S, N or G, more preferably D, N or G, even more preferably N, at amino acid position 87;

(t) A, V, L or F, more preferably A, V or F, even more preferably V, at amino acid position 89;

(u) F, S, H, D or Y, more preferably F, S, H or D, at amino acid position 90;

(v) D, Q or E, more preferably D or Q, even more preferably D, at amino acid position 92;

(w) G, N, T or S, more preferably G, N or T, even more preferably G, at amino acid position 95;

(x) T, A, P, F or S, more preferably T, A, P or F, even more preferably F, at amino acid position 98;

(y) R, Q, V, I, M, F, or L, more preferably R, Q, I, M, F or L, even more preferably Y, even more preferably L, at amino acid position 103; and

(z) N, S or A, more preferably N or S, even more preferably N, at amino acid position 107.

Additionally or alternatively, one or more of the following substitutions can be made in the variable region of the light chain of the immunobinding molecule of the invention:

(aa) Q, D, L, E, S, or I, more preferably L, E, S, or I, even more preferably L or E, at amino acid position 1;

(bb) S, A, Y, I, P or T, more preferably A, Y, I, P or T, even more preferably P or T at amino acid position 2;

(cc) Q, V, T or I, more preferably V, T or I, even more preferably V or T, at amino acid position 3;

(dd) V, L, I or M, more preferably V or L, at amino acid position 4;

(ee) S, E or P, more preferably S or E, even more preferably S, at amino acid position 7;

(ff) T or I, more preferably I, at amino acid position 10;

(gg) A or V, more preferably A, at amino acid position 11;

(hh) S or Y, more preferably Y, at amino acid position 12;

(ii) T, S or A, more preferably T or S, even more preferably T, at amino acid position 14;

(jj) S or R, more preferably S, at amino acid position 18;

(kk) T or R, more preferably R, at amino acid position 20;

(ll) R or Q, more preferably Q, at amino acid position 24;

(mm) H or Q, more preferably H, at amino acid position 46;

(nn) K, R or I, more preferably R or I, even more preferably R, at amino acid position 47;

(oo) R, Q, K, E, T, or M, more preferably Q, K, E, T or M, at amino acid position 50;

(pp) K, T, S, N, Q or P, more preferably T, S, N, Q or P, at amino acid position 53;

(qq) I or M, more preferably M, at amino acid position 56;

(rr) H, S, F or Y, more preferably H, S or F, at amino acid position 57;

(ss) I, V or T, more preferably V or T, R, even more preferably T, at amino acid position 74;

(tt) R, Q or K, more preferably R or Q, even more preferably R, at amino acid position 82;

(uu) L or F, more preferably F, at amino acid position 91;

(vv) G, D, T or A, more preferably G, D or T, even more preferably T, at amino acid position 92;

(xx) S or N, more preferably N, at amino acid position 94;

(yy) F, Y or S, more preferably Y or S, even more preferably S, at amino acid position 101; and

(zz) D, F, H, E, L, A, T, V, S, G, or I, more preferably H, E, L, A, T, V, S, G, or I, even more preferably A or V, at amino acid position 103.

In other embodiments, the immunobinding molecules of the invention comprise one or more stability enhancing mutations described in US Provisional Application Serial No. 61/075692, entitled "Solubility Optimization of Immunobinders", filed June 25, 2008. In certain preferred embodiments, the immunobinding molecule comprises a solubility enhancing mutation at an amino acid position selected from the group of amino acid positions of the heavy chain consisting of 12, 103 and 144 (AHo numbering convention). In one preferred embodiment, the immunobinding molecule contains one or more substitutions selected from the group consisting of: (a) serine (S) at amino acid position of heavy chain 12; (b) serine (S) or threonine (T) at the amino acid position of heavy chain 103; and (c) serine (S) or threonine (T) at the amino acid position of heavy chain 144. In another embodiment, the immunobinding molecule contains the following substitutions: (a ) serine (S) at amino acid position of heavy chain 12; (b) serine (S) or threonine (T) at the amino acid position of heavy chain 103; and (c) serine (S) or threonine (T) at the amino acid position of heavy chain 144.

In certain preferred embodiments, the immunobinding molecule comprises stability enhancing mutations in residues of the scaffold of the acceptor scaffold of the light chain in at least one of positions 1, 3, 4, 10, 47, 57, 91, and 103 of the variable region of the light chain in accordance with the AHo numbering system . In a preferred embodiment, the light chain acceptor framework contains one or more substitutions selected from the group consisting of (a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c) leucine (L) at position 4; (d) serine (S) at position 10; (e) arginine (R) at position 47; (e) serine (S) at position 57; (f) phenylalanine (F) at position 91 and (g) valine (V) at position 103.

To obtain a humanized antibody containing a mutation as described above, any of the many available methods can be used.

Thus, the present invention relates to an immunobinding molecule humanized by the method described herein.

In certain preferred embodiments, the target antigen of the specified immunobinding molecule is VEGF or TNFα.

The polypeptides described in the present invention or obtained by the method of the present invention can be synthesized, for example, by methods known in the art. Alternatively, nucleic acid molecules encoding the desired variable regions can be synthesized and the polypeptides prepared by recombinant methods.

For example, after the sequence of the humanized variable region is determined, the variable region or polypeptide containing it can be obtained by methods of molecular biology known in the art. More specifically, recombinant DNA methods can be used to produce a wide range of polypeptides by transforming the host cell with a nucleic acid sequence (e.g., a DNA sequence encoding a desired variable region (e.g., modified heavy or light chains; their variable domains, or other antigen-binding fragments thereof)) )

In one embodiment, an expression vector can be prepared comprising a promoter operably linked to a DNA sequence encoding at least V _H or V _L. If necessary or desired, a second expression vector can be obtained containing a promoter that is operably linked to a DNA sequence encoding a complementary variable domain (i.e., when the original expression vector encodes V _H , the second expression vector encodes V _L and vice versa). Then, one or both expression vectors can transform the cell line (e.g., an immortalized mammalian cell line) and cultivate under conditions that allow expression of the chimeric variable domain or chimeric antibody (see, for example, international patent application No. PCT / GB85 / 00392 Neuberger et. Al .

In one embodiment, the implementation can be obtained variable regions containing the amino acid sequence of the CDR of the donor and FR of the acceptor, and then for the implementation of amino acid substitutions of the CDR to make changes in the nucleic acid molecule.

Typical industry-recognized methods for producing a nucleic acid molecule encoding a variant of the amino acid sequence of a polypeptide include, but are not limited to, obtaining site specific (or mediated by oligonucleotides) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously obtained DNA encoding a polypeptide.

Site-specific mutagenesis is the preferred method for producing substitutional variants. This method is well known in the art (see, for example, Carter et al., Nucleic Acids Res. 13: 4431-4443 (1985) and Kunkel et al., Proc. Natl. Acad. Sci. USA 82: 488 (1987 )). Briefly, when conducting site-specific DNA mutagenesis, the source DNA is altered by first hybridizing the oligonucleotide encoding the desired mutation with a single strand of such source DNA. After hybridization, DNA polymerase is used to synthesize the whole second strand using a hybridizing oligonucleotide as a primer and using a single strand of the original DNA as a template. Thus, an oligonucleotide encoding the desired mutation is introduced into the resulting double-stranded DNA.

PCR mutagenesis is also suitable for obtaining amino acid sequence variants of polypeptides. See Higuchi, in PCR Protocols, pp. 177-183 (Academic Press, 1990); and Vallette et al., Nuc. Acids Res. 17: 723-733 (1989). In summary, when small amounts of template DNA are used as the starting material for PCR, primers that are slightly different in sequence from the corresponding region in template DNA can be used to produce relatively large amounts of a specific DNA fragment that differs from the template sequence only in positions, where the primers differ from the matrix.

Another method for producing variants, cassette mutagenesis, is based on the method described by Wells et al., Gene 34: 315-323 (1985). The starting material is a plasmid (or other vector) containing DNA for mutation. The codon (s) in the source DNA for mutation are identified. Each side of the identified mutation site (s) must have a unique restriction endonuclease recognition site. If such restriction sites do not exist, they can be obtained using the oligonucleotide-mediated mutagenesis described above, by introducing them at suitable positions of the DNA polypeptide encoding the polypeptide. Plasmid DNA is cut into these sections for its linearization. By standard methods, a double-stranded oligonucleotide encoding a DNA sequence between restriction sites but containing the desired mutation (s) is synthesized, where two oligonucleotide chains are synthesized separately and then hybridized together using standard methods. This double-stranded oligonucleotide is called a cassette. This cassette is designed to have 5'- and 3'-ends that are compatible with the ends of the linearized plasmid so that it can be directly ligated into the plasmid. Now this plasmid contains a mutated DNA sequence.

The variable region obtained by the methods of the invention can be rearranged and further altered to further enhance antigen binding. Thus, before or after the steps described above, it is possible to carry out steps including, for example, affinity maturation. In addition, empirical binding data can be used for further optimization.

One skilled in the art will appreciate that the polypeptides of the invention can be further modified so that they differ in amino acid sequence but still retain the desired activity. For example, for a protein, additional nucleotide substitutions can be made, leading to amino acid substitutions in “non-essential” amino acid residues. For example, a non-essential amino acid residue in an immunoglobulin polypeptide can be replaced with another amino acid residue from the same family of side chains. In another embodiment, the amino acid region can be replaced with a structurally similar region that differs in the order and / or composition of the side chain family, i.e., a conservative substitution can be made in which the amino acid residue is replaced by an amino acid residue with a similar side chain.

Families of amino acid residues with similar side chains are defined in this area, including major side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromas side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine).

In addition to amino acid substitutions, the present invention provides other modifications, for example, in the amino acid sequences of the Fc region to obtain a variant of the Fc region with altered effector function. For example, one or more amino acid residues of the Fc region can be removed to weaken or enhance binding to FcR. In one embodiment, to obtain such an Fc region variant, one or more residues of the Fc region can be modified. Typically, in accordance with this embodiment of the invention, no more than one to about ten residues of the Fc region are removed. The Fc region of this document, containing one or more deletions of amino acids, preferably retains at least about 80%, and preferably at least about 90%, and most preferably at least about 95% of the original Fc region or natural sequence Human Fc regions.

It is also possible to obtain variants of Fc regions with insertions of amino acids, where the effector function of these variants is altered. For example, next to one or more positions in the Fc region identified herein as affecting the binding of FcR, at least one amino acid residue can be introduced (e.g., from one to two amino acid residues and typically no more than ten residues ) By “near” is meant within the range of one to two amino acid residues from the remainder of the Fc region identified herein. Such variants of Fc regions may exhibit increased or decreased binding of FcRn.

Such variants of the Fc region, as a rule, contain at least one modified amino acid in the Fc region. In one embodiment, amino acid modifications may be combined. For example, an Fc region variant may contain two, three, four, five, etc. substitutions, for example, in specific provisions in the Fc region identified herein. In another embodiment, the polypeptide may have altered binding to FcRn and to another Fc receptor.

In one embodiment, the polypeptides described in the present invention or obtained by the method of the present invention, for example, humanized Ig variable regions and / or polypeptides containing humanized Ig variable regions, can be produced by recombinant methods. For example, a polynucleotide sequence encoding a polypeptide can be inserted into a suitable expression vector for recombinant expression. When the polypeptide is an antibody, polynucleotides encoding additional variable regions of the light and heavy chains, optionally connected to constant regions, can be inserted into the same or a different expression vector. To facilitate subsequent purification, an affinity tag sequence (e.g., His tag (6)) may optionally be attached to a polypeptide sequence, or an affinity tag sequence may be included in the polypeptide sequence. Plots of DNA encoding immunoglobulin chains are operably linked to control sequences in expression vector (s) that allow expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcriptional termination sequences. Preferably, expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. After introducing the vector into a suitable host, the host is maintained under conditions suitable for a high level of expression of nucleotide sequences and assembly and purification of the polypeptide.

These expression vectors, as a rule, are replicated in the host organisms as an episom or as an integral part of the host chromosomal DNA. Typically, expression vectors contain selective markers (for example, ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of cells transformed with the desired DNA sequences (see, for example, US patent No. 4704362).

One particularly suitable prokaryotic host for cloning polynucleotides (e.g., DNA sequences) of the present invention is E. coli . Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus , and other Enterobacteriaceae , such as Salmonella , Serratia, and various Pseudomonas species.

Other microbes, such as yeast, are also suitable for expression. Examples of yeast hosts are Saccharomyces and Pichia , with corresponding vectors with expression control sequences (eg, promoters), regions of origin of replication, termination of the sequence, and the like, as desired. Typical promoters include promoters of 3-phosphoglycerate kinase and other glycolysis enzymes. Inducible yeast promoters include, among others, alcohol dehydrogenase, isocytochrome C and enzymes responsible for the utilization of methanol, maltose and galactose.

Within the scope of the present invention, the preferred host cells are E. coli and S. cerevisiae .

In addition to microorganisms, tissue culture of mammals can be used to express and produce the polypeptides of the present invention (for example, polynucleotides encoding immunoglobulins or fragments thereof). See Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987). In fact, eukaryotic cells are preferred because a number of suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in this area, and it includes CHO cell lines, various Cos cell lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells, and hybridomas. The expression vectors for these cells may include expression control sequences, such as a replication start site, promoter and enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and informational sites necessary for processing, such as ribosome binding sites , RNA splicing sites, polyadenylation sites and transcription termination sequences. Preferred expression control sequences are promoters derived from genes for immunoglobulins, SV40, adenoviruses, cattle papilloma virus, cytomegalovirus, and the like. See Co et al., J. Immunol 148: 1149 (1992).

Vectors containing polynucleotide sequences of interest (for example, sequences encoding heavy and light chains and controlling expression of the sequence) can be introduced into the host cell by well-known methods that vary depending on the type of host cell. For example, for prokaryotic cells, transfection with calcium chloride is typically used, while for other host cells, calcium phosphate treatment, electroporation, lipofection, biolistic or virus-based transfection can be used. (mainly see Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989). Other methods used to transform mammalian cells include polybrene, fusion of protoplasts, liposomes, electroporation and microinjections (mainly see Sambrook et al., supra.) To obtain transgenic animals, transgenes can be introduced via microinjection into fertilized eggs or can be introduced into the genome of embryonic stem cells, and the nuclei of such cells can be transplanted into devoid egg nuclei.

The subject polypeptide can also be inserted into transgenes for insertion into the genome of a transgenic animal and subsequent expression, for example, in the milk of a transgenic animal (see, for example, Deboer et al. 5741957; Rosen 5304489 and Meade 5849992. Suitable transgenes include coding sequences for lungs and / or heavy chains in functional communication with the promoter and enhancer of a breast-specific gene, such as casein or beta-lactoglobulin.

Polypeptides can be expressed using one vector or two vectors. For example, antibody heavy and light chains can be cloned into separate expression vectors and co-transfected into cells.

In one embodiment, signal sequences may be used to facilitate expression of the polypeptides of the invention.

After expression, the polypeptides can be purified by standard methods for the art, including ammonium sulfate precipitation, affinity columns (e.g., protein A or protein G), column chromatography, HPLC purification, gel electrophoresis, and the like. (mainly see Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).

Humanized Ig variable regions or polypeptides containing them can be expressed in host cells or in cell lines in culture. They can also be expressed in cells in vivo . A cell line that is transformed (e.g., transfected) to produce an altered antibody may be an immortalized mammalian cell line, such as cell lines of lymphoid origin (e.g., myeloma, hybridoma, trioma or quadromic cell lines). The cell line may also include normal lymphoid cells, such as B cells, immortalized by transformation with a virus (e.g., Epstein-Barr virus).

Although, as a rule, the cell line used to produce the polypeptide is a mammalian cell line, cell lines from other sources (such as bacteria and yeast) can also be used. In particular, bacterial strains derived from E. coli can be used, in particular, for example, phage display.

Some immortalized lymphoid cell lines, such as myeloma cell lines, secrete individual Ig light or heavy chains in their normal state. If such a cell line is transformed with a vector expressing an altered antibody obtained by the method of the invention, the remaining steps of the method are not necessary, provided that the normally secreted chain is complementary to the variable domain of the Ig chain encoded by the previously obtained vector.

If the immortalized cell line does not secrete or does not secrete a complementary chain, it is necessary to introduce into the cells a vector encoding the corresponding complementary chain or its fragment.

In the event that an immortalized cell line secrets a complementary light or heavy chain, a transformed cell line can be obtained, for example, by transforming a suitable bacterial cell with a vector and then merging the bacterial cell with an immortalized cell line (for example, by fusion of spheroplasts). Alternatively, DNA can be introduced directly into the immortalized cell line by electroporation.

In one embodiment, the humanized Ig variable region, as described in the present invention or obtained by the method of the present invention, may be present in the antigen binding fragment of any antibody. Fragments can be produced recombinantly and constructed, synthesized or prepared by cleavage of the antibody with a proteolytic enzyme. For example, the fragment may be a Fab fragment; papain cleavage separates the antibody in the region before the interchain (i.e., V _H -V _H ) disulfide bond that connects the two heavy chains. This leads to the formation of two identical fragments containing the light chain and the V _H and C _H 1 domains of the heavy chain. Alternatively, the fragment may be a fragment of F (ab ') ₂ . These fragments can be obtained by cleaving the antibody with pepsin, which cleaves the heavy chain after an interchain disulfide bond and leads to the formation of a fragment containing both antigen-binding sites. Another alternative is to use a single chain antibody. Single chain Fv fragments (scFv) can be produced in a variety of ways. For example, the C-terminus of V _H can be attached to the N-cone of V _L. Typically, a linker is placed between V _H and V _L (e.g., (GGGGS) ₄ ). However, the order in which the chains can be connected can be reversed, and labels can be included that facilitate detection or purification (e.g., Myc, His or FLAG- labels) (labels such as these can be attached to any antibody or antibody fragment at invention; their use is not limited to scFv). Thus, and as noted below, labeled antibodies are included in the scope of the present invention. In alternative embodiments, the antibodies described herein or prepared by the methods described herein can be heavy chain dimers or light chain dimers. In addition, you can use the light or heavy chain antibodies, or parts thereof, for example, single-domain antibodies (DAb).

In another embodiment, the humanized Ig variable region, as described in the present invention or obtained by the method of the present invention, is present in a single chain antibody (scFv) or minaret (see, for example, US Pat. No. 5,837,821 or WO 94 / 09817A1). Minobodies are dimeric molecules derived from two polypeptide chains, each of which contains an scFv molecule (a separate polypeptide containing one or more antigen-binding sites, for example, the V _L domain connected by a movable linker to the V _H domain fused to the C _H 3 domain by connecting peptide). ScFv molecules can be constructed in the V _H -linker-V _L orientation or in the V _L -linker-V _H orientation. The movable hinge that connects the V _L and V _H domains forming the antigen binding site preferably contains from about 10 to about 50 amino acid residues. An illustrative connecting peptide for this purpose is (Gly ₄ Ser) ₃ (Huston et al., (1988). PNAS, 85: 5879). Other connecting peptides are known in the art.

Methods for producing single chain antibodies are well known in the art, for example, Ho et al., (1989), Gene, 77:51; Bird et al., (1988) Science 242: 423; Pantoliano et al., (1991), Biochemistry 30: 10117; Milenic et al., (1991), Cancer Research, 51: 6363; Takkinen et al., (1991), Protein Engineering 4: 837. Minobodies can be obtained by constructing the scFv component and attaching the peptide-CH ₃ component by methods described in the art (see, for example, US Pat. No. 5,837,821 or WO 94 / 09817A1). These components can be isolated from individual plasmids as restriction fragments, and then ligated and re-cloned into a suitable vector. The corresponding construct can be verified by restriction digestion and DNA sequence analysis. In one of the embodiments, the minidoll according to the invention contains a connecting peptide. In one embodiment, the implementation of the connecting peptide contains a Gly / Ser linker, for example, GGGSSGGGSGG.

In another embodiment, a tetravalent minitool can be constructed. The tetravalent minitel can be constructed in the same way as the minitel, except that two scFv molecules are coupled using, for example, the amino acid sequence (G ₄ S) ₄ G ₃ AS using a movable linker.

In another embodiment, a humanized variable region, as described in the present invention or obtained by the method of the present invention, may be present in the die. Diabodies are similar to scFv molecules, but typically contain a short (less than 10, and preferably 1-5) linker of amino acid residues connecting both variable domains so that the V _L and V _H domains on the same polypeptide chain cannot interact. Instead, the V _L and V _H domains of one polypeptide chain interact with the V _H and V _L domains (respectively) on the second polypeptide chain (WO 02/02781).

In another embodiment, the humanized variable region of the invention may reside in an immunoreactive fragment or portion of an antibody (e.g., an scFv molecule, an amino acid, a tetravalent amino acid or a diabody) operably linked to the FcR binding portion. In an illustrative embodiment, the FcR binding portion is an entire Fc region.

Preferably, the humanization methods described herein produce Ig variable regions in which the affinity for the antigen is substantially unchanged from that of the donor antibody.

In one embodiment, the implementation of the polypeptides containing the variable domains of the present invention bind to antigens with a binding affinity of greater (or equal) affinity with a Ka association constant of approximately 10 ⁵ M ^-1 , 10 ⁶ M ^-1 , 10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 or 10 ¹² M ^-1 (including the affinities between these values).

Affinity, avidity and / or specificity can be measured in many ways. Typically, and regardless of the particular method by which affinity is determined or measured, the methods of the invention when they produce an antibody that in any aspect of its clinical use is superior to the antibody (or antibodies) from which it was obtained (for example, the methods of the invention are considered effective or successful, when the modified antibody can be administered at a lower dose or less often or in a more convenient route of administration than the antibody (or antibodies) from which it was obtained) improve the affinity of the antibody.

More specifically, the affinity of an antibody for the antigen to which it binds can be measured by various assays, including, for example, an ELISA assay, a BiaCore assay, or a KinExA ™ 3000 assay (available from Sapidyne Instruments (Boise, ID)). Briefly, sepharose granules are coated with an antigen (the antigen used in the methods of the invention may be any antigen of interest (e.g., a cancer antigen; a cell surface protein or a secreted protein; a pathogen organism antigen (e.g., a bacterial or viral antigen (e.g. , HIV antigen, influenza antigen or hepatitis antigen)) or allergen) by covalent binding. Antibody dilutions are prepared for testing and each dilution is added as defined well, a detecting antibody (for example, a goat anti-human IgG antibody conjugate with HRP) was added, followed by the addition of a chromogenic substrate (for example, HRP), The plate was then read in an ELISA spectrophotometer to read the tablets at 450 nM and calculated EC ₅₀ values. (however, it should be understood that the methods described herein are generally applicable; they are not limited to the production of antibodies that bind any particular antigen or class of antigens.)

Those skilled in the art will recognize that the determination of affinity is not always as simple as it looks in the simplest case. Since antibodies have two arms, their apparent affinity is usually much higher than the intrinsic affinity of the variable region for an antigen (this is believed to be due to avidity). Native affinity can be measured using scFv or Fab fragments.

In another aspect, the present invention relates to bespecifically molecules containing a humanized rabbit antibody or a fragment thereof according to the invention. Derivatives can be formed with the antibody of the invention or its antigen-binding parts, or they can be combined with another functional molecule, for example, another peptide or protein (for example, another antibody or receptor ligand) to produce a bispecific molecule that binds at least two different binding sites or two target molecules. Derivatives can be formed with the antibody of the invention, or it can be coupled to more than one other functional molecule to produce multispecific molecules that bind more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be included in the term "bispecific molecule" as used herein. To obtain a bispecific molecule of the invention, the antibody of the invention can be functionally linked (for example, by chemical binding, genetic fusion, non-covalent binding or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, tumor-specific or pathogen-specific antigens, peptidomimetic or a binding simulator such that a bispecific molecule is formed. Thus, the present invention relates to bispecific molecules containing at least one first binding molecule with specificity for the first target and a second binding molecule with specificity for one or more additional target epitopes.

In one embodiment, the bispecific molecules of the invention comprise, as a specific binding molecule, at least one antibody, or a fragment thereof, including, for example, Fab, Fab ', F (ab') ₂ , Fv, or single chain Fv. The antibody may also be a light chain or heavy chain dimer, or a minimal fragment thereof, such as Fv or a single chain construct, as described in Ladner et al., US Pat. No. 4,946,778, the contents of which are expressly incorporated by reference.

Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules of the invention are mouse monoclonal antibodies, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present invention can be obtained by conjugating constituent specific binding molecules known in the art. For example, each specific binding molecule of a bispecific molecule can be obtained separately and then conjugated to each other. When the specific binding molecules are proteins or peptides, a number of binding or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetylthioacetate (SATA), 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidimidyl-3- ( 2-pyridyldithio) propionate (SPDP) and sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky et al., (1984) J. Exp. Med. 160: 1686 ; Liu, MA et al., (1985) Proc. Natl. Acad. Sci. USA 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al., (1985) Science 229: 81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

When the specific binding molecules are antibodies, they can be conjugated by the formation of sulfhydryl bonds between the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified so that it contains an odd amount of sulfhydryl residues, preferably one, before conjugation.

Alternatively, both specific binding molecules can be encoded on a single vector and expressed and assembled in a single host cell. This method is particularly suitable when the bispecific molecule is a fusion protein mAb × mAb, mAb × Fab, Fab × F (ab ') ₂ or a ligand × Fab. The bispecific molecule of the invention may be a single chain molecule containing one single chain antibody and one binding determinant, or a single chain bispecific molecule containing two binding determinants. Bespecifically molecules may contain at least two single-stranded molecules. Methods for producing bispecific molecules are described, for example, in US Pat. No. 5,260,203; US patent No. 5455030; US patent No. 4881175; U.S. Patent No. 5,132,405; U.S. Patent No. 5,091,513; U.S. Patent No. 5,476,786; U.S. Patent No. 5013653; US patent No. 5258498 and US patent No. 5482858.

The binding of bispecific molecules to their specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry, based on the sorting of single cells (e.g., FACS analysis), biological analysis (e.g., growth inhibition), or Western blot analysis. In each of these assays, the presence of protein-antibody complexes of particular interest is typically determined using a labeling reagent (eg, antibody) specific for the complex of interest. For example, antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to antibody-VEGF complexes. Alternatively, complexes can be detected using any of a variety of other immunological assays. For example, an antibody can be radioactively labeled and used in radioimmunological analysis (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, incorporated herein by as a link). A radioactive isotope can be detected by methods such as the use of a γ counter or scintillation counter or by autoradiography.

In another aspect, the present invention relates to a humanized rabbit antibody or fragment thereof conjugated to a therapeutic molecule, such as a cytotoxin, a drug (e.g., an immunosuppressor), or a radiotoxin. Such conjugates are referred to herein as “immunoconjugates”. Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that harms (e.g. kills) cells. Examples include taxol, cytochalazine B, gramicidin D, ethidium bromide, emetin, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracidione, mitramidocinocortocortin, dextroricin-tetrocortocortin, dextrin-tetracortocortin, tetracycidocortin, tetracycidocortin, tetracycidocortin, dextrin , lidocaine, propranolol and puromycin and their analogues or homologs. Therapeutic agents may also include, for example, antimetabolites (e.g. methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g. mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine l (BSN) CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g. daunorubicin (formerly daunomycin) and doxorubicin (e.g. d) , bleomycin, mitramycin and anthramycin in (AMC)) and antimitotic agents (e.g. vincristine and vinblastine).

Other preferred examples of therapeutic cytotoxins that can be conjugated to an antibody of the invention include duocarmycins, calicheamicins, maytansines and auristatins and their derivatives. An example conjugate of calicheamicin with an antibody is commercially available (milotarg ™; Wyeth-Ayerst).

Cytotoxins can be conjugated to the antibodies of the invention using linker technology available in the art. Examples of types of linkers that have been used to conjugate cytotoxin to an antibody include, but are not limited to, hydrazone, thioethers, esters, disulfides, and peptide-containing linkers. You can choose a linker that, for example, is sensitive to cleavage at low pH under lysosomal compartment conditions or is sensitive to cleavage by proteases, such as proteases, primarily expressed in tumor tissue, such as cathepsins (e.g., cathepsins B, C, D).

For additional information on types of cytotoxins, linkers, and methods for conjugating therapeutic agents to antibodies, also see Saito, G. et al., (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P.A. et al., (2003) Cancer Immunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3: 1089-1091; Senter, P.D. and Springer, C.J. (2001) Adv. Drug Deliv. Rev. 53: 247-264.

The antibodies of the present invention can also be conjugated to a radioactive isotope to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ^90, and lutetium ¹⁷⁷ . A method for producing radio immunoconjugates is widely known in the art. Examples of radio immunoconjugates are commercially available, including Zevalin ™ (IDEC Pharmaceuticals) and Bexar ™ (Corixa Pharmaceuticals), and similar methods can be used to produce radio immunoconjugates using antibodies of the invention.

The antibody conjugates of the invention can be used to modify an existing biological response, and the drug molecule should not be construed as being limited by classical chemical therapeutic agents. For example, the drug molecule may be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, an enzymatically active toxin or an active fragment thereof, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin; a protein, such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as, for example, lymphokines, interleukin-1 ("IL-1), interleukin-2 (" IL-2 "), interleukin-6 (" IL-6 "), granulocyte-macrophage colony stimulating factor ( "GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Methods for conjugating such therapeutic molecules to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., (Eds.), Pp. . 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al., (Eds.), Pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., (Eds.), Pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al., (Eds.), Pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982).

In one aspect, the invention relates to pharmaceutical compositions comprising humanized rabbit antibodies for treating a disease. The term "pharmaceutical composition" refers to preparations that are in a form that allows a certain effective biological activity of the antibody or derivative of the antibody, and which do not contain additional components that are toxic to individuals who are administered this composition. "Pharmaceutically acceptable" excipients (carriers, additives) are excipients that can be reasonably administered to the mammal in question to provide an effective dose of the active ingredient used.

A “stable” formulation is one in which the antibody or antibody derivative substantially retains its physical stability and / or chemical stability and / or biological activity during storage. Available in the art and reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993) various analytical methods for measuring protein stability. Stability can be measured at a selected temperature for a selected period of time. Preferably, the composition is stable at room temperature (approximately 30 ° C) or at 40 ° C for at least 1 month and / or stable at approximately 2-8 ° C for at least 1 year, for at least 2 years. In addition, the composition is preferably stable after freezing (for example, to -70 ° C) and thawing of the composition.

An antibody or antibody derivative "retains its physical stability" in a pharmaceutical composition if it does not show aggregation, precipitation and / or denaturation by visual examination of color and / or transparency, or as measured by scattering of UV light or by gel chromatography.

An antibody or antibody derivative "retains its chemical stability" in the pharmaceutical composition if the chemical stability at a given time is such that the protein is considered to still retain biological activity, as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. A chemical change may include a change in size (e.g., compression), which can be evaluated, for example, using gel chromatography, SDS-PAGE and / or time-of-flight mass spectrometry with laser desorption / ionization using a matrix (MALDI / TOF MS). Other types of chemical change include a charge change (for example, resulting from deamidation), which can be evaluated, for example, by ion exchange chromatography.

An antibody or antibody derivative "retains its biological activity" in the pharmaceutical composition if the biological activity of the antibody at a given time is within about 10% (within the range of the analysis error) of the biological activity demonstrated at the time when the pharmaceutical composition was obtained, as determined, for example, in an antigen binding assay. Other analyzes of the "biological activity" of antibodies are indicated below in this document.

By “isotonic” is meant that the composition of interest has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of approximately 250 to 350 mOsm. Isotonicity can be measured using, for example, a steam pressure osmometer or cryoscopic type.

"Polyol" is a substance with several hydroxyl groups and includes sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids. Preferred polyols herein have a molecular weight of less than about 600 kDa (for example, in the range of about 120 to about 400 kDa). “Reducing sugar” is a sugar containing a semi-acetal group that can reduce metal ions or covalently react with lysine and other amino groups in proteins, and “non-reducing sugar” is a sugar that does not have these reducing sugar properties. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melicytosis and raffinose. Examples of sugar alcohols are mannitol, xylitol, erythritol, threitol, sorbitol and glycerin. In relation to sugar acids, they include L-gluconate and its metal salts. When it is desired that the composition be stable to freeze-thaw, the polyol is preferably a polyol that does not crystallize at freezing temperatures (for example, -20 ° C) since this destabilizes the antibody in the composition. Preferred polyols herein are non-reducing sugars, such as sucrose and trehalose, where trehalose is more preferred than sucrose due to the better stability of trehalose in solutions.

As used herein, “buffer” refers to a buffer solution that inhibits pH changes by the action of its conjugated base acid components. the pH of the buffer of the present invention is in the range of from about 4.5 to about 6.0; preferably from about 4.8 to about 5.5; and most preferably, the pH is about 5.0. Examples of buffers that maintain a pH in this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. When a freeze-thaw stable formulation is desired, the buffer is preferably not phosphate.

In a pharmacological sense, in the context of the present invention, a “therapeutically effective amount” of an antibody or antibody derivative refers to an amount effective to prevent or treat a disorder in which the antibody or antibody derivative is effective. A “disease / disorder” is any condition that can be beneficially treated with an antibody or antibody derivative. It includes chronic and acute disorders or diseases, including pathological conditions that predispose the mammal to the corresponding violation.

"Preservative" is a compound that can be included in the composition to significantly reduce the action of bacteria in it, thus, for example, facilitating the preparation of the composition for repeated use. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative herein is benzyl alcohol.

The present invention also relates to pharmaceutical compositions comprising compounds of one or more antibodies or antibody derivatives with at least one physiologically acceptable carrier or excipient. The pharmaceutical compositions may contain, for example, one or more of water, buffers (e.g., neutral physiological saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethyl sulfoxide, carbohydrates (e.g. glucose, mannose, sucrose or dextran) , mannitol, proteins, adjuvants, polypeptides or amino acids, such as glycine, antioxidants, chelating agents such as EDTA or glutathione, and / or preservatives. As indicated above, other active ingredients may may be included in the pharmaceutical compositions provided herein.

A carrier is a substance that can be associated with an antibody or antibody derivative prior to administration to a patient, often with the aim of regulating the stability or bioavailability of the compound. Carriers for use in such formulations are typically biocompatible and may also be biodegradable. Carriers include, for example, monovalent or polyvalent molecules, such as serum albumin (e.g., human or bovine), egg albumin, peptides, polylysine and polysaccharides, such as aminodextran and polyamidoamines. Carriers also include solid carrier materials such as granules and microparticles containing, for example, polylactate-polyglycolate, a lactide-glycolide copolymer, polyacrylate, latex, starch, cellulose or dextran. The carrier can carry the compounds in a variety of ways, including covalent binding (directly or via a linker group), non-covalent interaction or mixing.

The pharmaceutical compositions can be formulated for any suitable route of administration, including, for example, topical oral, nasal, rectal or parenteral administration. In certain embodiments, compositions in a form suitable for oral administration are preferred. Such forms include, for example, pills, tablets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules or syrups or elixirs. In other embodiments, the compositions provided herein can be formulated as a lyophilisate. As used herein, the term parenteral includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal and intraperitoneal injection, as well as any similar injection or infusion method.

Compositions intended for oral administration can be obtained by any method known in the art of preparing pharmaceutical compositions, and they may contain one or more agents, such as sweeteners, flavors, colorants and preservatives, to impart attractiveness and palatability to the preparations. Tablets contain the active ingredient in admixture with physiologically acceptable excipients suitable for the preparation of tablets. Such excipients include, for example, inert diluents (e.g. calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating and disintegrating agents (e.g. corn starch or alginic acid), binders (e.g. starch, gelatin or gum arabic ) and lubricants (e.g. magnesium stearate, stearic acid or talc). The tablets may be uncoated, or they may be coated by known methods to delay disintegration and absorption in the gastrointestinal tract and thus provide a prolonged action for a longer period. For example, you can use a substance to delay time, such as glyceryl monostearate or glyceryl distearate.

Oral formulations may also be provided in the form of hard gelatin capsules, where the active ingredient is mixed with a solid inert diluent (e.g., calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules, where the active ingredient is mixed with an aqueous or oily medium ( e.g. peanut butter, paraffin oil or olive oil). Aqueous suspensions contain the antibody or antibody derivative in admixture with excipients suitable for preparing aqueous suspensions. Such excipients include suspending agents (for example, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum arabic) and dispersing agents or humectants (for example, natural phospholipids, such as acids, butyric acids, , condensation products of ethylene oxide with long chain aliphatic alcohols, such as heptadecaethylenoxyketanol, condensation products ethylene oxide with partial esters derived from fatty acids and hexite, such as polyoxyethylene sorbitan monooleate; or condensation products of ethylene oxide with partial esters derived from fatty acids and hexite anhydrides such as polyethylene sorbitan monooleate). Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl-p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin. Syrups and elixirs can be formulated with sweeteners such as glycerin, propylene glycol, sorbitol, or sucrose. Such formulations may also contain one or more of an irritant reducing agent, preservative, flavoring and / or coloring agent.

Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil (e.g., peanut oil, olive oil, sesame oil or coconut oil) or in a mineral oil such as paraffin oil. The oily suspensions may contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweeteners, such as the sweeteners mentioned above and / or flavors, may be added to provide a pleasant taste for oral preparations. Such suspensions can be prevented by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparing an aqueous suspension by adding water provide the delivery of the active ingredient in admixture with a dispersing agent or humectant, suspending agent and one or more preservatives. Suitable dispersing agents or humectants and suspending agents are illustrated by the agents already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions may also be in the form of oil-in-water emulsions. The oil phase may be a vegetable oil (e.g., olive oil or peanut oil), mineral oil (e.g., paraffin oil), or a mixture thereof. Suitable emulsifiers include natural gums (e.g. gum arabic or tragacanth gum), natural phospholipids (e.g. soy, lecithin and esters or partial esters derived from fatty acids and hexite), anhydrides (e.g. sorbitan monooleate) and partial ester condensation products derived from fatty acids and hexite with ethylene oxide (e.g. polyoxyethylene sorbitan monooleate). The emulsion may also contain one or more sweeteners and / or flavors.

The pharmaceutical composition can be obtained in the form of a sterile injectable aqueous or oily suspension in which, depending on the carrier used and the concentration, the modulator is either suspended or dissolved in the carrier. Such a composition can be formulated in accordance with the prior art using suitable dispersing agents, humectants and / or suspending agents, such as the above agents. Among the acceptable carriers and solvents that can be used are water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fatty oils may be used as a solvent or suspension medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids can be used to prepare injectable compositions, as oleic acid, and excipients such as local anesthetics, preservatives and / or buffers can be dissolved in a carrier.

The pharmaceutical compositions can be formulated as sustained release formulations (i.e., a formulation, such as a capsule, for sustained release of the modulator after administration). Typically, such formulations can be prepared using well-known techniques and administered, for example, by oral, rectal or subcutaneous administration, or by administration to a desired target site. Carriers for use in such formulations are biocompatible and may also be biodegradable; preferably, the composition provides a relatively constant level of release of the modulator. The amount of antibody or antibody derivative contained in a sustained release formulation depends, for example, on the site of administration, rate and expected duration of release, and the nature of the disease / disorder to be treated or prevented.

Antibodies or antibody derivatives provided herein are typically administered in an amount to achieve a concentration in body fluid (e.g., blood, plasma, serum, CSF, synovial fluid, lymph, intercellular fluid, tears or urine), which sufficient for detectably binding to a target, for example, such as VEGF, and to prevent or suppress diseases / disorders mediated by such a target, for example, VEGF-mediated diseases / disorders. The dose is believed to be effective if it leads to visible improvements in the patient, as described herein. A preferred range of systemic doses is from about 0.1 mg to about 140 mg per kilogram of body weight per day (from about 0.5 mg to about 7 g to one patient per day), with oral doses generally of about 5- 20 times higher than intravenous doses. The amount of antibody or antibody derivative that can be combined with carrier substances to form a single dosage form varies depending on the organism to be treated and the particular route of administration. Unit dosage forms typically contain from about 1 mg to about 500 mg of the active ingredient.

The pharmaceutical compositions can be packaged to treat conditions that respond to an antibody or antibody derivative directed, for example, to VEGF. Packed pharmaceutical compositions may include a container containing an effective amount of at least one antibody or antibody derivative, as described herein, and instructions (eg, a label) indicating that the composition should be used to treat a disease / disorder that responds to this one antibody or antibody derivative after administration to a patient.

Antibodies or antibody derivatives of the present invention can also be chemically modified. Preferred modifying groups are polymers, for example, optionally substituted straight or branched chain polyalkenyl, polyalkenylene or polyoxyalkylene polymers or unbranched or branched polysaccharides. Such an effector group can increase the half-life of an antibody in vivo . Specific examples of synthetic polymers include optionally substituted poly (ethylene glycol) (PEG), poly (propylene glycol), straight or branched chain poly (vinyl alcohol), or derivatives thereof. Specific natural polymers include lactose, amylose, dextran, glycogen, or derivatives thereof. The size of the polymer can be varied as desired, but, as a rule, its average molecular weight is in the range from 500 Da to 50,000 Da. For topical application, when the antibody is designed to penetrate tissue, the preferred molecular weight of the polymer is approximately 5000 Da. The polymer molecule can be attached to an antibody, in particular, to the C-terminus of the heavy chain of a Fab fragment via a covalently linked hinge peptide, as described in WO0194585. Regarding the attachment of PEG molecules, reference may be made to "Poly (ethyleneglycol) Chemistry, Biotechnological and Biomedical Applications", 1992, J. Milton Harris (ed), Plenum Press, New York and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998 , M. Aslam and A. Dent, Grove Publishers, New York.

After obtaining the antibody of interest or an antibody derivative as described above, a pharmaceutical composition containing it is obtained. The antibody for formulation is not subjected to previous lyophilization and the composition of interest in this document is an aqueous composition. Preferably, the antibody or antibody derivative in the composition is an antibody fragment, such as scFv. The therapeutically effective amount of the antibody in the composition is determined, for example, by taking into account the desired dose volumes and the method (s) of administration. An exemplary antibody concentration in the composition is from about 0.1 mg / ml to about 50 mg / ml, preferably from about 0.5 mg / ml to about 25 mg / ml, and most preferably from about 2 mg / ml to about 10 mg / ml.

An aqueous composition is obtained containing an antibody or antibody derivative in a pH buffered solution. The pH of the buffer of the present invention is in the range of about 4.5 to about 6.0, preferably about 4.8 to about 5.5, and most preferably, the pH is about 5.0. Examples of buffers that maintain a pH in this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers. The concentration of the buffer may be from about 1 mm to about 50 mm, preferably from about 5 mm to about 30 mm, depending, for example, on the buffer and the desired isotonic composition. A preferred buffer is sodium acetate (approximately 10 mM), pH 5.0.

The composition includes a polyol, which acts as a tonicity agent and can stabilize the antibody. In preferred embodiments, the composition does not contain a tonic amount of salt, such as sodium chloride, as this may cause precipitation of the antibody or antibody derivative and / or may result in oxidation at low pH. In preferred embodiments, the polyol is a non-reducing sugar, such as sucrose or trehalose. The polyol is added to the composition in an amount that can be varied in accordance with the desired isotonicity of the composition. Preferably, the aqueous composition is isotonic, in which case suitable concentrations of the polyol in the composition are, for example, in the range of about 1% to about 15% w / v, preferably in the range of about 2% to about 10% w / about. However, hypertonic or hypotonic formulations may also be suitable. The amount of added polyol can also be changed in accordance with the molecular weight of the polyol. For example, less monosaccharide (e.g. mannitol) can be added compared to a disaccharide (such as trehalose).

A surfactant is also added to the composition of the antibody or antibody derivative. Examples of surfactants include nonionic surfactants such as polysorbates (e.g., polysorbates 20, 80, etc.) or poloxamers (e.g., poloxamer 188). The amount of surfactant added is such that it reduces the aggregation of the formulated antibody / antibody derivative, and / or minimizes the formation of particles in the composition, and / or reduces adsorption. For example, a surfactant may be present in an amount from about 0.001% to about 0.5%, preferably from about 0.005% to about 0.2%, and most preferably from about 0.01% to about 0, one%.

In one embodiment, the composition comprises the above agents (i.e., an antibody or antibody derivative, buffer, polyol and surfactant) and essentially does not contain one or more preservatives, such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium chloride. In another embodiment, a preservative may be included in the formulation, especially when the formulation is a multiple dosage formulation. The concentration of preservative may be in the range of from about 0.1% to about 2%, most preferably from about 0.5% to about 1%. The composition may include one or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 21st edition, Osol, A. Ed. (2006), provided that they do not adversely affect the desired characteristics of the composition. Acceptable carriers, excipients or stabilizers are nontoxic to the recipient at the dosages and concentrations used and include additional buffering agents; auxiliary solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; complex compounds with metals (for example, Zn-protein complexes); biodegradable polymers such as polyesters; and / or salt forming counterions, such as sodium.

Formulations for use for in vivo administration must be sterile. This is easily accomplished by filtration through sterile filter membranes before or after preparation.

The composition is administered to a mammal in need of antibody treatment, preferably a human, by known methods, such as intravenous administration as a bolus or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasinovial, intrathecal, oral, topical or inhalation . In preferred embodiments, the composition is administered to the mammal via intravenous administration. To this end, the composition can be injected using, for example, a syringe or dropper.

A suitable dose (“therapeutically effective amount”) of an antibody depends, for example, on the condition to be treated, the severity and course of the condition, whether the antibody is administered for prophylactic or therapeutic purposes, prior treatment, the patient’s medical history and its response to the antibody, such as the antibody used, solution the attending physician. It is advisable to administer the antibody or antibody derivative to the patient once or through several administrations and subsequently can be administered to the patient at any time for diagnosis. An antibody or antibody derivative may be administered as a single treatment method or in combination with other drugs or treatment methods suitable for treating a corresponding disease.

As a general suggestion, a therapeutically effective amount of an administered antibody or derivative of an antibody is in the range of about 0.1 to about 50 mg / kg body weight of the patient with one or more administrations, for example, with the usual range of antibody used, administered daily, from about 0 3 to about 20 mg / kg, more preferably from about 0.3 to about 15 mg / kg. However, other dosage regimens may also be suitable. The progress of this treatment is easily controlled by conventional methods.

In another embodiment, the invention relates to an industrial product comprising a container containing a pharmaceutical composition of the present invention, preferably an aqueous composition, and optionally containing instructions for its use. Suitable containers include, for example, bottles, ampoules, and syringes. The container can be obtained from a number of materials, such as glass or plastic. An example of a container is a glass ampoule for single use with a volume of 3-20 cm ³ . Alternatively, for multi-dose formulations, the container may be a 3-100 cm ³ glass ampoule. The container contains the composition, and the label on the container, or attached to it, may contain instructions for use. The industrial product may further include materials desirable from a commercial and user point of view, including other buffers, diluents, filters, needles, syringes and package inserts with instructions for use.

In certain preferred embodiments, the industrial product comprises a lyophilized immunobinding molecule, as described herein, or prepared by the methods described herein.

Examples

The present description is further illustrated by the following examples, which should not be construed as an additional limitation. The contents of all figures and all references, patents, and published patent applications cited throughout the entire application are hereby expressly incorporated by reference in their entirety.

Materials and methods

Basically, in the practical implementation of the present invention, unless otherwise indicated, conventional methods of chemistry, molecular biology, recombinant DNA technology, immunology (in particular, for example, antibody technology) are used and standard methods for producing polypeptides. See, for example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C.S.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons (1992). Methods for transplanting CDRs from rabbit monoclonal antibodies and other non-human monoclonal antibodies to selected human antibody scaffolds are described in detail above. Examples of experiments on such a transplant are given below.

For the purpose of better understanding, transplants designated as “min” are transplants where the CDRs are transplanted to frame 1.4 or its variable domain, while transplants designated as “max” are transplants where CDRs are transplanted to frame 1.4 or its variable domain and where the framework further comprises donor framework residues that interact with the antigen.

Example 1: Construction rFW1.4

1.1. Primary sequence analysis and database searches

1.1.1. Collection of rabbit immunoglobulin sequences

The sequences of the variable domains of the mature antibodies and the germ line of the rabbit were collected from various public databases (for example, the Kabat database and IMGT) and entered into their own database in the form of a single-letter code of amino acid sequences. For a complete analysis, the authors used only part of the amino acids corresponding to the V (variable) region. Sequences in the KDB database containing less than 70% of the complete sequence or containing many undefined residues in the framework regions were deleted. Sequences that are more than 95% identical to other sequences in the database are also excluded to avoid random interference in the analysis.

1.1.2. Alignment and numbering of rabbit sequences

The rabbit antibody sequences were aligned using a traditional sequence alignment tool based on the Needleman-Wunsch algorithm and Blossum matrices. The introduction of gaps and the designation of the positions of the residues was carried out in accordance with the AHo numbering system for the variable domains of immunoglobulins (Honegger and Pluckthun, 2001). The Kabat numbering scheme was also used in parallel, as it represents the most widely accepted standard for numbering residues in an antibody. Kabat numbering was assigned using the SUBIM program. This program analyzes the variable regions of an antibody sequence and numbers the sequence according to a system established by Kabat and colleagues (Deret et al 1995).

Frame regions and CDRs were determined by the Kabat definition, which is based on sequence variability and is the most commonly used. However, the designation CDR H1 was a compromise between various definitions, including AbM, Kabat, average contact data obtained by analyzing the contacts of the antibody and antigen of a subset of three-dimensional complex structures (MacCallum et al., 1996) and Chotia, which is based on determining the position of structural regions loops (described above and shown in Fig. 1).

1.1.3. Frequency and conservativeness of residual positions

The amino acid sequence variability was analyzed using a set of 423 rabbit sequences from the Kabat database. The frequency of residues, f (r), for each position, i, in mature rabbit sequences was calculated by the number of times that a particular residue was observed in a data set divided by the total number of sequences. The degree of conservatism for each position, i, was calculated using the Simpson index, which takes into account the number of different amino acids encountered, as well as the relative content of each residue.

where: N represents the total number of amino acids, r represents the number of different amino acids encountered at each position, and n represents the number of residues of a particular type of amino acid.

1.1.4. Genealogical analysis of the rabbit V region

To study the repertoire of the rabbit, phylogenetic analysis tools were used. V-region amino acid sequences were clustered using a clustering algorithm and a topological algorithm. For the entire array, a distance matrix was calculated and used as an indicator of the use of the germ line. The consensus sequence of the entire cluster was calculated and the closest variant of the rabbit germ line sequence was identified. Also from the complete set of sequences, a general consensus sequence was obtained.

1.1.5. The distribution of human subgroups.

For each characteristic rabbit sequence from different clusters, the most homologous human subgroup was identified using the implementation of EXCEL sequence analysis algorithms and classification methods based on analysis of the repertoire of human antibodies (Knappik et al., 2000).

1.2. Human acceptor framework design

With a copy of the rabbit's repertoire from the sequence analysis described above, residues in the scaffold, typically involved in the location of the rabbit CDR, were determined. Among the frameworks with high homology with the rabbit repertoire and with the corresponding clusters, one with good biophysical properties was selected from the totality of fully human sequences. The framework selected to serve as an acceptor framework belongs to the variable region of the light chain of the Kappa 1 subgroup and the variable region of the heavy chain of the Kappa 1 subgroup ID ESBATech KI27, a43, respectively. This stable and soluble antibody framework was identified in the screening of the human spleen scFv library using a yeast-based screening method called the "quality control" system (Auf der Maur et al., 2004) and designated as "FW1.4". Although the stable and soluble framework sequence of FW1.4 shows high homology, it is not the most homologous sequence available. The identified residue was introduced into said acceptor scaffold to give rFW1.4.

With information on the amino acid sequence variability, the use of the germ line and the structural characteristics of rabbit antibodies, the authors analyzed FW1.4 for compatibility of residual positions necessary to maintain the CDR conformation in the new human framework. The authors examined the variable regions of FW1.4 for compatibility by the following characteristics:

i. Residues that are part of the canonical sequences for loop-like structures.

ii. Frame residues located on the contact surface V _L / V _H.

iii. Base of residues located directly below the CDR

iv. Remains of the upper and lower core

v. Subtype-Defining Wireframe Residues

1.3. Rabbit CDR transplant

Transplants were obtained by simply combining CDR sequences (as defined above) from a single antibody with the FW1.4 or rFW1.4 frame sequence. Identified residues potentially involved in the binding. For each sequence of the rabbit variable domain, the closest germ line variant was determined. If the closest germ line was not established, the sequence was compared with the consensus of the subgroup or the consensus of rabbit sequences with a high percentage of similarity. Rare framework residues were considered as a possible result of somatic hypermutation and, thus, playing a role in binding. Thus, such residues were transplanted into an acceptor framework.

1.4 Results

By analyzing the repertoire of rabbit antibodies, based on the structure, variability of amino acid sequences and using the germ line, 5 positions of residues in the light chain FW1.4 were found, which were modified to maintain the conformation of the rabbit CDR loops. These positions in rabbit antibodies are highly conserved. For these 5 positions, consensus residues were obtained from the rabbit repertoire and introduced into the human acceptor framework 1.4. Upon modification of these conservative positions, this framework actually became compatible with all six complementarity determining regions (CDRs) of any rabbit CDRs. Baseline rFW1.4, containing various rabbit CDRs, is well expressed and well produced unlike individual wild-type chains. 16 representatives were obtained, obtained on the basis of a combination of this scaffold and rabbit CDR, a detailed characterization demonstrated functionality.

Example 2: B-cell screening system

To select B-cells that bind a target of interest through their B-cell receptors (BCR), an FACS (single cell sorting flow cytometry based) cytometry screening system was adapted for ESBATech. For example, one of the targets was a soluble protein, namely, a single chain antibody (ESBA903) labeled with a fluorescent dye (PE and PerCP). A suspension of lymphocytes was obtained from the spleen of rabbits immunized with a recombinant target. Cells were then incubated with labeled PE and PerCP ESBA903, as well as antibodies specific for IgG (labeled APC) or IgM (labeled FITC). ESBA903-positive B cells that express IgG but not IgM on their surface are sorted into 96-well plates (Figure 3; Table 2). Using the thymoma auxiliary cell line (EL4-B5: see Zubler et al, 1985, J. Immunol, 134 (6): 3662-3668), the selected B cells proliferated, differentiated into plasma cells and secreted antibodies. The affinity of these IgGs for the target was confirmed by ELISA and Biacore measurements. The kinetic parameters for the seven selected clones are shown in Table 1. These clones from a total of ≈200 sorted cells demonstrate binding affinities in the range of low concentrations from nanomolar to picomolar. Finally, mRNA was isolated from 6 clones of interest and the CDRs were transplanted into the FW1.4 single-stranded framework.

Table 1
Kinetic values for 7 supernatants of B-cell cultures B cell clone k _a  [Ms ^-one ] k _d  [sec ^-one ] K _D  [M] SG2 2.91E + 06 2,95E-04 1.01E-10 SE11 3.63E + 05 3.81E-04 1,05E-09 2E-03 8.34E + 05 3,53E-04 4.23E-10 9E-03 8.66E + 05 6.47E-04 7.47E-10 7D-03 3.97E + 05 3.04E-04 7,65E-10 12B-02 1,08E + 06 1,10E-04 1.01E-10

table 2
Statistical Sort Options Group Number of events % original % Total All events 100,000 #### 100.0 Lymphocytes 86585 86.6 86.6 Single lymphocytes 1 86013 99.3 86.0 Single lymphocytes 2 85523 99,4 85.5 B-cells of memory? 5450 6.4 5,4 Sorted cells 16 0.3 0,0 Binding 903 cells 160 2.9 0.2

Example 3: Detection of the interaction between granules coated with antibodies against TNF-alpha and CHO cells expressing membrane-bound TNF-alpha.

To evaluate whether the flow disrupts at high pressure during flow cytometry or not, non-covalent binding of two cells was carried out, the following experiment was performed. CHO cells stably transfected with membrane-bound TNF-alpha (B-220 cells) were incubated with beads coated with PE-labeled anti-TNF antibodies. Under these conditions, the granules imitated memory B cells (they have more or less the same size). Non-transfected CHO cells were used as a negative control, and APC labeled granules coated with non-binding antibody (anti-CD19). After 2 hours of incubation at 4 ° C with stirring, the suspension of granule cells was analyzed by FACS (using 130 μm nozzles). Figure 4 shows that the specific binding between granules with antibodies against TNF-alpha and CHO cells transfected with TNF-alpha is clearly detectable by FACS. Indeed, in this sample (upper panel), approximately two-thirds of the granules are bound to cells (585 connected relative to 267 unbound). In contrast, in control samples (middle and lower panels), the granules hardly bind to CHO cells. In addition, both sets of granules (with antibodies against TNF-alpha-PE and antibodies against CD19-APC) were mixed together with CHO cells transfected with TNF-alpha. Figure 5 and table 4 show that approximately half of the granules with anti-TNF-alpha antibodies bind to CHO cells, while the vast majority of granules with anti-CD19 antibodies remain unbound. The percentage of granule binding to the cells in each sample is detailed in Table 5. Thus, a demonstration was made that specific selection of single B cells that bind to the integral membrane target protein via their B cell receptor using flow cytometry is possible.

Table 3a
Statistical sorting parameters (also see Fig. 4a) Group Number of events % original % Total All events 10,000 ### 100.0 P1 9692 96.9 96.9 P3 585 6.0 5.9 P4 one 0,0 0,0 P2 267 2.7 2.7

Table 3b
Statistical sorting parameters (also see Fig. 4b) Group Number of events % original % Total All events 10,000 ### 100.0 P1 9399 94.0 94.0 P3 3 0,0 0,0 P4 6 0.1 0.1 P2 550 5,6 5,6 Table 3c
Statistical sorting parameters (also see Fig. 4c) Group Number of events % original % Total All events 10,000 ### 100.0 P1 9001 90.0 90.0 P3 13 0.1 0.1 P4 7 0.1 0.1 P2 811 8.1 8.1

Table 4
Statistical sorting parameters (also see FIG. 5) Group Number of events % original % Total All events 10,000 ### 100.0 P1 9096 91.0 91.0 P3 401 4.4 4.0 P4 2 0,0 0,0 P2 856 8.6 8.6

Table 5
The proportion of granules associated with CHO cells in each sample Cells pellet mAb % bound pellets Sample 1 CHO-TNFα (B220) anti-TNFα antibody 68.0 Sample 2 CHO-TNFα (B220) anti-CD19 antibody 0.9 Sample 3 CHO wild type anti-TNFα antibody 1,5 Sample 4 CHO-TNFα (B220) anti-TNFα antibody 47.0 anti-CD19 antibody 0.4

Example 4: CDR Transplant and Functional Humanization of Rabbit Anti-TNFα Donor Antibodies

Four rabbit antibodies against TNFα "Rabmab" were selected for CDR transplantation (EPI-1, EPI-15, EP-34, EP-35 and EP-42). The general design of a CDR transplant experiment, humanization, and preliminary characterization of humanized rabbit donor antibodies was performed as described. Unlike traditional humanization methods that use the acceptor framework of a human antibody having the highest sequence homology with a non-human donor antibody, the rabbit CDR was transplanted into a human framework (FW1.4), which was preselected based on the desired functional properties (solubility and stability ) using the analysis of "quality control" (WO0148017). This stable and soluble framework sequence showed high homology with RabMab.

For each RabMab, CDR transplants were obtained using the principle described herein. At the min transplants, only rabbit CDRs were transplanted from the V _L and V _H domains of the rabbit donor antibody into the human acceptor framework FW1.4. Max transplants refer to a rabbit CDR transplant in rFW1.4.

ScFvs described and characterized herein were prepared as follows. Humanized V _L sequences were coupled to humanized V _H sequences using the linker SEQ ID NO: 8 to give scFv in the following orientation: NH ₂ -V _L -linker-V _H -COOH. In many cases, DNA sequences encoding various scFvs were synthesized de novo from an Entelechon GmbH service provider (www.entelechon.com). The resulting DNA inserts were cloned into the bacterial expression vector pGMP002 via the Nco I and Hind III restriction sites introduced at the 5 ′ and 3 ′ ends of the scFv DNA sequence, respectively. A BamH I site is located between DNA sequences of the V _L domain and the V _H domain. In some cases, the DNA encoding scFv was not synthesized de novo , and the constructs expressing scFv were cloned by domain permutation. Thus, the V _L domains were excised and inserted into the new constructs through the restriction sites Nco I and BamH I, and the V _H domains through the restriction sites BamH I and Hind III. In other cases, point mutations were introduced into the V _H and / or V _L domains using current state of the art PCR assembly methods. Cloning of GMP002 is described in Example 1 of WO2008006235. Obtaining scFv was carried out similarly to ESBA105, as described in example 1 of WO2008006235.

Table 3 summarizes the detailed characteristics of the four rabbit monoclonal antibodies (EP6, EP19, EP34, EP35, and EP43) and their variants with transplanted CDRs. Although CDR transplants in BIACore binding assays and in LF29 TNFα-mediated cytotoxicity assays showed a wide range of activity, 3 out of 4 maximal (max) transplants showed therapeutically significant activity. EP43 max showed the most suitable binding affinity (K _d 0.25 nM) and excellent EC ₅₀ in the analysis of cytotoxicity. These data demonstrate that FW1.4 (SEQ ID NO: 1 and 2) is an illustrative soluble and stable human acceptor framework region for rabbit CDR transplantation.

Performance analysis

The neutralizing activity of binding antibodies against TNFα was evaluated in the analysis of TNFα mediated cytotoxicity L929. The toxicity of mouse actinomycin-treated L929 fibroblast cells induced human recombinant TNF (hTNF). It was determined that 90% of the maximum hTNF-induced cytotoxicity is achieved at a TNF concentration of 1000 pg / ml. All L929 cells were cultured in RPMI 1640 with phenol red, with L-glutamine supplemented with fetal calf serum (10% v / v). The neutralizing activity of binding antibodies against TNFα was evaluated in RPMI 1640 without phenol red and 5% fetal calf serum. To L929 cells in the presence of 1000 pg / ml hTNF to determine the concentration at which the antagonistic effect reached half the maximum inhibition (% EC ₅₀ ), various concentrations (0-374 ng / ml) of anti-TNF binding antibodies were added. The dose response curve was approximated by non-linear sigmoid regression with a variable slope and an EC _{50 was} calculated.

Anti-TNF Biacore scFv binding assay

To measure the binding affinity, surface plasmon resonance measurements were performed on a BIAcore ™ -T100 using an NTA sensor chip and labeled His TNF (manufactured by ESBATech). The surface of the NTA sensor chip consists of a carboxymethylated dextran matrix pre-immobilized with nitrile triacetic acid (NTA) to capture histidine-labeled molecules by chelating Ni ²⁺ NTA. N-his human TNFα trimers (5 nM) are captured by nickel through their N-terminal tags his and, in various concentrations ranging from 30 nM to 0.014 nM, are injected with ESBA105 (analyte) in 3-fold serial dilution steps. At the regeneration stage, the complex formed by nickel, ligand and the analyte is washed off. This allows the use of the same regeneration conditions for different samples. The response signal is obtained by surface plasmon resonance (SPR) technology and measured in resonance units (RU). All measurements are carried out at 25 ° C. Sensograms are obtained for each of the anti-TNF scFv samples after an integrated correction to the reference cells, followed by subtraction of the buffer sample. The apparent dissociation rate constant (k _d ), the apparent association rate constant (k _a ), and the apparent equilibrium dissociation constant (K _D ) were calculated using the one-to-one Langmuir binding model using BIAcore T100 version 1.1 analysis software.

Table 3
Second Generation of TNF-alpha Binding Molecules Description ID L929 * k _straight k _arr K _D FT-IRTM ° C RF output ** EP1_min 1071 but.*** - - - - 2 EP6_min 673 but.*** 4.67E + 04 4.94E-03 1,06E-07 50,2 35 EP15_min 1073 but.*** 1,57E + 05 4,10E-02 2.62E-07 - 41.5 EP19_min 616 but.*** - - - - - EP34_min 643 but.*** - - - - - EP35_min 1075 but.*** - - - - one EP42_min 1076 but.*** 1.42E + 05 8.35E-03 5.87E-08 - 3 EP43_min 705 but.*** 5.38E + 03 2.98E-02 5.54E-06 70,2 30,0 EP1_max 1072 but.*** 1,11E + 04 6.30E-04 5.69 E-08 - 44 EP6_max 674 1,1 2.84E + 05 1,45E-04 5,12E-10 48.1 12

EP15_max 1074 0.39 1.53E + 06 2,26E-03 1.48E-09 68.6 57.8 EP19_max 1007 0.6 2.25E + 04 6,54E-05 2.91E-09 53.5 52 EP34_max 791 10.5 5.86E + 05 1,68E-05 2.86E-11 72,4 4.05 EP35_max 1089 5.20 7.72E + 05 1,50E-04 1.94E-10 - 0.66 EP42_max 1077 but.*** 1.21E + 05 4,19E-04 3.46E-09 - 47.6 EP43_max 676 6.4 1.78E + 05 4.48E-05 2.51E-10 74.3 21.73 EP34min_C-His 790 0.2 EP19max_C-His 789 1.9 * L929 [EC ₅₀ -E ₁₀₅ / EC ₅₀ -X], compared in mass units [ng / ml] relative to the effectiveness of ESBA105 (WO06 / 131013)
** (mg / l of solution for refolding); *** Not determined

Example 5: CDR Transplant and Functional Humanization of Rabbit VEGF Donor Antibodies

Eight Rabmabs against VEGF (375, 435, 509, 511, 534, 567, 578, and 610) were selected for CDR transplantation. Unlike traditional methods of humanization, which use the acceptor framework of a human antibody having the highest sequence homology with a non-human donor antibody, the rabbit CDR was transplanted into the human acceptor framework FW1.4 (SEQ ID NO: 1 and 2), which was previously selected on based on the desired functional properties (solubility and stability) using the analysis of "quality control" (WO0148017).

For each of RabMab (rabbit antibodies), several CDR transplants were obtained using the principle described herein (see Example 4). Min transplants included a minimal transplant, where only rabbit CDRs were transplanted from the V _L and V _H domains of the rabbit donor antibody into the human acceptor framework FW1.4 (SEQ ID NO: 1). Max transplants contained not only rabbit CDRs from V _L and V _H , but also some additional frame residues from rabbit donor antibodies, for which a prediction was made that they are important for antigen binding. In the case of 578 max, the FW1.4 framework region of the heavy chain variable domain contained additional amino acid changes at the positions of Kabat 23H, 49H, 73H, 78H and 94H.

Table 4 summarizes the detailed characteristics of the “min” and “max” options with transplanted CDRs. Their effectiveness as VEGF inhibitors is described, which is measured using a competitive VEGFR ELISA and / or HUVEC assay. These data demonstrate that FW1.4 (SEQ ID NO: 1 and 2) is an illustrative soluble and stable human acceptor framework region for rabbit CDR transplantation.

ScFv binding assay against VEGF Biacore

Biacore scFv binding ability was tested and binding affinity was measured using an exemplary surface plasmon resonance method on a BIAcore ™ -T100. The VEGF proteins that were tested for binding by these scFv candidates in this example and further examples include purified Escherichia coli expressed recombinant human VEGF ₁₆₅ (PeproTech EC Ltd.), recombinant human VEGF ₁₂₁ (PeproTech EC Ltd.), recombinant human VEGF ₁₁₀ ( ESBATech AG), recombinant mouse VEGF ₁₆₄ (PeproTech EC Ltd.), recombinant rat VEGF ₁₆₄ (Biovision), recombinant rabbit VEGF ₁₁₀ (ESBATech AG) and recombinant human PLGF (PeproTech EC Ltd.). For the experiment with surface plasmon resonance, carboxymethylated dextran biosensor chips (CM4, GE Healthcare) were activated with N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide hydrochloride according to the supplier's instructions. Each of 6 different forms of VEGF, as illustrated above, was coupled to different flow cells on a CM4 sensor chip in an amount of 1 to 4 by a standard amine binding method. The range of responses obtained with these immobilized VEGF molecules after coupling and blocking was ≈250-500 response units (RU) for hVEGF _165, ≈200 RU for hVEGF _110, hVEGF _121, mouse VEGF _164, VEGF ₁₆₄ and VEGF ₁₁₀ rat rabbit, and ≈400 RU for PLGF. The 4th flow cell of each chip was treated in a similar manner, except that no proteins were added before blocking and the flow cell was used as an integrated reference. Different concentrations of anti-VEGF scFv were injected into the flow cells (e.g. 90 nM, 30 nM, 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM and 0.04 nM) in HBS- buffer EP (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% P20 surfactant) at a flow rate of 30 μl / min for 5 minutes. Dissociation of scFv to VEGF and VEGF on a CM4 chip allowed passage for 10 min at 25 ° C. Sensograms are obtained for each of the scFv samples to VEGF after an integrated correction to the reference cells, followed by subtraction of the sample buffer. The apparent dissociation rate constant (k _d ), the apparent association rate constant (k _a ), and the apparent equilibrium dissociation constant (K _D ) were calculated using the one-to-one Langmuir binding model using BIAcore T100 version 1.1 analysis software.

HUVEC analysis of VEGF inhibition

HUVEC analysis is a method of measuring the effectiveness of the described scFv to VEGF as candidates for VEGF inhibitors.

Human umbilical vein endothelial cells (HUVEC) (Promocell) were used, combined from several donors, from passage 2 to passage 14. Cells were seeded at 1000 cells / well in 50 μl of complete medium for growing endothelial cells (ECGM) (Promocell) containing 0 , 4% ECGS / H, 2% fetal calf serum, 0.1 ng / ml epidermal growth factor, 1 μg / ml hydrocortisone, 1 ng / ml basic fibroblast factor and 1% penicillin / streptomycin (Gibco). After a period of 7 to 8 hours, 50 μl of depleted medium (ECGM without additives containing 0.5% heat-inactivated FCS and 1% penicillin / streptomycin) was added to the cells, and the cells were maintained on depleted medium for a period of 15 to 16 hours. In a depleted medium, 3-fold serial dilutions of scFv VEGF (0.023-150 nM) and one of the following: recombinant human VEGF ₁₆₅ (0.08 nM), recombinant mouse VEGF ₁₆₄ (0.08 nM) or recombinant rat VEGF ₁₆₄ ( 0.3 nM), and pre-incubated for 30-60 minutes at room temperature. Used various concentrations of VEGF to compensate for their different relative biological activity. Used concentrations that stimulate submaximal induced VEGF proliferation (EC ₉₀ ). 100 μl of the mixtures was added to 96-well tissue culture plates containing a HUVEC suspension and incubated for 4 days in a wet incubator with 37 ° C / 5% CO ₂ . HUVEC proliferation was evaluated by measuring the optical density at 450 nm (620 nm was used as the reference wavelength) after adding 20 μl / well of WST-1 cell proliferation reagent (Roche) using a Sunrise plate microspectrophotometer (Tecan). Data were analyzed using an approximation of a 4-parameter logistic curve, and scFv to VEGF concentrations needed to inhibit HUVEC proliferation by 50% (EC ₅₀ ) were determined from the inhibition curves.

Table 4 ID No. of protein Relative activity of hVEGR2 in competitive ELISA [EC _{50 luc} [nM] / EC _{50 test} [nM]) Relative activity of hVEGR1 in a competitive ELISA [EC _{50 luc} [nM] / EC _{50 test} [nM]) Biacore Measurements hVEGF ₁₆₅ k _a  (1 / M sec) k _d  (1 / sec) K _D  (M) 375 min 857 0.3 but. 9.27E + 05 5,01E-03 5.41E-09 375-max 873 0.6 but. 2.44E + 06 6.55E-03 2.68E-09 375-max C-His 877 0.4 but. 2.93E + 05 8,75E-04 2.98E-09 509 min 854 1,0 2.9 6.23E + 05 1,14E-03 1.82E-09 509-max 855 4.1 13 2.26E + 06 2.72E-03 1,21E-09 509-maxII 856 0.6 0.09 8.38E + 05 2.82E-03 3.37E-09 511 min 801 4.9 0.7 5,05E + 05 1,28E-03 2,53E-09 511-max 802 8.7 8 6.59E + 05 4,40E-05 6.67E-11 534-min C-His 807 0.1 but. 2.71E + 05 9.21E-03 3.41E-08 534-max 793 1,1 but. 1.88E + 06 1.73E-02 9.21E-09 567 min 884 9.7 57 2.01E + 06 4.61E-04 2,30E-10 567-max 874 4.1 15.7 / 54.5 1,20E + 06 2,26E-04 1.88E-10 578 min 820 4.1 4.8 1.14E + 06 1,03E-02 9,01E-09 578-max 821 9.6 35.5 / 51.6 7,00E + 05 3.07E-04 4.39E-10 610 min 882 0.1 but. 2.51E + 05 2,65E-03 1,06E-08 610-max 883 0.4 but. 5.09E + 05 6,01E-04 1,18E-09 435 min 944 but. but. but. but. but. 435-max 945 7.6 but. 1,67E + 05 7.55E-04 4,53E-09

EQUIVALENTS

Numerous modifications and alternative embodiments of the present invention will be apparent to those skilled in the art, taking into account the above description. Thus, this description should be considered only as illustrative and intended to indicate to specialists in this field the best way to implement the present invention. The details of the structure can essentially vary without deviating from the essence of the invention, and the exclusive use of all modifications falling within the scope of the attached claims is reserved. It should be understood that the present invention is limited only to the extent required by the attached claims and applicable law.

All literary and similar documents cited in this application, including patents, patent applications, articles, books, monographs, dissertations, web pages, figures and / or applications, regardless of the format of such literary and similar documents, are explicitly included in the full volume as a reference. If one or more of the included literary and similar documents differs from or contradicts this description, including certain terms, the use of terms, the described methods or the like, should be guided by this application.

Claims (10)

1. The acceptor framework of the variable region of the heavy chain of a human antibody or a humanized antibody to transplant a CDR from a rabbit antibody, where the framework contains
threonine (T) at position 24,
alanine (A) or glycine (G) at position 56,
threonine (T) or asparagine (N) at position 84 and
leucine (L) or valine (V) at position 89 (AHo numbering). 2. The antibody acceptor framework according to claim 1, wherein the amino acid at position 12, 103 and / or 144 (AHo numbering) is further substituted with a hydrophilic amino acid. 3. The acceptor framework of the antibody according to claim 2, further having solubility enhancing substitutions
(a) serine (S) at position 12,
(b) serine (S) or threonine (T) at position 103 and / or
(c) serine (S) or threonine (T) at position 144 (AHo numbering) in the heavy chain framework. 4. The acceptor framework of the antibody according to claim 1, further having
glutamic acid (E) at position 1,
valine (V) at position 3,
leucine (L) at position 4,
serine (S) at position 10,
arginine (R) at position 47,
serine (S) at position 57,
phenylalanine (F) at position 91 and / or
valine (V) at position 103 in the framework of the variable region of the light chain (AHo numbering). 5. The acceptor scaffold of an antibody according to claim 4, further comprising scaffold donor residues involved in antigen binding. 6. The acceptor framework of the antibody according to claim 1, further comprising
valine (V) at position 25,
lysine (K) at position 82,
arginine (R) at position 108 and / or
glycine (G) at position 141 of the heavy chain variable region (AHo numbering). 7. The use of the acceptor framework of the antibody according to any one of paragraphs. 1-6 for transplantation of the rabbit CDR. 8. The selected nucleic acid encoding the acceptor framework of the antibody according to any one of paragraphs. 1-6. 9. An expression vector containing a nucleic acid according to claim 8. 10. A host cell containing the expression vector according to claim 9 for the production of the acceptor scaffold of an antibody according to any one of claims. 1-6.

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