JP2019065050A - Humanization of rabbit antibodies using universal antibody framework - Google Patents

Abstract

To provide humanization of rabbit antibodies using a universal antibody framework.SOLUTION: The present invention relates to the universal antibody acceptor framework and to methods for grafting non-human antibodies, e.g., rabbit antibodies, using the universal antibody acceptor framework. Antibodies generated by the methods of the invention are useful in a variety of diagnostic and therapeutic applications. The invention provides methods for grafting rabbit and other non-human CDRs, into the soluble and stable light chain and/or heavy chain human antibody framework sequences disclosed herein, thereby generating humanized antibodies with superior biophysical properties. In particular, immunobinders generated by the methods of the invention exhibit superior functional properties such as solubility and stability.SELECTED DRAWING: Figure 1

Description

(Related information)
This application claims priority to US 61/075, 697 filed on June 25, 2008, and further US 61/155, 041 filed on February 24, 2009 and February 24, 2009. Claim priority to US 61 / 155,105.

BACKGROUND OF THE INVENTION
Monoclonal antibodies, their conjugates and derivatives are of great commercial importance as therapeutic and diagnostic agents. Non-human antibodies usually elicit a strong immune response in patients after a single low dose injection (Schroff, 1985, Cancer Res, 45: 879-85, Shawler, J Immunol, 1985, 135). 1553-5, Dillman, Cancer Biother, 1994, 9, 17-28). Thus, several methods have been developed to reduce the immunogenicity of murine and other rodent antibodies, as well as techniques for generating fully human antibodies using, for example, transgenic mice or phage display. . Chimeric antibodies that combine rodent variable regions with human constant regions (eg, Boulianne Nature, 1984, 312, 643-6) have been genetically engineered to reduce immunogenicity issues considerably (see For example, LoBuglio, Proc Natl Acad Sci, 1989, 86, 4220-4; Clark, Immunol Today, 2000, 21, 397-402). Humanized antibodies have also been constructed. In humanized antibodies, the variable region itself rodent sequences have been genetically engineered to be as close to human sequences as possible while preserving at least the original CDRs, or alternatively, the CDRs of the rodent antibody are human antibodies (For example, Riechmann, Nature, 1988, 332, 323-7; Patent Document 1). Rabbit polyclonal antibodies are widely used in biological assays such as ELISA or Western blot. Polyclonal rabbit antibodies are often preferred over polyclonal rodent antibodies as they usually have much higher affinity. In addition, rabbits can often elicit good antibody responses against poorly immunogenic antigens in mice and / or antigens that do not yield good binders when used in phage display. Because of these well-known advantages of house rabbit antibodies, house rabbit antibodies are ideal for use in the discovery and development of therapeutic antibodies. The reason why this is not commonly done is mainly the technical difficulties in the generation of monoclonal rabbit antibodies. Myeloma-like tumors are not known in rabbits, and conventional hybridoma technology that produces monoclonal antibodies is not applicable to rabbit antibodies. The pioneering work of providing fusion cell line partners for rabbit antibody-expressing cells was performed by Knight et al. (Spieker-Polet et al., PNAS, 1995, 92, 9348-52), and improved fusion partner cell lines were It described by Pytela et al. In 2005 (see, for example, Patent Document 2). However, this technology is not widely used because the corresponding know-how is basically controlled by a single research group. Alternative methods for the generation of monoclonal antibodies with cloning of antibodies from selected antibody-expressing cells via RT-PCR have been described in the literature, but success has been reported for home rabbit antibodies once. Absent.

  Domestic rabbit antibodies, like the mouse antibodies, are expected to elicit a strong immune response if used for human therapy. Thus, rabbit antibodies need to be humanized before they can be used clinically. However, because of the structural differences between the rabbit and mouse antibodies and between the rabbit and human antibodies, it is possible to apply the method used to make the humanized rodent antibody to the rabbit antibody. Not easy. For example, the light chain CDR3 (CDR L3) is often much longer than the CDR L3 from previously known human or mouse antibodies.

  Although there are only a few domestic rabbit antibody humanization approaches described in the prior art, they are not classical fusion approaches in which non-human donor CDRs are grafted onto human acceptor antibodies. US Pat. No. 5,956,015 describes a so-called "resurfacing" strategy. The goal of "resurfacing" strategies is to reconstitute solvent accessible residues of non-human frameworks so that they are more human-like. Similar humanization techniques for domestic rabbit antibodies, as described in US Pat. Both Patents 4 and 5 disclose methods of humanizing a rabbit rabbit monoclonal antibody, including comparison of the amino acid sequence of the parent rabbit antibody with the amino acid sequence of a similar human antibody. Subsequently, the amino acid sequence of the parent rabbit antibody is modified so that the framework regions of the parent rabbit antibody are more similar in sequence to the equivalent framework regions of similar human antibodies. In order to obtain good binding ability, it is necessary to carry out laborious development efforts individually for each immunobinder.

  A potential problem with the above approach is that the human framework is not used and that the rabbit framework is genetically engineered to look more human-like. Such an approach carries the risk that amino acid stretches embedded in the core of the protein may still contain immunogenic T cell epitopes.

  So far, applicants have not identified humanized rabbit antibodies by applying the state of the art fusion approach. This can be explained by the fact that rabbit rabbit CDRs can be quite different from human or rodent CDRs. As is known in the art, many rabbit rabbit VH chains have an additional pair of cysteines as compared to their murine and human counterparts. In addition to the conserved disulfide bridge formed between cys22 and cys92, there is also a cys21-cys79 bridge, and furthermore, in some rabbit chains, the last residue of CDRH1 and the first residue of CDRH2 There is also an inter-CDR SS bridge formed between Moreover, paired cysteine residues are often found in CDR-L3. Furthermore, many rabbit antibody CDRs do not belong to any previously known standard structure. In particular, CDR-L3 is often much longer than its human or murine counterpart, CDR-L3.

  Thus, fusion of non-human CDR antibodies into human frameworks is a major protein engineering challenge. The transition of the antigen binding loop from the naturally evolved framework to a different artificially chosen human framework must be done to preserve the natural loop conformation for antigen binding. Antigen binding affinity is often greatly reduced or eliminated after loop fusion. The use of carefully selected human frameworks in the fusion of antigen binding loops maximizes the probability of retaining binding affinity in the humanized molecule (Roguzka et al., 1996). Although many of the fusion experiments available in the literature give a rough guide to CDR fusion, it is not possible to generalize the pattern. A typical problem consists in losing specificity, stability or productivity after fusing the CDR loops.

U.S. Pat. No. 5,693,761 U.S. Pat. No. 7,429,487 WO 04/016740 WO 08/144757 WO 05/016950

  Thus, there is an urgent need for improved methods for reliably and rapidly humanizing house rabbit antibodies for use as therapeutic and diagnostic agents. Furthermore, there is a need for human acceptor frameworks to ensure humanization of rabbit antibodies, which provides functional antibodies and / or antibody fragments with drug-like biophysical properties.

(Summary of the Invention)
Surprisingly, highly soluble and stable humans identified by quality control (QC) assays (as disclosed in WO0148017 and Auf der Maur et al. (2001) FEBS Lett, 508, 407-412). Antibody frameworks have been found to be particularly suitable for housing CDRs from other non-human animal species, such as those of rabbits. Thus, in a first aspect, the invention provides light and heavy chain variable regions of certain human antibodies (so-called "FW1.4" antibodies). The antibodies are particularly suitable as universal acceptors for CDRs from various antibodies of different binding specificities, in particular house rabbit antibodies, regardless of the presence or absence of disulfide bridges in the CDRs. Furthermore, the invention provides two variant sequences of said particular human antibody framework, namely rFW1.4 and rFW1.4 (V2). Both are particularly suitable frameworks as universal acceptor frameworks for the fusion of rabbit CDRs. In another aspect, the present invention provides motifs of framework residues that provide a human framework suitable for housing CDRs from other non-human animal species, particularly those of rabbits.

  The humanized immunobinder produced by fusing the rabbit rabbit CDRs into these highly compatible variable light and heavy chain frameworks consistently aligns the spatial orientation of the rabbit rabbit antibody (from which the donor CDRs are derived) And hold it securely. Thus, it is not necessary to introduce structurally important positions of the donor immunobinder into the acceptor framework. Because of these advantages, high-throughput humanization of rabbit antibodies can be achieved with little or no optimization of binding capacity.

Thus, in another aspect, the invention fuses rabbit rabbit CDRs and other non-human CDRs into the soluble and stable light and / or heavy chain human antibody framework sequences disclosed herein, and Provides a method of generating humanized antibodies with superior biophysical properties. In particular, the immunobinder produced by the method of the present invention exhibits excellent functional properties such as solubility and stability.
The present invention provides, for example, the following items.
(Item 1)
An immunobinder specific for a desired antigen, comprising: (i) rabbit variable light chain CDRs; and (ii) a human variable heavy chain framework having at least 50% identity to SEQ ID NO: 4.
(Item 2)
The immunobinder acceptor framework according to claim 1, wherein said variable heavy chain framework is selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 6.
(Item 3)
The immunobinder acceptor framework according to any one of the preceding items, further comprising a variable light chain framework having at least 85% identity to SEQ ID NO: 2.
(Item 4)
The immunobinder acceptor framework according to claim 3, comprising threonine (T) at position 87 (AHo numbering) of the light chain variable framework.
(Item 5)
The immunobinder acceptor framework according to any one of the preceding items, comprising a variable heavy chain framework and a variable light chain framework, both linked by a linker, in particular the linker of SEQ ID NO: 8.
(Item 6)
A framework having at least 70% identity to SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, in particular comprising SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: The immunobinder acceptor framework according to any one of the preceding items, comprising a framework that is 7.
(Item 7)
Threonine at position 24 (T), valine at position 25 (V), alanine (A) at position 56 or glycine (G), lysine at position 82 (K), threonine at position 84 (T), valine at position 89 V) A human or humanized immunobinder variable heavy chain acceptor framework comprising at least three amino acids of the group consisting of V and arginine (R) at position 108 (AHo numbering).
(Item 8)
The immunobinder acceptor framework according to any one of the preceding items, wherein the amino acid at position 12, 103 and / or 144 (AHo numbering) is replaced by a hydrophilic amino acid.
(Item 9)
(A) serine (S) at position 12 in the heavy chain framework,
(B) Serine (S) or threonine (T) at position 103, and / or (c) serine (S) or threonine (T) at position 144
The immunobinder acceptor framework according to item 8, having (AHo numbering).
(Item 10)
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; The immunobinder acceptor framework according to any one of the preceding items, having serine (S), phenylalanine (F) at position 91 and / or valine (V) at position 103 (AHo numbering).
(Item 11)
Any of the preceding items further comprising donor immunobinder, in particular heavy chain CDR1s, CDR2s and CDR3s derived from mammalian immunobinders, preferably house rabbit immunobinder, and / or light chain CDR1s, CDR2s and CDR3s Immunobinder acceptor framework as described.
(Item 12)
The immunobinder acceptor framework according to claim 11, further comprising donor framework residues involved in antigen binding.
(Item 13)
The immunobinder acceptor framework according to any one of the preceding items further comprising glycine (G) (AHo numbering) at position 141 of the variable heavy chain.
(Item 14)
14. Use of an immunobinder acceptor framework according to any one of items 1 to 13 for the fusion of rabbit rabbit CDRs.
(Item 15)
An immunobinder comprising the immunobinder acceptor framework according to any one of items 1 to 13.
(Item 16)
The immunobinder according to item 15, which is a scFv antibody.
(Item 17)
One or more binding molecules, in particular therapeutic agents such as cytotoxic agents, cytokines, chemokines, growth factors or other signaling molecules, imaging agents, or second proteins, in particular transcription activators or DNA binding domains The immunobinder according to any one of items 15 to 16, further comprising.
(Item 18)
19. Use of an immunobinder according to any one of items 15 to 17 in diagnostic application, therapeutic application, target evaluation or gene therapy.
(Item 19)
14. An immunobinder acceptor framework according to any one of items 1-13 or a nucleic acid encoding an immunobinder according to any one of items 15-17, in particular an isolated nucleic acid.
(Item 20)
A vector comprising the nucleic acid according to item 19.
(Item 21)
21. A host cell comprising the vector according to item 20.
(Item 22)
20. Use of the nucleic acid of item 19 or the vector of item 20 in gene therapy.
(Item 23)
An immunobinder acceptor framework according to any one of items 1 to 13, an immunobinder according to any one of items 15 to 17, a nucleic acid according to item 19, or a vector according to item 20 Composition.
(Item 24)
A method of humanizing a rabbit rabbit immunobinder, wherein said rabbit rabbit immunobinder comprises heavy chain CDR1, CDR2 and CDR3 sequences and / or light chain CDR1, CDR2 and CDR3 sequences, said method comprising
(I) fusing at least one heavy chain CDR of the group consisting of CDR H1, CDR H2 and CDR H3 sequences within a human heavy chain acceptor framework having at least 50% identity to SEQ ID NO: 1, and And / or (ii) a human light chain acceptor framework having at least 50% identity to SEQ ID NO: 2, ie at least one of the group consisting of CDR L1, CDR L2 and CDR L3 sequences within a human light chain framework Fusing the light chain CDRs.
(Item 25)
(I) said human heavy chain framework comprises the framework amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 6,
(Ii) said human light chain framework comprises the framework amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 9
The method according to item 24.
(Item 26)
The method according to item 24 or 25, further comprising the step of replacing a framework residue with a framework residue of the rabbit immunobinder involved in antigen binding.
(Item 27)
By substitution at least one of the heavy chain amino positions 12, 103 and 144 (AHo numbering), in particular by substitution with hydrophilic amino acids, preferably (a) Serine (S), (b) position at position 12 26. From step 24 further comprising replacing framework residues of the heavy chain acceptor framework by a substitution selected from the group consisting of threonine 103 (T) and (c) threonine (T) at position 144 26. The method according to any one of 26.
(Item 28)
In the stability-enhancing substitution at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the light chain variable region by the AHo numbering system, in particular (a) glutamic acid (E) at position 1; (B) valine at position 3 (V), (c) leucine at position 4 (L), (d) serine at position 10 (S), (e) arginine at position 47 (R), (e) at position 57 A substitution selected from the group consisting of serine (S), (f) phenylalanine (F) at position 91, and (g) valine (V) at position 103, replacing the framework residue of the light chain acceptor framework 26. A method according to any one of items 24 to 27, further comprising the steps of
(Item 29)
Humanized immunobinder by the method according to any one of items 24 to 28.
(Item 30)
The immunobinder of claim 29, wherein said target antigen is VEGF or TNFα.
(Item 31)
a) incubating the rabbit B cells with the target antigen, b) selecting the rabbit B cells that bind to the target antigen, and c) identifying the CDRs of the antibody variable domain generated by the B cells. 29. A method according to any one of items 24 to 28, further comprising the identification of donor CDRs of antibody variable domains generated by B cells.
(Item 32)
The method according to item 31, wherein the B cells are isolated from rabbits immunized against the target antigen, preferably by DNA vaccination.
(Item 33)
33. The item according to any one of items 31 or 32, wherein the B cells and the target antigen are stained differently and the coexistence of the two stains is positively selected by flow cytometry based single cell sorting. Method.
(Item 34)
34. The method according to any one of items 31 to 33, wherein the target antigen is a soluble protein.
(Item 35)
34. A method according to any one of items 31 to 33, wherein said target antigen is expressed on the surface of cells, in particular said target antigen is a transmembrane protein, in particular a multiple transmembrane protein.
(Item 36)
36. The method according to item 35, wherein the target antigen is indirectly stained by staining the cells expressing the target antigen with a fluorescent dye in the cell.
(Item 37)
37. The method according to any one of items 31 to 36, wherein the B cells are stained with one or more labeled antibodies specific for B cell surface markers.
(Item 38)
The method according to any one of items 31 to 37, wherein B cells are stained with a labeled anti-IgG antibody.
(Item 39)
A method according to item 38, wherein B cells are stained with a labeled anti-IgG antibody and a differently labeled anti-IgM antibody.
(Item 40)
The method according to item 39, wherein the B cells stained with the labeled anti-IgM antibody are negatively selected.
(Item 41)
From 31 further comprising culturing the B cells selected in step b) according to paragraph 31 such that the generated antibodies are secreted into the medium, in particular in the presence of a helper cell line 40. The method of any one of 40.
(Item 42)
42. A method according to any one of items 41, further comprising the step of testing said secreted antibody for specific binding to said target antigen.
(Item 43)
The CDRs of said antibody are amplified, in particular by RT-PCR, fused in an acceptor framework to produce an immunobinder and subsequently tested for specific binding to said target antigen. 42. The method according to 42.
(Item 44)
For fusion of rabbit rabbit CDRs of an immunobinder acceptor framework comprising a sequence having at least 90% identity to SEQ ID NO: 1 and / or a sequence having at least 85% identity to SEQ ID NO: 2 use.
(Item 45)
45. The use according to item 44, wherein said framework comprises SEQ ID NO: 1 and SEQ ID NO: 2 both connected by a linker, in particular by a linker having SEQ ID NO: 8.

FIG. 1 shows the definition of CDR H1 used herein to fuse the antigen binding site from a rabbit rabbit monoclonal antibody into a highly soluble and stable human antibody framework. FIG. 2 schematically shows a fluorescent antibody 2 labeled B cell 1 that interacts with target expressing cell 3 stained with intracellular dye 4. Selected targets: 5; BCR: 6. FIG. 3 shows the FACS selection process of rabbit B cells binding to ESBA 903 soluble target. FIG. 3A: Lymphocytes are gated by forward and side scatter. FIG. 3B: Among them, IgG + IgM-cells (possibly memory B cells) are selected (red gate). FIG. 3C: Cells double-stained with ESBA903-PE and ESBA903-PerCP (green gate) are predicted to encode high affinity IgG for ESBA903. The cells showing the brightest fluorescence (pink gate) were sorted into 96 well plates. Figure 4: Anti-TNFα antibody (PE labeled) coated beads bind to TNFα transfected CHO cells (upper panel), Anti CD19 antibody (APC labeled) control beads coated with TNFα transfected CHO cells Not binding to (middle panel), showing that beads coated with anti-TNFα antibody (PE label) do not bind to wild type (wt) CHO cells (lower panel). The dot plot on the left shows forward and side scatter, which indicate the size and granularity of the event, respectively. A single bead (about 3 μm) population is gated to P2. The CHO cells (approximately 30 μm) finally bound to the beads are gated on P1. The central dot plot shows P1 events (CHO cells) for PE or APC staining. Thus, if cells interact with anti-TNFα beads, they are shown on the P3 gate, and if they interact with anti-CD19 beads, they appear on the P4 gate. The right side shows the details of the statistics for each sample. FIG. 5 shows that anti-TNFα-PE coated beads and anti-CD 19 APC coated beads are mixed with TNFα-transfected CHO cells, and CHO cells are gated (P1), among them coated with anti-TNFαPE Cells that bind either to the coated beads or to the anti-CD19-APC coated beads are shown at gates P3 and P4, respectively, indicating that non-bound beads appear to be gate P2. FIG. 6 is an analysis of the rabbit antibody sequence extracted from the Kabat database, which confirms that CDR3 of the variable heavy chain is usually 3 amino acids longer than its mouse counterpart.

(Detailed Description of the Invention)
(Definition)
For the purpose of which the present invention may be more readily understood, certain terms are defined as follows. Additional definitions are set forth throughout this detailed description.

The term "antibody" refers to whole antibodies and any antigen binding fragment. The terms "antigen binding polypeptide" and "immunobinder" are used simultaneously herein. An "antibody" refers to a protein or antigen binding portion thereof that is optionally glycosylated, comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as V _H ) and a heavy chain constant region. The heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region comprises one domain, ie, CL. The V _H and V _L regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDR), and more conserved regions, termed framework regions (FR), interspersed with hypervariable regions. Each V _H and V _L is composed of three CDRs and four FRs, arranged from amino to carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy and light chain variable regions contain binding domains that interact with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" (or simply "antibody portion") 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 has been shown that the antigen binding function of antibodies can be performed by fragments of full length antibodies. Examples of binding fragments encompassed by the term "antigen-binding portion" of antibodies include (i) Fab fragments which are monovalent fragments consisting of V _L , V _H , CL and CH1 domains, (ii) disulfides of the hinge region A bivalent fragment F (ab ') ₂ fragment containing two Fab fragments linked by cross-linking, (iii) Fd fragment consisting of V _H and CH 1 domains, (iv) V _L and V _{H of one} arm of antibody Fv fragment consisting of domains, (v) single domain or dAb fragment consisting of one V _H domain (Ward et al. (1989) Nature 341: 544-546), and (vi) isolated complementarity Optionally, by a decision region (CDR), or (vii) a synthetic linker Included are combinations of two or more isolated CDRs that may be linked. Furthermore, although the two domains of the Fv fragment, namely V _L and V _H , are encoded by separate genes, they use recombinant methods with synthetic linkers that allow them to be made as a single protein chain In this single protein chain, the V _L and V _H regions are paired and known as a monovalent molecule (single chain Fv (scFv); see, for example, Bird et al. (1988) Science, vol. 242, And Huston et al. (1988), Proc. Natl. Acad. Sci. USA, Vol. 85, 5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art and screened for utility in the same manner as intact antibodies. The antigen binding moiety may be produced by recombinant DNA techniques or produced by enzymatic or chemical cleavage of intact immunoglobulin. The antibodies may be of different isotypes, eg, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

The term "immunobinder" refers to a molecule containing all or part of the antigen binding site of an antibody, eg all or part of a heavy chain variable domain and / or a light chain variable domain, thus an immunobinder is Specifically recognize the target antigen. Non-limiting examples of immunobinders are full-length immunoglobulin molecules and scFvs, but not limited to: (i) Fab fragments, ie, V _L domains, V _H domains, C _L domains and C _H 1 A monovalent fragment consisting of domains; (ii) a F (ab ') ₂ fragment, ie a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) essentially Fab 'fragments are Fab with part of the hinge region (FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd Edition, see 1993)); (iv) Fd fragment consisting of the _{V H} and _{C H} 1 domains; ( v) antibody single arm V _L domain and Fv fragment consisting of _{V.sub.H and V.sub.H} domains; (vi) Dab fragment consisting of _V.sub.H or V _L domains (Ward et al. (1989) Nature 341: 544-546), camel antibody (Hamers-Casterman et al., Nature 363: 446-448 (1993), and Dumulin et al., Protein Science 11: 500-515 (2002), or shark antibodies (e.g., Shark Ig-NAR Nanobodies <(R)>). Etc .; and (vii) antibody fragments, including a heavy chain variable region containing a single variable domain and a nanobody that is two constant domains.

"Single-chain antibodies", the term "single-chain Fv" or "scFv" antibody heavy chain variable domain are linked by a linker (or region; V _H) and an antibody light chain variable domain (or region; V _L Means a molecule containing 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 linkers in the state of the art consist of the repetitive GGGGS amino acid sequence or variants thereof. In a preferred embodiment of the invention, a (GGGGS) ₄ linker of the amino acid sequence as set forth in SEQ ID NO: 8 is used, but also variants of 1-3 repeats are possible (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA, 90, 6444 to 6448). Other linkers that can be used in the present invention are described by Alfthan et al. (1995), Protein Eng. 8, pp. 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. Org. Mol. Biol. 293: 41-56 and Roovers et al. (2001) Cancer Immunol. Is described by.

  As used herein, the term "functional property" is for the person skilled in the art, for example, in order to improve the manufacturing properties or therapeutic efficacy of the polypeptide (eg, compared to conventional polypeptides) Properties of a polypeptide (eg, an immunobinder) for which improvement is desirable and / or advantageous. In one embodiment, the functional property is stability (eg, thermal stability). In another embodiment, the functional property is solubility (eg, under cellular conditions). In yet another embodiment, the functional property is aggregation behavior. In yet another embodiment, the functional property is protein expression (eg, in a prokaryotic cell). In yet another embodiment, the functional property is resolubilization behavior after solubilization of the inclusions in the manufacturing process. In certain embodiments, the functional property is not an improvement in antigen binding affinity. In another preferred embodiment, the improvement in one or more functional properties does not substantially affect the binding affinity of the immunobinder.

  The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody primarily responsible for antigen binding. One of the most commonly used definitions for the six CDRs is described by Kabat E. et al. A. (1991) Sequences of proteins of immunological interest. , NIH Publication, pages 91 to 3242. As used herein, the Kabat definition of CDRs refers to light chain variable domain CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) and heavy chain variable domain Only applies to CDR2 and CDR3 (CDR H2, CDR H3 or H2, H3). However, as used herein, CDR1 (CDR H1 or H1) of the heavy chain variable domain is defined by the position of the residue beginning at position 26 and ending before position 36 (Kabat numbering). This is basically a fusion of CDR H1 defined differently by Kabat and Chotia (see also FIG. 1 for illustration).

  The term "antibody framework" as used herein refers to the portion of the variable domain of either VL or VH that serves as the backbone for the variable domain's antigen binding loop (CDR). In essence, this is a variable domain without CDRs.

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

The terms "specific binding", "selective binding", "selectively binding" and "specifically binding" refer to antibody binding to an epitope of a given antigen. Ordinarily, the antibody will be less than about ^{10 -8} M, such as about ^{10 -9} M or less than about ^{10 -10} M or even less than low values, it binds with an affinity of less than about ^{10 -7} M _{(K D).}

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

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

  The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. The vectors disclosed herein may be capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Alternatively, it may be integrated into the host cell's genome upon introduction into the host cell, thereby being able to replicate with the host genome (eg, non-episomal mammalian vectors).

  The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells include bacterial cells, microbial cells, plant cells or animal cells, preferably, Escherichia coli, Bacillus subtilis; Saccharomyces cerevisiae, Pichia pastoris, CHO (Chinese hamster ovary cell line) or NS0 cells.

  The term "lagomorph" refers to taxonomically members of the Lagomorpha order and includes the Leporidae (eg, Hare and rabbit) and the Ochotonidae (pika). In the most preferred embodiment, the rabbit is a rabbit. The term "home rabbit" as used herein refers to an animal belonging to the leporidae family.

  "Identity," as used herein, refers to sequences that match between two polypeptides, between molecules, or between two nucleic acids. Where a position in both of the two sequences being compared is occupied by the same base or the same amino acid monomer subunit (eg, if a position in each of the two polypeptides is occupied by lysine), respectively The molecules of are identical at that position. The "percent identity" between the two sequences is shared by those sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap It is a function of the number of identical positions. Generally, comparisons are made when the two sequences are aligned to give the greatest identity. Such alignments may be, for example, either a Blossum 62 matrix or a PAM 250 matrix, as well as gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6. It can be provided using the method of the algorithm of Needleman and Wunsch (J. Mol. Biol., (48), pages 444-453 (1970)) incorporated into the GAP program in the GCG software package. .

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

  Various aspects of the invention are described in further detail in the following subsections. It is understood that the various embodiments, choices, and ranges can be arbitrarily combined. Further, depending on the particular embodiment, selected definitions, embodiments or ranges may not apply.

  Unless otherwise stated, amino acid positions are indicated according to the AHo numbering scheme. The AHo numbering system is described by Honegger, A. et al. And Pluckthun, A. (2001); Mol. Biol. 309, 657-670). Instead, see Kabat et al. (Kabat, E. A. et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242). The Kabat numbering system described in more detail can be used. A conversion table of two different numbering systems used to identify amino acid residue positions within antibody heavy and light chain variable regions is given in A.I. Honegger, J.M. Mol. Biol. 309 (2001), 657-670.

  In a first aspect, the invention provides a universal acceptor framework for fusing CDRs from other animal species, such as rabbits. It has previously been described that antibodies or antibody derivatives comprising human frameworks identified in the so-called "quality control" screening (WO0148017) are generally characterized by high stability and / or solubility. Human single chain framework FW 1.4 (combination of SEQ ID NO: 1 (named a43 in WO 03/097697) and SEQ ID NO 2 (named K 127 in WO 03/097697) is a quality control assay Although apparently low performance, it was surprisingly found that this framework has a high degree of intrinsic thermodynamic stability and good reproducibility even in combination with various different CDRs. It was issued. The stability of this molecule can be largely attributed to its framework region. It has further been shown that FW 1.4 is inherently highly compatible with the antigen binding site of the rabbit antibody. Thus, FW 1.4 is a scaffold suitable for constructing stable humanized scFv antibody fragments resulting from the fusion of the rabbit rabbit loop. Thus, in one aspect, the invention comprises a VH sequence having at least 90% identity to SEQ ID NO: 1 and / or a VL sequence having at least 85% identity to SEQ ID NO: 2, more preferably a rabbit rabbit CDR At least 60%, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95% of the sequence of FW 1.4 (SEQ ID NO: 3) for the fusion of Provided is an immunobinder acceptor framework comprising sequences having identity.

  Furthermore, it has been found that FW1.4 can be optimized by substituting several residue positions of the FW1.4 heavy chain and / or by substituting one position in the FW1.4 light chain. It was done. Thereby, it has surprisingly been found that the loop conformation of a broad range of rabbit rabbit CDRs in VH can be fully maintained, largely independently of the sequence of the donor framework. The residue in the heavy chain of FW 1.4 and one position in the light chain are conserved in the rabbit antibody. The consensus residue at the position in the heavy chain and one position in the light chain was deduced from the rabbit rabbit repertoire and was introduced into the sequence of the human acceptor framework.

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

  Thus, the present invention provides the variable heavy chain framework of SEQ ID NO: 1 further comprising one or more amino acid residues that generally support the conformation of the CDRs from the rabbit immunobinder. In particular, said residues are present at one or more amino acid positions selected from the group consisting of 24H, 25H, 56H, 82H, 84H, 89H and 108H (AHo numbering). These positions have been found to affect CDR conformations, and thus mutations to accommodate donor CDRs are contemplated. The one or more residues are threonine at position 24 (T), valine at position 25 (V), glycine or alanine at position 56 (G or A), lysine at position 82 (K), threonine at position 84 Preferably, it is selected from the group consisting of (T), valine at position 89 (V) and arginine at position 108 (R) (AHo numbering). It is preferred that at least 3 residues, more preferably 4, 5, 6 residues, most preferably all 7 residues are present. It has been surprisingly found that the presence of the above mentioned residues improves the stability of the immunobinder.

  In a preferred embodiment, the invention relates to threonine at position 24 (T), valine at position 25 (V), alanine at position 56 (A) or glycine (G), threonine at position 84 (T), lysine at position 82 (K), valine (V) at position 89, and arginine (R) at position 108 (AHo numbering), at least one residue, more preferably at least 3 residues, more preferably 4, 5, 6 At least 50%, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, with the proviso that the residue, most preferably 7 residues, is present. Provided is an immunobinder acceptor framework comprising VH with 90%, 95% and even more preferably 100% identity. In a preferred embodiment, the immunobinder acceptor framework is an immunobinder acceptor framework for a rabbit rabbit CDR.

  In a preferred embodiment, the variable heavy chain framework is SEQ ID NO: 4 or SEQ ID NO: 6 or comprises SEQ ID NO: 4 or SEQ ID NO: 6. Both variable heavy chain frameworks may be combined, for example, with any suitable light chain framework.

Therefore, the present invention
(I) a variable heavy chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID NO: 4 and / or (ii) a sequence Provided is an immunobinder acceptor framework comprising a variable light chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably at least 95% identity to the number 2.

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

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

In a preferred embodiment, the immunobinder acceptor framework comprises
(I) a variable heavy chain framework selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6, and / or (ii) a variable light chain framework of SEQ ID NO: 2 or SEQ ID NO: 9.

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

  In another preferred embodiment, the immunobinder acceptor framework is a sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID NO: 5, On the other hand, the sequence is preferably not SEQ ID NO: 3. More preferably, the immunobinder acceptor framework comprises or is SEQ ID NO: 5.

  In another preferred embodiment, the immunobinder acceptor framework is a sequence having at least 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID NO: 7, On the other hand, the sequence is preferably not SEQ ID NO: 3. More preferably, the immunobinder acceptor framework comprises or is SEQ ID NO: 7.

  Furthermore, it is surprisingly found that the presence of the above-mentioned amino acid motif makes the framework, preferably a human framework, particularly suitable for housing CDRs from other non-human animal species, in particular rabbit rabbit CDRs. It was issued. The motif does not negatively impact the stability of the immunobinder. The CDRs appear in a conformation similar to their natural spatial orientation in the rabbit immunobinder, so it is not necessary that any structurally significant positions be fused to the acceptor framework. Thus, the human or humanized immunobinder acceptor framework is a threonine at position 24 (T), a valine at position 25 (V), an alanine at position 56 (A) or a glycine (G), a lysine at position 82 (K) , At least 3 amino acids in the group consisting of threonine (T) at position 84, valine (V) at position 89 and arginine (R) at position 108 (AHo numbering), preferably 4, 5, 6 amino acids, more preferably 7 Contains amino acids.

  The immunobinder acceptor frameworks described herein may comprise a solubility promoting substitution on the heavy chain framework, preferably at positions 12, 103 and 144 (AHo numbering). Preferably, hydrophobic amino acids are replaced by more hydrophilic amino acids. Examples of hydrophilic amino acids include 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 is (a) serine (S) at position 12; (b) serine (S) or threonine (T) at position 103; and / or (c) serine (S) at position 144. Or contains threonine (T).

  In addition, stability enhancing amino acids may be present at one or more of positions 1, 3, 4, 10, 47, 57, 91 and 103 (AHo numbering) of the variable light chain framework. The variable light chain framework consists of glutamic acid at position 1 (E), valine at position 3 (V), leucine at position 4 (L), serine at position 10 (S); arginine at position 47 (R), position 57 It is more preferred to include serine (S), phenylalanine (F) at position 91 and / or valine (V) at position 103.

  In another preferred embodiment, the VH comprises glycine (G) at position 141, as glutamine (Q) is prone to deamination. This substitution may improve the long term storage of the protein.

For example, the acceptor frameworks disclosed herein can be used to generate human or humanized antibodies that retain the binding properties of the non-human antibody from which the non-human CDRs are derived. Thus, in a preferred embodiment, the present invention provides a donor immunobinder, preferably a mammalian immunobinder, more preferably a rabbit immunobinder, most preferably heavy chain CDR1, CDR2 and CDR3 from rabbit and / or light chain CDR1, It includes the immunobinder acceptor framework disclosed herein further comprising CDR2 and CDR3. Thus, in one embodiment, the present invention
(I) rabbit variable light chain CDRs,
(Ii) at least 50%, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, most preferably 100% identity to SEQ ID NO: 4 Provides an immunobinder specific for the desired antigen, including a human variable heavy chain framework having

  In a preferred embodiment, threonine (T) at position 24, valine (V) at position 25, alanine (A) at position 56 or glycine (G) at position 84, in said human variable heavy chain framework sequence There is a requirement that at least one amino acid is present in the group consisting of (T), 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 immunobinder comprises heavy chain CDR1s, CDR2s and CDR3s derived from the donor immunobinder, and light chain CDR1s, CDR2s and CDR3s.

  As known in the art, many rabbit rabbit VH chains have an additional pair of cysteines as compared to their murine and human counterparts. In addition to the conserved disulfide bridge formed between cys22 and cys92, there is also a cys21-cys79 bridge, and furthermore, in some rabbit chains, the last residue of CDRH1 and the first residue of CDRH2 There is also an inter-CDR SS bridge formed between In addition, in CDR-L3, a pair of cysteine residues are often found. Furthermore, many rabbit antibody CDRs do not belong to any previously known canonical structure. In particular, CDR-L3 is often much longer than its human or murine counterpart, CDR-L3.

As mentioned above, fusion of non-human CDRs to the frameworks disclosed herein yields a molecule in which the CDRs appear in the proper conformation. If necessary, the affinity of the immunobinder can be improved by fusing the antigen interaction framework residues of the non-human donor immunobinder. These positions are, for example,
(I) identifying each germline precursor sequence, or alternatively, using a consensus sequence in the case of highly homologous framework sequences,
(Ii) may be identified by generating a sequence alignment of the germline precursor sequence or consensus sequence of step (i) with the donor variable domain sequence, and (iii) identifying the residues which are different.

  Distinct residues on the surface of the molecule were often mutated during the affinity generation process in vivo, presumably to generate affinity for the antigen.

  In another aspect, the invention provides an immunobinder comprising an immunobinder acceptor framework as described herein. For example, the immunobinder may be a scFv antibody, full length immunoglobulin, Fab fragment, Dab or nanobody.

  In a preferred embodiment, the immunobinder comprises one or more molecules, for example, cytotoxic agents, therapeutic agents such as cytokines, chemokines, growth factors or other signaling molecules, contrast agents, or transcriptional activators or DNA binding. It is bound to a second protein such as a domain.

  The immunobinder disclosed herein can be used, for example, in diagnostic applications, therapeutic applications, target evaluation or gene therapy.

  The invention further provides an isolated nucleic acid encoding an immunobinder acceptor framework as disclosed herein or an immunobinder (s) as herein disclosed.

  In another embodiment, a vector comprising the nucleic acid disclosed herein is provided.

  The nucleic acids or vectors disclosed herein can be used, for example, in gene therapy.

  The invention further encompasses host cells comprising the vectors and / or nucleic acids disclosed herein.

  Further, there is provided a composition comprising an immunobinder acceptor framework as disclosed herein, an immunobinder as disclosed herein, an isolated nucleic acid as disclosed herein or a vector as disclosed herein. .

  The sequences disclosed herein are as follows (X residue is the CDR insertion site).

In another aspect, the invention provides methods of humanizing non-human antibodies by fusing the CDRs of the non-human donor antibody to a stable and soluble antibody framework. In a particularly preferred embodiment, the CDR stems and frameworks from the rabbit antibody are as described above.

  A general method of fusing CDRs into a human acceptor framework is disclosed by Winter in US Pat. No. 5,225,539, and by Queen et al. In WO 9007861 A1 in US Pat. No. 5,225,539, which is incorporated herein by reference in its entirety. The general strategy for fusing CDRs from rabbit rabbit monoclonal antibodies to selected frameworks is related to those of Winter et al. And Queen et al., But differs in certain major respects. In particular, the methods of the present invention are exemplary Winter and Queen known in the art in that the human antibody frameworks disclosed herein are particularly suitable as universal acceptors for human or non-human donor antibodies. It is different from the methodology. Thus, unlike the general methods of Winter and Queen, the framework sequences used in the humanization method of the invention do not necessarily have the highest sequence similarity to the sequence of the non-human (e.g., rabbit) antibody from which the donor CDRs are derived. It is not a framework arrangement that indicates gender. In addition, framework residue fusions from donor sequences are not required to support CDR conformations. At most, antigen binding amino acids located in the framework or other mutations that occurred during somatic hypermutation may be introduced.

  Specific details of the fusion method to generate humanized rabbit-derived antibodies with high solubility and stability are described below.

Thus, the present invention provides methods of humanizing a rabbit rabbit CDR donor immunobinder comprising heavy chain CDR1, CDR2 and CDR3 sequences and / or light chain CDR1, CDR2 and CDR3 sequences. This method is
(I) on the heavy chain 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 Fusing at least one, preferably two, more preferably three CDRs of the group consisting of CDR1, CDR2 and CDR3 sequences into a human heavy chain acceptor framework having the affinity, and / or (ii) At least 50%, preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID NO: 2 on the light chain At least one, preferably two, of the groups consisting of CDR1, CDR2 and CDR3 sequences in the human light chain acceptor framework, preferably Or including the step of fusing the three CDRs.

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

  In a highly preferred embodiment, the method comprises an immunobinder having at least 75%, 80%, 85%, 90%, more preferably at least 95% identity to SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 (I) fusing the heavy chain CDR1, CDR2 and CDR3 sequences into the heavy chain, and (ii) fusing the light chain CDR1, CDR2 and CDR3 sequences into the light chain. More preferably, the immunobinder is SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7 or comprises SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7.

  In another embodiment, to improve antigen binding, the method may further comprise replacing the acceptor framework residue by a donor residue involved in antigen binding.

  In an exemplary embodiment of the method of the present invention, the amino acid sequences of CDR donor antibodies are first identified and the sequences are aligned using conventional sequence alignment tools (eg, Needleman-Wunsch algorithm and Blossum matrix). Introduction of gaps and nomenclature of residue positions can be performed using conventional antibody numbering systems. For example, the AHo numbering system can be used for immunoglobulin variable domains. The Kabat numbering scheme is also applicable since it is the most widely adopted standard for numbering residues in antibodies. Kabat numbering can be assigned, for example, using the SUBIM program. This program analyzes the variable regions of antibody sequences and numbers the sequences according to the system established by Kabat et al. (Deret et al., 1995). The framework and CDR regions are usually defined according to the Kabat definition, which is based on sequence variability and is most commonly used. However, for CDR-H1, such designations are as defined by Kabat, average contact data generated by analysis of contacts between antibodies of a subset of 3D complex structures and antigen (MacCallum et al., 1996), Preferably, it is a combination of the definition of Chotia based on the position of the structural loop region (see also FIG. 1). The conversion tables of two different numbering systems used to identify amino acid residue positions of antibody heavy and light chain variable regions are given in A.A. Honegger, J.M. Mol. Biol. 309 (2001), 657-670. The Kabat numbering system is described in Kabat et al. (Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U. S. Department of Health and Human Services, NIH Publication No. 91-3242). , Has been described in more detail. The AHo numbering system is described by Honegger, A. et al. And Pluckthun, A. (2001); Mol. Biol. 309, 657-670, are described in more detail.

  The variable domains of a rabbit monoclonal antibody can be classified, for example, into corresponding human subgroups, using, for example, EXCEL implementation of sequence analysis algorithms and classification methods based on analysis of human antibody repertoires (Knappik et al. 2000, J Mol Biol., Feb. 11, 296 (1), 57-86).

  CDR conformations can be assigned to the donor antigen binding region and, subsequently, residue positions necessary to maintain various canonical structures can also be identified. The standard structures of five CDRs of the six antibody hypervariable regions (L1, L2, L3, H1 and H2) of the rabbit antibody are determined using the definition of Chothia (1989).

  In a preferred embodiment, CDRs are generated, identified and isolated according to the following method: B cells (preferably rabbit B cells) are either (i) target antigen (preferably purified) or (ii) on the surface Incubate with cells expressing the target antigen.

  In the case of (ii), said cells which express the target antigen are, for example, mammalian cells which naturally express the selected target or are transformed to express the target protein on the surface, preferably CHO or It may be HEK 293 cells, yeast cells, preferably yeast spheroblasts, or bacterial cells. During expression, the target antigen may be incorporated into the cell membrane or may be bound to the cell membrane and expressed on the cell surface. The cells may, for example, be cultured as an isolate in cell culture or isolated from their natural environment, eg, a tissue, an organ or an organism.

  When the target antigen is expressed on the cell surface, ie (ii), transmembrane proteins are particularly preferred, multiple transmembrane proteins such as GPCRs (G protein coupled receptors) or ion channels, or recombinant expression and Further preferred are any other proteins which are difficult to maintain their natural conformation upon purification. Traditional immunization with recombinant proteins is not recommended in these cases due to the loss of the native conformation of the integral membrane protein / complex during the purification process or the insufficient amount of pure protein Or impossible. In a preferred embodiment of the invention, a mammal, more preferably a rabbit, is immunized with DNA instead of the recombinant protein, for example according to the DNA vaccination protocol disclosed in WO / 2004/087216. DNA vaccination induces a rapid immune response to natural antigens. Since no recombinant protein is required, this technique is, on the one hand, very cost-effective and, on the other hand, more importantly, the method is integral to the integral membrane complex and / or multiple transmembranes. Allows natural expression of membrane proteins. The B cells may be isolated from the immunized mammal, preferably the rabbit, or alternatively may be naive B cells.

  In a subsequent step of the method, B cells, preferably memory B cells, are isolated from the lymph vessels (such as spleen or lymph nodes) of immunized animals, preferably immunized rabbits. These B cells are incubated in cells expressing the antigen on the surface, or in a mixture with a fluorescently labeled soluble antigen. Target specific antibodies are expressed on their surface, such that B cells are isolated that bind to the target antigen or target antigen expressed on the cell surface. In a highly preferred embodiment, B cells and / or target cells are stained to allow isolation by flow cytometry-based B cell / target cell complex or B cell / antigen complex sorting. Flow cytometry usually measures the fluorescence emitted from a single cell as it crosses the laser beam. However, some researchers have already demonstrated cell-cell interactions, such as cadherin (Panorchan et al., 2006, J. Cell Science, 119: 66-74; Leong et Hibma, 2005, J. Immunol. Methods, Volume 302, 116-124) or cytometers are used to investigate adhesions mediated by integrins (Gawaz et al., 1991, J. Clin. Invest, 88, 1128-1134). Thus, in (ii), cells expressing the selected target are stained with an intracellular fluorescent dye (eg, calcein). B cells are stained with fluorescent antibodies that bind to cell surface specific markers. Thus, it is possible to select a two-color "event" present in two cells adhering to one another via B cell receptor-target interactions (see Figure 2).

  Since IgG usually has higher affinity than IgM, it is preferable to select positive B cells which express IgG but not IgM (which is characteristic of memory B cells). For this purpose, it is preferred to use multicolor stains in which an antibody specific for IgG and an antibody specific for IgM are differentially labeled, eg APC and FITC, respectively.

  In a particular embodiment, the read-out for B cell sorting can also be selected for the ability of the interaction to functionally block or activate receptor signaling. For example, B cells can be incubated with cells that functionally express a GPCR (G protein coupled receptor). Agonists that signal through GPCRs can be added to the mixture to induce Ca2 + efflux from the GPCR-mediated endoplasmic reticulum. If the antibodies presented to B cells functionally block agonist signaling, as a result, this cell-cell interaction also blocks Ca 2+ efflux. Ca 2+ efflux can be measured quantitatively by flow cytometry. Thus, only B cell / target cell aggregates that show either an increase or a decrease in Ca 2+ efflux are sorted.

  Following the selection step, B cells are cultured under conditions suitable for the antibody to be secreted into the medium. The antibodies produced are monoclonal antibodies. Culturing may require the use of helper cell lines, such as a plethysmocyte helper cell line (see, eg, EL 4-B5, Zubler et al., 1985, J. Immunol., 134 (6), 3662-6368). For example, in order to remove an antibody produced against a protein expressed on the cell surface other than the target protein, a verification step is preferably performed to test the generation of an antibody that specifically binds to the target. For example, CELISA, a modified enzyme linked immunosorbent assay (ELISA) in which the coating step is performed on whole cells, is suitable for the purpose. The method allows assessment of the selectivity and ability of the antibody to compete with the ligand.

  The antibodies generated in the above steps are then analyzed to identify the CDRs of said antibodies. This may require steps such as purification of the antibodies, elucidation of their amino acid sequences and / or nucleic acid sequences.

  Finally, the CDRs can be fused to the acceptor framework, preferably the acceptor framework described above, for example by gene synthesis using the oligo extension method. In one embodiment, art-recognized methods of CDR fusion can be used to transfer the donor CDRs into the acceptor framework. In most cases, all three CDRs from the heavy chain are grafted from the donor antibody to a single acceptor framework and all three CDRs from the light chain are grafted to another acceptor framework. Some of the CDRs may not be involved in binding to the antigen, and CDRs with different sequences (and the same length) may have the same fold (hence, despite the different sequences, the antigen It is expected that not all of these CDRs will need to be grafted, as the contact from the to main chain contact may be retained). In fact, even single domains (Ward et al., 1989, Nature 341, 544-546) or even single CDRs (R. Taub et al., 1989, J. Biol Chem, 264, 259-265) alone. Can have antigen binding activity. However, the intent of CDR fusion is to graft an identical or nearly identical antigen binding site from an animal to a human antibody, such as whether all CDRs are grafted or only a few CDRs are grafted (e.g. U.S. Pat. No. 5,225,539 (Winter)).

  In another embodiment, the CDRs of the donor antibody may be modified before or after being incorporated into the acceptor framework.

  Instead, antibody characterization is performed only in their final immunobinder format. For this approach, CDR sequences of antibodies expressed on sorted B cells can be derived from cultures of sorted B cells, or directly from selected single B cells by RT-PCR. to recover. For the purpose, the proliferation of B cells and / or the verification step described above and / or the analysis step described above may be unnecessary. One-step PCR procedure by combination of two pools of oligonucleotides with partial overlap, wherein one oligonucleotide pool encodes the CDRs and the second one encodes the framework region of a suitable immunobinder scaffold Production of a humanized immunobinder. Instead of characterizing the IgG secreted into the cell culture supernatant, high-throughput sequencing, cloning and production make it possible to perform clonal selection based on the performance of the purified humanized immunobinder. In its preferred embodiment, the immunobinder is a scFv.

  However, fusion of the CDRs can result in the loss of affinity of the generated immunobinder to the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of somatic hypermutation. Thus, fusion of such donor framework amino acids to the framework of the humanized antibody may still be necessary. Amino acid residues of the non-human immunobinder involved in antigen binding can be identified by inspection of the domestic rabbit monoclonal antibody variable region sequence and structure. Each residue in the CDR donor framework that is different from the germline can be important. If the closest germline can not be determined, the sequences can be compared to the subgroup consensus or to the consensus of the rabbit sequence with a high percentage of similarity. Rare framework residues may be the result of somatic hypermutation and are therefore thought to play a role in binding.

  The antibodies of the invention can be further optimized to exhibit enhanced functional properties, such as enhanced solubility and / or stability. In a specific embodiment, the antibody of the invention is a PCT application number entitled “Sequence Based Engineering and Optimization of Single Chain Antibodies”, filed March 12, 2008, which is incorporated herein by reference. It is optimized according to the "functional consensus" method disclosed in PCT / EP2008 / 001958.

  For example, comparing the immunobinder of the invention with a database of functionally selected scFvs used to identify amino acid residue positions that are more or less resistant to variability than the corresponding positions in the immunobinder, It can thereby be shown that such identified residue positions may be suitable for manipulation to improve functionality such as stability and / or solubility.

Exemplary framework residue positions for substitution and exemplary framework substitutions are described in PCT Application No. PCT / June 25, 2008, entitled "Methods of Modifying Antibodies, and Modified Antibodies with Improved Functional Properties". No. CH 2008/000285 and PCT Application No. PCT / CH2008 / 000284, filed June 25, 2008, entitled "Sequence Based Engineering and Optimization of Single Chain Antibodies". For example, one or more of the following substitutions may be introduced at amino acid positions in the heavy chain variable region of the immunobinder of the invention (AHo numbering is referred to for each amino acid position listed below) :
(A) Q or E at amino acid position 1;
(B) Q or E at amino acid position 6;
(C) at amino acid position 7, T, S or A, more preferably T or A, even more preferably T;
(D) A, T, P, V or D at amino acid position 10, more preferably T, P, V or D,
(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 at amino acid position 14, more preferably R, M, E or Q, even more preferably R or E;
(H) L or V, more preferably L at amino acid position 19;
(I) R, T, K or N at amino acid position 20, more preferably R, T or N, even more preferably N;
(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) at amino acid position 55, M or I, more preferably I;
(O) at amino acid position 77, K or R, more preferably K;
(P) A, V, L or I at amino acid position 78, more preferably A, L or I, even more preferably A;
(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 at amino acid position 89, more preferably A, V or F, even more preferably V;
(U) F, S, H, D or Y at amino acid position 90, more preferably F, S, H or D;
(V) at amino acid position 92, D, Q or E, more preferably D or Q, even more preferably D;
(W) G, N, T or S at amino acid position 95, more preferably G, N or T, even more preferably G;
(X) T, A, P, F or S at amino acid position 98, more preferably T, A, P or F, even more preferably F;
(Y) R, Q, V, I, M, F or L at amino acid position 103, more preferably R, Q, I, M, F or L, still more preferably Y, still more preferably L And (z) N, S or A at amino acid position 107, more preferably N or S, even more preferably N.

Additionally or alternatively, one or more of the following substitutions may be introduced into the light chain variable region of the immunobinder of the invention:
(Aa) of amino acid position 1, Q, D, L, E, S or I, more preferably L, E, S or I, even more preferably L or E;
(Bb) S, A, Y, I, P, or T at amino acid position 2, more preferably A, Y, I, P, or T, even more preferably P or T;
(Cc) of amino acid position 3, Q, V, T or I, more preferably V, T or I, even more preferably V or T;
(Dd) V, L, I, or M at amino acid position 4, more preferably V or L;
(Ee) S, E or P at amino acid position 7, more preferably S or E, even more preferably S;
(Ff) at amino acid position 10, T or I, more preferably I;
(Gg) at amino acid position 11, A or V, more preferably A;
(Hh) S or Y, more preferably Y at amino acid position 12;
(Ii) at amino acid position 14, T, S or A, more preferably T or S, even more preferably T;
(Jj) S or R, more preferably S, of amino acid position 18;
(Kk) at amino acid position 20, T or R, more preferably R;
(Ll) R or Q, more preferably Q at amino acid position 24;
(Mm) H or Q at amino acid position 46, more preferably H;
(Nn) K, R or I at amino acid position 47, more preferably R or I, even more preferably R;
(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 at amino acid position 53, more preferably T, S, N, Q, or P;
(Qq) I or M, more preferably M at amino acid position 56;
(Rr) H, S, F or Y at amino acid position 57, more preferably H, S or F;
(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 at amino acid position 82, more preferably R or Q, even more preferably R;
(Uu) L or F at amino acid position 91, more preferably F;
(Vv) at amino acid position 92, G, D, T or A, more preferably G, D or T, even more preferably T;
(Xx) S or N, more preferably N of amino acid position 94;
(Yy) F, Y or S at amino acid position 101, more preferably Y or S, and even more preferably S; and D, F, H, E, L, A, T at amino acid position 103 , V, S, G or I, more preferably H, E, L, A, T, V, S, G or I, even more preferably A or V.

  In another embodiment, the immunobinder of the present invention is described in US Provisional Application No. 61 / 075,692, filed Jun. 25, 2008, entitled "Solubility Optimization of Immunobinders". Contains one or more mutations that enhance In one preferred embodiment, the immunobinder comprises a solubility enhancing mutation at an amino acid position selected from the group of heavy chain amino acid positions consisting of 12, 103, and 144 (AHo numbering method). In a preferred embodiment, the immunobinder is (a) serine (S) at heavy chain amino acid position 12; (b) serine (S) or threonine (T) at heavy chain amino acid position 103; and (c) heavy chain amino acid It contains one or more substitutions selected from the group consisting of serine (S) or threonine (T) at position 144. In another embodiment, the immunobinder comprises the following substitutions: (a) serine (S) at heavy chain amino acid position 12; (b) serine (S) or threonine (T) at heavy chain amino acid position 103; c) Serine (S) or threonine (T) at heavy chain amino acid position 144.

  In certain preferred embodiments, the immunobinder comprises a light chain acceptor framework at least one of positions 1, 3, 4, 10, 47, 57, 91 and 103 of the light chain variable region according to the AHo numbering system. Containing framework enhancement residues at framework residues. In a preferred embodiment, the light chain acceptor framework comprises (a) glutamate at position 1 (E), (b) valine at position 3 (V), (c) position 4 leucine (L), (d) position 10 serine (S), (e) arginine (R) at position 47, (e) serine (S) at position 57, (f) phenylalanine (F) at position 91, and (g) valine at position 103 (V) And one or more substitutions selected from the group consisting of

  Any of the various available methods of producing humanized antibodies, including the mutations described above, can be used.

  Thus, the present invention provides an immunobinder humanized according to the methods described herein.

  In certain preferred embodiments, the target antigen of the immunobinder is VEGF or TNFα.

  For example, the polypeptides described in the present invention or the polypeptides produced by the methods of the present invention can be synthesized using techniques known in the art. Alternatively, nucleic acid molecules encoding the desired variable regions can be synthesized and polypeptides can be produced by recombinant methods.

  For example, once the sequence of the humanized variable region has been determined, the variable region or a polypeptide comprising it can be generated by techniques well known in the art of molecular biology. More specifically, recombinant DNA techniques can be used to produce a wide variety of polypeptides by transforming host cells with nucleic acid sequences (eg, a DNA sequence encoding a desired variable region (eg, For example, modified heavy or light chains; variable domains or other antigen binding fragments thereof)).

In one embodiment, an expression vector can be prepared that includes a promoter operably linked to a DNA sequence encoding at least _VH or _VL . If necessary or desirable, a second expression vector can be prepared which contains a promoter operably linked to the DNA sequence encoding the complementary variable domain (ie, the parent expression vector encodes V _H , When the two expression vectors encode V _L and vice versa). Thereafter, a cell line (eg, an immortalized mammalian cell line) can be transformed with one or both of the expression vectors and cultured under conditions that allow expression of the chimeric variable domain or chimeric antibody (eg, See International Patent Application No. PCT / GB85 / 00392 of Neuberger et al.

  In one embodiment, a variable region comprising the amino acid sequences of the donor CDR and acceptor FR may be made, and then changes may be introduced into the nucleic acid molecule to effect CDR amino acid substitutions.

  Exemplary methods recognized in the art for making nucleic acid molecules encoding amino acid sequence variants of the polypeptide include site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and the polypeptide. Preparations by cassette mutagenesis of pre-prepared DNA encoding include but are not limited to these.

  Site-directed mutagenesis is a preferred method for preparing substitution variants. This technique is well known in the art (e.g., Carter et al., Nucleic Acids Res., 13: 4431-4443 (1985), Kunkel et al., Proc. Natl. Acad. Sci. USA, vol. 82, 488). Volume (1987)). Briefly, in performing site-directed mutagenesis of DNA, modification of the parental DNA is first performed by hybridizing a single strand of the parental DNA to an oligonucleotide encoding the desired mutation. Following hybridization, the entire second strand is synthesized using DNA polymerase, using the hybridised oligonucleotide as a primer and using a single strand of the parental DNA as a template. Thereby, the oligonucleotide encoding the desired mutation is incorporated into the resulting double stranded DNA.

  PCR mutagenesis is also suitable for producing amino acid sequence variants of the polypeptide. Higuchi, PCR Protocols, pages 177-183 (Academic Press, 1990); Vallette et al., Nuc. Acids Res. 17, pp. 723-733 (1989). Briefly, when a small amount of template DNA is used as a starting material in PCR, the primers differ from the template sequence only at positions where the primers differ from the template, using primers that differ slightly in sequence from the corresponding regions of the template DNA. Relatively large amounts of specific DNA fragments can be generated.

  Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al., Gene, 34, 315-323 (1985). The starting material is a plasmid (or other vector) containing the DNA to be mutagenized. The codon or codons in the parent DNA to be mutagenized are identified. There must be unique restriction endonuclease sites on each side of the identified mutation site (s). If such a restriction enzyme recognition site is not present, it can be generated using the above-described oligonucleotide-mediated mutagenesis method which introduces a restriction enzyme recognition site at an appropriate position in the DNA encoding the polypeptide. . The plasmid DNA is cut at these sites to linearize it. Double stranded oligonucleotides encoding the sequence of DNA between restriction enzyme recognition sites, but containing the desired mutation (s), are synthesized using standard procedures, wherein two strands of the oligonucleotide Are separately synthesized using standard techniques and then hybridized to each other. This double stranded oligonucleotide is called a cassette. This cassette is designed to have 5 'and 3' ends compatible with the ends of the linearized plasmid so as to be directly ligated to the plasmid. This plasmid now contains the mutagenized DNA sequence.

  The variable regions generated by the methods of the invention can be remodeled to further modify them to further enhance antigen binding. Thus, the above-described steps can precede or follow additional steps, including, for example, affinity maturation. In addition, empirical binding data can also be used for further optimization.

  One skilled in the art will appreciate that the polypeptides of the invention can be further modified to differ in amino acid sequence but not in the desired activity. For example, additional nucleotide substitutions may be added to the protein leading to amino acid substitutions at "non-essential" amino acid residues. For example, a nonessential amino acid residue in an immunoglobulin polypeptide can be substituted with another amino acid residue of the same side chain family. In another embodiment, stretches of amino acids can be substituted with structurally similar stretches that differ in order and / or composition of side chain family members, ie, amino acid residues have similar side chains Conservative substitutions may be made that are substituted with amino acid residues.

  Families of amino acid residues having similar side chains have been defined in the art and include basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), Uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branched Side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine) can be mentioned.

  Apart from amino acid substitutions, the present invention also contemplates other modifications to the Fc region amino acid sequence, eg, to generate Fc region variants with altered effector function. For example, one or more amino acid residues of the Fc region can be removed to reduce or enhance binding to the FcR. In one embodiment, one or more Fc region residues can be modified to generate such Fc region variants. Generally, according to this embodiment of the invention, 1 to about 10 or less Fc region residues are removed. The Fc region comprising one or more amino acid deletions herein preferably retains at least about 80%, preferably at least about 90%, most preferably at least about 95% of the starting Fc region or native sequence human Fc region. Do.

  Amino acid inserted Fc region variants with altered effector function can also be generated. For example, at least one amino acid residue adjacent to one or more Fc region positions identified herein as impacting FcR binding (eg, 1 to 2 amino acid residues, usually 10 residues or less) Can be introduced. By "adjacent" is meant within 1 to 2 amino acid residues of the Fc region residues identified herein. Such Fc region variants may exhibit enhanced or reduced FcRn binding.

  Such Fc region variants usually contain at least one amino acid modification within the Fc region. In one embodiment, amino acid modifications may be combined. For example, the variant Fc region may comprise within it, for example, substitutions of 2, 3, 4, 5, etc. among the specific Fc region positions identified herein. In another embodiment, the polypeptide may have altered binding to FcRn and binding to another Fc receptor.

  In one embodiment, the polypeptide described in the present invention, or the polypeptide produced by the method of the present invention, eg, a humanized Ig variable region and / or a polypeptide comprising a humanized Ig variable region is recombinant It may be one produced by the method. For example, a polynucleotide sequence encoding a polypeptide can be inserted into an expression vector suitable for recombinant expression. When the polypeptide is an antibody, polynucleotides encoding additional light and heavy chain variable regions, optionally linked to a constant region, can be inserted into the same or different expression vectors. Affinity tag sequences (eg, His (6) tags) can optionally be bound or included in the polypeptide sequence to facilitate downstream purification. The DNA segment encoding the immunoglobulin chain is operably linked to control sequences in the expression vector (s) that ensure expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, a promoter (eg, a naturally associated or heterologous promoter), a signal sequence, an enhancer element and a transcription termination sequence. The expression control sequence is preferably a eukaryotic cell promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the polypeptide.

  These expression vectors are usually replicable in the host organism either as an episome or as a part integrated into the host chromosomal DNA. In general, expression vectors will contain a selectable marker (eg, ampicillin resistance, hygromycin resistance, tetracycline resistance or neomycin resistance) to allow detection of cells transformed with the desired DNA sequence. (See, eg, US Pat. No. 4,704,362).

  E. E. coli is one of the prokaryotic host particularly useful for cloning the polynucleotides (eg, DNA sequences) of the present invention. 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 useful for expression. Saccharomyces and Pichia are exemplary yeast hosts, with suitable vectors optionally having expression control sequences (eg, a promoter), an origin of replication, termination sequences and the like. Exemplary promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and promoters from enzymes responsible for methanol, maltose and galactose utilization.

  Within the scope of the present invention, E.I. coli and S. coli. S. cerevisiae is a preferred host cell.

  In addition to microorganisms, mammalian tissue cultures can also be used to express and produce the polypeptides of the invention (eg, polynucleotides encoding immunoglobulins or fragments thereof). Winnacker, From Genes to Clones, VCH Publishers, N .; Y. , N. See Y (1987). Because many suitable host cell lines capable of secreting heterologous proteins (eg, intact immunoglobulins) have been developed in the art, eukaryotic cells are virtually preferred, including CHO cell lines, various Cos cells, etc. And HeLa cells, 293 cells, myeloma cell lines, transformed B cells and hybridomas. Expression vectors for these cells include expression control sequences such as origin of replication, promoters and enhancers (Queen et al., Immunol. Rev., Vol. 89, 49 (1986)), and ribosome binding site, RNA splicing site It may contain the necessary processing information site, such as a polyadenylation site, a transcription terminator sequence, etc. Preferred expression control sequences are promoters obtained from immunoglobulin genes, SV40, adenovirus, bovine papilloma virus, cytomegalovirus and the like. Co et al. Immunol. 148, 1149 (1992).

  Vectors containing the polynucleotide sequences of interest (eg, sequences encoding heavy and light chains and expression control sequences) can be introduced into host cells by well-known methods (depending on the type of cellular host). For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistics or virus based transfection can be used for other cellular hosts (general) Sambrook et al., Molecular Cloning: A Laboratory Manual (see Cold Spring Harbor Press, Second Edition, 1989) Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion. (See, generally, Sambrook et al., Supra) for the production of transgenic animals. , That microinjected transgene into fertilized oocytes, or incorporated into the genome of embryonic stem cells, it can be introduced the nuclei of such cells into enucleated intracellularly.

  The polypeptide of interest can also be incorporated into the transgene for introduction into the genome of the transgenic animal and subsequent expression, eg, in the milk of the transgenic animal (eg, Deboer et al., 5, 741, 957; Rosen, 5, 304, 489; and Meade, 5, 849, 992). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer from a mammary gland specific gene such as casein or beta-lactoglobulin.

  The polypeptide can be expressed using a single vector or can be expressed using two vectors. For example, antibody heavy and light chains may be cloned into separate expression vectors and co-transfected into cells.

  In one embodiment, a signal sequence can be used to facilitate expression of a polypeptide of the invention.

  Once expressed, the polypeptides are standard procedures in the art including ammonium sulfate precipitation, affinity columns (eg, protein A or protein G), column chromatography, HPLC purification, gel electrophoresis, etc. (General (See, eg, Scopes, Protein Purification (Springer-Verlag, N.Y. (1982))).

  The humanized Ig variable regions or polypeptides comprising them can be expressed by host cells or cultured cell lines. They can also be expressed in cells in vivo. Cell lines that are transformed (eg, transfected) to produce modified antibodies can be immortalized mammalian cell lines, eg, those of lymphoid origin (eg, myelomas, hybridomas, etc.) , Trioma or quadroma cell lines). The cell line may also include normal lymphoid cells, such as B cells, immortalized by transformation with a virus (eg, Epstein-Barr virus).

  The cell lines used to produce the polypeptide are usually mammalian cell lines, although cell lines from other sources (such as bacteria and yeast) can also be used. In particular, E.I. Bacterial strains derived from E. coli can be used, inter alia, for example, phage display can be used.

  Some immortalized lymphoid cell lines, such as myeloma cell lines, in their normal state, secrete isolated Ig light or heavy chains. When such a cell line is transformed with a vector expressing a modified antibody prepared in the method of the present invention, the normally secreted chain is the variable domain of the Ig chain encoded by the vector previously prepared. It is not necessary to carry out the remaining steps of the method, provided that the strand is complementary to.

  If the immortalized cell line does not secrete or secrete the complementary strand, it is necessary to introduce into the cell a vector encoding the appropriate complementary strand or fragment thereof.

  If the immortalized cell line secretes a complementary light or heavy chain, the transformed cell line may, for example, transform a suitable bacterial cell with the vector, and then the bacterial cell with the immortalized cell line (eg, , By spheroplast fusion). Alternatively, DNA can be introduced directly into immortalized cell lines by electroporation.

In one embodiment, the humanized Ig variable regions described herein, or the humanized Ig variable regions produced by the methods of the invention, may be present in an antigen binding fragment of any antibody. These fragments can be produced recombinantly as well as manipulated, synthesized or produced by digesting the antibody with a proteolytic enzyme. For example, the fragment may be a Fab fragment, and digestion with papain cleaves the antibody in the region prior to the interchain (i.e. V _H -V _H ) disulfide bond linking the two heavy chains. This results in the formation of two identical fragments containing the light chain and the V _H and C _H 1 domains of the heavy chain. Alternatively, this fragment may be an F (ab ') ₂ fragment. These fragments can be generated by digesting the antibody with pepsin. Pepsin cleaves the heavy chain after an interchain disulfide bond, resulting in a fragment containing both antigen binding sites. Yet another alternative is to use "single chain" antibodies. Single chain Fv (scFv) fragments can be constructed in a variety of ways. For example, the C-terminus of V _H can be linked to the N-terminus of V _L. Typically, a linker (eg (GGGGS) ₄ ) is placed between V _H and V _L. However, the order in which the chains can be linked can be reversed and can include tags (eg, Myc tags, His tags or FLAG tags) to facilitate detection or purification (tags such as these of the invention It can be added to any antibody or antibody fragment; their use is not limited to scFv). Thus, as described below, tagged antibodies are within the scope of the present invention. In alternative embodiments, the antibodies described herein or antibodies produced by the methods described herein may be heavy chain dimers or light chain dimers. In addition, antibody light or heavy chains or portions thereof, such as single domain antibodies (DAb) can also be used.

In another embodiment, the humanized Ig variable region according to the present invention or the humanized Ig variable region produced by the method of the present invention is present in a single chain antibody (ScFv) or a minibody (minibody) See, for example, U.S. Patent No. 5,837,821 or WO 94/09817 A1). The minibodies are each a ScFv molecule (a single polypeptide comprising one or more antigen binding sites, eg, a V _L domain linked by a flexible linker to the V _H domain fused to the CH 3 domain via a connecting peptide) A dimeric molecule made of two polypeptide chains containing ScFv molecules can be constructed in the V _H -linker-V _L orientation or in the V _L -linker-V _H orientation. Preferably, the flexible hinge linking the V _L and V _H domains making up the antigen binding site comprises about 10 to about 50 amino acid residues. An exemplary 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. (1991) Biochemistry 30: 10117; Milenic et al. (1991) Cancer Research 51: 6363; Takkinen et al. (1991) Protein Engineering 4: 837. Minibodies can be generated by constructing the ScFv component and the connecting peptide-CH ₃ component using methods described in the art (see, eg, US Pat. No. 5,837,821 or WO 94 / 09817A1) . These components can be isolated as restriction fragments from separate plasmids and then ligated and recloned into an appropriate vector. Proper assembly can be confirmed by restriction enzyme digestion and DNA sequence analysis. In one embodiment, the minibody of the invention comprises a connecting peptide. In one embodiment, the connecting peptide comprises a Gly / Ser linker, such as GGGSSGGGSGG.

In another embodiment, tetravalent minibodies can be constructed. For example, building a tetravalent minibody in the same manner as the minibody except that two ScFv molecules are linked using a flexible linker having the amino acid sequence (G ₄ S) ₄ G ₃ AS it can.

In another embodiment, the humanized variable regions described herein or the humanized variable regions produced by the methods of the present invention may be present in diabodies. Diabodies are similar to scFv molecules, but usually have a short (less than 10, preferably 1 to 5) amino acid residue linker connecting both variable domains and therefore V on the same polypeptide chain _L domain and V _H domain can not interact. Instead, the V _L and V _H regions of one polypeptide chain (respectively) interact with the V _H and V _L domains on the second polypeptide chain (WO 02/02781).

  In another embodiment, a humanized variable region of the invention is an immunoreactive fragment or antibody of an antibody (eg, scFv molecule, minibody, tetravalent minibody, or diabody) operably linked to an FcR binding moiety. May be present in parts. In an exemplary embodiment, the FcR binding moiety is a complete Fc region.

  Preferably, the humanization methods described herein result in an Ig variable region in which the affinity of the antigen is substantially unchanged as compared to the donor antibody.

In one embodiment, a polypeptide comprising a variable domain of the invention is about 10 ⁵ M ^-1 , about 10 ⁶ M ^-1 , about 10 ⁷ M ^-1 , about 10 ⁸ M ^-1 , about 10 ⁹ M ^-1. Bind an antigen with a binding affinity with a binding constant Ka greater than (or equal to) about 10 ¹⁰ M ⁻¹ , about 10 ¹¹ M ⁻¹ or about 10 ¹² M ⁻¹ (intermediate affinity of these values including).

  Affinity, avidity and / or specificity can be measured in various ways. Generally, regardless of the exact method by which affinity is defined or measured, the method of the invention produces an antibody that is superior in any aspect of clinical application to the original antibody (or antibodies) , The method improves antibody affinity (eg, where the modified antibody can be administered at a lower dose or less frequent or convenient route of administration than the antibody (or antibodies) from which it is produced, The method of the invention is considered to be effective or successful).

More specifically, the affinity between the antibody and antigen to which it binds, for example, various, including ELISA assays, BiaCore assay or ^KinExA TM 3000 assay (available from Sapidyne Instruments (Boise (ID)) ) Can be measured by various assays. Briefly, Sepharose beads are covalently attached to an antigen (the antigen used in the method of the present invention may be any antigen of interest (eg, cancer antigen; cell surface protein or secreted protein; pathogen antigen (eg, bacteria or Coat with viral antigen (eg, HIV antigen, influenza antigen or hepatitis antigen), which may be an allergen) Prepare dilutions of the antibodies to be tested and add each dilution to designated wells in the plate. Then add detection antibody (eg, goat anti-human IgG-HRP conjugate) to each well, followed by substrate for chromogenic (eg, HRP), then plate at 450 nM with ELISA plate reader Read, calculate EC50 values (the method described here is generally applicable; Comprising particular it is understood as not being limited to the production of antigens or antibodies that bind to the class of antigens).

  One skilled in the art recognizes that affinity determination is not always as simple as always looking at a single number. Since antibodies have two arms, their apparent affinity is usually much higher than the intrinsic affinity between the variable region and the antigen (which is believed to be due to avidity). Intrinsic affinity can be measured using scFv or Fab fragments.

  In another aspect, the invention features a bispecific molecule comprising the humanized rabbit antibody or fragment thereof of the invention. An antibody of the invention, or an antigen-binding portion thereof, is derivatized or linked to another functional molecule, such as, for example, another peptide or protein (eg, a ligand for another antibody or receptor), at least 2 Bispecific molecules can be produced that bind to two different binding sites or target molecules. The antibodies of the invention may be derivatized or linked to two or more other functional molecules to produce multispecific molecules that bind to three or more different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecules" as used herein. To generate a bispecific molecule of the invention, an antibody of the invention is used as one of another antibody, an antibody fragment, a tumor specific or pathogen specific antigen, a peptide, or a binding mimetic. Alternatively, it may be operably linked (eg, by chemical coupling, gene fusion, noncovalent association, or otherwise) such that a bispecific molecule is generated to a plurality of other binding molecules. Thus, the present invention is bispecific comprising at least one first binding molecule having specificity for a first target, and a second binding molecule having specificity for one or more additional target epitopes. Contains sex molecules.

  In one embodiment, the bispecific molecule of the invention has at least one antibody, or antibody fragment thereof (e.g., Fab, Fab ', F (ab') 2, Fv, or single chain Fv) as binding specificity. Including). The antibody may be a light chain or heavy chain dimer, or any minimal fragment thereof, as described in Ladner et al., US Pat. No. 4,946,778, the contents of which are expressly incorporated by reference. It may be Fv or a single chain construct, etc.).

  While human monoclonal antibodies are preferred, other antibodies that can be used for the bispecific molecules of the invention are murine, chimeric and humanized monoclonal antibodies.

  The bispecific molecules of the invention can be prepared by conjugating the binding specificities of the components using methods known in the art. For example, each binding specificity of the bispecific molecule may be produced separately and then conjugated to one another. If the binding specificity is a protein or peptide, various coupling or crosslinking agents can be used for covalent conjugation. Examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis (2-nitrobenzoic acid) (DNTB), o-phenylene dimaleimide ( o PDM), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). Karpovsky et al. (1984) J. Exp. Med., 160, 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82: 8648). As another method, Paulus (1985) Behring Ins. Mitt. 78: 118-132; Brennan et al. (1985) Science 229: 81-83, and Glennie et al. Immunol. 139, pp. 2368-2375. Preferred conjugating agents are SATA and sulfo-SMCC, both of which are Pierce Chemical Co. Available from (Rockford, IL).

  Where the binding specificity is an antibody, they may be conjugated by sulfhydryl binding of the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number, preferably one, of sulfhydryl residues prior to conjugation.

Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful where the bispecific molecule is a mAb × mAb, mAb × Fab, Fab × F (ab ′) ₂ , or a ligand × Fab fusion protein. The bispecific molecule of the invention may be a single chain molecule comprising one single chain antibody and a binding determinant, or a single chain bispecific molecule comprising two binding determinants. Bispecific molecules may comprise at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in US Pat. Nos. 5,260,203, 5,455,030, 4,881,175, 5, 132,405, U.S. Pat. No. 5,091,513, U.S. Pat. No. 5,476,786, U.S. Pat. No. 5,013,653, U.S. Pat. No. 5,258,498, and U.S. Pat. , 482, 858.

  Binding to their specific targets by bispecific molecules can be performed, for example, by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry based single cell sorting (eg FACS analysis), bioassays (Eg, growth inhibition), or can be confirmed by Western blot assay. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by using a labeled reagent (eg, an antibody) specific for the complex of interest. For example, antibody complexes can be detected, for example, using an enzyme linked antibody or antibody fragment that recognizes and specifically binds to the antibody-VEGF complex. Alternatively, the complex can be detected using any of various other immunoassays. For example, the antibody may be radioactively labeled and may be used in a radioimmunoassay (RIA) (e.g. Weintraub, B. Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay, which is incorporated herein by reference) See Techniques, The Endocrine Society, March 1986). Radioactive isotopes can be detected by such means as the use of a gamma counter, or a scintillation counter, or by autoradiography.

  In another aspect, the invention features a humanized rabbit antibody, or fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (eg, an immunosuppressant), or a radiotoxin. Such complexes are referred to herein as "immunoconjugates". Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." Cytotoxic or cytotoxic agents include any agents that are harmful (eg, kill) to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and their analogs or homologs. As a therapeutic agent, for example, antimetabolite (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agent (eg, mechlorethamine, thiotepa), chlorambucil , Melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP), cisplatin), anthra, Cyclins (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg dactinomycin (previous acti Mycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine) can be cited also.

Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the invention include duocarmycin, calicheamicin, maytansine, and auristatin, and derivatives thereof. An example of a calicheamicin antibody conjugate is commercially available ^{(Mylotarg TM, Wyeth-Ayerst)} .

  Cytotoxins can be conjugated to the antibodies of the invention using linker technology available in the art. Examples of types of linkers used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers. The linker is cleaved by a protease such as, for example, one that is susceptible to cleavage by low pH within the lysosomal compartment, or a protease that is preferentially expressed in tumor tissue, such as cathepsins (eg cathepsin B, C, D) An easy one may be selected.

  For further discussion of cytotoxins, types of linkers, and methods for conjugating therapeutic agents to antibodies, see Saito, G., et al. Et al. (2003) Adv. Drug Deliv. Rev. 55, 199-215; Trail, P., et al. A. (2003) Cancer lmmuno1. Immunother. 52: 328-337; Payne, G., et al. (2003) Cancer Cell, Vol. 3, pp. 207-212; Allen, T. et al. M. (2002) Nat. Rev. Cancer 2, pp. 750-763; And Kreitman, R. et al. J. (2002) Curr. Opin. Investig. Drugs, 3, 1089-1091; Senter, P., et al. D. And Springer, C. J. (2001) Adv. Drug Deliv. Rev. 53: 247-264.

The antibodies of the present invention can also be conjugated to radioactive isotopes to produce cytotoxic radiopharmaceuticals, also called radioimmunoconjugates. Examples of radioactive isotopes that can be conjugated to antibodies for use in diagnosis or therapy include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ , and lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radio immunoconjugates are commercially available, including Zevalin ^TM (IDEC Pharmaceuticals) and ^Bexxar TM (Corixa Pharmaceuticals), using the same method to prepare radio immunoconjugates using an antibody of the present invention Can. The antibody conjugates of the invention can be used to modulate a given biological response and drug moieties should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety may be a protein or polypeptide having the desired biological activity. Such proteins include, for example, enzymatically active toxins, or active fragments thereof, such as, for example, abrin, ricin A, Pseudomonas exotoxins, or diphtheria toxins; proteins such as tumor necrosis factor or interferon-γ; Biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophages Colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors may be mentioned.

  Techniques for conjugating such therapeutic moieties to antibodies are well known and are described, for example, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Editor), pages 243-56, (Alan R. Liss, Inc. 1985). Hellstrom et al., In "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy"; Controlled Drug Delivery (Second Edition), Robinson et al. (Editor), pages 623-53 (Marcel Dekker, Inc., 1987). "Antibodies For Drug Delivery"; Monoclon l Antibodies '84: Biological And Clinical Applications, Pinchera et al. (Editor), pp. 475-506 (1985), Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review"; Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (Editor), pp. 303-16 (Academic Press 1985), "Analysis, Results, And Future Prospect Of The Therapeutic Use Of Radiolabe. led Antibody In Cancer Therapy ", and Thorpe et al.," The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates ", Immunol. Rev. 62: 119-58 (1982).

  In one aspect, the invention provides a pharmaceutical formulation comprising a humanized rabbit antibody for the treatment of a disease. The term "pharmaceutical formulation" is in a form that allows for the biological activity of the antibody or antibody derivative to be clearly effective, and prepared without additional components that are toxic to the subject to whom the formulation has been administered. I say a thing. A "pharmaceutically acceptable" excipient (vehicle, excipient) is one that can be reasonably administered to a subject mammal to achieve an effective dose of the active ingredient employed.

  A "stable" formulation is one in which the antibody or antibody derivative in the formulation essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art, see, for example, Peptide and Protein Drug Delivery, pages 247-301, edited by Vincent Lee, Marcel Dekker, Inc. New York, N .; Y. , Pubs. (1991) and Jones, A. et al. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature for a selected period of time. Preferably, the formulation is stable at room temperature (about 30 ° C.) or 40 ° C. for at least one month and / or stable at about 2-8 ° C. for at least one year or at least two years. Moreover, the formulation is preferably stable after freezing (e.g. up to -70 <0> C) and thawing of the formulation.

  An antibody or antibody derivative is pharmaceutical if it shows no signs of aggregation, precipitation and / or degeneration as determined by visual inspection of color and / or clarity, or as measured by UV light scattering or size exclusion chromatography. "Retain its physical stability" in the formulation.

  The antibody or antibody derivative may be “in its chemical formulation” in a pharmaceutical formulation so long as the chemical stability at a given time is still considered to retain the biological activity of the protein as defined below. Stability. " Chemical stability can be assessed by detecting and quantifying the morphology of the chemically altered protein. Chemical changes can be assessed using, for example, size exclusion chromatography, SDS-PAGE and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS), resizing (eg clipping) Can be involved in Other types of chemical changes include, for example, charge changes that can be assessed by, for example, ion exchange chromatography (e.g., resulting from deamidation).

  The antibody or antibody derivative is within about 10% or less of the biological activity exhibited when the biological activity of the antibody at a given time is prepared, eg, as a pharmaceutical formulation is determined by an antigen binding assay Within the error), "retain its biological activity" in a pharmaceutical formulation. Other "biological activity" assays for antibodies are described in detail herein below.

  By "isotonic" is meant that the formulation of interest has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice-freezing type osmometer.

  A "polyol" is a substance with multiple hydroxyl groups and includes sugars (reduced and non-reduced sugars), sugar alcohols and sugar acids. Preferred polyols herein have a molecular weight of less than about 600 kD (eg, in the range of about 120 to about 400 kD). A "reducing sugar" is one that contains a hemiacetal group that can reduce metal ions or that can be covalently reacted with lysine and other amino groups in proteins, and a "non-reducing sugar" is a reducing sugar It does not have these characteristics. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol and glycerol are examples of sugar alcohols. For sugar acids, this includes L-gluconate and its metal salts. Where it is desired that the formulation be stable to freeze-thaw, the polyol preferably does not crystallize at a freezing temperature (eg -20 ° C) such that the antibodies in the formulation become unstable. It is. Non-reducing sugars such as sucrose and trehalose are preferred polyols herein, with trehalose being preferred over sucrose because of the excellent solution stability of trehalose.

  As used herein, "buffer" refers to a buffer solution that resists changes in pH due to the action of the acid-base conjugation component. Buffers of the invention have a pH in the range of about 4.5 to about 6.0, preferably about 4.8 to about 5.5, and most preferably about pH 5.0. Examples of buffers that control pH in this range include acetate (eg, sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers. It can be mentioned. If a formulation stable to freeze-thaw is desired, the buffer is preferably not phosphate.

  In the pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of an antibody or antibody derivative refers to an amount effective for the prevention or treatment of a disorder in connection with the treatment for which the antibody or antibody derivative is effective. A "disease / disorder" is any condition that would benefit from treatment with an antibody or antibody derivative. This includes chronic and acute disorders or diseases, including those pathological conditions which cause the mammal to suffer from the disorder in question.

  A "preservative" is a compound that can be included in the formulation and can essentially reduce the action of bacteria there, thus facilitating, for example, the preparation of multi-use formulations. it can. Examples of possible preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (mixture of alkyl benzyl dimethyl ammonium chlorides, compounds with long alkyl chain alkyl groups), and benzethonium chloride. Be Other types of preservatives include phenol, aromatic alcohols such as 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 invention also provides a pharmaceutical composition comprising one or more antibodies or antibody derivative compounds together with at least one physiologically acceptable carrier or excipient. Pharmaceutical compositions include, for example, one or more of water, buffer (eg, neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oil, dimethylsulfoxide, carbohydrate (eg, glucose, mannose) Sucrose or dextran), mannitol, a protein, an adjuvant, a polypeptide, or an amino acid such as glycine, an antioxidant, a chelating agent such as EDTA or glutathione, and / or a preservative. As mentioned above, other active ingredients can (although not necessarily) be included in the pharmaceutical compositions provided herein.

  Carriers are substances that may be associated with the antibody or antibody derivative prior to administration to a patient, often for the purpose of controlling the stability or bioavailability of the compound. Carriers for use within such formulations are generally biocompatible and may also be biodegradable. Carriers include, for example, serum albumin (eg, human or bovine), egg albumin, peptides, monovalent or multivalent molecules such as polylysine, and polysaccharides such as aminodextran and polyamidoamines. Carriers also include solid carrier materials such as beads and microparticles including, for example, polylactate, polyglycolate, poly (lactide-co-glycolide), polyacrylate, latex, starch, cellulose or dextran. The carrier may have the compound in a variety of ways, including covalent bonding (either directly or through a linker group), non-covalent interactions or admixtures.

  The pharmaceutical composition can be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, rectal or parenteral administration. In certain embodiments, compositions in a form suitable for oral use are preferred. Such forms include, for example, pills, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Within yet other embodiments, the compositions provided herein can be formulated as a lyophilizate. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (eg intravenous), intramuscular, spinal cord, intracapsular, intrathecal and intraperitoneal injections, and any similar Injection or infusion techniques.

  Compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and sweeteners to provide attractive and savory preparations One or more agents such as flavoring agents, coloring agents, and preservatives can be included. Tablets contain the active ingredient in admixture with physiologically acceptable excipients that are suitable for the manufacture of tablets. Such excipients include, for example, inert diluents (eg, calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate), granulating agents and disintegrants (eg, corn starch or alginic acid), Binders such as starch, gelatin or acacia, as well as lubricants such as magnesium stearate, stearic acid or talc are included. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide sustained action over an extended period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be used.

  Formulations for oral use may be as hard gelatine capsules in which the active ingredient is mixed with inert solid diluents such as calcium carbonate, calcium phosphate or kaolin, or in vehicles in which the active ingredient is water or oil It may be provided as a soft gelatine capsule mixed with (eg, peanut oil, liquid paraffin, or olive oil). Aqueous suspensions contain the antibody or antibody derivative in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents (eg, sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia), and dispersing or wetting agents (eg, , Naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides such as polyoxyethylene stearate with fatty acids, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethylene oxysetanol, polyoxyethylene Condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols such as sorbitol monooleate, or polyethylene Condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as sorbitan monooleate). Aqueous suspensions may contain one or more preservatives (eg, ethyl or n-propyl p-hydroxybenzoic acid), one or more coloring agents, one or more flavoring agents, and one or more flavoring agents. Sweetening agents such as sucrose or saccharin. Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also include one or more demulcents, preservatives, flavors, and / or coloring agents.

  Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents and / or flavoring agents such as those described above can be added to provide a palatable oral preparation. Such suspensions may be preserved by the addition of an anti-oxidant such as ascorbic acid.

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

  The pharmaceutical composition may be in the form of an oil-in-water emulsion. The oily phase can be a vegetable oil, such as olive oil or peanut oil, a mineral oil, such as liquid paraffin, or a combination of these. Suitable emulsifying agents include naturally occurring gums (eg, acacia gum or tragacanth gum), naturally occurring phosphatides (eg, soy, lecithin, and esters or partial esters derived from fatty acids and hexitols), anhydrides (eg, Sorbitan monooleate) and condensation products of fatty acid and a partial ester derived from hexitol with ethylene oxide (e.g. polyoxyethylene sorbitan monooleate). The emulsions may also contain one or more sweetening and / or flavoring agents.

  The pharmaceutical composition can be prepared as a sterile injectable aqueous or oily suspension, wherein the modulator is suspended or dissolved in a vehicle depending on the vehicle and concentration used. Such compositions can be formulated according to the known art using suitable dispersing agents, moisturizing agents and / or suspending agents such as those mentioned above. Among the acceptable vehicles and solvents that may be employed are water, 1,3-butanediol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectables, and adjuvants such as local anesthetics, preservatives and / or buffers can be dissolved in the vehicle.

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

  The antibodies or antibody derivatives provided herein generally bind detectably to a target such as, for example, VEGF to prevent or inhibit target-mediated diseases / disorders, such as, for example, VEGF-mediated diseases / disorders. In an amount to achieve a concentration in body fluid (eg, blood, plasma, serum, CSF, synovial fluid, lymph, interstitial fluid, tears or urine). The dose is considered to be effective as it provides a recognizable patient benefit as described herein. The preferred systemic dose is in the range of about 0.1 mg to about 140 mg per kilogram of body weight per day (about 0.5 mg to about 7 g per patient per day) and oral doses are generally Approximately 5 to 20 times more than the intravenous dose. The amount of antibody or antibody derivative that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms will generally contain between about 1 mg and about 500 mg of the active ingredient.

  The pharmaceutical composition can be packaged to treat conditions responsive to, for example, an antibody or antibody derivative directed to VEGF. The packaged pharmaceutical composition comprises a container holding an effective amount of at least one antibody or antibody derivative as described herein, and a disease / disorder responsive to one antibody or antibody derivative after administration to a patient. Instructions (eg, a label) may be included to indicate the use of the contained composition to treat

  The antibodies or antibody derivatives of the invention can also be chemically modified. Preferred modifying groups are, for example, polymers such as optionally substituted linear or branched polyalkenes, polyalkenylene or polyoxyalkylene polymers, or branched or unbranched polysaccharides. Such effector groups can increase the half life of the antibody in vivo. Specific examples of synthetic polymers include optionally substituted linear or branched poly (ethylene glycol) (PEG), poly (propylene glycol), poly (vinyl alcohol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. The size of the polymer can be varied as desired, but generally the average molecular weight is in the range of 500 Da to 50000 Da. When the antibody is designed to penetrate tissue for local application, the preferred molecular weight of the polymer is about 5000 Da. The polymer molecule can be attached to the C-terminal end of the heavy chain of the Fab fragment via an antibody, in particular via a covalently linked hinge peptide as described in WO 0194 585. For attachment of the PEG moiety, see "Poly (ethylene glycol) Chemistry, Biotechnological and Biomedical Applications", 1992, J. Org. Milton Harris (ed.), Plenum Press, New York, and "Bioconjugation Protein Coupling Techniques for the Biomedical Sciences", 1998, M. et al. Aslam and A. See Dent, Grove Publishers, New York.

  After preparing the desired antibody or antibody derivative as described above, a pharmaceutical formulation containing it is prepared. The antibody to be formulated is not subjected to prior lyophilization, and the formulation of interest herein is an aqueous formulation. Preferably, the antibody or antibody derivative in the formulation is an antibody fragment such as a scFv. The therapeutically effective amount of antibody present in the formulation is determined, for example, taking into account the desired dose volume and mode of administration (s). About 0.1 mg / ml to about 50 mg / ml, preferably about 0.5 mg / ml to about 25 mg / ml, and most preferably about 2 mg / ml to about 10 mg / ml are typical antibody concentrations in the formulation It is.

  Aqueous formulations are prepared comprising the antibody or antibody derivative in pH buffered solution. Buffers of the invention have a pH in the range of about 4.5 to about 6.0, preferably about 4.8 to about 5.5, and most preferably about pH 5.0. Examples of buffers that control pH within this range include acetate (eg sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers Can be mentioned. The concentration of buffer may be about 1 mM to about 50 mM, preferably about 5 mM to about 30 mM, and can be determined, for example, by the desired tonicity of the buffer and the formulation. The preferred buffer is sodium acetate (about 10 mM), pH 5.0.

  Polyols that can act as tonicifiers and stabilize antibodies can be included in the formulation. In a preferred embodiment, the formulation does not include a salt such as sodium chloride in a tonicifying amount, as it may cause precipitation of the antibody or antibody derivative and / or oxidation at low pH. In a preferred embodiment, the polyol is a non-reducing sugar such as sucrose or trehalose. The polyol is added to the formulation in an amount that is variable to the desired tonicity of the formulation. Preferably, the aqueous formulation is isotonic, in which case a suitable polyol concentration in the formulation is, for example, in the range of about 1% to about 15% w / v, preferably about 2% to about 10% whv Range. However, hypertonic or hypotonic formulations may also be appropriate. The amount of polyol added can also vary with the molecular weight of the polyol. For example, smaller amounts of monosaccharides (eg, mannitol) can be added as compared to disaccharides (such as trehalose).

  Surfactants are also added to the antibody or antibody derivative formulations. Exemplary surfactants include nonionic surfactants such as polysorbates (eg, polysorbate 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of surfactant added is that amount which reduces aggregation of the formulated antibody / antibody derivative and / or minimizes the formation of particles in the formulation and / or reduces adsorption. For example, the surfactant may be present in an amount of about 0.001% to about 0.5%, preferably about 0.005% to about 0.2%, and most preferably about 0.01% to about 0.1%. May be present in the formulation.

  In one embodiment, the formulation comprises the above-identified agents (ie, antibodies or antibody derivatives, buffers, polyols and surfactants), essentially benzyl alcohol, phenol, m-cresol, chlorobutanol and It does not contain one or more preservatives such as benzethonium chloride. In another embodiment, preservatives can be included in the formulation, particularly if the formulation is a multi-dose formulation. The concentration of preservative may range from about 0.1% to about 2%, most preferably from about 0.5% to about 1%. Remington's Pharmaceutical Sciences 21st edition, Osol, A. et al. Condition that one or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Ed. (2006), do not adversely affect the desired characteristics of the formulation Can be included in the formulation. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and may include additional buffering agents, cosolvents, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA Metal complexes (e.g. Zn-protein complexes), biodegradable polymers such as polyesters, and / or salt forming counterions such as sodium.

  The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following preparation of the formulation.

  The formulation may be intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical to mammals, preferably humans, in need of treatment with the antibody. Or administration according to known methods, such as by bolus injection or intravenously by continuous infusion over time. In a preferred embodiment, the formulation is administered to a mammal by intravenous administration. For such purpose, the formulation can be injected, for example using a syringe or via an IV line.

  Appropriate dosages of the antibody ("therapeutically effective amount") can be, for example, the condition to be treated, the severity and course of the condition, whether the antibody is to be administered for prophylactic or therapeutic purposes, prior therapy, patient history and It depends on the response to the antibody, the type of antibody used, and the discretion of the attending physician. The antibody or antibody derivative may be suitably administered to the patient over a single or series of treatments and may be administered to the patient at any time after diagnosis. The antibody or antibody derivative can be administered as a sole treatment or in conjunction with other drugs or treatments useful for the treatment of the medical condition in question.

  As a general proposition, the therapeutically effective amount of the antibody or antibody derivative to be administered will be in the range of about 0.1 to about 50 mg / kg of the patient's body weight, whether single dose or multiple doses. A typical range of antibodies used is, for example, about 0.3 to about 20 mg / kg, more preferably about 0.3 to about 15 mg / kg, on a daily basis. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques.

  In another embodiment of the invention, an article of manufacture is provided, comprising a container holding a pharmaceutical formulation of the invention, preferably an aqueous formulation, and instructions for its use are optionally provided. Suitable containers include, for example, bottles, vials and syringes. The container can be formed of various materials such as glass or plastic. A typical container is a 3-20 cc single-use glass vial. Alternatively, for multi-dose formulations, the container may be a 3-100 cc vial. The container holds the formulation and can indicate directions for use by a label on the container or by a label associated with the container. The article of manufacture can further include other substances desired from the commercial and user point of view, including other buffers, diluents, filters, needles, syringes, and inserts with instructions for use Can be mentioned.

  In certain preferred embodiments, the article of manufacture comprises a lyophilised immunobinder as described herein, or a lyophilised immunobinder produced by a method as described herein.

(Example)
The present disclosure is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference in their entirety.

(Materials and methods)
In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, eg, antibody technology), and standard techniques of polypeptide preparation. See, eg, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), Volume 510, Paul, S. et al. Antibody Engineering: A Practical Approach (Practical Approach Series, Volume 169), edited by McCafferty, Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al., C. Biol. S. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, Ausubel et al. Ed., John Wiley & Sons (1992). Methods of fusion of CDRs to selected human antibody frameworks from rabbit and other non-human monoclonal antibodies are described in detail above. An example of such a fusion experiment is shown below.

  For the purpose of better understanding, the fusion designated "min" is one in which the CDRs are fused to Framework 1.4 or its variable domain, while the fusion designated "max" is , CDRs are fused to framework 1.4 or its variable domain, and the framework further comprises donor framework residues that interact with the antigen.

(Example 1: Design of rFW1.4)
(1.1. Primary Sequence Analysis and Database Search)
(1.1.1. Collection of rabbit rabbit immunoglobulin sequences)
The variable domain and germline sequences of the mature rabbit antibody were collected from various open source databases (eg, the Kabat database and IMGT) and entered into the customized database as a one-letter code amino acid sequence. For all analyses, we used only the amino acid portion corresponding to the V (variable) region. Sequences in the KDB database were discarded that contained less than 70% completeness, or multiple unresolved residues within the framework region. Sequences with greater than 95% identity to any other sequences in the database were also removed to avoid random noise in the analysis.

(1.1.2. Alignment and numbering of rabbit sequences)
The rabbit antibody sequences were aligned using the Needleman-Wunsch algorithm and a conventional sequence alignment tool based on Blossum matrix. Gap introduction and residue position nomenclature were performed according to the immunoglobulin variable domain AHo numbering system (Honegger and Pluckthun, 2001). The Kabat numbering scheme was also used since it is the most widely adopted standard method for numbering residues in antibodies. Kabat numbering was assigned using the SUBIM program. This program analyzes the variable regions of antibody sequences and numbers the sequences according to the system established by Kabat et al. (Deret et al., 1995).

  Definitions of framework and CDR regions are made according to the Kabat definition, which is based on sequence variability and is most commonly used. Nevertheless, the designation of CDR-H1 is the definition of AbM, the definition of kabat, the average contact data generated by analysis of contacts between antibody and antigen of a subset of 3D complex structures (MacCallum et al., 1996) And a compromise between various definitions, including the definition of Chotia based on the position of the structural loop region (shown above and shown in FIG. 1).

(1.1.3. Residue position frequency and conservation)
Amino acid sequence diversity was analyzed using a set of 423 rabbit sequences obtained from the kabat database. The residue frequency f (r) at each position i in the mature rabbit sequence was calculated by dividing the number of times a particular residue is observed in the data set by the total number of sequences. The degree of conservation of each position i was calculated using Simpson's index, taking into account the number of different amino acids present and the relative abundance of each residue.

In the formula, N is the total number of amino acids, r is the number of different amino acids present at each position, and n is the number of residues of a specific amino acid type.

(1.1.4. Serial analysis of V region of rabbits)
A phylogenetic analysis tool was used to investigate the rabbit repertoire. The amino acid sequences of the V region were clustered using both cluster and topological algorithms. A distance matrix was calculated for the entire array and used as an indicator of germline use. The consensus sequence of each cluster was calculated to identify the closest rabbit germline sequence counterparts. A comprehensive consensus sequence was also obtained from the entire set of sequences.

(1.1.5. Assignment of human subgroups)
Identification of the most homologous human subgroups using EXCEL implementation of sequence analysis algorithms and classification based on analysis of human antibody repertoire (Knappik et al., 2000) for each rabbit representative sequence of various clusters did.

(1.2. Design of human acceptor framework)
Blueprints of the rabbit repertoire from the sequence analysis described above were used to identify residues in the framework that are commonly involved in the localization of the rabbit CDRs. Among the frameworks and their respective clusters with high homology to the rabbit repertoire, those with good biophysical properties were selected from a pool of one fully human sequence. The frameworks selected for use as acceptor frameworks belong to variable light chain subgroup kappa 1 and variable heavy chain subgroup III, corresponding to ESBATech's ID KI 27, a43. This stable and soluble antibody framework was identified by screening a human spleen scFv library using a yeast-based screening method (Auf der Maur et al., 2004) named "Quality Control" system It was named "FW 1.4". Although the stable and soluble framework sequence FW1.4 shows high homology, it was not the highest homology sequence available. The identified residues were incorporated into the acceptor framework to generate rFW1.4.

Using information about the amino acid sequence diversity, germline usage and structural features of the rabbit antibody, we adapt the residue positions necessary to preserve CDR conformation in the novel human framework We analyzed FW 1.4 for. We examined the variable region of FW 1.4 for the suitability of the following features:
i. Residues that are part of the loop sequence reference sequence ii. Framework residues located at the VL / VH interface iii. Platform for residues just below the CDRs iv. Upper and lower core residues v. Framework residues that define the subtype.

(1.3. Fusion of rabbit CDRs)
Fusion fragments were generated by simply combining the CDR sequences from 1 antibody (as defined above) with the framework sequences of FW1.4 or rFW1.4. Residues potentially involved in binding were identified. For each rabbit variable domain sequence, the closest germline counterpart of the rabbit was identified. If the closest germline could not be established, the sequences were compared against the consensus of subgroups or the consensus of rabbit sequences with high percent similarity. Rare framework residues could be the result of somatic hypermutation and were therefore considered to play a role in binding. Thus, such residues were fused to the acceptor framework.

(1.4. Results)
By analyzing the rabbit rabbit antibody repertoire with respect to structure, amino acid sequence diversity and germline use, we found five residue positions in the light chain of FW1.4, modified to maintain the loop conformation of the rabbit rabbit CDRs. The These positions are highly conserved in rabbit antibodies. Consensus residues at these five positions were deduced from the rabbit repertoire and were introduced into human acceptor framework 1.4. The modifications of these conserved positions made the framework compatible with virtually all six complementarity determining regions (CDRs) of any rabbit rabbit CDRs. Master rFW 1.4 containing various rabbit CDRs was well expressed and well produced as opposed to a wild type single chain. Sixteen members were generated from the combination of this framework with the rabbit CDRs and functionality was shown by detailed characterization.

(Example 2: B cell screening system)
A FACS (flow cytometry based single cell sorting) based screening system has been established at ESBATech to select B cells that bind via their B cell receptor (BCR) to the target of interest. One target was, for example, a soluble protein, ie a single chain antibody (ESBA 903) labeled with a fluorochrome (PE and PerCP). Lymphocyte suspensions were prepared from the spleens of rabbit targets immunized with recombinant targets. The cells were then incubated with PE and PerCP labeled ESBA 903 and an antibody specific for IgG (APC labeled) or an antibody specific for IgM (FITC labeled). ESBA 903 positive B cells that express IgG but not IgM on their surface were sorted into 96 well plates (Figure 3; Table 2). B which grew and differentiated into plasma cells and secreted antibodies by plethysmoid helper cell line (EL4-B5: see Zubler et al., 1985, J. Immunol, 134 (6), 3662-6368). The cells were selected. The affinity of these IgGs for the target was assessed by ELISA and Biacore measurement. The kinetic parameters of 7 selected clones are shown in Table 1. These clones, obtained from a pool of about 200 sorted cells, exhibit binding affinities in the low nanomolar to picomolar range. Finally, mRNA was isolated from the 6 clones of interest and the CDRs fused to single chain framework FW1.4.

Example 3: Detection of the interaction between anti-TNF alpha antibody coated beads and CHO cells expressing membrane bound TNF alpha
The following experiments were performed to assess whether high pressure in the flow cytometry stream breaks non-covalent bonds between the two cells. Membrane-bound TNFα stably transfected CHO cells (B-220 cells) were incubated with beads coated with PE-labeled anti-TNFα antibody. In this setup, the beads mimic memory B cells (they have approximately the same size). As negative controls, non-transfected CHO cells and beads coated with APC labeled irrelevant antibody (anti-CD19) were used. After incubation for 2 hours at 4 ° C. with agitation, cell bead suspensions were analyzed by FACS (using a 130 μm nozzle). FIG. 4 shows that specific binding between anti-TNFα beads and TNFα-transfected CHO cells is clearly detectable by FACS. In fact, in this sample (upper panel) about 2/3 of the beads are bound to the cells (585 bound vs. 267 unbound). In contrast, most of the beads do not bind to CHO cells in control samples (middle and lower panels). In addition, both bead populations (anti-TNFα-PE and anti-CD19-APC) were mixed with TNFα-transfected CHO cells. FIG. 5 and Table 4 show that about half of the anti-TNFα beads bind to CHO cells, while the majority of anti-CD19 beads remain unbound. The percent of cell bound beads in each sample is detailed in Table 5. Thus, we demonstrate that specific selection of single B cells that bind to integral membrane target proteins via their B cell receptors is possible using flow cytometry.

Example 4 CDR Fusion and Functional Humanization of Anti-TNF.alpha.
Four anti-TNF [alpha] rabbit antibody "Rabmab" (EPI-1, EPI-15, EP-34, EP-35 and EP-42) were selected for CDR fusion. The general experimental scheme of CDR fusion, humanization and preliminary characterization of humanized rabbit donor antibody was performed as outlined in the detailed description. Unlike traditional humanization methods that utilize human antibody acceptor frameworks that share maximum sequence homology with non-human donor antibodies, using quality control assays (WO0148017), the desired functional properties (solubility and stability) The rabbit rabbit CDRs were fused to a human framework (FW 1.4) preselected for sex. These stable and soluble framework sequences showed high homology to RabMab.

  CDR fusions were generated for each of the RabMabs using the methodology described herein. In the "Min" fusion, only the rabbit rabbit CDRs were grafted from the VL and VH domains of the rabbit rabbit donor antibody into human acceptor framework FW1.4. The "Max" fusion piece refers to the fusion of the rabbit CDRs to rFW1.4.

The scFv described and characterized herein were produced as follows. The humanized VL sequence was connected to the humanized VH sequence via the linker of SEQ ID NO: 8 resulting in an scFv with an orientation of NH ₂ -VL-linker-VH-COOH. In many cases, DNA sequences encoding various scFv were synthesized de novo at the service provider Entelechon GmbH (www.entelechon.com). The resulting DNA insert fragment was cloned into the bacterial expression vector pGMP002 via the NcoI and HindIII restriction enzyme recognition sites introduced at the 5 'and 3' ends of the scFv DNA sequence, respectively. A BamHI restriction enzyme recognition site is located between the DNA sequence of the VL domain and the DNA sequence of the VH domain. In some cases, scFv encoding constructs were cloned by domain shuffle, without newly synthesizing scFv encoding DNA. Therefore, the VL domain was excised and introduced into a new construct via the NcoI and BamHI restriction sites, the VH domain was excised and introduced via the BamHI and HindIII restriction sites. In other cases, point mutations were introduced into the VH and / or VL domains using assembly PCR methods of advanced technology. The cloning of GMP 002 is described in Example 1 of WO2008006235. The production of scFv was carried out similarly for ESBA 105 described in Example 1 of WO2008006235.

  Table 3 summarizes the detailed characterization data of four rabbit rabbit monoclonals (EP6, EP19, EP34, EP35 and EP43) and their CDR fusion variants. These CDR fusions showed broad activity in BIACore binding assay and L929, TNFα mediated cytotoxicity assay, but 3 out of 4 max ("max") fusions show therapeutically important activity The EP 43 max showed the most favorable binding affinity (Kd of 0.25 nM) and an excellent EC50 in the cytotoxicity assay. This data indicates that FW 1.4 (SEQ ID NOS: 1 and 2) is an exemplary soluble and stable human acceptor framework region for rabbit rabbit CDR fusion.

(Efficacy assay)
The neutralizing activity of the anti-TNFα binder was evaluated in the L929 TNFα mediated cytotoxicity assay. The toxicity of actinomycin-treated mouse L929 fibroblasts was induced with recombinant human TNF (hTNF). 90% of maximal hTNF induced cytotoxicity was determined to be a TNF concentration of 1000 pg / ml. All L929 cells were cultured in RPMI 1640 with phenol red in L-glutamine medium supplemented with fetal bovine serum (10% v / v). The neutralizing activity of the anti-TNFα binder was evaluated in RPMI 1640 without phenol red, 5% fetal bovine serum. Various concentrations (0 to 374 ng / mL) of anti-TNF binder are added to L929 cells in the presence of 1000 pg / ml hTNF to determine the concentration at which the antagonist action reaches half maximal inhibition (EC 50%). Dose response curves were fitted to non-linear sigmoidal regression with variable slope and EC50s were calculated.

(Biacore binding analysis of anti-TNF scFv)
For binding affinity measurements, surface plasmon resonance measurements using a BIAcore ^TM -T100, it was utilized with NTA sensor chip and His-tagged TNF (produced at ESBATech). The surface of the NTA sensor chip consists of a carboxymethylated dextran matrix pre-immobilized with nitrilotriacetic acid (NTA) to capture histidine tagged molecules via Ni2 + NTA chelation. Human TNFα N-his trimer (5 nM) is captured by nickel via their N-terminal his tag and ESBA 105 (analytical at several concentrations ranging from 30 nM to 0.014 nM in 3-fold serial dilution steps Inject the In the regeneration step, the complex formed by the nickel, the ligand and the analyte is washed away. This makes it possible to use the same regeneration conditions for different samples. Reaction signals are generated by surface plasmon resonance (SPR) techniques and measured in resonance units (RU). All measurements are performed at 25 ° C. After inline reference cell correction and subsequent subtraction of buffer samples, sensorgrams were generated for each anti-TNF scFv sample. Apparent dissociation rate constant (k _d ), apparent association rate constant (k _a ), and apparent dissociation equilibrium constant (k _D ), 1 to 1 Langmuir binding using BIAcore T100 evaluation software version 1.1 Calculated using the model.

(Example 5: CDR fusion and functional humanization of anti-VEGF rabbit rabbit donor antibody)
Eight anti-VEGF Rabmabs (375, 435, 509, 511, 534, 567, 578 and 610) were selected for CDR fusion. Unlike traditional humanization methods that utilize human antibody acceptor frameworks that share maximum sequence homology with non-human donor antibodies, using quality control assays (WO0148017), the desired functional properties (solubility and stability) The rabbit rabbit CDRs were fused to human acceptor framework FW 1.4 (SEQ ID NO: 1 and 2) preselected for sex.

  Using the methodology described herein, a number of CDR fusions were generated for each of the RabMabs (rabbits) (see Example 4). The "Min" fusion piece contained the smallest fusion piece. In this, only the rabbit rabbit CDRs were grafted from the VL and VH domains of the rabbit rabbit donor antibody into human acceptor framework FW 1.4 (SEQ ID NO: 1). The "Max" fusion fragment contained not only the rabbit rabbit CDRs for VL and VH, but also some additional framework residues from rabbit rabbit donors that were predicted to be important for antigen binding. For 578 max, the heavy chain variable domain framework region of FW 1.4 has additional amino acid modifications at Kabat positions 23H, 49H, 73H, 78H and 94H.

  Table 4 summarizes the detailed characterization data for "Min" and "Max" CDR fusion variants. Shown are their efficacy as VEGF inhibitors as measured using VEGFR competitive ELISA and / or HUVEC assays. This data indicates that FW 1.4 (SEQ ID NOS: 1 and 2) is an exemplary soluble and stable human acceptor framework region for rabbit rabbit CDR fusion.

(Biacore binding analysis of anti-VEGF scFv)
tested Biacore binding ability of scFv, the binding affinity, by BIAcore ^TM -T100, was measured using the exemplary surface plasmon resonance method. The VEGF proteins tested for binding by these scFv candidates in this and subsequent 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 surface plasmon resonance experiments, carboxymethylated dextran biosensor chip (CM4, GE Healthcare) was prepared according to the supplier's instructions N-ethyl-N '-(3-dimethylaminopropyl) carbodiimide hydrochloride and N -Activated with hydroxysuccinimide. The six different VEGF forms exemplified above were coupled to one of four different flow cells on a CM4 sensor chip using a standard amine coupling procedure. The range of responses obtained with these immobilized VEGF molecules after coupling and blocking is approximately 250-500 response units (RU) for hVEGF ₁₆₅ , hVEGF ₁₁₀ , hVEGF ₁₂₁ , mouse VEGF ₁₆₄ , rat VEGF ₁₆₄ , and It was about 200 RU for rabbit rabbit VEGF ₁₁₀ and about 400 RU for PLGF. The fourth flow cell of each chip was treated the same, except that the protein was not immobilized prior to blocking, and this flow cell was used as an in-line reference. Various concentrations of anti-VEGF scFv (eg 90 nM, 30 nM) in HBS-EP buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20) , 10 nM, 3.33 nM, 1.11 nM, 0.37 nM, 0.12 nM and 0.04 nM) were injected into the flow cell at a flow rate of 30 μl / min for 5 minutes. Dissociation of anti-VEGF scFv from VEGF on CM4 chip could be allowed to proceed for 10 minutes at 25 ° C. After subtraction of buffered samples following in-line reference cell correction, sensorgrams were generated for each anti-VEGF scFv sample. Apparent dissociation rate constant (k _d ), apparent association rate constant (k _a ), and apparent dissociation equilibrium constant (k _D ), using BIAcore T100 evaluation software version 1.1, one to one Langmuir binding model Calculated using.

(HUVEC assay of VEGF inhibition)
The HUVEC assay is a method of measuring the efficacy of the disclosed anti-VEGF scFv candidate as a VEGF inhibitor.

Human umbilical vein endothelial cells (HUVECs) (Promocell) pooled from several donors were used at passages 2-14. The cells were 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 The cells were seeded at 1000 cells / well in 50 μl of complete endothelial cell growth medium (ECGM) (Promocell), containing (Gibco). Seven to eight hours later, 50 μl of starvation medium (ECGM without supplement with 0.5% heat inactivated FCS and 1% penicillin / streptomycin) is added to the cells and the cells are starved for 15 to 16 hours The 3-fold serially diluted anti-VEGF scFv (0.023-150 nM), and recombinant human VEGF ₁₆₅ (0.08 nM), recombinant mouse VEGF ₁₆₄ (0.08 nM) or recombinant rat VEGF ₁₆₄ (0.3 nM) One was prepared in starvation medium and preincubated for 30-60 minutes at room temperature. Different concentrations of VEGF were used to compensate for their different relative biological activities. A concentration (EC ₉₀ ) that stimulates submaximal VEGF-induced proliferation was used. 100 μl of the mixture was added to a 96-well tissue culture plate containing HUVEC suspension and incubated for 4 days in a 37 ° C./5% CO ₂ humidified incubator. After adding 20 μl / well WST-1 cell proliferation reagent (Roche) using Sunrise microplate reader (Tecan), measure the growth of HUVEC by measuring the absorbance at 450 nm (using 620 nm as reference wavelength) Evaluated by. Data were analyzed using a four parameter logistic curve fit (curve-fit) and the concentration of anti-VEGF scFv required for 50% HUVEC growth inhibition (EC ₅₀ ) was derived from the inhibition curve.

(Equivalent)
Many modifications and alternative embodiments of the present invention will be apparent to those skilled in the art in view of the foregoing description. Accordingly, this description is to be construed as illustrative only and is for the purpose of teaching those skilled in the art the best mode of carrying out the present invention. The details of the structure may be varied substantially without departing from the spirit of the invention, and possess exclusive use of all modifications which fall within the scope of the appended claims. It is intended that the present invention be limited only to the extent required by the appended claims and the applicable rules of law.

  All documents and analogues cited in this application, including patents, patent applications, editorials, books, articles, discourses, web pages, figures and / or appendices, are referred to regardless of the format of such documents and analogues Are expressly incorporated by reference in their entirety. In cases where one or more of the incorporated documents and analogues differ or conflict with this specification, including the defined terms, the usage of the terms, the described techniques or the like, the present specification will have priority.

Claims (1)

Humanization of house rabbit antibodies using a universal antibody framework.