JP2020114260A - 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 June 25, 2008, and also US 61/155,041 filed 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). , 1530-5, Dillman, Cancer Biother, 1994, vol. 9, p. 17-28). Therefore, several methods have been developed to reduce the immunogenicity of murine and other rodent antibodies, as well as techniques for producing fully human antibodies, for example using transgenic mice or phage display. .. A chimeric antibody having a rodent variable region and a human constant region together (for example, Boulianne Nature, 1984, 312, 643-6) was constructed by genetic engineering to significantly reduce the problem of immunogenicity ( 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 the humanized antibody, the rodent sequence of the variable region itself has been genetically engineered to be as close as possible to the human sequence while preserving at least the original CDR, or the CDR of the rodent antibody is a human antibody. (E.g., 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 because they usually have much higher affinity. In addition, rabbits are often able to elicit good antibody responses to antigens that are poorly immunogenic in mice and/or that do not provide a good binder when used in phage display. These well-known advantages of rabbit antibodies make them ideal for use in the discovery and development of therapeutic antibodies. The reason this is not generally done is mainly due to technical difficulties in generating 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 in providing a fusion cell line partner 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 have been identified. It was described by Pytela et al. in 2005 (see, for example, Patent Document 2). However, this technology is not widely spread, as the corresponding know-how is basically controlled by a single research group. Alternative methods for the production of monoclonal antibodies involving the cloning of antibodies from selected antibody-expressing cells via RT-PCR have been described in the literature, but the rabbit antibody has never been reported to be successful. Absent.

The rabbit antibody is expected to elicit a strong immune response when used in human therapy, similar to the mouse antibody. Therefore, rabbit antibodies need to be humanized before they can be used clinically. However, due to the structural differences between the rabbit and mouse antibodies, and between the rabbit and human antibodies, respectively, it is not possible to apply the methods used to make humanized rodent antibodies to the rabbit antibody. It's not easy. For example, the light chain CDR3 (CDRL3) is often much longer than previously known human or mouse antibody-derived CDRL3.

Although there are only a few rabbit antibody humanization approaches described in the prior art, they are not the classical fusion approach in which non-human donor CDRs are grafted onto human acceptor antibodies. US Pat. No. 6,037,058 describes a so-called “resurfacing” strategy. The goal of the "resurfacing" strategy is to reconstruct the solvent accessible residues of the non-human framework so that they are more human-like. Similar humanization techniques for rabbit antibodies, described in US Pat. Both US Pat. Nos. 5,837,639 and 5,037,058 disclose methods for humanizing rabbit rabbit monoclonal antibodies, which include comparing the amino acid sequence of a parental rabbit antibody with the amino acid sequence of a similar human antibody. The amino acid sequence of the parental rabbit antibody is then modified so that the framework regions of the parental rabbit antibody are more similar in sequence to the equivalent framework regions of similar human antibodies. In order to obtain good binding capacity, it is necessary to carry out laborious development efforts for each immunobinder individually.

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

To date, Applicants have not identified humanized rabbit antibodies by applying state of the art fusion approaches. This can be explained by the fact that the rabbit CDRs can be quite different from the human or rodent CDRs. As is known in the art, many rabbit VH chains have an additional pair of cysteines compared to their mouse and human counterparts. In addition to the conserved disulfide bridge formed between cys22 and cys92, there is also a cys21-cys79 bridge, and in some rabbit chains, the last residue of CDRH1 and the first residue of CDRH2 There are also inter-CDR SS bridges formed between the two. Moreover, paired cysteine residues are often found in CDR-L3. Moreover, 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 mouse counterpart, CDR-L3.

Therefore, fusion of non-human CDR antibodies into the human framework is a major protein engineering challenge. The transfer of antigen binding loops from naturally evolved frameworks to different artificially selected human frameworks must be done so that the natural loop conformation is retained for antigen binding. Antigen binding affinity is often greatly diminished or abolished after loop fusion. The use of a carefully selected human framework in the fusion of the antigen binding loop maximizes the probability of retaining binding affinity in the humanized molecule (Roguzka et al., 1996). Although many fusion experiments available in the literature provide a rough guide to CDR fusion, it is not possible to generalize the patterns. A typical problem is the loss of specificity, stability or productivity after fusing the CDR loops.

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

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

(Outline of the invention)
Surprisingly, highly soluble and stable humans identified by quality control (QC) assays (such as those disclosed in WO0148017 and Auf der Maur et al. (2001), FEBS Lett, 508, 407-412). It has been found that the antibody framework is particularly suitable for housing CDRs from other non-human animal species, such as rabbit CDRs. Thus, in a first aspect, the invention provides the light and heavy chain variable regions of certain human antibodies (so-called "FW1.4" antibodies). The antibody is particularly suitable as a universal acceptor for CDRs from various antibodies with different binding specificities, especially rabbit antibodies, regardless of whether disulfide bridges are present 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 versatile acceptor frameworks for fusion of rabbit CDRs. In another aspect, the invention provides motifs of framework residues that result in a human framework suitable for housing CDRs from other non-human animal species, particularly CDRs of rabbit.

Humanized immunobinders generated by fusing the rabbit CDRs into these highly compatible variable light and heavy chain frameworks consistently align the spatial orientation of the rabbit antibody, from which the donor CDRs are derived. And hold it securely. Therefore, it is not necessary to introduce a structurally important position 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.

Accordingly, in another aspect, the invention provides fusion of a rabbit CDR and other non-human CDRs into a soluble and stable light and/or heavy chain human antibody framework sequences disclosed herein, comprising: Thereby provide a method for producing a humanized antibody 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 the following items, for example.
(Item 1)
An immunobinder specific for a desired antigen comprising (i) a rabbit variable light chain CDR, and (ii) a human variable heavy chain framework having at least 50% identity to SEQ ID NO:4.
(Item 2)
The immunobinder acceptor framework of item 1, wherein the 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 of item 3, comprising a 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, particularly 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: 7. The immunobinder acceptor framework of any one of the preceding items, including the framework of 7.
(Item 7)
Threonine (T) at position 24, Valine (V) at position 25, Alanine (A) or Glycine (G) at position 56, Lysine (K) at position 82, Threonine (T) at position 84, Valine at position 89 ( V) and at least three amino acids of the group consisting of Arginine (R) at position 108 (AHo numbering), a human or humanized immunobinder variable heavy chain acceptor framework.
(Item 8)
The immunobinder acceptor framework according to any one of the preceding items, wherein the amino acids at positions 12, 103 and/or 144 (AHo numbering) have been replaced by hydrophilic amino acids.
(Item 9)
(A) 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
Item 9. The immunobinder acceptor framework according to item 8, which has (AHo numbering).
(Item 10)
In the variable light chain framework 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), at position 57 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)
A donor immunobinder, especially a mammalian immunobinder, preferably a heavy chain CDR1, CDR2 and CDR3 from a rabbit immunobinder, and/or a light chain CDR1, CDR2, and CDR3, further comprising any one of the preceding items. The described immunobinder acceptor framework.
(Item 12)
12. The immunobinder acceptor framework of item 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)
Use of the immunobinder acceptor framework according to any one of items 1 to 13 for fusion of rabbit CDRs.
(Item 15)
An immunobinder comprising the immunobinder acceptor framework according to any one of Items 1 to 13.
(Item 16)
Item 16. The immunobinder according to item 15, which is an scFv antibody.
(Item 17)
One or more binding molecules, in particular a therapeutic agent such as a cytotoxic agent, cytokine, chemokine, growth factor or other signaling molecule, an imaging agent, or a second protein, especially a transcriptional activator or DNA binding domain. The immunobinder according to any one of Items 15 to 16, further comprising:
(Item 18)
Use of the immunobinder according to any one of items 15 to 17 in diagnostic applications, therapeutic applications, targeted evaluation or gene therapy.
(Item 19)
A nucleic acid encoding the immunobinder acceptor framework according to any one of items 1 to 13 or the immunobinder according to any one of items 15 to 17, particularly an isolated nucleic acid.
(Item 20)
A vector comprising the nucleic acid according to Item 19.
(Item 21)
A host cell containing the vector of item 20.
(Item 22)
Use of the nucleic acid according to item 19 or the vector according to item 20 in gene therapy.
(Item 23)
The immunobinder acceptor framework according to any one of Items 1 to 13, the immunobinder according to any one of Items 15 to 17, the nucleic acid according to Item 19, or the vector according to Item 20. Composition.
(Item 24)
A method of humanizing a rabbit immunobinder, wherein the 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 into a human heavy chain acceptor framework having at least 50% identity to SEQ ID NO:1; and /Or (ii) a human light chain acceptor framework having at least 50% identity to SEQ ID NO: 2, ie, within the human light chain framework at least one of the group consisting of CDR L1, CDR L2 and CDR L3 sequences A method comprising fusing a light chain CDR.
(Item 25)
(I) the human heavy chain framework comprises the framework amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 4 or SEQ ID NO: 6,
(Ii) the 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)
26. The method according to item 24 or 25, further comprising the step of substituting a framework residue with a framework residue of the rabbit immunobinder involved in antigen binding.
(Item 27)
Substitutions in at least one of the heavy chain amino positions 12, 103, and 144 (AHo numbering), particularly by substitution with hydrophilic amino acids, preferably in the serine (S), (b) position at (a) position 12; From item 24, further comprising the step of substituting framework residues of the heavy chain acceptor framework by a substitution selected from the group consisting of threonine (T) at 103, and (c) threonine (T) at position 144. 27. The method according to any one of 26.
(Item 28)
A 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 (V) at position 3, (c) leucine (L) at position 4, (d) serine (S) at position 10, (e) arginine (R) at position 47, (e) at position 57 Substitution of framework residues of the light chain acceptor framework with a substitution selected from the group consisting of serine (S), phenylalanine (F) at position 91, and valine (V) at position (g) 103. 28. The method according to any one of items 24-27, further comprising:
(Item 29)
A humanized immunobinder according to the method according to any one of items 24 to 28.
(Item 30)
30. The immunobinder according to item 29, wherein the target antigen is VEGF or TNFα.
(Item 31)
a) incubating the rabbit B cells with a target antigen, b) selecting the rabbit B cells that bind to the target antigen, and c) identifying the CDRs of the antibody variable domains produced by the B cells, 29. The method of any of paragraphs 24-28, further comprising identifying donor CDRs of antibody variable domains produced by B cells.
(Item 32)
32. The method of item 31, wherein the B cells are isolated from rabbits immunized against the target antigen, preferably by DNA vaccination.
(Item 33)
33. Item 31 or 32, wherein the B cells and the target antigen are stained differently and the co-localization 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. The method according to any one of items 31 to 33, wherein the target antigen is expressed on the surface of cells, in particular the target antigen is a transmembrane protein, in particular a multiple transmembrane protein.
(Item 36)
Item 36. The method according to Item 35, wherein the target antigen is indirectly stained by staining the cells expressing the target antigen with an intracellular fluorescent dye.
(Item 37)
37. The method of any of paragraphs 31-36, wherein the B cells are stained with one or more labeled antibodies specific for B cell surface markers.
(Item 38)
38. The method of any of paragraphs 31-37, wherein the B cells are stained with a labeled anti-IgG antibody.
(Item 39)
39. The method of item 38, wherein the B cells are stained with a labeled anti-IgG antibody and a differently labeled anti-IgM antibody.
(Item 40)
40. The method of item 39, wherein B cells stained with the labeled anti-IgM antibody are negatively selected.
(Item 41)
From item 31, further comprising the step of culturing the B cells selected in step b) according to item 31, such that the produced antibody is secreted into the medium, especially in the presence of a helper cell line. 40. The method according to any one of 40.
(Item 42)
42. The method of any of paragraphs 41, further comprising testing the secreted antibody for specific binding to the target antigen.
(Item 43)
The CDRs of the antibody are amplified, fused by an RT-PCR, fused into an acceptor framework to produce an immunobinder, and subsequently tested for specific binding to the target antigen. 42. The method according to 42.
(Item 44)
For the fusion of a rabbit CDR, 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. 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 a linker having SEQ ID NO: 8.

FIG. 1 shows the definition of CDR H1 used here to fuse the antigen binding site from a rabbit monoclonal antibody into a highly soluble and stable human antibody framework. FIG. 2 schematically shows B cells 1 labeled with a fluorescent antibody 2 that interact with target expressing cells 3 stained with intracellular dye 4. Selected targets: 5; BCR: 6. FIG. 3 shows the FACS selection process of rabbit B cells binding to ESBA903 soluble target. Figure 3A: Lymphocytes are gated by forward and side scatter. Figure 3B: Among them IgG+IgM- cells (probably memory B cells) are selected (red gate). Figure 3C: Cells double stained with ESBA903-PE and ESBA903-PerCP (green gate) are predicted to encode high affinity IgG for ESBA903. The cells with the brightest fluorescence (pink gate) were sorted into 96-well plates. FIG. 4 shows that beads coated with anti-TNFα antibody (PE-labeled) bind to TNFα-transfected CHO cells (upper panel), control beads coated with anti-CD19 antibody (APC-labeled) are TNFα-transfected CHO cells. It does not bind (middle panel) and beads coated with anti-TNFα antibody (PE labeled) do not bind to wild type (wt) CHO cells (lower panel). The dot plots on the left show forward and side scatter, which show the size and granularity of the event, respectively. Single bead (approximately 3 μm) populations are gated on P2. Finally, the CHO cells (about 30 μm) 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 at the P3 gate, and if they interact with anti-CD19 beads, they appear at the P4 gate. On the right side, the statistical details for each sample are shown. FIG. 5 shows that anti-TNFα-PE coated beads and anti-CD19APC coated beads were mixed with TNFα-transfected CHO cells and CHO cells were gated (P1), among which anti-TNFαPE was coated. Cells binding either to the coated beads or to the beads coated with anti-CD19-APC are shown at gates P3 and P4, respectively, indicating that unbound beads appear at gate P2. FIG. 6 is an analysis of rabbit antibody sequences extracted from the Kabat database, confirming that the 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 making the present invention more easily understandable, specific terms are defined as follows. Further definitions are given throughout this detailed description.

The term "antibody" refers to whole antibodies and any antigen binding fragments. The terms "antigen binding polypeptide" and "immunobinder" are used herein simultaneously. An "antibody" refers to a protein or antigen-binding portion thereof that comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds and is optionally glycosylated. Each heavy chain comprises a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, 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, CL. The _VH and _VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) and more conserved regions called framework regions (FRs) interspersed with hypervariable regions. Each V _H and V _L is composed of 3 CDRs and 4 FRs, arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from amino-terminus to carboxy-terminus. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of an antibody may mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (Clq).

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 an antigen (eg, TNF). It has been shown that the antigen binding function of the antibody can be performed by fragments of the full length antibody. Examples of binding fragments encompassed by the term “antigen-binding portion” of an antibody include (i) Fab fragments that are monovalent fragments consisting of V _L , V _H , CL and CH1 domains, (ii) hinge region disulfides. F(ab′) ₂ fragment, a bivalent fragment containing two Fab fragments linked by a bridge, (iii) Fd fragment consisting of V _H and CH1 domains, (iv) single arm _VL and V _H of antibody. Fv fragment consisting of a domain, (v) a single domain or dAb fragment consisting of one _VH domain (Ward et al. (1989) Nature 341:544-546), and (vi) isolated complementarity. Determinant regions (CDRs), or (vii) a combination of two or more isolated CDRs optionally linked by synthetic linkers. Furthermore, the two domains of the Fv fragment, _VL and _VH , are encoded by separate genes, but they use recombinant methods with synthetic linkers that allow them to be made into 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); eg Bird et al. (1988) Science, 242, 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA, 85, 5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art and screened for utility in the same manner as intact antibodies. The antigen-binding portion may be produced by recombinant DNA technology or may be produced by enzymatic or chemical cleavage of intact immunoglobulin. Antibodies can be of different isotypes, eg, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

The term "immunobinder" refers to a molecule that contains 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, and thus an immunobinder is It specifically recognizes the target antigen. Non-limiting examples of immunobinders include full-length immunoglobulin molecules and scFv, as well as, but not limited to, (i) Fab fragments, ie, V _L domain, V _H domain, C _L domain and C _H 1. A monovalent fragment consisting of a domain; (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′ fragment that is a Fab having part of the hinge region (see FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd edition, 1993)); (iv) Fd fragment consisting of V _H domain and C _H 1 domain; v) an Fv fragment consisting of the single-arm _VL and _VH domains of the antibody; (vi) a Dab fragment consisting of the _VH or _VL domain (Ward et al. (1989) Nature 341:544-546). , Camel antibodies (see Hamers-Casterman et al., Nature 363: 446-448 (1993), and Dumoulin et al., Protein Science 11: 500-515 (2002)), or shark antibodies (eg. , A single domain antibody such as shark Ig-NAR Nanobodies®; and (vii) an antibody fragment comprising a heavy chain variable region containing a single variable domain and two constant domains, Nanobodies. To be

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

As used herein, the term “functional property” refers to those skilled in the art (eg, as compared to a conventional polypeptide), eg, to improve the manufacturing properties or therapeutic efficacy of the polypeptide. It is a property of a polypeptide (eg, an immunobinder) that is desirable and/or advantageous for improvement. In one embodiment, the functional characteristic is stability (eg, thermal stability). In another embodiment, the functional property is solubility (eg, under cellular conditions). In yet another embodiment, the functional characteristic is aggregation behavior. In yet another embodiment, the functional property is protein expression (eg, in prokaryotic cells). In yet another embodiment, the functional property is refolding behavior after solubilization of inclusion bodies in the manufacturing process. In certain embodiments, the functional property is not improved 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" means one of the six hypervariable regions within the variable domain of an antibody that contribute primarily to antigen binding. One of the most commonly used definitions for the six CDRs is Kabat E. et al. A. Et al. (1991) Sequences of proteins of immunological interest. , NIH Publication, pp. 91-324. As used herein, the definition of Kabat for CDRs refers to the light chain variable domains CDR1, CDR2, and CDR3 (CDR L1, CDR L2, CDR L3, or L1, L2, L3) and heavy chain variable domains. Only applies to CDR2 and CDR3 (CDR H2, CDR H3, or H2, H3). However, as used herein, the CDR1 of the heavy chain variable domain (CDR H1 or H1) 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 which is defined differently by Kabat and Chotia (see also Figure 1 for illustration).

As used herein, the term "antibody framework" refers to that portion of the variable domain, either VL or VH, which acts as a scaffold for the antigen binding loops (CDRs) of the variable domain. In essence, it is a CDR-free variable domain.

The term “epitope” or “antigenic determinant” refers to the site on an antigen that an immunoglobulin or antibody specifically binds (eg, a specific site on a TNF molecule). Epitopes usually include 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”, Volume 66, G.I. E. See Morris, edited (1996).

The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to antibody binding to an epitope of a given antigen. Generally, antibodies bind with an affinity (K _D ) of less than about 10 ⁻⁷ M, such as less than about 10 ⁻⁸ M, less than about 10 ⁻⁹ M or less than about 10 ⁻¹⁰ M or even lower.

The term "K _D" or "Kd" refers to a particular antibody - refers to the dissociation equilibrium constant of antigen interaction. Generally, 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, as determined using, for example, the Surface Plasmon Resonance (SPR) technique of the BIACORE device. It binds to TNF with a dissociation equilibrium constant (K _D ) of less than about 10 ⁻⁷ M, such as the value.

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

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it 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 can be ligated. In another embodiment, the vector is a viral vector, in which case additional DNA segments may be ligated into the viral genome. The vectors disclosed herein may be those 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 one which is integrated into the genome of the host cell upon introduction into the host cell and is thereby able to replicate with the host genome (eg a non-episomal mammalian vector).

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 a taxonomic member of the order Lagomorpha and includes the families Leporidae (eg, hares and rabbits) and Ochotonidae (pika). In the most preferred embodiment, the rabbit is a rabbit. The term "rabbit" as used herein refers to an animal belonging to the family Leporidae.

As used herein, "identity" means a sequence that matches between two polypeptides, molecules, or between two nucleic acids. If a position in both compared two sequences is occupied by the same base or the same amino acid monomer subunit (eg, a position in each two polypeptides is occupied by lysine), respectively, The molecules of are identical at that position. The "percent identity" between two sequences is shared by those sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences. It is a function of the number of identical positions. Generally, the comparisons are made when the two sequences are aligned for maximum identity. Such an alignment may be, for example, either a Blossum62 matrix or a PAM250 matrix, and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6. Can be provided using the algorithmic method of Needleman and Wunsch (J. Mol. Biol., (48), 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 trial of the present invention, suitable methods and materials are those described below. In case of conflict, the present specification, including definitions, will control. Additionally, 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 combined in any combination. Furthermore, selected definitions, embodiments, or ranges may not apply in some particular embodiments.

Unless otherwise stated, amino acid positions are indicated according to the AHo numbering scheme. The AHo numbering system is described by Honegger, A.; And Pluckthun, A.; (2001), J. Mol. Biol. 309, pp. 657-670). Instead, Kabat, et al. The Kabat numbering system described in more detail can be used. A conversion table for the two different numbering systems used to identify amino acid residue positions within antibody heavy and light chain variable regions is provided in A. Honegger, J.; Mol. Biol. 309 (2001), pp. 657-670.

In a first aspect, the invention provides a universal acceptor framework for fusing CDRs from other animal species, eg, rabbit. It has been previously described that antibodies or antibody derivatives comprising the human framework identified in so-called "quality control" screens (WO0148017) are generally characterized by high stability and/or solubility. The human single-chain framework FW1.4 (a combination of SEQ ID NO: 1 (designated a43 in WO 03/097697) and SEQ ID NO: 2 (designated K127 in WO 03/097697)) was used in quality control assays. Despite its apparently poor performance, it was surprisingly found that the framework had a high degree of intrinsic thermodynamic stability and good reproducibility when combined with different CDRs. Was issued. The stability of this molecule can be attributed, in large part, to its framework regions. It has further been shown that FW1.4 is essentially compatible with the antigen binding site of the rabbit antibody. Therefore, FW1.4 is a suitable scaffold for constructing stable humanized scFv antibody fragments resulting from fusion of rabbit loops. 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 CDR A sequence of FW1.4 (SEQ ID NO:3) for the fusion of at least 60%, more preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 95% of SEQ ID NO:3. An immunobinder acceptor framework is provided that comprises sequences having identity.

Furthermore, it was found that FW1.4 can be optimized by substituting some residue positions in the heavy chain of FW1.4 and/or by substituting one position in the light chain of FW1.4. Was done. Thereby, it was surprisingly found that the loop conformation of the extensive rabbit CDRs in VH can be completely maintained, largely independent of the sequences of the donor framework. The residue in the heavy chain of FW1.4 and one position in the light chain are conserved in the rabbit antibody. A consensus residue at that position in the heavy chain and one position in the light chain was deduced from the rabbit repertoire and 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. Furthermore, rFW1.4 containing various rabbit CDRs was well expressed and produced well as opposed to the rabbit wild-type single chain, yet almost completely retained the affinity of the original donor rabbit antibody. To do.

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

In a preferred embodiment, the invention provides threonine (T) at position 24, valine (V) at position 25, alanine (A) or glycine (G) at position 56, threonine (T) at position 84, 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, and more preferably 4, 5, 6 At least 50%, more preferably at least 55%, 60%, 65%, 70%, 75%, 80%, 85% in SEQ ID NO: 4, provided that there are residues, most preferably 7 residues. An immunobinder acceptor framework comprising VHs having 90%, 95%, and even more preferably 100% identity is provided. In a preferred embodiment, the immunobinder acceptor framework is the immunobinder acceptor framework for rabbit CDRs.

In a preferred embodiment, the variable heavy chain framework is or comprises SEQ ID NO:4 or SEQ ID NO:6. Both of the variable heavy chain frameworks can 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 An immunobinder acceptor framework comprising a variable light chain framework having at least 70%, preferably at least 75%, 80%, 85%, 90%, and more preferably at least 95% identity to number 2 is provided.

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

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

In a preferred embodiment, the immunobinder acceptor framework is
(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. , Volume 8, pages 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.

Moreover, it has been surprisingly found that the presence of the above-mentioned amino acid motifs makes the framework, preferably the human framework, particularly suitable for the accommodation of CDRs from other non-human animal species, in particular rabbit CDRs. Was issued. The motif does not have a negative impact on the stability of the immunobinder. The CDRs appear in a conformation that resembles their natural spatial orientation in the rabbit immunobinder, so that no structurally significant position need be fused to the acceptor framework. Thus, a human or humanized immunobinder acceptor framework has threonine (T) at position 24, valine (V) at position 25, alanine (A) or glycine (G) at position 56, lysine (K) at position 82. , Threonine (T) at position 84, valine (V) at position 89 and arginine (R) at position 108 (AHo numbering), at least 3 amino acids, preferably 4, 5, 6 amino acids, more preferably 7 amino acids. Contains amino acids.

The immunobinder acceptor frameworks described herein may include solubility-enhancing substitutions at the heavy chain frameworks, preferably positions 12, 103 and 144 (AHo numbering). Hydrophobic amino acids are preferably replaced by more hydrophilic amino acids. Hydrophilic amino acids include, for example, arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glycine (G), histidine (H), lysine (K), serine (S) and threonine. (T). More preferably, the heavy chain framework is (a) serine (S) at position 12, (b) serine (S) or threonine (T) at position 103, and/or (c) serine (S) at position 144. Alternatively, it 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 is: 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 at 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 contains a glycine (G) at position 141 because glutamine (Q) is prone to deamination. This substitution may improve 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 were derived. Thus, in a preferred embodiment, the invention provides a heavy chain CDR1, CDR2 and CDR3, and/or a light chain CDR1 from a donor immunobinder, preferably a mammalian immunobinder, more preferably a rabbit immunobinder, most preferably a rabbit. Includes the immunobinder acceptor frameworks disclosed herein, further comprising CDR2, and CDR3. Thus, in one embodiment, the present invention provides
(I) a variable light chain CDR of rabbit;
(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 And a human variable heavy chain framework having an immunobinder specific for a desired antigen.

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

Preferably the rabbit is a rabbit. More preferably, the immunobinder comprises heavy chain CDR1, CDR2 and CDR3 from the donor immunobinder and light chain CDR1, CDR2 and CDR3.

As is known in the art, many rabbit VH chains have additional paired cysteines compared to their mouse and human counterparts. In addition to the conserved disulfide bridge formed between cys22 and cys92, there is also a cys21-cys79 bridge, and in some rabbit chains, the last residue of CDRH1 and the first residue of CDRH2 There are also inter-CDR SS bridges formed between the two. Moreover, in CDR-L3, counter-cysteine residues are often found. Moreover, 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 mouse counterpart, CDR-L3.

As mentioned above, fusion of a non-human CDR to the frameworks disclosed herein yields a molecule in which the CDR appears in the proper conformation. If required, 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,
It can be identified by (ii) generating a sequence alignment of the germline precursor or consensus sequence of step (i) with the donor variable domain sequence, and (iii) identifying the residues that differ.

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

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

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

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

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

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 containing the vectors and/or nucleic acids disclosed herein.

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

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

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

General methods of fusing CDRs into the human acceptor framework are disclosed by Winter in US Pat. No. 5,225,539, by Queen et al. in WO9007861 A1, which is incorporated herein by reference in its entirety. The general strategy for fusing the CDRs from the rabbit monoclonal antibody to the framework of choice relates to that of Winter et al. and Queen et al., but differs in certain key respects. In particular, the method of the present invention is particularly suited to the human antibody frameworks disclosed herein as a universal acceptor for human or non-human donor antibodies, in that typical Winter and Queen known in the art. Different from the methodology. Thus, unlike the general method of Winter and Queen, the framework sequences used in the humanization methods of the invention do not necessarily have the best sequence similarity to the sequence of the non-human (eg, rabbit) antibody from which the donor CDRs were derived. It is not a framework sequence showing sex. In addition, no framework residue fusions from the donor sequence are required to support the CDR conformation. At most, antigen binding amino acids located in the framework or other mutations that occurred during somatic hypermutation can be introduced.

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

Accordingly, the invention provides a method of humanizing a rabbit CDR donor immunobinder comprising heavy chain CDR1, CDR2 and CDR3 sequences, and/or light chain CDR1, CDR2 and CDR3 sequences. This method
(I) 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 on the heavy chain. Fusion of at least one, preferably two, and more preferably three CDRs of the group consisting of CDR1, CDR2 and CDR3 sequences into a human heavy chain acceptor framework having sex properties, and/or (ii) Have 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 Fusing at least one, preferably two, and more preferably three CDRs of the group consisting of CDR1, CDR2, and CDR3 sequences into the human light chain acceptor framework.

In a preferred embodiment, the variable chain acceptor framework is (i) a human heavy chain framework comprising a framework amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, and SEQ ID NO:6; And 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 the immunobinder having at least 75%, 80%, 85%, 90%, and 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 more preferably 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 residues with a donor residue involved in antigen binding.

In an exemplary embodiment of the methods of the invention, the amino acid sequence of the CDR donor antibody is 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 done 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 as it is the most widely adopted standard method for numbering residues in antibodies. Kabat numbering can be assigned using, for example, 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 Kabat's definition, which is based on sequence variability and is the most commonly used. However, for CDR-H1, such a designation is based on the Kabat definition, mean contact data generated by analysis of contacts between antibodies and antigens of a subset of 3D complex structures (MacCallum et al., 1996), And a combination of the definitions of Chotia based on the position of structural loop regions (see also FIG. 1). A conversion table for two different numbering systems used to identify amino acid residue positions in the variable regions of antibody heavy and light chains is provided in A. Honegger, J.; Mol. Biol. 309 (2001), pp. 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, US Department of Health N. H. Num. , In more detail. The AHo numbering system is described by Honegger, A.; And Pluckthun, A.; (2001), J. Mol. Biol. , 309, pp. 657-670, in more detail.

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

CDR conformations can be assigned to the donor antigen binding regions and subsequently the residue positions required to maintain the various canonical structures identified. The canonical structure of 5 of the 6 antibody hypervariable regions (L1, L2, L3, H1 and H2) of the rabbit antibody is determined using the definition of Chothia (1989).

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

In the case of (ii), the cell expressing the target antigen may be, for example, a mammalian cell, preferably CHO, which naturally expresses the selected target or has been transformed to express the target protein on its surface. It can be a HEK293 cell, a yeast cell, preferably a yeast spheroplast, or a bacterial cell. Upon expression, the target antigen may be incorporated into the cell membrane or bound to the cell membrane and expressed on the cell surface. The cells may be cultured as isolates, eg in cell culture, or isolated from their natural environment, eg a tissue, organ or organism.

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

In a subsequent step of the method, B cells, preferably memory B cells, are isolated from the lymphatic vessels (such as the spleen or lymph nodes) of the immunized animal, preferably the immunized rabbit. These B cells are incubated in cells expressing the antigen on the surface or in a mixture with soluble, fluorescently labeled antigen. B cells that express target-specific antibodies on their surface and thus bind the target antigen, or the target antigen expressed on the cell surface, are isolated. 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 by a single cell as it traverses a laser beam. However, some investigators have already reported that cell-cell interactions such as cadherin (Panorchan et al., 2006, J. Cell Science, 119, 66-74; Leong et Hibma, 2005, J. Immunol. Methods, 302, pp. 116-124) or integrins (Gawaz et al., 1991, J. Clin. Invest, 88, pp. 1128-1134) using a cytometer. Therefore, in the case of (ii), cells expressing the selected target are stained with an intracellular fluorescent dye (eg, calcein). B cells are stained with a fluorescent antibody that binds to a cell surface specific marker. Therefore, one can select a two-color "event" that resides in two cells adhering to each other via a B cell receptor-target interaction (see Figure 2).

Since IgG usually has a higher affinity than IgM, it is preferable to select positive B cells that express IgG rather than IgM on their surface, which is characteristic of memory B cells. For this purpose, it is preferred to use multicolor stains in which antibodies specific for IgG and IgM are differentially labeled, eg with APC and FITC, respectively.

In a particular embodiment, a read-out for B cell sorting can also be selected for its ability to interact 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 the GPCR can be added to the mixture to induce GPCR-mediated Ca2+ efflux from the endoplasmic reticulum. When antibodies presented to B cells functionally block agonist signaling, this cell-cell interaction also blocks Ca2+ efflux. Ca2+ efflux can be quantitatively measured by flow cytometry. Therefore, only B cell/target cell aggregates showing either increased or decreased Ca2+ efflux are sorted.

Following the selection step, B cells are cultured under conditions suitable for the antibody to be secreted into the medium. The antibody produced is a monoclonal antibody. Culturing may require the use of a helper cell line, such as the pleurocytoma helper cell line (see, eg, EL4-B5, Zubler et al., 1985, J. Immunol., 134(6), 3662-3668). For example, in order to exclude antibodies raised against proteins expressed on the cell surface other than the target protein, it is preferable to perform a verification step of testing the production of antibodies that specifically bind 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 this purpose. The method allows evaluation of the selectivity and ability of antibodies to compete with ligand.

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

Finally, the CDRs can be fused to an acceptor framework, preferably the above-mentioned acceptor frameworks, 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 will be grafted from the donor antibody into a single acceptor framework and all three CDRs from the light chain will be grafted into another acceptor framework. Some CDRs may not be involved in binding to the antigen and CDRs with different sequences (and the same length) may have the same folding (thus despite different sequences the antigen It is expected that there will not always be a need to graft all of these CDRs, since contact can be retained from to the backbone contact). In fact, even a single domain (Ward et al., 1989, Nature, 341, 544-546) or even a single CDR (R. Taub et al., 1989, J. Biol Chem, 264, 259-265) alone. Can have antigen-binding activity. However, whether all CDRs or only some CDRs are transplanted, the intent of CDR fusion is to transfer the same or nearly the same antigen binding site from an animal to a human antibody (eg, , US Pat. No. 5,225,539 (Winter)).

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

Instead, antibody characterization is done only in their final immunobinder format. For this approach, the CDR sequences of the antibody expressed on the sorted B cells were directly analyzed by RT-PCR from a culture of the sorted B cells or from a single cell of the sorted B cells. to recover. For that purpose, B cell proliferation and/or the validation steps described above and/or the analysis steps described above may be unnecessary. A one-step PCR procedure with a combination of two pooled oligonucleotides with partial overlap, one oligonucleotide pool encoding the CDRs and a second pool encoding the framework regions of a suitable immunobinder scaffold. It is possible to produce a humanized immunobinder in. Instead of characterizing the secreted IgG in cell culture supernatants, high throughput sequencing, cloning, and production allow performance-based clonal selection of purified humanized immunobinder. In its preferred embodiment, the immunobinder is a scFv.

However, the fusion of the CDRs can result in a loss of affinity of the generated immunobinder for the antigen due to framework residues that are in contact with the antigen. Such interactions may be the result of somatic hypermutation. Therefore, fusion of such donor framework amino acids to the framework of the humanized antibody may still be necessary. The non-human immunobinder amino acid residues involved in antigen binding can be identified by examination of the rabbit monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that is different from the germline can be considered significant. If the closest germline cannot be established, the sequences can be compared against a subgroup consensus or a consensus of rabbit sequences 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.

Antibodies of the invention can be further optimized to exhibit enhanced functional properties, such as enhanced solubility and/or stability. In certain embodiments, the antibodies of the invention are PCT application numbers entitled "Sequence Based Engineering and Optimization of Single Chain Antibodies," filed March 12, 2008, which is hereby incorporated by reference. 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 be shown thereby that such identified residue positions may be suitable for manipulation to improve functionality such as stability and/or solubility.

Exemplary framework residue positions for the substitutions and exemplary framework substitutions are described in "Applications for Modifying Antibiotics with Improved Functional Properties," filed on June 25, 2008, PCT, entitled "Methods of Modifying Antibodies, and Modified Antibiotic Functional Properties." 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 can be introduced at amino acid positions in the heavy chain variable region of the immunobinder of the invention (see AHo numbering 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) T, S, or A at amino acid position 7, more preferably T or A, even more preferably T;
(D) A, T, P, V, or D at amino acid position 10, more preferably T, P, V, or D,
(E) L or V at amino acid position 12, more preferably L,
(F) V, R, Q, M, or K at amino acid position 13, more preferably V, R, Q, or M;
(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 at amino acid position 19, more preferably L;
(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 at amino acid position 21, more preferably I, F, or L, even more preferably I or L;
(K) R or K at amino acid position 45, more preferably K;
(L) T, P, V, A or R at amino acid position 47, more preferably T, P, V, or R, even more preferably R;
(M) K, Q, H, or E at amino acid position 50, more preferably K, H, or E, even more preferably K;
(N) M or I at amino acid position 55, more preferably I;
(O) K or R at amino acid position 77, 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 at amino acid position 82, more preferably E, T, or A, even more preferably E;
(R) T, S, I, or L at amino acid position 86, more preferably T, S, or L, even more preferably T;
(S) D, S, N, or G at amino acid position 87, more preferably D, N, or G, even more preferably N;
(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) D, Q, or E at amino acid position 92, 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, even more preferably Y, even 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 can be introduced into the light chain variable region of the immunobinder of the invention:
(Aa) Q, D, L, E, S, or I at amino acid position 1, 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) Q, V, T, or I at amino acid position 3, 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) T or I at amino acid position 10, more preferably I;
(Gg) A or V at amino acid position 11, more preferably A;
(Hh) S or Y at amino acid position 12, more preferably Y;
(Ii) T, S, or A at amino acid position 14, more preferably T or S, even more preferably T;
(Jj) S or R at amino acid position 18, more preferably S;
(Kk) T or R at amino acid position 20, more preferably R;
(Ll) R or Q at amino acid position 24, more preferably Q;
(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 at amino acid position 50, more preferably Q, K, E, T or M;
(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 at amino acid position 56, more preferably M;
(Rr) H, S, F, or Y at amino acid position 57, more preferably H, S, or F;
(Ss) I, V, or T at amino acid position 74, more preferably V or T, R, even more preferably T;
(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) G, D, T, or A at amino acid position 92, more preferably G, D, or T, even more preferably T;
(Xx) S or N at amino acid position 94, more preferably N;
(Yy) F, Y, or S at amino acid position 101, more preferably Y or S, even more preferably S; and (zz) at amino acid position 103, D, F, H, E, L, A, T. , V, S, G, or I, more preferably H, E, L, A, T, V, S, G, or I, even more preferably A or V.

In another embodiment, the immunobinder of the present invention has a stability described in US Provisional Application No. 61/075,692, filed June 25, 2008, entitled “Solubility Optimization of Immunobinders”. One or more mutations that enhance In certain preferred embodiments, 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) a serine (S) at heavy chain amino acid position 12; (b) a serine (S) or threonine (T) at heavy chain amino acid position 103; and (c) a heavy chain amino acid. It contains one or more substitutions at position 144 selected from the group consisting of serine (S) or threonine (T). In another embodiment, the immunobinder has 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; and ( c) Contains a heavy chain amino acid position 144 at serine (S) or threonine (T).

In certain preferred embodiments, the immunobinder has a light chain acceptor framework at 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. Contains a stability-enhancing mutation at a framework residue of. In a preferred embodiment, the light chain acceptor framework is (a) glutamic acid (E) at position 1, (b) valine (V) at position 3, (c) leucine (L) at position 4, (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 (V) at position 103. ) Including one or more substitutions selected from the group consisting of:

Any of a variety of available methods for producing humanized antibodies containing the mutations described above can be used.

Accordingly, the present invention provides immunobinders that are humanized according to the methods described herein.

In certain preferred embodiments, the immunobinder target antigen 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, the nucleic acid molecule encoding the desired variable region can be synthesized and the polypeptide produced by recombinant methods.

For example, once the humanized variable region sequence has been determined, the variable region or a polypeptide containing it can be produced 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 a host cell with a nucleic acid sequence (eg, a DNA sequence encoding a desired variable region ( Modified heavy or light chains; variable domains or other antigen binding fragments thereof)).

In one embodiment, expression vectors can be prepared that include a promoter operably linked to a DNA sequence encoding at least _VH or _VL . If necessary or desired, a second expression vector containing a promoter operably linked to the DNA sequence encoding the complementary variable domain can be prepared (ie, the parental expression vector encodes _VH and 2 expression vectors encode _VL , and vice versa). A cell line (eg, an immortalized mammalian cell line) can then be transformed with one or both of the expression vectors and cultured under conditions that allow expression of the chimeric variable domain or antibody (eg, See International Patent Application No. PCT/GB85/00392 to Neuberger et al.).

In one embodiment, variable regions can be created that include the amino acid sequences of the donor CDRs and the acceptor FRs, and then changes introduced into the nucleic acid molecule to result in CDR amino acid substitutions.

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

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

PCR mutagenesis is also suitable for making amino acid sequence variants of the polypeptide. Higuchi, PCR Protocols, pp. 177-183 (Academic Press, 1990); Vallette et al., Nuc. Acids Res. , 17:723-733 (1989). Briefly, when a small amount of template DNA is used as a starting material in PCR, a primer having a sequence slightly different from the corresponding region of the template DNA is used, and the primer is different from the template sequence only at a position different from the template. It is possible to produce relatively large amounts of specific DNA fragments.

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 mutagenized DNA. Identify the codon(s) in the mutagenized parental DNA. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If such a restriction enzyme recognition site does not exist, it can be generated using the above-described oligonucleotide-mediated mutagenesis method of introducing the 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. A double-stranded oligonucleotide encoding the sequence of DNA between the restriction enzyme recognition sites, but containing the desired mutation(s), was synthesized using standard procedures, wherein the two strands of the oligonucleotide are Are synthesized separately 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 that are compatible with the ends of the linearized plasmid so that it can be directly ligated to the plasmid. This plasmid now contains the mutagenized DNA sequence.

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

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

A family of amino acid residues having similar side chains has been defined in the art, including basic side chains (e.g. lysine, arginine, histidine), acidic side chains (e.g. aspartic acid, glutamic acid), Uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta branching Examples include side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine).

Apart from amino acid substitutions, the present invention also contemplates other modifications to the Fc region amino acid sequence, eg, to produce Fc region variants with altered effector function. For example, one or more amino acid residues in the Fc region can be removed to reduce or enhance binding to FcR. In one embodiment, one or more Fc region residues can be modified to produce 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. To do.

An amino acid insertion Fc region variant having a modified effector function can also be produced. For example, at least one amino acid residue adjacent to one or more Fc region positions identified herein as those that impact FcR binding (eg, 1-2 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 in the Fc region. In one embodiment, amino acid modifications can be combined. For example, a variant Fc region may have substitutions therein, for example 2, 3, 4, 5 etc. of particular Fc region positions identified herein. In another embodiment, the polypeptide may have altered binding to FcRn and to another Fc receptor.

In one embodiment, the polypeptides described in the present invention, or the polypeptides produced by the methods of the present invention, eg, humanized Ig variable regions and/or polypeptides comprising humanized Ig variable regions, are recombinant. Can be produced by a method. For example, the polynucleotide sequence encoding the polypeptide can be inserted into an expression vector suitable for recombinant expression. When the polypeptide is an antibody, additional light chain variable region and heavy chain variable region encoding polynucleotides, optionally linked to constant regions, can be inserted into the same or different expression vectors. Affinity tag sequences (eg, His(6) tags) can optionally be attached or included in the polypeptide sequence to facilitate downstream purification. The DNA segment encoding the immunoglobulin chain is operably linked to the control sequences in the expression vector(s) that ensure the expression of the immunoglobulin polypeptide. Expression control sequences include, but are not limited to, promoters (eg, naturally associated or heterologous promoters), signal sequences, enhancer elements and transcription termination sequences. The expression control sequence is preferably a eukaryotic 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 nucleotide sequences and collection and purification of the polypeptide.

These expression vectors are usually replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Generally, the expression vector contains 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 cells that is particularly useful for cloning the polynucleotides (eg, DNA sequences) of the 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 microorganisms such as yeast are also useful for expression. Saccharomyces and Pichia are exemplary yeast hosts, suitable vectors optionally containing expression control sequences (eg, promoters), origins of replication, termination sequences, and the like. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, inter alia, promoters from alcohol dehydrogenase, isocytochrome C, and promoters from the enzyme group responsible for methanol, maltose and galactose utilization.

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

In addition to microorganisms, mammalian tissue culture 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.M. Y. , N.; See Y (1987). Since 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. Lines, HeLa cells, 293 cells, myeloma cell lines, transformed B cells and hybridomas. Expression vectors for these cells include replication origins, expression control sequences such as promoters and enhancers (Queen et al., Immunol. Rev., 89, 49 (1986)), as well as a ribosome binding site, an RNA splicing site. , A polyadenylation site, a transcriptional terminator sequence, and other necessary processing information sites. Preferred expression control sequences are promoters derived 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, heavy and light chain coding sequences and expression control sequences) can be introduced into host cells by well-known methods (depending on the type of cell 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 cell hosts (general Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd edition, 1989). Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion. , Liposomes, electroporation, and microinjection (see, generally, Sambrook et al., supra.) Transgenic animal production includes microinjection of the transgene into fertilized oocytes, or It can be incorporated into the genome of embryonic stem cells and the nucleus of such cells can be introduced into enucleated oocytes.

The polypeptide of interest can also be incorporated into a transgene for introduction into the genome of the transgenic animal and subsequent expression in, for example, 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 promoters and enhancers from mammary gland-specific genes such as casein or beta-lactoglobulin.

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

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

Once expressed, the polypeptide can be expressed using standard procedures in the art, including ammonium sulfate precipitation, affinity columns (eg Protein A or Protein G), column chromatography, HPLC purification, gel electrophoresis, etc. In general, see Scopes, Protein Purification (Springer-Verlag, NY (1982))).

Humanized Ig variable regions or polypeptides containing them can be expressed by host cells or cultured cell lines. They can also be expressed in cells in vivo. The cell line transformed (eg, transfected) to produce the modified antibody can be an immortalized mammalian cell line, eg, of lymphoid origin (eg, myeloma, hybridoma). , Trioma or quadroma cell lines). The cell line can also include normal lymphoid cells, such as B cells, which have been immortalized by transformation with a virus (eg, Epstein-Barr virus).

The cell lines used to produce the polypeptides are typically mammalian cell lines, although cell lines from other sources, such as bacteria and yeast, can also be used. In particular, E. Bacterial strains 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, secrete isolated Ig light or heavy chains in their normal state. When such cell lines are transformed with a vector expressing a modified antibody prepared during the method of the invention, the normally secreted chain is the variable domain of the Ig chain encoded by the previously prepared vector. 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 does not 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, be transformed into a suitable bacterial cell with the vector, and then the bacterial cell may be transformed with the immortalized cell line (eg, , By spheroplast fusion). Alternatively, DNA can be introduced directly into the immortalized cell line by electroporation.

In one embodiment, the humanized Ig variable regions described in this invention, or the humanized Ig variable regions produced by the methods of this invention, may be present in an antigen binding fragment of any antibody. These fragments can be produced recombinantly, as well as engineered, synthesized or produced by digesting the antibody with proteolytic enzymes. For example, the fragment can be a Fab fragment and digestion with papain cleaves the antibody in the region preceding the interchain (ie, _VH - _VH ) disulfide bond joining the two heavy chains. This results in the formation of two identical fragments containing the V _H and C _H 1 domains of the light chain and 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 various ways. For example, connecting the C-terminus of _{V H} to the N-terminus of the _{V L.} Generally, a linker (eg (GGGGS) ₄ ) is placed between V _H and V _L. However, the order in which the strands can be linked can be reversed and can include tags that facilitate detection or purification (eg, Myc tags, His tags or FLAG tags) (such tags as those of the present 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 invention. In alternative embodiments, the antibodies described herein or produced by the methods described herein can be heavy chain dimers or light chain dimers. In addition, antibody light or heavy chains or parts thereof, such as single domain antibodies (DAbs) can also be used.

In another embodiment, the humanized Ig variable region according to the invention, or the humanized Ig variable region produced by the methods of the invention, is present in a single chain antibody (ScFv) or minibody (minibody). See, for example, US Pat. No. 5,837,821 or WO94/09817A1). Each minibody is a ScFv molecule (a single polypeptide containing one or more antigen binding sites, eg, a _VL domain linked by a flexible linker to a _VH domain fused to a CH3 domain via a connecting peptide). Is a dimeric molecule made up of two polypeptide chains containing ScFv molecules can be constructed in a _VH -linker- _VL orientation or a _VL -linker- _VH orientation. The flexible hinge joining the _VL and _VH domains that make up the antigen binding site preferably comprises from about 10 to about 50 amino acid residues. An exemplary connecting peptide for this purpose is (Gly4Ser)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 Gene 77, 51; Bird et al. 1988 Science 242, 423; Pantoliano. Et al. (1991), Biochemistry, 30: 10117; Milenic et al. (1991), Cancer Research, 51: 6363; Takkinen et al. (1991), Protein Engineering, 4: 837. Minibodies can be generated by constructing ScFv components and connecting peptide-CH ₃ components using methods described in the art (see, eg, US Pat. No. 5,837,821 or WO94/09817A1). .. These components can be isolated as restriction fragments from separate plasmids and then ligated and recloned into a suitable vector. Proper assembly can be verified by restriction enzyme digestion and DNA sequence analysis. In one embodiment, a minibody of the invention comprises a connecting peptide. In one embodiment, the connecting peptide comprises a Gly/Ser linker, eg GGGSSGGGSGG.

In another embodiment, tetravalent minibodies can be constructed. For example, a tetravalent minibody can be constructed in the same manner as a 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 in this invention, or the humanized variable regions produced by the methods of this invention, can be present in diabodies. Diabodies are similar to scFv molecules, but usually have a short (less than 10 and preferably 1-5) amino acid residue linker connecting both variable domains, and therefore V on the same polypeptide chain. _{The L} domain and _VH domain cannot interact. Instead, the _VL and _VH regions of one polypeptide chain interact with the _VH and _VL domains on the second polypeptide chain (respectively) (WO 02/02781).

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

The humanization methods described herein preferably result in an Ig variable region with substantially unchanged antigen affinity as compared to the donor antibody.

In one embodiment, the variable domain-containing polypeptides of the invention are 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. , Binds to an antigen with a binding affinity Ka of greater than (or equal to) about 10 ¹⁰ M ^-1 , about 10 ¹¹ M ^-1 or about 10 ¹² M ^-1 (intermediate affinity between these values). including).

Affinity, avidity and/or specificity can be measured in various ways. In general, regardless of the precise way in which affinity is defined or measured, the methods of the invention produce antibodies that are superior to the antibody (or antibodies) from which they are made in any aspect of clinical application. , The method is one that improves antibody affinity (eg, where the modified antibody can be administered at a lower dose or less frequently than the source antibody (or antibodies) or by a convenient route of administration, The method of the present invention is considered to be effective or successful).

More particularly, the affinity between an antibody and the antigen it binds to varies with, for example, an ELISA assay, a BiaCore assay or a KinExA ^™ 3000 assay (available from Sapidyne Instruments (Boise (ID))). It can be measured by various assays. Briefly, the sepharose beads are covalently bound to an antigen (an antigen used in the methods of the invention can be any antigen of interest (eg, cancer antigen; cell surface or secreted protein; pathogen antigen (eg, bacterial or Coat with viral antigens (eg HIV antigens, influenza antigens or hepatitis antigens); which may be allergens) Prepare dilutions of the antibody to be tested and add each dilution to designated holes in the plate. Detection antibody (eg, goat anti-human IgG-HRP conjugate) is then added to each well, followed by chromogenic substrate (eg, HRP).The plates are then at 450 nM in an ELISA plate reader. Read and calculate EC50 values (methods described herein are generally applicable; it is understood that they are not limited to the production of antibodies that bind to any particular antigen or class of antigens).

Those skilled in the art recognize that the determination of affinity is not simple enough to always look 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 affinities can be measured using scFv or Fab fragments.

In another aspect, the invention features a bispecific molecule that comprises a humanized rabbit antibody or fragment thereof of the invention. The antibody of the invention, or antigen binding portion thereof, is derivatized or linked to another functional molecule, eg, another peptide or protein (eg, a ligand for another antibody or receptor), to form at least 2 Bispecific molecules can be produced that bind to three different binding sites or target molecules. Antibodies of the invention can be derivatized or linked to two or more other functional molecules to produce a multispecific molecule that binds to three or more different binding sites and/or target molecules. Yes, such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To produce the bispecific molecule of the present invention, the antibody of the present invention may be combined with one antibody, such as another antibody, antibody fragment, tumor-specific or pathogen-specific antigen, peptide, or binding mimetic. Alternatively, it may be operably linked (eg, by chemical coupling, gene fusion, non-covalent association, or otherwise) to a plurality of other binding molecules to yield a bispecific molecule. Accordingly, the invention provides a 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. Including sex molecules.

In one embodiment, the bispecific molecule of the invention has as binding specificity at least one antibody, or antibody fragment thereof (eg, Fab, Fab′, F(ab′)2, Fv, or single chain Fv). Including). An antibody is a dimer of light or heavy chains, 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. Fv or single chain constructs, etc.).

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

The bispecific molecule of the present invention can be prepared by conjugating the binding specificity 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 each other. When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Examples of the cross-linking agent include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis(2-nitrobenzoic acid) (DNTB), o-phenylenedimaleimide ( oPDM), 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). Other methods include Paulus (1985) Behring Ins. Mitt. 78, 118-132; Brennan et al. (1985) Science, 229, 81-83, and Glennie et al. (1987) J. Am. Immunol. , 139, pp. 2367-2375. Preferred conjugating agents are SATA and Sulfo-SMCC, both of which are from Pierce Chemical Co. (Rockford, IL).

If 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 of sulfhydryl residues, preferably one, before 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 when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab') ₂ , or ligand x Fab fusion protein. The bispecific molecule of the present 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 include at least two single chain molecules. Methods for preparing bispecific molecules are described, for example, in US Pat. No. 5,260,203, US Pat. No. 5,455,030, US Pat. No. 4,881,175, US Pat. 132,405, US Pat. No. 5,091,513, US Pat. No. 5,476,786, US Pat. No. 5,013,653, US Pat. No. 5,258,498, and US Pat. , 482,858.

Binding to their specific target by bispecific molecules is eg described by enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), flow cytometry based single cell sorting (eg FACS analysis), bioassay. (Eg growth inhibition), or by Western blot assay. Each of these assays generally uses a labeled reagent (eg, an antibody) specific for the complex of interest to detect the presence of the protein-antibody complex of particular interest. For example, antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds the antibody-VEGF complex. Alternatively, the complex can be detected using any of a variety of other immunoassays. For example, the antibody may be radiolabeled and used in a radioimmunoassay (RIA) (eg, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radian Radiology, incorporated herein by reference). See Techniques, The Endocrine Society, March 1986). Radioisotopes can be detected by such means as the use of gamma counters, or scintillation counters, 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, drug (eg, immunosuppressant drug), or radiotoxin. Such a complex is referred to herein as an "immunoconjugate." Immunoconjugates that include one or more cytotoxins are called "immunotoxins." Cytotoxic or cytotoxic agents include any agent that is detrimental to (eg kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mitramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologues thereof. Examples of the therapeutic agent include antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiotepa), chlorambucil. , Melphalan, carmustine (BSNU), and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP), cisplatin), anthra. Cyclins (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)), and anti-mitotic agents (eg, , Vincristine and vinblastine).

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 ^™ , Wyeth-Ayerst).

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

For further discussion of cytotoxins, linker types, 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.; A. Et al. (2003) Cancer lmmunol. Immunother. 52, 328-337; Payne, G.; (2003) Cancer Cell, Vol. 3, pp. 207-212; Allen, T.; M. (2002) Nat. Rev. Cancer Volume 2, 750-763; Pastan, I.; And Kreitman, R.; J. , (2002) Curr. Opin. Investig. Drugs, vol. 3, pp. 1089-1091; Center, P.M. D. And Springer, C.I. J. (2001) Adv. Drug Deliv. Rev. , 53, pages 247-264.

The antibodies of the invention can also be conjugated to radioisotopes to produce cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to, iodine ¹³¹ , indium ¹¹¹ , yttrium ⁹⁰ , and lutetium ¹⁷⁷ . Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available including Zevalin ^™ (IDEC Pharmaceuticals) and Bexxar ^™ (Corixa Pharmaceuticals) and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention. You can The antibody conjugates of the invention can be used to modulate a given biological response and the drug moiety should not be construed as limited to classical chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide that has the desired biological activity. Such proteins include, for example, enzymatically active toxins, or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxins; proteins such as tumor necrosis factor or interferon-γ; or 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 factor may be mentioned.

Techniques for conjugating such therapeutic moieties to antibodies are well known, for example in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (edited), pp. 243-56, (Alan R. Liss, Inc. 1985). Arn et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy"; "Antibodies for Drug Delivery"; Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al., (eds.), pp. 475-506 in (1985), Thorpe, "Antibody Carriers of Cytotoxic Agents in Cancer Therapy: A Review"; Monoclonal Antibodies for Cancer Detection and Therapy, Baldwin et al., (eds.), at pp. 303~16 (Academic Press 1985 years), "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled 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 disease. The term "pharmaceutical formulation" refers to a preparation that is such a form that allows for the biological activity of the antibody or antibody derivative that is demonstrably effective and that does not include additional components that are toxic to the subject to whom the formulation is administered. Say something. “Pharmaceutically acceptable” excipients (vehicles, additives) are those 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 inherently retains its physical and/or chemical stability and/or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art, eg, Peptide and Protein Drug Delivery, pages 247-301, edited by Vincent Lee, Marcel Dekker, Inc. , New York, N.; Y. Pubs. (1991), and Jones, A.; 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 1 month, and/or stable at about 2-8°C for at least 1 year or at least 2 years. Moreover, the formulation is preferably stable after freezing (eg, up to -70°C) and thawing the formulation.

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

An antibody or antibody derivative is said to have "in its chemical form in a pharmaceutical formulation, so long as its chemical stability at a given time is such that the protein is still believed to retain its biological activity as defined below. Keeps stability.” Chemical stability can be assessed by detecting and quantifying chemically altered protein morphology. 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), resize (eg, clipping). Can be involved. Other types of chemical changes include charge changes that can be assessed, for example, by ion exchange chromatography (eg, due to deamidation).

An antibody or antibody derivative is within about 10% of the biological activity exhibited by the antibody's biological activity at a given time, eg, when the pharmaceutical formulation is prepared as determined in an antigen binding assay (assay). Error”), “retain its biological activity” in the pharmaceutical formulation. Other "biological activity" assays for antibodies are detailed 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 that has multiple hydroxyl groups and includes sugars (reducing and non-reducing sugars), sugar alcohols and sugar acids. Preferred polyols herein have a molecular weight of less than about 600 kD (eg, in the range of about 120 to about 400 kD). The "reducing sugar" includes a hemiacetal group capable of reducing a metal ion or covalently reacting with lysine and other amino groups in a protein, and "non-reducing sugar" means a reducing sugar. It does not have these characteristics of. 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. If it is desired that the formulation is freeze-thaw stable, the polyol preferably does not crystallize at freezing temperatures (eg, -20°C) such that the antibodies in the formulation are unstable. Is. Non-reducing sugars such as sucrose and trehalose are the 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 acid-base conjugation components. The buffers of the present 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 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. Can be mentioned. If a freeze-thaw stable formulation is desired, the buffer is preferably not phosphate.

In the pharmacological sense, a “therapeutically effective amount” of an antibody or antibody derivative, in the context of the present invention, refers to an amount effective in preventing or treating a disorder, in relation to the treatment for which the antibody or antibody derivative is effective. “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 pathological conditions that cause mammals to have the disorder in question.

A "preservative" is a compound that can be included in a formulation and essentially reduce the action of bacteria there, thus facilitating the manufacture of, for example, a multi-use formulation. it can. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chloride where the alkyl group is a long chain compound), and benzethonium chloride. To be Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative herein is benzyl alcohol.

The invention also provides pharmaceutical compositions comprising one or more antibody or antibody derivative compounds, together with at least one physiologically acceptable carrier or excipient. The pharmaceutical composition can be, for example, one or more of water, buffers (eg, neutral buffered saline or phosphate buffered saline), ethanol, mineral oil, vegetable oils, dimethylsulfoxide, carbohydrates (eg glucose, mannose). , Sucrose or dextran), mannitol, proteins, adjuvants, polypeptides, or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, and/or preservatives. As mentioned above, other active ingredients may, but need not, be included in the pharmaceutical compositions provided herein.

A carrier is a substance that may be associated with the antibody or antibody derivative prior to administration to the 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), ovalbumin, peptides, mono- or polyvalent 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), polyacrylates, latex, starch, cellulose or dextran. The carrier may carry the compound in a variety of ways, including covalent bonding (either directly or via a linker group), non-covalent interaction or admixture.

The pharmaceutical composition can be formulated for any suitable mode of administration, including, for example, topical, oral, nasal, rectal or parenteral administration. For certain embodiments, compositions 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, intracranial, intrathecal and intraperitoneal injection, and any analogs. Injection or infusion techniques.

Compositions intended for oral use may be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, in order to provide an attractive and palatable preparation, a sweetening agent may be provided. One or more agents such as flavoring agents, colorants, and preservatives. 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 and disintegrating agents (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 mentioned. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over an extended period of time. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

Formulations for oral use may be as a hard gelatin capsule in which the active ingredient is mixed with an inert solid diluent (eg, calcium carbonate, calcium phosphate, or kaolin), or a vehicle in which the active ingredient is water or oil. It may be given as a soft gelatin capsule, mixed with (for example 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 (for example sodium carboxymethylcellulose, methylcellulose, hydropropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and acacia gum), and dispersants or humectants (for example , Naturally occurring phosphatides such as lecithin, condensation products of alkylene oxides such as polyoxyethylene stearate with fatty acids, condensation products of ethylene oxide such as heptadecaethyleneoxycetanol with long-chain aliphatic alcohols, polyoxyethylene A condensation product of a partial ester derived from a fatty acid such as sorbitol monooleate and hexitol with ethylene oxide, or a condensation product of a partial ester derived from a fatty acid such as polyethylene sorbitan monooleate and an anhydride of hexitol with an ethylene oxide). To be Aqueous suspensions include one or more preservatives (eg, ethyl or n-propyl p-hydroxybenzoic acid), one or more colorants, one or more flavoring agents, and one or more Sweeteners such as sucrose or saccharin may also be included. Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain one or more demulcents, preservatives, flavoring agents, 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 mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and/or flavoring agents may be added to provide a palatable oral preparation. Such suspensions may be preserved by the addition of an antioxidant such as ascorbic acid.

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

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

The pharmaceutical compositions may, depending on the vehicle and concentration used, be prepared as a sterile injectable aqueous or oleaginous suspension in which the modulator is suspended or dissolved in the vehicle. Such compositions may be formulated according to the known art using suitable dispersing, wetting 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 saline. In addition, sterile fixed oils can be employed as the solvent or suspension medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be used in the preparation of injectable compositions 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 provides a sustained release of the modulator after administration). Such formulations can be prepared using commonly 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 may be biocompatible and biodegradable, preferably the formulation allows a relatively constant level of modulator release. The amount of antibody or antibody derivative contained within a 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 being treated or prevented.

The antibodies or antibody derivatives provided herein generally detectably bind to a target such as VEGF and prevent or inhibit a target mediated disease/disorder such as VEGF mediated disease/disorder. Is administered in an amount sufficient to reach a concentration in body fluids (eg, blood, plasma, serum, CSF, synovial fluid, lymph, interstitial fluid, tears or urine). A dose is considered to be effective when it produces a discernible patient benefit as described herein. A 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), with oral doses generally About 5 to 20 times higher than the intravenous dose. The amount of antibody or antibody derivative that can be combined with a 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 active ingredient.

The pharmaceutical composition can be packaged, for example, to treat a condition responsive to an antibody or antibody derivative directed against 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 that responds to the one antibody or antibody derivative after administration to a patient. Instructions (e.g., a label) indicating that the containing composition is used for treating the can be included.

The antibody or antibody derivative of the present invention can also be chemically modified. Preferred modifying groups are, for example, optionally substituted linear or branched polyalkenes, polyalkenylene or polyoxyalkylene polymers, or polymers such as 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 ranges from 500 Da to 50,000 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 portion of the heavy chain of the Fab fragment via an antibody, particularly a covalently linked hinge peptide, as described in WO0194585. Regarding the attachment of the PEG moiety, “Poly(ethyleneglycol) Chemistry, Biotechnological and Biomedical Applications”, 1992, J. Am. Milton Harris (eds.), Plenum Press, New York, and "Bioconjugation Protein Coupling Technologies for the Biomedical Sciences", 1998, M.A. 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 formulated antibody has not been 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 scFv. The therapeutically effective amount of the antibody present in the formulation is determined taking into account, for example, the desired dose volume and mode(s) of administration. 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. Is.

Aqueous formulations are prepared containing the antibody or antibody derivative in a pH buffered solution. The buffers of the present 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 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. Are listed. The buffer concentration can be from about 1 mM to about 50 mM, preferably from about 5 mM to about 30 mM, and can be determined, for example, by the buffer and the desired isotonicity of the formulation. A preferred buffer is sodium acetate (about 10 mM), pH 5.0.

A polyol that acts as a tonicifier and can stabilize the antibody is included in the formulation. In a preferred embodiment, the formulation does not include salts such as sodium chloride in tonicifying amounts, as it can cause precipitation of the antibody or antibody derivative and/or can cause 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 can vary for the desired isotonicity of the formulation. Preferably, the aqueous formulation is isotonic, where 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. Is the range. However, hypertonic or hypotonic formulations may also be suitable. The amount of polyol added can also be varied with respect to the molecular weight of the polyol. For example, smaller amounts of monosaccharides (eg, mannitol) can be added compared to disaccharides (such as trehalose).

Surfactants are also added to the antibody or antibody derivative formulation. Typical surfactants include nonionic surfactants such as polysorbates (eg, polysorbate 20, 80, etc.) or poloxamers (eg, poloxamer 188). The amount of surfactant added is such that it 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 is 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%. Can be present in the formulation at

In one embodiment, the formulation comprises the above-identified agents (ie, antibody or antibody derivative, buffer, polyol and surfactant), and is essentially benzyl alcohol, phenol, m-cresol, chlorobutanol and It does not contain one or more preservatives such as benzethonium chloride. In another embodiment, a preservative can be included in the formulation, especially if the formulation is a multiple dose formulation. The preservative concentration 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.; One or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Ed. (2006), provided that they do not adversely affect the desired characteristics of the formulation. And can be included in the formulation. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, additional buffers, cosolvents, antioxidants including ascorbic acid and methionine, chelating agents such as EDTA. , Metal complexes (eg Zn-protein complexes), biodegradable polymers such as polyesters, and/or salt-forming counterions such as sodium.

The formulations used for in vivo administration must be sterile. This is easily accomplished by filtration through a sterile filtration membrane before or after preparing the formulation.

The formulation may be administered intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarticularly, intrasynovially, intrathecally, orally, topically to a mammal, preferably human, in need of treatment with the antibody. Alternatively, it is administered according to known methods such as intravenous administration by the inhalation route, as a bolus or by continuous infusion over a period of time. In a preferred embodiment, the formulation is administered to mammals by intravenous administration. For such purposes, the formulation may be injected, for example, with a syringe or via the IV line.

An appropriate dosage of antibody (a "therapeutically effective amount") can be, for example, the condition to be treated, the severity and course of the condition, whether the antibody is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical 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 treatment 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 the sole treatment or in combination with other drugs or therapies useful in the treatment of the condition in question.

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

In another embodiment of the invention, an article of manufacture is provided that comprises a container for holding a pharmaceutical formulation of the invention, preferably an aqueous formulation, and optionally instructions for its use. Suitable containers include, for example, bottles, vials and syringes. The container can be formed from 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 glass vial. The container holds the formulation and the label on the container or the label associated with the container may indicate directions for use. The article of manufacture may further include other materials desired from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. Are listed.

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

(Example)
The present disclosure is further described 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 entireties.

(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 (particularly antibody technology) and standard techniques for preparing polypeptides. For example, Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Pain, Protocols (Methods in Bol, 10). , Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, Volume 169), McCafferty et al. S. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, Ausubel et al., Ed., John Wiley & Sons (1992). Methods for fusion of CDRs to selected human antibody frameworks from rabbit and other non-human monoclonal antibodies have been described in detail above. An example of such a fusion experiment is shown below.

For better understanding, the fusion fragment named "min" is the CDR fused to framework 1.4 or its variable domain, while the fusion fragment named "max" is , The 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 immunoglobulin sequences)
Variable domains and germline sequences of mature rabbit antibodies were collected from various open source databases (eg, Kabat database and IMGT) and entered as a one-letter coded amino acid sequence into a customized database. For all analyses, we used only the amino acid portion corresponding to the V (variable) region. Sequences in the KDB database that were less than 70% complete or contained multiple undetermined residues within the framework regions were discarded. Sequences with greater than 95% identity to any other sequence 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 a conventional sequence alignment tool based on the Needleman-Wunsch algorithm and Blossum matrix. Introduction of gaps and nomenclature of residue positions was done according to the AHo numbering system of immunoglobulin variable domains (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).

The framework and CDR regions are defined according to Kabat's definition, which is based on sequence variability and is the most commonly used. Nevertheless, the CDR-H1 designation is based on the AbM definition, the kabat definition, the average contact data generated by analysis of contacts between antibodies and antigens of a subset of the 3D complex structure (MacCallum et al., 1996). ) And the definition of Chotia based on the position of structural loop regions (see above and in FIG. 1).

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

Where 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 particular amino acid type.

(1.1.4. Serial analysis of V region of rabbit)
The rabbit repertoire was investigated using a phylogenetic analysis tool. Both cluster and topological algorithms were used to cluster V region amino acid sequences. A distance matrix was calculated for the entire array and used as an indicator of germline use. The consensus sequence for each cluster was calculated and the closest rabbit germline sequence counterpart was identified. The overall consensus sequence was also obtained from the entire set of sequences.

(1.1.5. Assignment of human subgroup)
For each rabbit representative sequence of the various clusters, the most homologous human subgroups were identified using a taxonomy based on the EXCEL implementation of a sequence analysis algorithm and analysis of the human antibody repertoire (Knappik et al., 2000). did.

(1.2. Design of human acceptor framework)
The blueprint of the rabbit repertoire from the sequence analysis described above was used to identify residues in the framework that are commonly involved in the localization of the rabbit CDRs. Of the frameworks and their respective clusters with high homology to the rabbit repertoire, one with good biophysical properties was selected from a pool of fully human sequences. The frameworks selected for use as acceptor frameworks belong to the variable light chain subgroup kappa 1 and the variable heavy chain subgroup III, corresponding to ESBATech ID KI27, a43. This stable and soluble antibody framework was identified by screening human splenic scFv libraries using a yeast-based screening method termed the "quality control" system (Auf der Maur et al., 2004). FW 1.4". The stable and soluble framework sequence FW1.4 shows high homology, but it was not the highest homology sequence available. The identified residues were incorporated into the acceptor framework to generate rFW1.4.

Using information on amino acid sequence diversity, germline usage and structural features of rabbit antibodies, we find the compatibility of residue positions required to preserve CDR conformations in the novel human framework. Was analyzed for FW1.4. We examined the variable regions of FW1.4 for suitability of the following features:
i. Residues that are part of the reference sequence of the loop structure ii. Framework residues located at the VL/VH interface iii. The platform of residues immediately below the CDRs iv. Upper and lower core residues v. Framework residues that define the subtype.

(1.3. Fusion of rabbit CDR)
The fusion fragment was generated by simply combining the CDR sequences from one 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 to a subgroup consensus or a consensus of rabbit sequences with a high percentage of similarity. Rare framework residues could be the result of somatic hypermutation and therefore thought to play a role in binding. Therefore, such residues were fused to the acceptor framework.

(1.4. Results)
By analyzing the rabbit antibody repertoire for structure, amino acid sequence diversity and germline usage, we found five residue positions in the light chain of FW1.4 that were modified to maintain the loop conformation of the rabbit CDRs. It was These positions are highly conserved with the rabbit antibody. The consensus residues at these 5 positions were deduced from the rabbit repertoire and introduced into the human acceptor framework 1.4. These modifications of conserved positions made the framework compatible with virtually all six complementarity determining regions (CDRs) of any rabbit CDRs. The master rFW1.4 containing various rabbit CDRs was well expressed and well produced as opposed to the wild type single chain. 16 members derived from the combination of this framework and rabbit CDRs were generated and detailed characterization demonstrated functionality.

(Example 2: B cell screening system)
A FACS (flow cytometry-based single cell sorting)-based screening system for selecting B cells that bind to their target of interest via their B cell receptors (BCRs) has been established at ESBATech. One target was, for example, a soluble protein, ie a single chain antibody (ESBA903) labeled with a fluorescent dye (PE and PerCP). Lymphocyte suspensions were prepared from spleens of rabbits immunized with the recombinant target. The cells were then incubated with PE and PerCP labeled ESBA903 and IgG specific antibody (APC labeled) or IgM specific antibody (FITC labeled). ESBA903-positive B cells that express IgG but not IgM on their surface were sorted into 96-well plates (Fig. 3; Table 2). The thymoma helper cell line (EL4-B5: Zubler et al., 1985, J. Immunol, 134(6), 3662-3668) proliferated, differentiated into plasma cells and secreted antibody B. Cells were selected. The affinity of these IgGs for the target was assessed by ELISA and Biacore measurements. The kinetic parameters of the 7 selected clones are shown in Table 1. These clones, obtained from a pool of approximately 200 sorted cells, show binding affinities in the low nanomolar to picomolar range. Finally, mRNA was isolated from 6 clones of interest and the CDR was fused to the single chain framework FW1.4.

(Example 3: Detection of interaction between beads coated with anti-TNFα antibody and CHO cells expressing membrane-bound TNFα)
The following experiments were performed to assess whether high pressure in the flow cytometry stream disrupts the non-covalent bond between two cells. CHO cells (B-220 cells) stably transfected with membrane-bound TNFα were incubated with PE-coated anti-TNFα antibody-coated beads. In this setup, the beads mimic memory B cells (they have about the same size). As negative controls, untransfected CHO cells and beads coated with an irrelevant antibody labeled with APC (anti-CD19) were used. After a 2 hour incubation 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 ⅔ of the beads were bound to cells (585 bound but 267 unbound). In contrast, in control samples (middle and lower panels) most beads do not bind to CHO cells. 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 approximately half of the anti-TNFα beads bound to CHO cells, while the majority of anti-CD19 beads remained unbound. The percentage 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 the integral membrane target protein via their B cell receptor is possible using flow cytometry.

Example 4: CDR Fusion and Functional Humanization of Anti-TNFα Rabbit Donor Antibody
Four anti-TNFα rabbit rabbit antibodies “Rabmab” (EPI-1, EPI-15, EP-34, EP-35 and EP-42) were selected for CDR fusion. A general experimental scheme for CDR fusion, humanization, and preliminary characterization of humanized rabbit donor antibodies was performed as outlined in the detailed description. Unlike traditional humanization methods that utilize the human antibody acceptor framework, which shares maximum sequence homology with non-human donor antibodies, a quality control assay (WO0148017) is used to achieve the desired functional properties (solubility and stability). The rabbit CDRs were fused to a human framework (FW1.4) preselected for sex). These stable and soluble framework sequences showed high homology to RabMab.

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

The scFv described and characterized herein were produced as follows. The humanized VL sequences via a linker of SEQ ID NO: 8 are connected to the humanized VH _sequences, resulted in scFv orientation of NH 2 -VL- linker -VH-COOH. Often, DNA sequences encoding various scFvs were de novo synthesized at the service provider Entelechon GmbH (www.entelechon.com). The resulting DNA insert 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 VL domain DNA sequence and the VH domain DNA sequence. In some cases, the DNA encoding the scFv was not newly synthesized and the scFv expression construct was cloned by domain shuffle. Therefore, the VL domain was excised and introduced into the new construct via the NcoI and BamHI restriction enzyme recognition sites, and the VH domain was excised and introduced via the BamHI and HindIII restriction enzyme recognition sites. In other cases, point mutations were introduced into the VH and/or VL domains using state-of-the-art assembly PCR methods. Cloning of GMP002 is described in Example 1 of WO2008006235. The production of scFv was performed similarly for ESBA105 as described in Example 1 of WO2008006235.

Table 3 outlines detailed characterization data for four rabbit monoclonals (EP6, EP19, EP34, EP35 and EP43) and their CDR fusion variants. These CDR fusions showed extensive activity in the BIACore binding assay and the L929, TNFα-mediated cytotoxicity assay, with 3 of the 4 maximal (“max”) fusions showing therapeutically significant activity. It was EP43max showed the most favorable binding affinity (Kd of 0.25 nM) and excellent EC50 in the cytotoxicity assay. This data indicates that FW1.4 (SEQ ID NOS: 1 and 2) is an exemplary soluble and stable human acceptor framework region for rabbit CDR fusions.

(Efficacy assay)
The neutralizing activity of anti-TNFα binders was evaluated in the L929 TNFα-mediated cytotoxicity assay. The toxicity of mouse L929 fibroblasts treated with actinomycin was induced with recombinant human TNF (hTNF). 90% of the 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 anti-TNFα binders was evaluated in RPMI 1640 without phenol red, 5% fetal bovine serum. To determine the concentration at which half-maximal inhibition of antagonism (EC50%) is reached, various concentrations (0-374 ng/mL) of anti-TNF binder are added to L929 cells in the presence of 1000 pg/ml hTNF. Dose response curves were fitted to a non-linear sigmoidal regression with variable slope and EC50 was calculated.

(Biacore binding analysis of anti-TNF scFv)
For binding affinity measurement, surface plasmon resonance measurement using BIAcore ^™ -T100 was used 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 trimers (5 nM) were captured by nickel via their N-terminal his-tag and ESBA105 (analyzed at several concentrations ranging from 30 nM to 0.014 nM in a 3-fold serial dilution step. Thing). In the regeneration step, the complex formed by nickel, the ligand and the analyte is washed away. This allows the same regeneration conditions to be used for different samples. Reaction signals are generated by surface plasmon resonance (SPR) technology and measured in resonance units (RU). All measurements are made at 25°C. Sensorgrams were generated for each anti-TNF scFv sample after in-line reference cell correction and subsequent buffer sample subtraction. The apparent dissociation rate constant (k _d ), the apparent association rate constant (k _a ), and the apparent dissociation equilibrium constant (K _D ) were calculated using the BIAcore T100 evaluation software version 1.1 for 1:1 Langmuir binding. Calculated using the model.

Example 5: CDR Fusion and Functional Humanization of Anti-VEGF 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 the human antibody acceptor framework, which shares maximum sequence homology with non-human donor antibodies, a quality control assay (WO0148017) is used to achieve the desired functional properties (solubility and stability). The rabbit CDRs were fused to the human acceptor framework FW1.4 (SEQ ID NO: 1 and 2) preselected for

A number of CDR fusion fragments were generated for each of the RabMabs (rabbit antibodies) using the methodology described herein (see Example 4). The "Min" fusion fragment contained the smallest fusion fragment. In this, only the rabbit CDRs were grafted from the VL and VH domains of the rabbit donor antibody into the human acceptor framework FW1.4 (SEQ ID NO: 1). The "Max" fusion fragment contained not only the rabbit CDRs for VL and VH, but also some additional framework residues from the rabbit donor that were predicted to be important for antigen binding. For 578max, the heavy chain variable domain framework region of FW1.4 has additional amino acid modifications at Kabat positions 23H, 49H, 73H, 78H and 94H.

Table 4 outlines detailed characterization data for the "Min" and "Max" CDR fusion variants. Figure 7 shows their efficacy as VEGF inhibitors as measured using VEGFR competitive ELISA and/or HUVEC assay. This data indicates that FW1.4 (SEQ ID NOs: 1 and 2) is an exemplary soluble and stable human acceptor framework region for rabbit CDR fusions.

(Biacore binding analysis of anti-VEGF scFv)
The scFv was tested for Biacore binding ability and binding affinity was measured using an exemplary surface plasmon resonance method with BIAcore ^™ -T100. In this and subsequent examples, VEGF proteins tested for binding by these scFv candidates included 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, a carboxymethylated dextran biosensor chip (CM4, GE Healthcare) was loaded with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride and N according to the supplier's instructions. -Activated with hydroxysuccinimide. Each of the six different VEGF forms exemplified above were coupled to one of four different flow cells on the CM4 sensor chip using standard amine coupling procedures. After coupling and blocking, the range of responses obtained with these immobilized VEGF molecules was about 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 VEGF ₁₁₀ and about 400 RU for PLGF. The fourth flow cell of each chip was treated similarly, 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% detergent 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 for 5 minutes at a flow rate of 30 μl/min. The dissociation of anti-VEGF scFv from VEGF on the CM4 chip could proceed for 10 minutes at 25°C. Sensorgrams were generated for each anti-VEGF scFv sample after buffer sample subtraction following in-line reference cell correction. The apparent dissociation rate constant (k _d ), the apparent association rate constant (k _a ), and the apparent dissociation equilibrium constant (K _D ) were calculated using the BIAcore T100 evaluation software version 1.1 with a 1:1 Langmuir binding model. Was calculated using.

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

Human umbilical vein endothelial cells (HUVEC) (Promocell) pooled from several donors were used from passage 2 to passage 14. Cells were treated with 0.4% ECGS/H, 2% fetal bovine serum, 0.1 ng/ml epidermal growth factor, 1 μg/ml hydrocortisone, 1 ng/ml basic fibroblast factor and 1% penicillin/streptomycin. Seed at 1000 cells/well in 50 μl complete endothelial cell growth medium (ECGM) (Promocell) containing (Gibco). After 7 to 8 hours, 50 μl of starvation medium (ECGM without supplement containing 0.5% heat-inactivated FCS and 1% penicillin/streptomycin) was added to the cells and allowed to starve for 15 to 16 hours. It was Three-fold serial dilutions of 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 activity. Submaximal VEGF-induced proliferation stimulating concentrations (EC ₉₀ ) were 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 humidified incubator at 37°C/5% CO ₂ . HUVEC proliferation is measured by absorbance at 450 nm (using 620 nm as reference wavelength) after addition of 20 μl/well WST-1 cell proliferation reagent (Roche) using a Sunrise Microplate Reader (Tecan). It was evaluated by The data were analyzed using a 4-parameter logistic 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 this invention will be apparent to those skilled in the art in light of the above description. Therefore, 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 invention. The details of construction may be varied substantially without departing from the spirit of the invention and are reserved for exclusive use of all modifications which come within the scope of the appended claims. It is intended that the invention be limited only to the extent required by the appended claims and the rules of applicable law.

All publications and analogs cited in this application, including patents, patent applications, editorials, books, papers, articles, web pages, figures and/or attachments, are referred to regardless of the format of such publications and analogs. Are explicitly incorporated in their entirety by. In the event that one or more of the incorporated documents and analogues differs or conflicts with this specification including defined terms, usage of terms, techniques described or the like, the specification is have priority.

Claims (1)

An immunobinder, immunobinder acceptor framework, nucleic acid, vector, host cell, composition, method, or use as described herein.