Improved anti-FLT3 antigen-binding protein

Abstract

The present invention provides novel human fms-related tyrosine kinase 3 (FLT3) antigen-binding proteins, such as antibodies, with improved FLT3 binding affinity and / or anti-tumor activity. The FLT3 antibodies of the present invention are generated by mutation of parental FLT3 antibodies and are tested in vitro in binding assays and in vivo in murine tumor models and human patient tumor samples. The antibodies of the present invention are provided in monospecific constructs or bispecific FLT3xCD3 antibody forms and exhibit excellent target affinity and / or tumor cell killing. The present invention also relates to methods for generating the antigen-binding proteins of the present invention, as well as nucleic acids encoding them, vectors expressing them, and host cells. The present invention further relates to methods of using the FLT3 antigen-binding proteins (ABPs) of the present invention for treating or diagnosing diseases such as leukemia.

Description

Field of the Invention

[0001] The present invention provides novel human fms-related tyrosine kinase 3 (FLT3) antigen-binding proteins, such as antibodies, having improved FLT3 binding affinity and / or anti-tumor activity. The FLT3 antibodies of the present invention are generated by mutation of parental FLT3 antibodies and are tested in vitro in binding assays and in vivo in murine tumor models and human patient tumor samples. The antibodies of the present invention are provided in monospecific constructs or bispecific FLT3xCD3 antibody forms and exhibit excellent target affinity and / or tumor cell killing. The present invention also relates to methods for generating the antigen-binding proteins of the present invention, nucleic acids encoding them, vectors expressing them, and host cells. The present invention further relates to methods of treating or diagnosing diseases such as leukemia using the FLT3 antigen-binding proteins (ABPs) of the present invention. Background of the Invention

[0003] Scientific work beginning in the 1980s has established that bispecific antibodies directed against tumor-associated antigens (TAAs) and the T cell receptor (TCR) / CD3 complex are capable of activating T cells, resulting in lysis of tumor cells expressing the TAA by the activated T cells (Staerz et al. Nature 1985, 314:628-631; Perez et al. Nature 1985, 316:354-356; Jung et al. Proc Natl Acad Sci USA 1986, 83:4479-4483). Since CD3 antibodies bind to Fc receptors (FcRs) through their Fc portions and are highly efficient in inducing T cell activation and cytokine release, which are harmful side effects, it is of great importance to construct Fc-depleted or attenuated bispecific TAAxCD3 antibodies to prevent FcR binding and allow target cell-restricted rather than FcR-mediated T cell activation (Jung et al. Immunol Today 1988; 9:257-260; Jung et al. Eur J Immunol 1991; 21:2431-2435).

[0004] The production of bispecific antibodies meeting such key prerequisites in terms of industrial quality and quantity remains a formidable challenge. Recently, a recombinant bispecific single-chain (bssc) antibody with CD19xCD3 specificity, called Blinatumomab, has shown rather high efficiency in treating ALL patients (Bargou et al. Science 2008, 321:974-977) and has received approval under the FDA's breakthrough designation. Notably, the drug is administered over several weeks by continuous 24-hour infusion due to its low serum half-life and rather high toxicity: the safety-applicable dose is 30 μg per patient per day, which is 10,000-fold lower than the safety-applicable doses used for treatment with established monospecific anti-tumor antibodies (Adams and Weiner. Nat Biotechnol 2005, 23:1147-57). The serum concentration of the resulting drug is below 1 ng / ml (Topp et al. J Clin Oncol 2011;29:2493-2498). This severe dose limitation, also observed in earlier clinical trials using different bispecific antibodies (Kroesen et al. Br J Cancer 1994;70:652-661; Tibben et al. Int J Cancer 1996;66:477-483), is due to off-target T cell activation, leading to systemic cytokine release. Clearly, this phenomenon precludes the optimal therapeutic activity of bispecific antibodies that stimulate the TCR / CD3 complex.

[0005] In principle, the dose-limiting off-target T cell activation and the resulting toxicity problems may be caused by two different mechanisms. T cell activation was not target cell-restricted at that time, that is, even the monovalent CD3 effector binding site in the bispecific antibody construct was able to induce some T cell activation in the absence of target cells to which the antibody binds with its targeting moiety. Strictly speaking, this represents off-target activation because cells carrying the target antigen are not required to induce this phenomenon. We have noted that this phenomenon changes considerably if different forms of different CD3 antibodies are used and if certain stimulatory bystander cells (SBCs) that provide co-stimulation for T cell activation, such as lymphoma cells (SKW6.4) or endothelial cells (HUVEC), are added. Therefore, the CD3 moiety that induces minimal "off-target" T cell activation should be selected for the construction of bispecific antibodies.

[0006] Since it binds to cells that normally express the TAA, the TAA targeted by the bispecific antibody is not entirely tumor-specific, leading to antibody-mediated T cell activation. Strictly speaking, this is not off-target activation as it is induced by antigen-expressing target cells (albeit "wrong"), i.e., normal cells rather than malignant cells. The above-mentioned bispecific CD19xCD3 antibody Blinatumomab certainly encounters this problem as its target antigen CD19 is expressed on normal B lymphocytes. Clearly, the specificity of the target antigen in malignant tissue is crucial for preventing such off-target T cell activation. Fms-like tyrosine kinase 3 (FLT3) is a hematopoietic class III receptor tyrosine kinase protein that shares homology with other class III family members, including the stem cell factor receptor (c-KIT), macrophage colony-stimulating factor receptor (FMS), and platelet-derived growth factor receptor (PDGFR). Upon binding to the FLT3 ligand, the FLT3 receptor undergoes homodimerization, leading to autophosphorylation of specific tyrosine residues in the juxtamembrane domain and downstream activation through the PI3K Akt, MAPK, and STAT5 pathways. Thus, FLT3 plays a crucial role as a signaling component in controlling the proliferation, survival, and differentiation of normal hematopoietic cells.

[0007] Human FLT3 is expressed in CD34+CD38- hematopoietic stem cells (HSCs) and subsets of dendritic cell precursors. It can also be detected in multipotent progenitors, such as CD34+CD38+CD45RA-CD123 |0W common myeloid progenitors (CMPs), CD34+CD38+CD45RA+CD123 |0W granulocyte-monocyte progenitors (GMPs), and CD34+CD38+CD10+CD19- common lymphoid progenitors (CLPs). Interestingly, FLT3 expression is almost absent in CD34+CD38-CD45RA-CD123- megakaryocyte-erythroid progenitors (MEPs). Thus, FLT3 expression is mainly restricted to early myeloid and lymphoid progenitors, with some expression in more mature monocyte lineage cells. In acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), the expression level of FLT3 is high. FLT3 is also expressed in blast crisis but not in the chronic phase of chronic myeloid leukemia (CML). Overall, FLT3 is expressed in approximately 98% of pre-B ALL patients and approximately 90% of AML patients.

[0008] Fifteen to 34% of AML patients show FLT3 / ITD mutations, with a lower frequency in children and a higher frequency in the elderly. Adult and pediatric AML patients with FLT3 / ITD mutations have a poor prognosis (Rombouts WJ, Blokland I, B, Ploemacher RE Leukemia. 2000 Apr; 14(4):675 - 83.), so FLT3 is a promising target for AML and also for ALL therapies. Mutated FLT3 is constitutively activated, and FLT3 signals through pathways including ras / MAP kinase, STAT5, and PI3 kinase / AKT, thereby contributing to the blockade of apoptosis and differentiation and the stimulation of proliferation. FLT3 can be targeted by antibody methods to treat both AML and ALL. Antibodies that bind FLT3 and inhibit FL - receptor binding have been developed. In a phase I study, the Imclone antibody IMC - EB10 was evaluated in relapsed AML patients, but the study was terminated due to lack of efficacy (ClinicalTrials.gov Identifier: NCT00887926). Thus, there is an urgent need to evaluate second - generation monoclonal antibodies, including bispecific antibodies for the treatment of AML.

[0009] In addition to T - cell activation induced by stimulation with authentic monomeric CD3, recent papers have also proposed an alternative mechanism of off - target activation that involves the targeting moiety of bispecific antibodies; if this moiety consists of single - chain fragments that induce clustering of the effector moiety of the bispecific antibody on the T - cell surface, it may induce strong signaling, leading to T - cell exhaustion (Long et al. Nat Med 2015; 6:581), which is hardly detectable by conventional short - term in vitro assays but severely affects in vivo efficacy. These observations have been obtained using T cells transfected with chimeric antigen receptors (CAR T cells). The chimeric T - cell receptor contains a single - chain antibody as the targeting moiety. It is highly likely that the results of Long et al. (2015) are equally applicable to bispecific antibodies with such targeting moieties, since these reagents, once bound to T cells, are functionally equivalent to T cells transfected with the corresponding CAR. It is well known in the art that most single - chain antibodies have a tendency to form multimers and aggregates (Worn et al. J Mol Biol 2001, 305:989 - 1010), so all but one of the CARs tested by Long et al. (2015) showed clustering and strong CD3 signaling, although to a variable extent (Long et al. 2015). The problems outlined in this article require a bispecific format that prevents multimerization and clustering through the targeting moiety.

[0010] Most bispecific formats have a very low serum half-life (1 - 3 hours) due to reduced molecular weight and lack of the CH3 domain. Thus, the prototype Blinatumomab antibody is applied by continuous 24-hour intravenous infusion over several weeks. Since off-target activation induced by the bivalent C-terminal CD3-binding portion may increase, the use of intact IgG-based formats with an extended serum half-life, such as Figure 1 the use of IgGsc depicted in

[0011] is considered inappropriate. Based on the above, there is a need in the art for improved ABPs targeting FLT3 that address at least one of the problems outlined above. SUMMARY OF THE INVENTION

[0013] Generally, the main aspects of the invention can be described as follows by a brief description:

[0014] In a first aspect, the invention relates to an antigen-binding protein (ABP) capable of binding to human fms-related tyrosine kinase 3 (FLT3), comprising:

[0015] (i) one, preferably two heavy chain variable domains, comprising a CDRH1 region shown in SEQ ID NO:01 (SYWMH), a CDRH2 region shown in SEQ ID NO:02 (EIDPSDSYKDYNQKFKD) (EIDPSDSYKDYNQKFKD), and a CDRH3 region shown in SEQ ID NO:03 (AITTTPFDF), or wherein in each case, CDRH1, CDRH2, and / or CDRH3 independently comprises a sequence having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion compared to SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03, respectively; and

[0016] (ii) one, preferably two light chain variable domains, comprising a CDRL1 region shown in SEQ ID NO:05 (RASQSISNNLH), a CDRL2 region shown in SEQ ID NO:06 (YASQSIS), and a CDRL3 region shown in SEQ ID NO:07 (QQSNTWPYT), or wherein in each case, CDRL1, CDRL2, and / or CDRL3 independently comprises a sequence having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion compared to SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07, respectively

[0017] characterized in that the one, preferably two heavy chain variable domains and the one, preferably two light chain variable domains each comprise an antibody framework region having at least a portion of the consensus framework sequence of a human antibody.

[0018] In a second aspect, the present invention relates to an antigen-binding protein (ABP) or an antigen-binding fragment thereof that is capable of binding to fms-related tyrosine kinase 3 (FLT3) and is capable of competing with the ABP of the first aspect for binding to FLT3.

[0019] In a third aspect, the present invention relates to a bispecific antigen-binding protein (ABP) that comprises a first antigen-binding domain capable of binding to the human fms-like tyrosine kinase 3 (FLT3) antigen and a second antigen-binding domain capable of binding to the human cluster of differentiation 3 (CD3) antigen, wherein the bispecific ABP:

[0020] a. binds FLT3 with an EC of less than 10 nM according to a binding assay of the bispecific ABP to FLT3-positive cells by flow cytometry analysis using a fluorescence-activated cell sorting (FACS) device; and 50 binds FLT3; and

[0021] b. binds CD3 with an EC of less than 200 nM according to a binding assay of the bispecific ABP to CD3-positive cells by flow cytometry analysis using a fluorescence-activated cell sorting (FACS) device. 50 binds CD3.

[0022] In a fourth aspect, the present invention relates to an isolated nucleic acid that comprises a sequence encoding the ABP or an antigen-binding fragment or monomer of the ABP, such as a heavy chain or a light chain, of the first or second aspect, or a sequence encoding the bispecific ABP according to the third aspect.

[0023] In a fifth aspect, the present invention relates to a nucleic acid construct (NAC) that comprises the nucleic acid of the fourth aspect and one or more other sequence features that permit the expression in a cell of the encoded antigen-binding protein (ABP) or bispecific ABP or a component of the ABP or bispecific ABP (such as an antibody heavy chain or light chain).

[0024] In a sixth aspect, the present invention relates to a recombinant host cell that comprises the nucleic acid of the fourth aspect or the nucleic acid construct (NAC) according to the second aspect.

[0025] In a seventh aspect, the present invention relates to a pharmaceutical composition that comprises: (i) the antigen-binding protein (ABP) or bispecific ABP of the first to third aspects, or (ii) the nucleic acid of the fourth aspect or the NAC according to the fifth aspect, or (iii) the recombinant host cell according to the sixth aspect, and a pharmaceutically acceptable carrier, stabilizer, and / or excipient.

[0026] In an eighth aspect, the invention relates to a component for use in medicine, wherein the component is selected from: (i) an antigen-binding protein (ABP) or bispecific ABP of the first to third aspects, or (ii) a nucleic acid of the fourth aspect or a NAC according to the fifth aspect, or (iii) a recombinant host cell according to the sixth aspect and a pharmaceutical composition according to the seventh aspect.

[0027] In a ninth aspect, the invention relates to a method of enhancing a cell-mediated immune response against human cells expressing human FLT3, which comprises contacting said cells in the presence of immune cells, such as T cells or natural killer (NK) cells, with: an antigen-binding protein (ABP) of the first or second aspect, or a bispecific ABP according to the third aspect or a nucleic acid encoding said ABP or bispecific ABP according to the fourth aspect, thereby enhancing the cell-mediated immune response against said human cells.

[0028] In a tenth aspect, the invention relates to a method for preventing and / or treating a proliferative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the component described in the eighth aspect; and wherein the proliferative disease is characterized by the expression of FLT3 in cells associated with the proliferative disorder. DETAILED DESCRIPTION OF THE INVENTION

[0030] In the following, the elements of the invention will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The description should be understood to support and cover embodiments that combine two or more explicitly described embodiments or that combine one or more explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, any arrangement and combination of all the elements described in the description of the present application should be considered to be disclosed in the present application.

[0031] In a first aspect, the present invention relates to an antigen-binding protein (ABP) capable of binding to human fms-related tyrosine kinase 3 (FLT3), comprising: (i) a heavy chain variable domain comprising a CDRH1 region shown as SEQ ID NO:01 (SYWMH), a CDRH2 region shown as SEQ ID NO:02 (EIDPSDSYKDYNQKFKD), and a CDRH3 region shown as SEQ ID NO:03 (AITTTPFDF), or in each case CDRH1, CDRH2, and / or CDRH3 independently comprises a sequence which has no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion as compared to SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03, respectively; or comprises a CDRH1, CDRH2, or CDRH3 sequence having at least 75% sequence identity or at least 80%, preferably 90% sequence identity with SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03; or (ii) a light chain variable domain comprising a CDRL1 region shown as SEQ ID NO:05 (RASQSISNNLH), a CDRL2 region shown as SEQ ID NO:06 (YASQSIS), and a CDRL3 region shown as SEQ ID NO:SEQ ID NO:07 (QQSNTWPYT) or wherein in each case CDRL1, CDRL2, and / or CDRL3 independently comprises a sequence which has no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion as compared to SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07, respectively; or comprises a CDRL1, CDRL2, or CDRL3 sequence having at least 75% sequence identity or at least 80% sequence identity with SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07, characterized in that the heavy chain variable region and the light chain variable region each comprise a human variable region framework sequence. Preferably, the heavy chain variable domain and the light chain variable domain each comprise an antibody framework region having at least a portion of the consensus framework sequence of a human antibody.

[0032] The present invention also provides novel, highly affinity and effective antibody constructs in humanized forms derived from the anti-FLT3 antibody 4G8 (a murine anti-FLT3 antibody fully disclosed in WO 2011 / 076922), which have been humanized for the first time herein by CDR grafting, meaning that the CDR regions of the murine antibody 4G8 are inserted into the framework regions of the heavy and light chains of a human antibody. However, the humanized 4G8 antibody is then subjected to extensive alterations of the form, constant regions and variable regions to obtain the ABP of the present invention. In principle, any variable human light chain and / or variable heavy chain can be used as a scaffold for CDR grafting. In an illustrative example of the humanized antibody of the present invention, the CDR regions of the light chain of antibody 4G8 (which means the CDR loops of SEQ ID NO: 5 to SEQ ID NO: 7) can be inserted into the human κ light sequence IGKV3-15*1 (variable domain) deposited under accession number M23090 in the IMGT / LIGM database. See also Ichiyoshi Y., Zhou M., Casali P. A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific 'germ-line' natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis' J. Immunol. 154(1):226-238(1995). In another illustrative example of the humanized antibody of the present invention, the CDR regions of the heavy chain of antibody 4G8 (meaning the CDR loops of SEQ ID NO: 1 to SEQ ID NO: 3) can be incorporated into the IGHV1-46*03 heavy chain sequence (variable domain) deposited under accession number L06612 in the IMGT / LIGM database. See also Watson C.T., et al. Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. Am. J. Hum. Genet. 92(4):530-546(2013).

[0033] As used herein, the term "antigen-binding protein" or "ABP" refers to a protein that specifically binds to a target antigen, such as one or more epitopes presented by or present on the target antigen. The antigen of the ABP of the present invention is FLT3, or in the case of a bispecific molecule, FLT3 and CD3. Typically, the antigen-binding protein is an antibody (or a fragment thereof), preferably a bispecific antibody; however, other forms of antigen-binding proteins are also contemplated by the present invention. For example, the ABP can be another (non-antibody) receptor protein derived from a small and robust non-immunoglobulin "scaffold", such as those having binding functions by using combinatorial protein design methods (Gebauer & Skerra, 2009; Curr Opin Chem Biol, 13:245). Specific examples of such non-antibody ABPs include: Affibody molecules Affibody based on the Z-domain of protein A (Nygren, 2008; FEBS J 275:2668); Affilins based on gamma-B crystallin and / or ubiquitin (Ebersbach et al, 2007; J MoBiol, 372:172); Affimers based on cystatin (Johnson et al, 2012; Anal Chem 84:6553); Affitins based on Sac7d from Sulfolobus acidcaldarius (Krehenbrink et al, 2008; J MolBiol 383:1058); and Affitins.Alphabodies based on triple - helical coiled - coils (Desmet et al, 2014; Nature Comms 5:5237); Anticalins based on lipocalins (Skerra, 2008; FEBS J 275:2677); Avimers based on the A - domains of various membrane receptors (Silverman et al, 2005; Nat Biotechnol 23:1556); DARPins based on ankyrin repeat motifs (Strumpp et al, 2008; Drug Discov Today, 13:695); Fynomers based on the SH3 - domain of Fyn (Grabulovski et al, 2007; J Biol Chem 282:3196); and Kunitz - domain peptides based on the Kunitz domain of various protease inhibitors (Nixon et al, Curr opin DrugDiscov Devel, 9:261) and Centyrins and Monobodies based on the fibronectin type III domain 10 (Diem etal., 2014; Protein Eng Des Sel 27:419 doi:10.1093 / protein / gzu016; Koide&Koide, 2007; Methods Mol Biol 352:95). In the case of the ABP of the present invention, the ABP is preferably provided in a bispecific form, which comprises antigen - binding domains of human FLT3 and human CD3.

[0034] As used herein, the term "complementary determining region" (or "CDR" or "hypervariable region") broadly refers to one or more hypervariable regions or complementary determining regions (CDRs) found in the variable regions of the light or heavy chain variable regions of an antibody. See, e.g., "IMGT", Lefranc et al, 20003, Dev Comp Immunol 27:55; Honegger & Plückthun, 2001, J Mol Biol 309:657, Abhinandan & Martin, 2008, Mol Immunol 45:3832, Kabat, et al. (1987): Sequences of Proteins of Immunological Interest National Institutes of Health, Bethesda, Md. These representations include hypervariable regions as defined by Kabat et al (1983) Sequences of Proteins of Immunological Interest, US Dept of Health and Human Services or hypervariable loops in the three-dimensional structure of an antibody (Chothia and Lesk, 1987; J Mol Biol 196:901). The CDRs in each chain are tightly held by framework regions and together with the CDRs in the other chain contribute to the formation of the antigen-binding site. Within the CDRs, there are selected amino acids that have been described as selective determining regions (SDRs), which represent the key contact residues used by the CDR in antibody-antigen interactions. (Kashmiri, 2005; Methods 36:25).

[0035] The term "antibody" generally refers to an immunoglobulin-based proteinaceous binding molecule. Typical examples of such antibodies are derivatives or functional fragments of immunoglobulins that retain binding specificity. Techniques for generating antibodies and antibody fragments are well known in the art. The term "antibody" also includes immunoglobulins (Igs) of different classes (i.e., IgA, IgG, IgM, IgD, and IgE) and subclasses (e.g., IgG1, IgG2, etc.). Also as described above, exemplary instances of antibody derivatives or molecules include Fab fragments, F(ab')2, Fv fragments, single-chain Fv fragments (scFv), diabodies, or domain antibodies (Holt LJ et al., Trends Biotechnol. 21(11), 2003, 484-490). Thus, the definition of the term "antibody" also includes embodiments such as chimeric, single-chain, and humanized antibodies.

[0036] As used herein, "ABP" may carry one or more domains having a sequence with at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the corresponding naturally occurring domains of immunoglobulin M, immunoglobulin G, immunoglobulin A, immunoglobulin D or immunoglobulin E. In this context, the term "about" or "approximately" as used herein means within 20% deviation, such as within 10% deviation or within 5% of a given value or range.

[0037] As used in the present invention, "percent sequence identity" means the percentage of paired identical residues relative to the number of residues in the longer of the two sequences after a homologous alignment of the sequence of a polypeptide of the present invention with the sequence being discussed. For the purpose of determining the percent amino acid sequence identity, the alignment can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring the alignment, including any algorithms required to achieve the maximum alignment over the full length of the sequences being compared. The same is true for the nucleotide sequences disclosed herein.

[0038] When used herein, "immunoglobulin" is generally a tetrameric glycosylated protein composed of two light (L) chains each of about 25 kDa and two heavy (H) chains each of about 50 kDa. Two types of light chains can be found in immunoglobulins, called lambda and kappa. Depending on the amino acid sequence of the heavy chain constant domain, immunoglobulins can be classified into five major classes: A, D, E, G and M, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. IgM immunoglobulins are composed of 5 basic heterotetrameric units as well as another polypeptide called the J chain and contain 10 antigen binding sites, while IgA immunoglobulins contain 2 - 5 basic 4-chain units which can polymerize and combine with the J chain to form multivalent assemblages. In the case of IgG, the 4-chain unit is typically about 150,000 daltons.

[0039] In IgG class immunoglobulins, there are several immunoglobulin domains in the heavy chain. The "immunoglobulin (Ig) domain" herein refers to an immunoglobulin region having different tertiary structures. In the context of IgG antibodies, the IgG isotypes each have three CH regions: according to the EU index in Kabat et al., "CH1" refers to positions 118 - 220, "CH2" refers to positions 237 - 340, and "CH3" refers to positions 341 - 447. The "hinge" or "hinge region" or "antibody hinge region" or "immunoglobulin hinge region" or "H" herein refers to a flexible polypeptide containing amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain terminates at EU position 220, and the IgG CH2 domain begins at EU position residue 237. Thus, for IgG, the hinge is defined herein to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1), where the numbering is according to the EU index in Kabat et al. The constant heavy chain as defined herein refers to the N-terminus of the CH1 domain to the C-terminus of the CH3 domain, and thus includes positions 118 - 447, where the numbering is according to the EU index.

[0040] The term "variable" refers to the part of an immunoglobulin domain that exhibits variability in its sequence and is involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variable domain"). The variability is not evenly distributed in the variable domain of an antibody; it is concentrated in subdomains in each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions", "HVRs", "HVs", or "complementary determining regions" (CDRs). The more conserved (i.e., non-hypervariable) parts of the variable domain are called "framework" regions (FRs). The variable domains of naturally occurring heavy and light chains each include four FR regions that mainly adopt a β-sheet configuration, which are connected by three hypervariable regions that form loops connecting the β-sheet structures and in some cases form parts of the β-sheet structures. The hypervariable regions in each chain are tightly bound together by the FRs and, together with the hypervariable regions in the other chain, contribute to the formation of the antigen-binding site (see Kabat et al., below). Generally, naturally occurring immunoglobulins include six CDRs (see below); three in VH (CDRH1, CDRH2, CDRH3) and three in VL (CDRL1, CDRL2, CDRL3). In naturally occurring immunoglobulins, CDRH3 and CDRL3 show the widest diversity among the six CDRs, and in particular, CDRH3 is considered to play a unique role in conferring excellent specificity on the immunoglobulin. The constant domains do not directly participate in antigen binding, but exhibit various effector functions, such as, for example, antibody-dependent, cell-mediated cytotoxicity and complement activation.

[0041] The terms "VH" (also referred to as VH) and "VL" (also referred to as VL) are used herein to refer, respectively, to the variable domains of the heavy and light chains of immunoglobulins. The variable region of an immunoglobulin light or heavy chain consists of "framework" regions interrupted by three hypervariable regions. Thus, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions include amino acid residues from "complementary determining regions" or "CDRs". There are three heavy-chain and three light-chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, "CDR" as used herein refers to all three heavy-chain CDRs (CDRH1, CDRH2, and CDRH3), or all three light-chain CDRs (CDRL1, CDRL2, and CDRL3), or both all heavy-chain and all light-chain CDRs (where appropriate). The three CDRs constitute the binding characteristics of the variable region of the light chain, while the three CDRs constitute the binding characteristics of the variable region of the heavy chain. The CDRs determine the antigen specificity of the immunoglobulin molecule and are separated by amino acid sequences that comprise the scaffold or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. The structure and protein folding of an antibody may mean that other residues are considered part of the antigen-binding region, and this should be understood by those skilled in the art. The CDRs provide most of the contact residues for the binding of an immunoglobulin to an antigen or epitope.

[0042] CDR3 is generally the largest source of molecular diversity within the antibody binding site. For example, H3 can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. Those skilled in the art will recognize that each subunit structure (e.g., CH, VH, CL, VL, CDR, FR structures) contains active fragments, such as portions of VH, VL, or CDR subunits that bind antigen, i.e., antigen-binding fragments, or portions of CH subunits that bind and / or activate, for example, Fc receptors and / or complement. CDR generally refers to Kabat CDR as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another criterion for characterizing the antigen-binding site refers to the hypervariable loops as described by Chothia. See, for example, Chothia, et al. (1992; J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Yet another criterion is the AbM definition used by Oxford Molecular's AbM modeling software. Generally, see, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Alternatively, similar descriptive relationships for Chothia hypervariable loops or AbM-defined loops are used to achieve embodiments described for Kabat CDR descriptions.

[0043] The corresponding immunoglobulin mu heavy chain, gamma heavy chain, alpha heavy chain, delta heavy chain, epsilon heavy chain, lambda light chain or kappa light chain can be of any species, such as mammalian species, including rodent species, amphibians, such as the subclass Lissamphibia, including, for example, frogs, toads, salamanders or newts, or invertebrates. Examples of mammals include, but are not limited to, rats, mice, rabbits, guinea pigs, squirrels, hamsters, hedgehogs, platypuses, pikas, armadillos, dogs, lemurs, goats, pigs, cows, opossums, horses, bats, groundhogs, orangutans, rhesus monkeys, woolly monkeys, macaques, chimpanzees, saguinusoedipus, marmosets or humans.

[0044] As described herein, an immunoglobulin is generally a glycoprotein or an antigen-binding portion thereof that includes at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain has a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. In some embodiments, the heavy chain constant region includes three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region includes one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). The CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen. Each VH and VL has three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigenic epitopes.

[0045] "Framework region" or "FR" residues are those variable domain residues other than the hypervariable regions. The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. Thus, a "human framework region" is a framework region that is substantially the same (about 85% or more, usually 90 - 95% or more) as the framework region of a naturally occurring human immunoglobulin. The framework regions of an antibody, i.e., the combined framework regions that make up the light and heavy chains, are used to position and align the CDRs. The CDRs are primarily responsible for binding to antigenic epitopes.

[0046] The terms "Fab", "Fab region", "Fab portion", or "Fab fragment" shall be understood to define a polypeptide comprising the VH, CH1, VL, and CL immunoglobulin domains. A Fab can refer to an isolated region of this type, or to this region in the context of an ABP, as well as to a full-length immunoglobulin or immunoglobulin fragment. Generally, the Fab region contains the entire light chain of an antibody. The Fab region can be used to define the "arms" of an immunoglobulin molecule. It contains the epitope-binding portion of the Ig. The Fab region of a naturally occurring immunoglobulin can be obtained as a proteolytic fragment by papain digestion. The "F(ab')2 portion" is a proteolytic fragment of a pepsin-digested immunoglobulin. The "Fab' portion" is the product generated by reducing the disulfide bonds of the F(ab')2 portion. As used herein, the terms "Fab", "Fab region", "Fab portion", or "Fab fragment" can further include a hinge region that defines the C-terminal end of the antibody arm. This hinge region corresponds to the hinge region found at the C-terminus of the CH1 domain in a full-length immunoglobulin, where the arm of the ABP can be used to define Y. The term hinge region is used in the art because immunoglobulins have a certain flexibility in this region. As used herein, a "Fab heavy chain" shall be understood to be the portion or polypeptide of a Fab fragment that comprises VH and CH1, while a "Fab light chain" as used herein shall be understood to be the portion or polypeptide of a Fab fragment that comprises VL and CL.

[0047] The terms "Fc region" or "Fc fragment" are used herein to define the C-terminal region of an immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The Fc portion mediates the effector functions of an antibody, such as activation of the complement system and activation of immune effector cells (e.g., NK cells) bearing Fc receptors. In a human IgG molecule, the Fc region is generated by papain cleavage at the N-terminus of Cys226. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during the production or purification of an ABP, or by recombinant engineering of the nucleic acid encoding the ABP heavy chain. Native sequence Fc regions include those of mammals, such as human or murine, IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. The Fc region contains two or three constant domains, depending on the class of the antibody. In embodiments where the immunoglobulin is IgG, the Fc region has CH2 and CH3 domains.

[0048] The term "single-chain variable fragment" (scFv) is used herein to define an antibody fragment in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are fused together by a short linker peptide of 10 to about 25 amino acids. For flexibility, the linker is typically rich in glycine, and for solubility, rich in serine or threonine, and can link the N-terminus of VH to the C-terminus of VL, or the N-terminus of VL to the C-terminus of VH. The scFv fragment retains a specific antigen-binding site but lacks immunoglobulin constant domains.

[0049] The term "epitope", also known as "antigenic determinant", refers to the part of an antigen that specifically binds to an antibody or T cell receptor to form a complex. Thus, the term "epitope" includes any molecular or protein determinant capable of specifically binding to an immunoglobulin or T cell receptor. The binding site (paratope) of the ABP described herein can specifically bind / interact with a conformational or continuous epitope that is polypeptide for the target structure. Antigenic determinants are usually composed of the chemically reactive surface groups of a molecule, such as amino acids or sugar side chains, and usually have specific three-dimensional structural features as well as specific charge features. In some embodiments, the epitope determinant includes the chemically reactive surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural features and / or specific charge features. With respect to polypeptide antigens, conformational or discontinuous epitopes are characterized by the presence of two or more discrete amino acid residues that are separated in the primary sequence but assemble into a consistent structure on the molecular surface when the polypeptide folds into its native protein / antigen (SeIa, M., Science (1969) 166, 1365-1374; Laver, W.G., et al. Cell (1990) 61, 553-556). The two or more discrete amino acid residues that contribute to the epitope can be present on separate portions of one or more polypeptide chains. These residues come together on the molecular surface when the polypeptide chains fold into a three-dimensional structure to form the epitope. In contrast, continuous or linear epitopes are composed of two or more discrete amino acid residues that are present in a single linear segment of the polypeptide chain.

[0050] In this context, the term "specific" or "specifically binds", also referred to as "directed to", means that, according to the present invention, an antibody or immunoreceptor fragment is capable of specifically interacting and / or binding with a specific antigen or ligand or a group of specific antigens or ligands, but substantially does not bind to other antigens or ligands. This binding can be illustrated by the specificity of the "lock and key principle". Antibodies are said to "bind the same epitope" if they cross-compete such that only one antibody can bind to the epitope at a given point in time, i.e., one antibody blocks the binding or modulating action of another antibody.

[0051] As used herein, the term "isolated ABP" refers to ABP that has been identified, isolated, and / or recovered from the components of its natural environment. The contaminant components in its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and can include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the ABP is purified to greater than 95% by weight antibody, e.g., greater than 99% by weight, as determined by the Lowry method. In some embodiments, the antibody is purified to homogeneity using Coomassie blue or preferably silver staining, under reducing or non-reducing conditions, as judged by SDS-PAGE. In some embodiments, the isolated ABP can be present within a recombinant cell in the absence of one or more components of the natural environment of the antibody. Typically, an isolated antibody is prepared by at least one purification step.

[0052] The (recombinant) ABP of the invention that binds FLT3 and / or FLT3-expressing cancer cells as described herein can be used in any suitable recombinant antibody format, such as an Fv fragment, scFv, a monovalent antibody lacking the hinge region, a microbody, a Fab fragment, a Fab′ fragment, an F(ab′)2 fragment. The recombinant ABP of the invention can also comprise constant domain(s), such as a human IgG constant domain, a CH1 domain (as in a Fab fragment), and / or the entire Fc region. Alternatively, the ABP of the invention can also be a full-length (whole) antibody, preferably in bispecific form.

[0053] There are multiple possible mechanisms of antibody-mediated cell effects, including anti-proliferation by blocking required growth pathways, intracellular signaling leading to apoptosis, enhanced receptor downregulation and / or turnover, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and promotion of an adaptive immune response (Cragg et al, 1999, Curr Opin Immunol 11 541-547, Glennie et al, 2000, Immunol Today 21 403-410). Antibody efficacy can be attributed to a combination of these mechanisms, and their relative importance in oncological clinical treatment appears to be cancer-dependent.

[0054] The importance of FcγR-mediated effector functions for the activity of certain antibodies has been demonstrated in mice (Clynes et al, 1998, Proc Natl Acad Sci U S A 95: 652-656, Clynes et al, 2000, Nat Med 6: 443-446), as well as from the observed correlation between clinical efficacy in humans and the allotypes of FcγRIIIa with high (V158) or low (F158) affinity polymorphisms (Cartron et al, 2002, Blood 99: 754-758, Weng & Levy, 2003, Journal of Clinical Oncology, 21: 3940-3947). These data together suggest that antibodies optimized for binding to certain FcγRs can better mediate effector functions and thus more effectively destroy target cells in patients. Therefore, a promising means of enhancing the anti-tumor potential of antibodies is to enhance their ability to mediate cytotoxic effector functions such as ADCC, ADCP, and CDC. In addition, antibodies can mediate anti-tumor mechanisms through growth inhibition or apoptosis signal transduction that can occur when the antibody binds to its target on tumor cells. Such signaling can be enhanced when the antibody is presented to tumor cells bound to immune cells via FcγR. Thus, an increase in the affinity of the antibody for FcγR can lead to enhanced anti-proliferative effects.

[0055] Some success has been achieved in modifying antibodies to provide enhanced effector functions by selectively enhancing binding to FcγR. Antibody engineering for optimized effector functions has been achieved using amino acid modifications (see, for example, US Patent Application US2004-0132101 or US Patent Application 2006-0024298).

[0056] The ABP of the present invention is capable of binding to human FLT3. The terms "fms-related tyrosine kinase 3" or "FLT3" are used interchangeably herein and include variants, isoforms, and species homologs of human FLT3. The UniProt accession number for the human FLT3 protein is P36888 (version of September 9, 2018). The gene for human FLT3 is located on chromosome 13 and has an HGNC accession name of HGNC:3765 (www.genenames.org – HGNC version of September 9, 2018). Human FLT3 is also known by the names CD135, FLK2, and STK1. However, in certain preferred cases, the antibodies of the present invention may not cross-react with FLT3 from species other than humans.

[0057] To determine epitopes, standard epitope mapping methods known in the art can be used. For example, fragments (peptides) of FLT3 that bind antibodies (e.g., synthetic peptides) can be used to determine whether a candidate antibody or its antigen-binding fragment binds to the same epitope. For linear epitopes, overlapping peptides of a defined length (e.g., 8 or more amino acids) are synthesized. The peptides can be offset by 1 amino acid, thereby preparing a series of peptides that cover every 8 amino acid fragments of the FLT3 protein sequence. Fewer peptides can be prepared by using a larger offset (e.g., 2 or 3 amino acids). Additionally, longer peptides (e.g., nonamers, decamers, or undecamers) can be synthesized. Standard methods can be used to determine the binding of the peptides to the antibody or antigen-binding fragment, including surface plasmon resonance (BIACORE) and ELISA assays. To examine conformational epitopes, larger FLT3 fragments can be used. Other methods for defining conformational epitopes using mass spectrometry have been described and can be (see, e.g., Baerga-Ortiz et al., Protein Science 11:1300-1308, 2002 and the references cited therein). Other methods for determining epitopes are provided in standard laboratory reference works, such as Current Protocols in Immunology, edited by Coligan et al., John Wiley & Sons, Unit 6.8 ("Phage Display Selection and Analysis of B-Cell Epitopes") and Unit 9.8 ("Identification of Epitopes Using Synthetic Peptide Combinatorial Libraries"). Epitopes can be determined by introducing point mutations or deletions into a known epitope and then testing the binding to one or more antibodies or antigen-binding fragments to determine which mutations reduce the binding of the antibody or antigen-binding fragment.

[0058] In some preferred embodiments, the ABP of the present invention comprises a heavy chain variable region in its first antigen-binding domain, and the heavy chain variable region comprises a human framework region of allele IGHV1-46, preferably IGHV1-46*3. Within the light chain, the ABP of the present invention comprises a variable region having the framework of IGKV3D-15*01. In some embodiments, the antibody of the present invention is a fully grafted humanized antibody, designated V0-V6, and comprises heavy and light chain variable domain sequences of SEQ ID NOs: 76 and 77, respectively. However, one achievement of the present invention is to provide mutant variants of humanized anti-FLT3 antibodies that have improved binding affinity, avidity and / or activity in the recruitment and activation of T cells and T cell-mediated anti-tumor cytotoxicity. Thus, according to the present invention, preferably, in the FLT3-specific first antigen-binding domain of the ABP of the present invention, the ABP comprises a heavy chain variable region that has a mutation at one or more positions selected from 16, 18, 19, 20, 22, 48, 57, 60, 69, 70, 75, 76, 78, 80, 81, 87 and 108, according to Kabat numbering. Most preferably, the mutations are any one or any combination or all of K16G, V18L, K19R, V20L, K22A, M48I, K57T, N60A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A and T108L, according to Kabat numbering. Additionally, the ABP of the present invention may further comprise a variable light chain sequence having one or more mutations selected from 49, 87 and 55, preferably Y49K, I55A and Y87F, in its FLT3-specific first antigen-binding domain, where the numbering is according to the Kabat system. In some embodiments, the ABP of the present invention may include heavy chain variable sequence mutations in its first antigen-binding domain, and preferably as the only mutations, include K16G, V18L, K19R, V20L, K22A, K57T, N60A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A and T108L, and a light chain variable sequence mutation of I55A, or no mutation in the variable light chain region. Or the ABP of the present invention may include K16G, V18L, K19R, V20L, K22A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A, T108L, preferably as the only mutations, and no mutation in the light chain variable sequence. Another example relates to an ABP that does not contain a mutation in the heavy chain variable sequence of the anti-FLT3 binding domain and contains an I55A mutation in the corresponding light chain variable sequence.Further preferred is the ABP of the present invention, which comprises in its anti-FLT3 first antigen-binding domain a heavy chain variable sequence having a mutation at position 48, preferably 48I, and mutations 49K and 87F in the corresponding light chain variable sequence. According to the Kabat system numbering.

[0059] Another embodiment of the present invention relates to an ABP, said ABP comprising in its first antigen-binding domain against FLT3 an antibody heavy chain variable region and an antibody light chain variable region, wherein said heavy chain variable region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 15, 17, 19, 21, 23 or 76, or in each case independently, optionally having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2 or 1 amino acid substitutions, insertions or deletions as compared to these sequences; and / or wherein the light chain variable region comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity with an amino acid sequence selected from SEQ ID NO: 16, 18, 20, 22, 24 or 77, or in each case independently, optionally having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2 or 1 amino acid substitutions, insertions or deletions as compared to these sequences. Wherein such amino acid substitutions are preferably conservative substitutions. Most preferably, the light and heavy chains in these ABPs are paired as disclosed herein in Table 1. Additionally, preferred ABPs according to the present invention include in the heavy chain variable region the amino acid positions 16, 18, 19, 20, 22, 48, 57, 60, 69, 70, 75, 76, 78, 80, 81, 87 and 108 provided in any one of SEQ ID NO: 15, 17, 19, 21 or 23; and / or include in the light chain variable region the amino acid positions 49, 55 and 87 provided in any one of SEQ ID NO: 16, 18, 20, 22 or 24; wherein the numbering is according to the Kabat system.

[0060] In some embodiments of the present invention, the heavy chain variable region of the ABP comprises an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 21, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 22.

[0061] In some embodiments of the present invention, the heavy chain variable region of ABP comprises an amino acid sequence having at least 85%, at least 90% or at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:76, and the light chain variable region comprises an amino acid sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:77.

[0062] In some specific embodiments of the present invention, ABP is preferred, wherein the heavy chain variable region comprises 48I, and wherein the light chain variable region comprises 87F, wherein the numbering is according to the Kabat system. Even more preferred is the ABP of the present invention, wherein the light chain variable region further comprises 49K, wherein the numbering is according to the Kabat system.

[0063] As explained herein, the ABP of the present invention is preferably a bispecific molecule, wherein the second antigen-binding domain binds to CD3, preferably wherein the second antigen-binding domain is fused to the heavy chain of the first antibody-binding domain. Preferably, the second antigen-binding domain comprises an scFv fragment, which scFv fragment comprises an amino acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% amino acid identity when compared with the sequences of SEQ ID NO:14, 25, 26 and 27, or independently in each case having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2 or 1 amino acid substitutions, insertions or deletions.

[0064] In some embodiments, the ABP according to the invention can be an ABP comprising at least one antibody heavy chain and / or an ABP comprising at least one antibody light chain, wherein the antibody heavy chain has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity with SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74 and 78, or has an amino acid sequence with no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, and preferably no more than 1 amino acid substitution, deletion or insertion; the antibody light chain has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity with SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75 and 79, or has an amino acid sequence with no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, and preferably no more than 1 amino acid substitution, deletion or insertion. In a preferred embodiment, the ABP of the invention is the ABP as described in this paragraph, provided that the light chain and the heavy chain are paired as shown in the antibodies disclosed herein in Table 1 below.

[0065] The ABP of the invention is preferably an ABP comprising one, preferably two antibody heavy chains and one, preferably two antibody light chains, each of which has at least 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%, preferably 100% sequence identity with the following, or has an amino acid sequence with no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, and preferably no more than 1 amino acid substitution, deletion or insertion:

[0066] a. Sequences selected from: SEQ ID NO: 28 of the heavy chain and SEQ ID NO: 29 of the light chain (antibody V1-V6);

[0067] b. Sequences selected from: SEQ ID NO: 30 of the heavy chain and SEQ ID NO: 31 of the light chain (antibody V2-V6);

[0068] c. Sequences selected from: SEQ ID NO: 32 of the heavy chain and SEQ ID NO: 33 of the light chain (antibody V3-V6);

[0069] d. Sequences selected from: SEQ ID NO: 34 of the heavy chain and SEQ ID NO: 35 of the light chain (antibody V4-V6);

[0070] e. A sequence selected from the following: SEQ ID NO:36 of the heavy chain and SEQ ID NO:37 of the light chain (antibody V5-V6);

[0071] f. A sequence selected from the following: SEQ ID NO:38 of the heavy chain and SEQ ID NO:39 of the light chain (antibody V1-V7);

[0072] g. A sequence selected from the following: SEQ ID NO:40 of the heavy chain and SEQ ID NO:41 of the light chain (antibody V2-V7);

[0073] h. A sequence selected from the following: SEQ ID NO:42 of the heavy chain and SEQ ID NO:43 of the light chain (antibody V3-V7);

[0074] i. A sequence selected from the following: SEQ ID NO:44 of the heavy chain and SEQ ID NO:45 of the light chain (antibody V4-V7);

[0075] j. A sequence selected from the following: SEQ ID NO:46 of the heavy chain and SEQ ID NO:47 of the light chain (antibody V5-V7);

[0076] k. A sequence selected from the following: SEQ ID NO:48 of the heavy chain and SEQ ID NO:49 of the light chain (antibody V1-V8);

[0077] l. A sequence selected from the following: SEQ ID NO:50 of the heavy chain and SEQ ID NO:51 of the light chain (antibody V2-V8);

[0078] m. A sequence selected from the following: SEQ ID NO:52 of the heavy chain and SEQ ID NO:53 of the light chain (antibody V3-V8);

[0079] n. A sequence selected from the following: SEQ ID NO:54 of the heavy chain and SEQ ID NO:55 of the light chain (antibody V4-V8);

[0080] o. A sequence selected from the following: SEQ ID NO:56 of the heavy chain and SEQ ID NO:57 of the light chain (antibody V5-V8);

[0081] p. A sequence selected from the following: SEQ ID NO:58 of the heavy chain and SEQ ID NO:59 of the light chain (antibody V1-V9);

[0082] q. A sequence selected from the following: SEQ ID NO:60 of the heavy chain and SEQ ID NO:61 of the light chain (antibody V2-V9);

[0083] r. selected from the following sequences: SEQ ID NO:62 of the heavy chain and SEQ ID NO:63 of the light chain (antibody V3-V9);

[0084] s. selected from the following sequences: SEQ ID NO:64 of the heavy chain and SEQ ID NO:65 of the light chain (antibody V4-V9);

[0085] t. selected from the following sequences: SEQ ID NO:66 of the heavy chain and SEQ ID NO:67 of the light chain (antibody V5-V9);

[0086] u. selected from the following sequences: SEQ ID NO:68 of the heavy chain and SEQ ID NO:69 of the light chain (antibody V6-V6);

[0087] v. selected from the following sequences: SEQ ID NO:70 of the heavy chain and SEQ ID NO:71 of the light chain (antibody V6-V7);

[0088] w. selected from the following sequences: SEQ ID NO:72 of the heavy chain and SEQ ID NO:73 of the light chain (antibody V6-V8);

[0089] x. selected from the following sequences: SEQ ID NO:74 of the heavy chain and SEQ ID NO:75 of the light chain (antibody V6-V9);

[0090] y. selected from the following sequences: SEQ ID NO:78 of the heavy chain and SEQ ID NO:79 of the light chain (antibody V0-V6).

[0091] Throughout this disclosure, preferred antibody variable chain variants are referred to by the name "Vx" or "Vx-Vy". The phrase "Vx", where x can be 0, 1, 2, 3, 4, 5, 6, 7, 8, or 9, refers to variable region variants disclosed in the present invention according to Table 1 or the Examples section, particularly as described in Example 3. In particular, the humanized 4G8 FLT3 antigen-binding domain is designated as variant 0 or V0. The terms V0, V1, V2, V3, V4, V5, and V6 can refer to variable region variants of the FLT3-binding site of the antibodies of the present invention. On the other hand, the terms V6, V7, V8, and V9 refer to certain variants of the CD3-binding site of the antibodies (bispecific) of the present invention. The term "Vx-Vy" is used herein to describe preferred bispecific FLT3xCD3 ABPs of the present invention, where Vx represents an anti-FLT3 binding site variant and Vy represents an anti-CD3 binding site variant. In this case, Vx can be one of V0, V1, V2, V3, V4, V5, and V6; and Vy can be one of V6, V7, V8, or V9. It should be understood that V6 can be used in the context of either the anti-FLT3 binding site variant or the anti-CD3 binding site variant of the present invention. Its usage is apparent from the context. From Table 1 below, the sequences and overall construct nomenclature of the variants also become apparent.

[0092] The heavy and light chains of the above-mentioned ABPs (a.) to (y.) preferably pair with each other. The preferred ABPs of the present invention are selected based on their ability to induce T cell-mediated killing and / or growth inhibition of leukemia cancer cells, for example, according to the data provided in the Examples section of this disclosure. For example, certain constructs such as V5-V9 exhibit the highest combined FLT3 and CD3 affinities. Although, in this regard, each construct containing the CD3 or FLT3 antigen-binding domain of V5-V9 is a preferred ABP, certain applications may require such strong FLT3 / CD3 binders. However, antibodies with slightly reduced affinities but thus better tolerability (humanized) or biological activity (anticancer activity) are also included as preferred constructs of the present invention. Thus, all of the above-described ABPs a. to x are preferred, particularly all humanized variants of the FLT3 (first) antigen-binding domain that do not contain V6-V6.

[0093] Then, another aspect of the present invention relates to a bispecific antigen-binding protein (ABP) that comprises a first antigen-binding domain capable of binding to the human fms-like tyrosine kinase 3 (FLT3) antigen and a second antigen-binding domain that binds to the human cluster of differentiation 3 (CD3) antigen. The bispecific ABP as described in this aspect may, in a preferred embodiment, include any one or combination of the embodiments of the ABP described previously herein. The ABP described previously herein can be the bispecific ABP as described hereinafter in all of its embodiments.

[0094] In certain embodiments, it is preferred that the bispecific ABP has an EC that is less than 10 nM, preferably less than 9 nM, more preferably less than 8 nM, more preferably less than 7 nM, more preferably less than 6 nM, or less than 5.5 nM 50 for binding to FLT3. Alternatively / additionally, the bispecific ABP of the present invention has an EC for binding to FLT3 that is greater than 0.5 nM, more preferably greater than 1 nM, more preferably greater than 1.3 nM, more preferably greater than 2 nM, more preferably greater than 3 nM or 4 nM, or greater than 4.5 nM 50 for binding to FLT3. Further alternatively / additionally, the bispecific ABP according to the present invention has an EC for binding to FLT3 that is less than 10 nM and greater than 0.5 nM, more preferably less than 9 nM and greater than 1 nM, preferably less than 8 nM and greater than 1.3 nM, preferably less than 7 nM and greater than 3 nM, preferably less than 6 nM and greater than 4 nM, preferably less than 5.5 nM and greater than 4.5 nM 50 for binding to FLT3. The EC of the binding of the bispecific ABP to FLT3 is determined by flow cytometry analysis of the binding of the bispecific ABP to FLT3-positive cells using a fluorescence-activated cell sorting (FACS) apparatus 50 ; preferably wherein the FLT3-positive cells are B-cell precursor leukemia cells, preferably NALM-16 cells (such as those deposited at DSMZ under ACC680); and / or preferably, wherein binding is detected using a fluorescently labeled secondary antibody; and / or wherein the bispecific ABP is incubated with the FLT3-positive cells for about 30 minutes prior to flow cytometry. In this embodiment, the present invention relates to antibodies, and it has surprisingly been found that certain preferred binding affinities to the target antigen-binding molecule can translate into improved therapeutic effects. In this regard, as shown in the examples, although certain antibodies have a lower affinity for FLT3 compared to other antibodies, they have a significantly improved effect in mediating cytotoxicity. In some preferred aspects and embodiments, antibodies that fall within such an affinity "window" as disclosed herein are preferred.

[0095] Alternatively / additionally, the bispecific ABP of the present invention has a k that is less than 50 μM, more preferably less than 20 μM, more preferably less than 10 μM, more preferably less than 5 μM, more preferably less than 1 μM DBind to FLT3; and / or bind FLT3 with a kD greater than 50 nM, more preferably greater than 100 nM, more preferably greater than 160 nM, more preferably greater than 200 nM, more preferably greater than 300 nM. Most preferably, the bispecific ABP of the present invention binds FLT3 with a kD within certain preferred ranges, such as less than 50 μM and greater than 50 nM, more preferably less than 20 μM and greater than 100 nM, more preferably less than 10 μM and greater than 160 nM, more preferably less than 5 μM and greater than 200 nM, more preferably less than 1 μm and greater than 300 nM; wherein the kD is measured by surface plasmon resonance, such as in a BIAcore affinity assay, as provided in the Examples section.

[0096] In another embodiment, the bispecific ABP according to the present invention binds CD3 with an EC50 of less than 200 nM, preferably less than 90 nM, more preferably less than 50 nM, more preferably less than 20 nM, more preferably less than 15 nM, and / or binds CD3 with an EC50 of greater than 1 nM, preferably greater than 2 nM, more preferably greater than 4.1 nM, more preferably greater than 6 nM, more preferably greater than 8 nM; and / or in some embodiments, certain ranges of the affinity for CD3 binding are preferred, such as binding CD3 with an EC50 of less than 200 nM and greater than 1 nM, preferably less than 200 nM and greater than 2 nM, more preferably less than 90 nM and greater than 4.1 nM, more preferably less than 20 nM and greater than 6 nM, more preferably less than 15 nM and greater than 8 nM. The EC50 of the bispecific ABP binding to CD3 is determined by flow cytometry analysis of the binding of the bispecific ABP to CD3-positive cells using a fluorescence-activated cell sorting (FACS) device; preferably wherein the CD3-positive cells are T cell leukemia cells, preferably Jurkat cells (deposited at DSMZ under ACC 282); and / or preferably, wherein a fluorescently labeled secondary antibody is used to detect the binding; and / or wherein the bispecific ABP is incubated with the CD3-positive cells for about 30 minutes before flow cytometry.

[0097] Alternatively / In addition, in some embodiments, the ABP of the present invention is characterized by their ability to kill cancer cells expressing human FLT3 such as ALL or AML cells or to inhibit their proliferation. Thus, preferred in the context of the present invention are bispecific ABP as described herein which inhibit the proliferation and / or viability of leukemic blood mononuclear cells of patients suffering from acute leukemia to equal to or less than 50%, more preferably equal to or less than 40%, more preferably equal to or less than 30%, and most preferably equal to or less than 25% in in vitro assays compared to an irrelevant control. Such preferred bispecific ABP of the present invention are exemplified in V3-V6, V3-V8, V3-V9, V4-V6, V4-V8, V4-V9, V5-V6, V5-V8 and V5-V9 and variants thereof disclosed herein.

[0098] In addition, some embodiments of the present invention relate to ABP which compete with the ABP of the present invention for binding to FLT3, e.g., competitively inhibit the binding of the antibodies of the present invention to FLT3. To determine competitive inhibition, a variety of assays known to those of ordinary skill in the art can be employed. For example, cross-competition assays can be used to determine whether an antibody or an antigen-binding fragment thereof competitively inhibits the binding of another antibody or an antigen-binding fragment thereof to FLT3. These include cell-based methods employing flow cytometry or solid-phase binding assays. Other assays can also be used which evaluate the ability of an antibody or an antigen-binding fragment thereof to cross-compete with FLT3 molecules not expressed on the cell surface in the solid phase or in solution.

[0099] The ABP according to the present invention can have two chains, a short chain (which can be a light chain in some embodiments) and a main chain (which can also be referred to as a heavy chain in some embodiments). The ABP is generally a dimer of these two chains.

[0100] The ABP of the present invention can preferably be a bispecific ABP. The bispecific ABP can comprise (i) a variable region which comprises a heavy chain variable domain and a light chain variable domain as defined in any one of the preceding claims, wherein said variable region comprises a first antigen-binding domain capable of binding human FLT3 and (ii) a heavy chain variable region and a light chain variable region of the ABP which comprise a second antigen-binding domain. It should be understood that the binding site of FLT3 is preferably the binding site of the FLT3-binding antibody of the present invention as described herein.

[0101] A "bispecific" or "bifunctional" ABP is an ABP having two different epitope / antigen-binding domains (or "sites") and thus having binding specificity for two different target epitopes. These two epitopes can be epitopes of the same antigen or, as is preferred in the present invention, of different antigens, e.g., different antigens FLT3 and CD3.

[0102] "Bispecific ABP" can be an ABP that binds one antigen or epitope with one of two or more binding arms defined by a first pair of heavy and light chains or a backbone and a shorter / smaller chain, and binds a different antigen or epitope on a second arm defined by a second pair of heavy and light chains or a backbone and a smaller chain. This embodiment of the bispecific ABP has two different antigen-binding arms both in terms of specificity and CDR sequences. Generally, the bispecific ABP is monovalent for each antigen it binds, that is, it binds the corresponding antigen or epitope with only one arm. However, bispecific antibodies can also be dimerized or multimerized, which is preferred in the context of the present invention. For example, in the dimeric IgGsc form as described herein, the antibody can have two binding sites for each antigen ( Figure 1 ). The bispecific antibody can be a hybrid ABP that can have a first binding region defined by a first light chain variable region and a first heavy chain variable region, and a second binding region defined by a second light chain variable region and a second heavy chain variable region. The present invention contemplates that one of these binding regions can be defined by a heavy chain / light chain pair. In the context of the present invention, the bispecific ABP can have a first binding site defined by the variable regions of the backbone and a smaller chain, and a second different binding site defined by the variable regions of the scFv fragment contained in the backbone of the ABP.

[0103] Methods for preparing bispecific ABP are known in the art, such as chemical conjugation of two different monoclonal antibodies, or chemical conjugation of two antibody fragments, such as two Fab fragments. Alternatively, bispecific ABP is prepared by the quadroma technique, that is, by fusing the hybridomas that produce the parental antibodies. Due to the random assortment of H and L chains, a potential mixture of ten different antibody structures is produced, and only one of them has the desired binding specificity.

[0104] The bispecific ABP of the present invention can act as a monoclonal antibody (mAb) against each target. In some embodiments, the antibody is chimeric, humanized or fully human. The bispecific ABP can be, for example, a bispecific tandem single-chain Fv, a bispecific Fab2 or a bispecific diabody.

[0105] Based on the domains included in the ABP of the present invention, the bispecific ABP of the present invention may comprise a Fab fragment, which generally may comprise a hinge region, a CH2 domain, and a single-chain Fv fragment. Such bispecific ABPs are referred to as "Fabsc"-ABPs and have been first described in international patent application WO 2013 / 092001. More specifically, the ABP in the form of "Fabsc" as used herein generally refers to the bispecific ABP of the present invention having a Fab fragment, which generally includes a hinge region located at the C-terminus of the Fab fragment connected to the N-terminus of the CH2 domain, and the C-terminus of the CH2 domain is in turn connected to the N-terminus of the scFv fragment. Such "Fabsc" does not contain or substantially does not contain a CH3 domain. In this context, "does not contain" or "substantially does not contain" means that the ABP does not contain a full-length CH3 domain. Preferably, it means that the ABP contains 10 or fewer, preferably 5 or fewer, preferably 3 or even fewer amino acids of the CH3 domain. In an illustrative embodiment, the Fabsc ABP of the present invention may comprise a CH2 domain that lacks the ability to dimerize via a disulfide bond formed by a cysteine residue at sequence position 226 of the hinge region and / or a cysteine residue at sequence position 229 of one of the hinge domains, according to Kabat numbering [EU-index]. Thus, in these embodiments, the cysteine residue at sequence position 226 and / or sequence position 229 is removed or replaced, for example, by a serine residue. Additionally / or, the "Fabsc" ABP of the present invention may also have a "Fc-attenuated" CH2 domain (which includes the hinge region). This "Fc attenuation" is achieved by deleting and / or substituting (mutating) at least one of the selected amino acid residues in the CH2 domain that are capable of mediating binding to the Fc receptor. In an illustrative embodiment, at least one amino acid residue in the hinge region or CH2 domain that is capable of mediating binding to the Fc receptor and is lacking or mutated is selected from the group consisting of sequence positions 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330 (sequence position numbers according to the EU index).In illustrative examples, such Fc-attenuated ABPs can include at least one mutation selected from the group consisting of: deletion of amino acid 228, deletion of amino acid 229, deletion of amino acid 230, deletion of amino acid 231, deletion of amino acid 232, deletion of amino acid 233, substitution of Glu233→Pro, substitution of Leu234→Val, deletion of amino acid 234, substitution of Leu235→Ala, deletion of amino acid 235, deletion of amino acid 236, deletion of amino acid 237, deletion of amino acid 238, substitution of Asp265→Gly, substitution of Asn297→Gln, substitution of Ala327→Gln and substitution of Ala330→Ser (numbered according to the sequence positions of the EU index, for example, also see International Patent Application WO2013 / 092001). Figure 1 O and Figure 1 P). In the case of bispecific antibodies that activate T cells, for example, against tumor cells, Fc attenuation may be required to prevent the antibody from binding to cells carrying Fc receptors, which may lead to unwanted off-target activation of T cells.

[0106] According to the publication of Coloma and Morrison (Nat Biotechnol 15:159-63, 1997), the bispecific ABPs of the present invention can also have a CH3 domain, which is usually arranged at the C-terminus of the CH2 domain. Such molecules are also referred to herein as "IgGsc" forms of ABPs and refer to the bispecific ABPs of the present invention having Fab fragments, which usually include a hinge region located at the C-terminus of the Fab fragment that is usually connected to the N-terminus of the CH2 domain, the C-terminus of the CH2 domain is usually connected to the N-terminus of the CH3 domain, and the C-terminus of the CH3 domain is usually connected to the N-terminus of the scFv fragment. Illustrative examples of IgGsc form ABPs are shown in Figure 1 In the context of the present invention, such bispecific ABP forms are preferred.

[0107] The common feature of the antibody formats Fabsc and IgGsc is that the N-terminal targeting moieties are composed of the "physiological" Fab or Fab2 regions, respectively, thus avoiding the use of single-chain moieties in this part of the molecule. If these formats are used for target cell-restricted T cell activation, Fc receptor (FcR) binding can be attenuated (if desired or required) to prevent FcR-mediated activation. For example, this can be achieved, for example, by introducing defined and well-known mutations in the CH2 domain of the molecule, as described above and in International Patent Application WO2013 / 092001 and Armour et al. Eur J Immunol 1999; 29:2613. Thus, the IgGsc ABP of the present invention can also have a CH2 domain (including the hinge region), wherein the lack or mutation of at least one amino acid residue in the hinge region or the CH2 domain that can mediate binding to the Fc receptor. As described above, the residues in the CH2 and hinge regions can be selected from the group consisting of sequence positions 228, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330 (sequence position numbering according to the EU index). However, due to the presence of the CH3 domain in the IgGsc molecule, two separate molecules will form a tetravalent molecule by (spontaneous) homodimerization through the CH3 domain (see again in this regard Figure 1 ). Therefore, it is not necessary to delete or mutate the cysteine residues at sequence position 226 and / or sequence position 229 in the hinge region. Thus, such tetravalent IgGsc ABPs of the present invention can have cysteine residues at sequence position 226 and / or sequence position 229 in one of the corresponding hinge domains, according to Kabat numbering [EU-index].

[0108] Consistent with the above disclosure of bispecific ABP comprising a set of CDR regions that mediate FLT3 binding and / or bind to leukemia cancer cells, the ABP of the present invention may comprise a second binding site that specifically binds to a receptor on an immune cell (such as a T cell or NK cell). The receptor present on the immune cell may be a receptor capable of activating the immune cell or stimulating the immune response of the immune cell. The induced immune response may preferably be a cytotoxic immune response. Such suitable receptors may be, for example, CD3, antigen-specific T cell receptor (TCR), CD28, CD16, NKG2D, Ox40, 4-1BB, CD2, CD5, programmed cell death protein 1 (PD-1), and CD95. Particularly preferred is an ABP in which the second binding site binds to CD3, TCR, or CD16. Most preferred is an ABP in which the second binding site specifically binds to CD3. A preferred ABP comprises a second binding site corresponding to the antigen-binding site of the anti-CD3 antibody OKT3. The amino acid sequences of the heavy chain variable domain and the light chain variable domain of the antibody OKT3 are also described, for example, in Arakawa et al J. Biochem. 120, 657-662 (1996) and International Patent Application WO 2015 / 158868 (see SEQ ID NOs: 17 and 18 in the sequence listing of WO 2015 / 158868). Another preferred ABP comprises a second binding site corresponding to the antigen-binding site of the anti-CD3 antibody UCHT1. The VH and VL sequences of the humanized UCHT1 antibody are described in International Patent Application WO 2013 / 092001. Other examples of CD3-binding ABP useful in the present invention include the ABP described in European Patent 2 155783B1 or European Patent EP 2 155 788 B1, which is capable of binding to epitopes of the CD3ε chain of humans and marmosets (Callithrix jacchus), cotton-top tamarins (Saguinus oedipus), or squirrel monkeys (Saimiri sciureus).

[0109] Thus, the bispecific ABP of the present invention may be a bispecific ABP, such as an IgGsc molecule comprising a Fab fragment and an scFv fragment as described herein. In this molecule, the first binding site may bind to FLT3 and may be contained in a Fab (or bivalent FLT3 F(ab)2 in the context of the IgGsc form) fragment as described herein, and the second binding site (which may bind to an immune receptor) may be contained in an scFv fragment, such as an scFv that specifically binds to CD3. Alternatively, the first binding site that binds to FLT3 is contained in a single-chain Fv fragment, and the second binding site (which may bind to CD3) is contained in a Fab fragment.

[0110] In some embodiments, after binding, such as binding to CD3, the bispecific ABP of the present invention does not itself activate immune cells, such as T cells. Instead, only when the two binding sites, such as the FLT3-specific binding site and the CD3-specific binding site, bind to the receptor on the T cell and FLT3 on the target cancer cell, can the former crosslink the activating receptor, thereby triggering effector cells to kill specific target cells. Standard functional assays can be established to evaluate the ability of lymphocytes to kill target cells in the presence and absence of the bispecific ABP of the present invention to evaluate and / or screen the ability of the ABP to activate the receptor to which it binds.

[0111] In some embodiments of the present invention, the bispecific ABP comprises an anti-CD3 antibody, such as the scFv of UCHT1 or its variant, as the second antigen-binding domain, which is disclosed, for example, in SEQ ID NOs: 14 and 25 to 27 of the present application.

[0112] It is noted in this context that the ABP may comprise one or more mutated amino acid residues within the scope of the present invention. The terms "mutated", "mutant" and "mutation" with respect to nucleic acids or polypeptides refer to an exchange, deletion or insertion of one or more nucleotides or amino acids compared to the nucleic acid or polypeptide that exists as "natural" or "parental" (if a reference is provided), i.e., the reference sequence that can be used to define the wild type. For example, the variable domain of the ABP of the present invention obtained by extensive mutagenesis of the parental 4G8 molecule and as described herein can be regarded as the parental sequence.

[0113] In this regard, it should be understood that the term "position", when used in accordance with the present invention, refers to the position of an amino acid within the amino acid sequence described herein. This position can be indicated relative to a similar natural sequence, such as the sequence of a naturally occurring IgG domain or chain. As used herein, the term "corresponding" also includes positions that are not necessarily or not only determined by the number of preceding nucleotides / amino acids. Thus, a given amino acid position that can be substituted can vary due to deletions or additions of amino acids elsewhere in the antibody chain.

[0114] Therefore, under "corresponding position" according to the present invention, it should be understood that the amino acids can be different in number but still have similar adjacent amino acids. The amino acids that can be exchanged, deleted or added are also covered by the term "corresponding position". To determine whether an amino acid residue in a given amino acid sequence corresponds to a position in the amino acid sequence of a naturally occurring immunoglobulin domain or chain, those skilled in the art can use means and methods well known in the art, such as alignment, manually or by using a computer program such as BLAST2.0 (which stands for Basic Local Alignment Search Tool) or ClustalW, or any other suitable program suitable for generating sequence alignments for alignment.

[0115] In some embodiments, the substitution (or replacement) is a conservative substitution. Conservative substitutions are generally substitutions that, listed according to the amino acid to be mutated, are each followed by one or more substitutions that can be considered conservative: Ala→Gly, Ser, Val; Arg→Lys; Asn→Gln, His; Asp→Glu; Cys→Ser; Gln→Asn; Glu→Asp; Gly→Ala; His→Arg, Asn, Gln; Ile→Leu, Val; Leu→Ile, Val; Lys→Arg, Gln, Glu; Met→Leu, Tyr, Ile; Phe→Met, Leu, Tyr; Ser→Thr; Thr→Ser; Trp→Tyr; Tyr→Trp, Phe; Val→Ile, Leu. Other substitutions are also permitted and can be determined empirically or in accordance with other known conservative or non-conservative substitutions. As a further guide, the following eight groups each contain amino acids that are commonly used to define conservative substitutions for one another:

[0116] - Alanine (Ala), Glycine (Gly);

[0117] - Aspartic acid (Asp), Glutamic acid (Glu);

[0118] - Asparagine (Asn), Glutamine (Gln);

[0119] - Arginine (Arg), Lysine (Lys);

[0120] - Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val);

[0121] - Phenylalanine (Phe), Tyrosine (Tyr), Tryptophan (Trp);

[0122] - Serine (Ser), Threonine (Thr); and

[0123] - Cysteine (Cys), Methionine (Met)

[0124] If such substitution results in a change in biological activity, more substantial changes can be introduced, such as those described below, or as further described with reference to the following amino acid classes, and the products are screened for the desired properties. Examples of such more substantial changes include: Ala→Leu, Ile; Arg→Gln; Asn→Asp, Lys, Arg, His; Asp→Asn; Cys→Ala; Gln→Glu; Glu→Gln; His→Lys; Ile→Met, Ala, Phe; Leu→Ala, Met, norleucine; Lys→Asn; Met→Phe; Phe→Val, Ile, Ala; Trp→Phe; Tyr→Thr, Ser; Val→Met, Phe, Ala.

[0125] In some embodiments, the ABP according to the invention comprises one or more amino acid residues, including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 amino acid residues, which are mutated to prevent dimerization through cysteine residues or to modulate Fc function (see above). In some of these embodiments, one or more amino acid residues in the CH2 domain and / or hinge region that are capable of mediating binding to the Fc receptor are mutated. If present, one or more amino acid residues capable of mediating binding to the Fc receptor can be amino acid residues capable of activating antibody-dependent cell cytotoxicity (ADCC) or complement-mediated cytotoxicity (CDC). In some embodiments, typically when the sequence is compared to the sequence of the corresponding naturally occurring domain in an immunoglobulin such as IgG, the corresponding amino acid residue capable of mediating binding to the Fc receptor is replaced by another amino acid. In some embodiments, typically such amino acid residues capable of mediating binding to the Fc receptor are deleted relative to the sequence of the corresponding naturally occurring domain in an immunoglobulin such as IgG.

[0126] In some embodiments, one or more mutations, such as substituted or deleted amino acid residues, are amino acids located at one of positions 226, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 265, 297, 327, and 330. Again, the amino acid numbering used corresponds to the sequence positions according to Kabat numbering [EU-index]. The corresponding deletions of amino acids can be, for example, the deletion of amino acid 228, usually proline in IgG, the deletion of amino acid 229, usually cysteine in IgG, the deletion of amino acid 230, usually proline in IgG, the deletion of amino acid 231, usually alanine in IgG, the deletion of amino acid 232, usually proline in IgG, the deletion of amino acid 233, usually glutamate in IgG, the deletion of amino acid 234, usually leucine in IgG, the deletion of amino acid 235, usually leucine in IgG, the deletion of amino acid 236, usually glycine in IgG, the deletion of amino acid 237, usually glycine in IgG, the deletion of amino acid 238, usually proline in IgG, and the deletion of amino acid 265, usually aspartic acid in IgG. The corresponding substitutions of amino acids can be, for example, the substitution of amino acid 226, usually cysteine in IgG, the substitution of amino acid 228, usually proline in IgG, the substitution of amino acid 229, usually cysteine in IgG, the substitution of amino acid 230, usually proline in IgG, the substitution of amino acid 231, usually alanine in IgG, the substitution of amino acid 232, usually proline in IgG, the substitution of amino acid 233, usually glutamate in IgG, the substitution of amino acid 234, usually leucine in IgG, the substitution of amino acid 235, usually leucine in IgG, the substitution of amino acid 265, usually aspartic acid in IgG, the substitution of amino acid 297, usually asparagine in IgG, the substitution of amino acid 327, usually alanine in IgG, and the substitution of amino acid 330, usually alanine in IgG. The respective substitutions can be one of Substitution Cys226→Ser, Substitution Cys229→Ser, Substitution Glu233→Pro, Substitution Leu234→Val, Substitution Leu235→Ala, Substitution Asp265→Gly, Substitution Asn297→Gln, Substitution Ala327→Gln, Substitution Ala327→Gly, and Substitution Ala330→Ser. As can be seen from the above, in some embodiments, one or both cysteine residues at positions 226 and 229 in the hinge region are substituted with another amino acid, such as substituted with a serine residue. This can prevent the formation of disulfide bonds with another backbone.In addition, and as explained below, deletion and / or substitution (mutation) of selected amino acid residues in the CH2 domain that are capable of mediating binding to Fc receptors can result in the ABP of the present invention having less or no activity in antibody-dependent cell-mediated cytotoxicity and complement fixation.

[0127] Another class of amino acid variants of the antibody alters the original glycosylation pattern of the ABP, if any. Alteration refers to the deletion of one or more carbohydrate moieties found in the antibody and / or the addition of one or more glycosylation sites not present in the antibody. Glycosylation of antibodies is generally N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are the recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. Addition of glycosylation sites to the antibody can be conveniently achieved by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Alteration can also be effected by adding one or more serine or threonine residues to the sequence of the original antibody or substituting one or more serine or threonine residues (for O-linked glycosylation sites).

[0128] In the context of the present invention, in some embodiments, the backbone portion of the ABP of the present invention that represents the immunoglobulin Fc region is generally inert or at least substantially low-impact with respect to binding to Fc receptors. As described, this is achieved by deletion and / or substitution (mutation) of at least one of the selected amino acid residues in the CH2 domain that are capable of mediating binding to Fc receptors. Such molecules are also referred to herein as "Fc-attenuated" ABP or "Fcko" ABP. Thus, for example, the portion of the antibody chain that can be regarded as representing the Fc fragment according to the present invention, i.e., the CH2 domain and the CH3 domain, if present, can define a "scaffold" without providing a specific biological function such as an effector function. However, it has been found in the present invention that this scaffold can provide significant advantages in the purification, production efficiency, and / or stability of the ABP of the present invention compared to known ABPs.

[0129] In some embodiments, the recognition of this Fc counterpart to a given Fc receptor and the corresponding binding are about 2-fold, about 5-fold, about 8-fold, about 10-fold, about 12-fold lower, about 15-fold, about 20-fold higher or lower than that of the Fc region of a naturally occurring immunoglobulin. In some embodiments, this Fc counterpart has no ability to bind Fc receptors at all. A person skilled in the art can readily determine the binding of an antibody to an Fc receptor, including determining the dissociation constant, using standard techniques such as surface plasmon resonance, for example measured using BiacoreTM. Any other method for measuring biomolecular binding can also be used, which can rely on spectroscopic, photochemical, photometric or radiological means, for example. Examples of corresponding detection methods are fluorescence correlation spectroscopy, photochemical crosslinking and the use of photoactive or radioactive labels, respectively. Some of these methods can include other separation techniques, such as electrophoresis or HPLC.

[0130] If desired, substitution or deletion of amino acid residues can be effected generally as explained above. For example, suitable mutations can be taken from Armour et al. (Eur. J. Immunol.

[1999] 29, 2613-2624). Other suitable positions for antibody chain sequence mutations can be obtained from the crystal structure data published on the complex between FcγRIII and the human IgG1 Fc fragment (Sondermann et al., Nature

[2000] 406, 267-273). In addition to measuring the binding affinity as described above to evaluate the level of "Fc attenuation" or loss of binding affinity, the ability (lack thereof) to mediate binding to Fc receptors can be functionally evaluated. In the case of an ABP that binds CD3 as a target, for example, binding can be evaluated by the mitogenity of such CD3-binding ABP on cells. Mitogenity is mediated by the binding of the CD3 antibody to Fc receptors on accessory cells such as monocytes. If the ABP of the invention having one CD3 binding site does not show any mitogenic effect, while the parental monoclonal anti-CD3 antibody having a functional Fc portion induces strong mitosis in T cells, it is evident that due to the lack of mitosis, the ABP of the invention lacks the ability to bind Fc and can thus be considered an "Fc knockout" molecule. Davis, Vida & Lipsky (J. Immunol (1986) 137, 3758) and Ceuppens, JL, & van Vaeck, F, (see J. Immunol. (1987) 139, 4067 or Cell. Immunol. (1989) 118, 136) have described exemplary examples of methods for evaluating anti-CD3-mediated mitogenity. Rosenthal-Allieri et al. (Rosenthal-Allieri MA, Ticcioni M, Deckert M, Breittmeyer JP, Rochet N, Rouleaux M, and Senik A, Bernerd A, Cell Immunol. 1995 163(1):88-95) and Grosse-Hovest et al. (Grosse-Hovest L, Hartlapp I, Marwan W, Brem G, Rammensee H-G, and Jung G, Eur J Immunol.

[2003] May; 33(5):1334-1340) have described further illustrative suitable examples of assays for evaluating the mitogenity of antibodies. Additionally, the lack of Fc binding can be evaluated by the ability of the ABP of the invention to mediate one or more known effector functions of the Fc portion.

[0131] As described above, cysteine residue substitutions or deletions can be made to introduce or remove one or more disulfide bonds, including introducing or removing potential or pre-existing disulfide bonds. Thus, the connection between the backbone of the ABP according to the invention and the lower weight / shorter length chains can be controlled, including established, strengthened or eliminated. By introducing or removing one or more cysteine residues, disulfide bonds can be introduced or removed. As an illustrative example, a tetrameric ABP according to the invention typically has one or more disulfide bonds connecting two dimeric ABPs. One such disulfide bond is typically defined by a cysteine in the backbone of the first dimeric ABP and a cysteine in the hinge region of the second dimeric ABP. In this regard, in some embodiments, the antibody according to the invention may include an amino acid substitution of a native cysteine residue with another amino acid residue at position 226 and / or 229 relative to the sequence of a human IgG immunoglobulin according to the Kabat numbering [EU-index].

[0132] Substitutions or deletions of amino acid residues such as arginine, asparagine, serine, threonine or tyrosine residues can also be made to modify the glycosylation pattern of the antibody. As an illustrative example, an IgG molecule has a single N-linked biantennary carbohydrate at Asn297 in the CH2 domain. IgG is heterogeneous with respect to the Asn297-linked carbohydrate for IgG from serum or produced in vitro in hybridomas or engineered cells. For human IgG, the core oligosaccharide typically consists of GlcNAc2Man3GlcNAc, with varying numbers of outer residues.

[0133] As noted, in addition to antigen / epitope binding, immunoglobulins are known to have further "effector functions", attributed to the biological activity of the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of the immunoglobulin and varying with immunoglobulin isotype. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Exerting the effector functions of an antibody typically involves recruitment of effector cells. Several immunoglobulin effector functions are mediated by Fc receptors (FcRs) that bind to the Fc region of the antibody. FcRs are defined by their specificity for immunoglobulin isotype; the Fc receptor for IgG antibodies is called FcγR, for IgE it is called FcεR, for IgA it is called FcαR, and so on. Any of these effector functions (or loss of such effector functions) such as CDC or ADCC can be used to assess whether the ABP according to the invention lacks the ability to bind Fc.

[0134] In this context, it should be noted that the term "Fc receptor" or "FcR" defines a receptor, typically a protein that is capable of binding to the Fc region of an antibody. Fc receptors are present on the surface of certain cells of the organism's immune system, such as natural killer cells, macrophages, neutrophils, and mast cells. In vivo, Fc receptors bind to immunoglobulins present on infected cells or invading pathogens that are fixed on them. Their activity stimulates phagocytes or cytotoxic cells to destroy microorganisms or infected cells through antibody-mediated phagocytosis or antibody-dependent cell-mediated cytotoxicity. Some viruses, such as flaviviruses, utilize Fc receptors to help them infect cells through a mechanism called antibody-dependent enhancement of infection. FcRs have been reviewed in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

[0135] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway begins with the binding of the first component of the complement system (C1q) to an antibody (appropriate subclass) that is bound to its associated antigen. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1997).

[0136] The term "complement system" is used in the art to refer to a number of small proteins found in the blood, called complement factors, which typically circulate in an inactive precursor (proprotein) form. The term refers to the ability of this immutable and non-adaptable system to "complement" the ability of antibodies and phagocytes to clear pathogens (such as bacteria) and antigen-antibody complexes from the organism. An example of a complement factor is the complex C1, which consists of C1q and two serine proteases, C1r and C1s. The complex C1 is a component of the CDC pathway. C1q is a hexavalent molecule with a molecular weight of approximately 460,000, whose structure resembles a bunch of tulips, where six collagenous "stems" are connected to six spherical heads. To activate the complement cascade, C1q must bind to at least two molecules of IgG1, IgG2, or IgG3.

[0137] "Antibody-dependent cell cytotoxicity" or ADCC refers to a form of cytotoxicity in which immunoglobulin molecules bound to Fc receptors (FcRs) are present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils, and macrophages - enabling these cytotoxic effector cells to specifically bind to target cells bearing an antigen and subsequently kill the target cells with cytotoxins. The antibody "arms" the cytotoxic cells and is required for killing the target cells by this mechanism. The major cell mediating ADCC, the NK cell, expresses only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. In some embodiments, the ADCC activity of a molecule of interest can be evaluated in vivo, e.g., in an animal model such as the animal model disclosed in Clynes et al., PNAS USA 95:652-656 (1998).

[0138] The ABP of the present invention can be produced using any known and recognized expression systems and recombinant cell culture techniques, e.g., by expression in a bacterial host (prokaryotic system) or a eukaryotic system such as yeast, fungi, insect cells, or mammalian cells. For example, when the ABP of the present invention is used in the "IgGsc" form, the ABP can (of course) be produced as described by Coloma and Morrison (Nat Biotechnol 15:159-63, 1997) or as described in the Examples section of the present application. Similarly, the ABP of the present invention in the "Fabsc" form can be produced as described in International Patent Application WO 2013 / 092001 or as described in the Examples section herein. The ABP of the present invention can also be produced in transgenic organisms such as goats, plants, or XENOMOUSE transgenic mice (engineered mice that have large segments of the human immunoglobulin locus and are defective in mouse antibody production). Antibodies can also be produced by chemical synthesis.

[0139] To generate the recombinant ABP of the present invention, polynucleotides encoding the antibody are typically isolated and inserted into a replicable vector such as a plasmid for further cloning (amplification) or expression. Illustrative examples of suitable expression systems are the glutamine synthetase systems (e.g., sold by Lonza Biologics), where the host cells are, for example, CHO or NS0. The polynucleotides encoding the antibody are readily isolated and sequenced using conventional methods. Vectors that can be used include plasmids, viruses, bacteriophages, transposons, minichromosomes, with plasmids being the typical embodiment. Generally, such vectors also include signal sequences operably linked to the light chain and / or heavy chain polynucleotides to facilitate expression, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence. The polynucleotides encoding the light chain and heavy chain can be inserted into separate vectors and transfected into the same host cell, or, if desired, both the heavy chain and the light chain can be inserted into the same vector for transfection into the host cell. The two chains can be arranged, for example, under the control of a dicistronic operon and expressed to produce a functional and correctly folded ABP, as described in Skerra, A. (1994) Use of the tetracycline promoter for the tightly regulated production of a murine antibody fragment in Escherichia coli, Gene 151, 131 - 135, or Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments, Gene 141, 79 - 8. Thus, according to one aspect of the present invention, there is provided a method of constructing a vector encoding the light chain and / or heavy chain of an antibody or an antigen-binding fragment thereof of the present invention, the method comprising inserting the light chain and / or heavy chain encoding the ABP of the present invention into a vector.

[0140] When using recombinant techniques, ABP can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium (see also Skerra 1994, supra). If the antibody is produced intracellularly, the first step is to remove particulate debris of the host cells or lysate fragments, for example by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe methods for isolating antibodies secreted into the periplasmic space of E. coli. Antibodies can also be produced in any oxidative environment. Such oxidative environments can be provided by the periplasm of Gram-negative bacteria such as E. coli, the extracellular environment of Gram-positive bacteria, or the lumen of the endoplasmic reticulum of eukaryotic cells, including animal cells such as insect or mammalian cells, and generally favor the formation of structural disulfide bonds. However, it is also possible to produce the ABP of the present invention in the cytosol of a host cell such as E. coli. In this case, the polypeptide can be obtained directly in a soluble and folded state, or it can be recovered in the form of inclusion bodies and then refolded in vitro. A further option is to use a specific host strain with an oxidative intracellular environment, and thus this can allow the formation of disulfide bonds in the cytosol (Venturi M, Seifert C, Hunte C. (2002) “High level production of functional antibody Fab fragments in an oxidizing bacterial cytoplasm.” J. Mol. Biol. 315, 1-8).

[0141] ABP produced by cells can be purified using any conventional purification technique, such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, where affinity chromatography is a preferred purification technique. ABP can be purified by affinity purification with a protein / ligand that specifically and reversibly binds to a constant domain, such as the CH1 or CL domain. Examples of such proteins are bacterial proteins that bind immunoglobulins, such as protein A, protein G, protein A / G, or protein L, where the binding of protein L is limited to ABP containing kappa light chains. An alternative method for purifying antibodies with kappa light chains is to use bead-conjugated anti-kappa antibodies (KappaSelect). The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma3 (Guss et al., EMBO J. 5:1567 1575 (1986)). The choice of purification method for a specific ABP of the present invention is within the knowledge of a person of ordinary skill in the art.

[0142] One or more affinity tags can also be equipped on one of the chains of the ABP of the present invention. Affinity tags, such as or II (Schmidt, T.G.M. et al. (1996) J. Mol. Biol. 255, 753-766), myc tags, FLAGTM tags, His6 tags or HA tags allow for easy detection of the recombinant ABP and also for simple purification.

[0143] Turning now to the nucleic acids of the present invention, the nucleic acid molecule encoding one or more chains of an antibody according to the present invention can be any nucleic acid of any possible configuration, such as single-stranded, double-stranded or a combination thereof. Nucleic acids include, for example, DNA molecules, RNA molecules, analogs of DNA or RNA using nucleotide analogs or using nucleic acid chemistry, locked nucleic acid molecules (LNA) and peptide nucleic acid molecules (PNA). The DNA or RNA can be of genomic or synthetic origin and can be single-stranded or double-stranded. Such nucleic acids can be, for example, mRNA, cRNA, synthetic RNA, genomic DNA, cDNA, synthetic DNA, copolymers of DNA and RNA, oligonucleotides, etc. In addition, the corresponding nucleic acids can contain unnatural nucleotide analogs and / or be linked to an affinity tag or a marker.

[0144] In some embodiments, a nucleic acid sequence encoding a chain, such as a heavy chain and / or a light chain, of an antibody according to the invention is included in a vector, such as a plasmid. Where substitutions or deletions will be included in the antibody chain when compared to the native domain or region of the antibody, the coding sequence of the corresponding native domain / region (such as included in an immunoglobulin sequence) can be used as a starting point for mutagenesis. To mutagenize selected amino acid positions, those skilled in the art can use various established standard methods for site-directed mutagenesis. A commonly used technique is to introduce mutations by PCR (polymerase chain reaction) using a mixture of synthetic oligonucleotides that have a degenerate base composition at the desired sequence position. For example, using the codons NNK or NNS (where N = adenine, guanine, cytosine, or thymine; K = guanine or thymine; S = adenine or cytosine) allows incorporation of all 20 amino acids plus the amber stop codon during the mutagenesis process, while the codon VVS limits the number of possible amino acids that can be incorporated to 12, as it excludes incorporation of the Cys, Ile, Leu, Met, Phe, Trp, Tyr, Val amino acids at the selected position in the polypeptide sequence; for example, using the codon NMS (where M = adenine or cytosine) limits the number of possible amino acids at the selected sequence position to 11, as it excludes incorporation of the Arg, Cys, Gly, Ile, Leu, Met, Phe, Trp, Val amino acids at the selected sequence position. In this regard, it should be noted that codons for other amino acids (different from the conventional 20 naturally occurring amino acids), such as selenocystein or pyrrolysine, can also be incorporated into the nucleic acid of the ABP. As described by Wang, L., et al. (2001) Science 292, 498 - 500 or Wang, L. and Schultz, P.G. (2002) Chem. Comm. 1, 1 - 11, it is also possible to use "artificial" codons (such as UAG) that are normally considered stop codons in order to insert other uncommon amino acids, such as ortho-methyl L-tyrosine or para-aminophenylalanine.

[0145] Using nucleotide building blocks with reduced base pair specificity, such as inosine, 8-oxo-2'-deoxyguanosine or 6(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimin-do-1,2-oxazine-7-one (Zaccolo et al. (1996) J. Mol. Biol. 255, 589-603), is another option for introducing mutations into a selected sequence segment. A further possibility is so-called triple mutagenesis. This method uses a mixture of different nucleotide triplets, each encoding an amino acid, for incorporation into the coding sequence ( B, et al., 1994 Nucleic Acids Res 22, 5600-5607).

[0146] Any suitable expression system can be used, such as in a suitable host cell or in a cell-free system, to express a nucleic acid molecule encoding a chain of an antibody according to the invention, such as a backbone and / or a smaller chain. The obtained ABP can be enriched by selection and / or isolation. Preferably, the nucleic acid of the invention is provided in the context of a genetic construct such as a vector / plasmid.

[0147] Also provided is a nucleic acid system or construct comprising such a nucleic acid of the invention, wherein the system of the invention comprises at least two nucleic acids of the invention, each encoding a monomer of the ABP of the invention, such as one nucleic acid encoding a heavy chain sequence and a second nucleic acid encoding a light chain sequence.

[0148] In some embodiments, the polypeptide of the ABP of the invention can be encoded by a nucleic acid for expression in vivo or in vitro. Thus, in some embodiments, an isolated nucleic acid encoding the ABP of the invention is provided. In some embodiments, the nucleic acid encodes a part or monomer of the ABP of the invention (such as one of the two (heavy and light) chains of an antibody), and / or another nucleic acid encodes another part or monomer of the ABP of the invention (such as the other of the two chains of an antibody). Such nucleic acids can be provided in combination or as a system together. In some embodiments, the nucleic acid encodes two or more ABP polypeptide chains, such as at least 2 antibody chains. The nucleic acid encoding multiple ABP chains can include a nucleic acid cleavage site between at least two chain sequences, can encode a transcription or translation start site between two or more chain sequences, and / or can encode a proteolytic target site between two or more ABP chains.

[0149] However, another aspect of the present invention provides a vector (e.g., an expression vector) that contains a nucleic acid encoding an ABP or a portion or monomer of an ABP as disclosed herein. For example, in some embodiments, where the ABP is a multimeric protein, the nucleic acid encodes only a single polypeptide chain of the antigen construct. Thus, to express such an antigen-binding construct, the expression vectors of the present invention can contain two or more nucleic acids, each encoding a different portion or monomer of the ABP, the combination of which will express the entire ABP. Similarly, the expression vectors of the present invention containing nucleic acids can be used in combination with other different expression vectors of the present invention that each encode different portions or monomers of the ABP, the nucleic acids of which encode only a portion or monomer of the antigen-binding construct. In other embodiments, the nucleic acid encodes multiple polypeptide chains of the ABP of the present invention. In some embodiments, the expression vector includes the pcDNA3.1TM / myc-His(-)A vector (Invitrogen, Inc.) or a variant thereof for mammalian expression. The pcDNA3.1 expression vector is characterized by a CMV promoter for mammalian expression and both mammalian (neomycin) and bacterial (ampicillin) selection markers. In some embodiments, the expression vector includes a plasmid. In some embodiments, the vector includes a viral vector, such as a retroviral or adenoviral vector. In an embodiment, the vector includes a cosmid, YAC or BAC.

[0150] In another related aspect, the present invention relates to a cell (e.g., a host cell and / or a recombinant host cell) that contains one or more nucleic acids of the present invention. Preferably, such a cell is capable of expressing the ABP (or a component thereof) encoded by the nucleic acid. For example, if the ABP of the present invention comprises two separate polypeptide chains (e.g., the heavy and light chains of IgG), the cell of the present invention can contain a first nucleic acid encoding (and capable of expressing) the heavy chain of such an ABP and a second nucleic acid encoding (and capable of expressing) the light chain of such an ABP; alternatively, the cell can contain a single nucleic acid encoding both chains of such an ABP. In these ways, such cells of the present invention will be capable of expressing the functional ABP of the present invention. The (host) cells of the present invention can be one of the mammalian, prokaryotic or eukaryotic host cells described elsewhere herein, particularly when the cell is a Chinese hamster ovary (CHO) cell.

[0151] In certain embodiments in this regard, the (host) cell is a human cell; in particular, it can be a human cell that has been sampled from a specific individual (e.g., autologous human cells). In such embodiments, such human cells can be propagated and / or manipulated in vitro to introduce the nucleic acids of the present invention. The utility of the manipulated human cells from a specific individual can give rise to the ABP of the present invention, including reintroducing such a population of manipulated human cells into a human subject, e.g., for treatment. In certain such uses, the manipulated human cells can be introduced into the same individual from whom they were first sampled; e.g., as autologous human cells.

[0152] The human cells subjected to such manipulation can be any germ or somatic cell type in vivo. For example, the donor cell can be a germ or somatic cell selected from the group consisting of: fibroblast, B cell, T cell, dendritic cell, keratinocyte, adipocyte, epithelial cell, epidermal cell, chondrocyte, cumulus cell, nerve cell, glial cell, astrocyte, heart cell, esophageal cell, muscle cell, melanocyte, hematopoietic cell, macrophage, monocyte, and mononuclear cell. The donor cell can be obtained from any organ or tissue in vivo. For example, it can be a cell from an organ selected from the group consisting of liver, stomach, intestine, lung, pancreas, cornea, skin, gallbladder, ovary, testis, kidney, heart, bladder, and urethra.

[0153] The present invention also provides a pharmaceutical composition comprising the ABP of the present invention and optionally a pharmaceutically acceptable excipient.

[0154] The ABP according to the present invention can be administered by any parenteral or non-parenteral (enteral) route effective for proteinaceous drug therapy. Parenteral administration methods include, for example, intradermal, subcutaneous, intramuscular, intratracheal, intranasal, intravitreal, or intravenous injection and infusion techniques, e.g., in the form of injection solutions, infusion solutions, or tinctures, as well as aerosol instillation and inhalation, e.g., in the form of aerosol mixtures, sprays, or powders. An overview of pulmonary drug delivery, i.e., by inhaled aerosols (which can also be used for intranasal administration) or by intratracheal instillation. For example, an overview of pulmonary drug delivery, i.e., by inhaled aerosols (which can also be used in intranasal administration) or by intratracheal instillation, is given by J.S. Patton et al. The lungs as a portal of entry for systemic drug delivery. Proc. Amer. Thoracic Soc. 2004 Vol. 1 pp. 338 - 344. Non-parenteral delivery modes are, for example, oral delivery, e.g., in the form of pills, tablets, capsules, solutions, or suspensions, or rectal delivery, e.g., in the form of suppositories. The ABP of the present invention can be administered systemically or locally in formulations containing conventional non-toxic pharmaceutically acceptable excipients or carriers, additives, and vehicles as required.

[0155] In one embodiment of the invention, the drug is administered parenterally to a mammal, particularly a human. Corresponding administration methods include, but are not limited to, for example, intradermal, subcutaneous, intramuscular, intratracheal or intravenous injection and infusion techniques, such as in the form of injection solutions, infusion solutions or tinctures, as well as aerosol instillation and inhalation forms, such as in the form of aerosol mixtures, sprays or powders. If the serum half-life of the compound is relatively short, a combination of intravenous and subcutaneous infusion and / or injection may be the most convenient. The pharmaceutical composition may be an aqueous solution, an oil-in-water emulsion or a water-in-oil emulsion.

[0156] In this regard, it should be noted that transdermal delivery techniques described in Meidan VM and Michniak BB 2004 Am. J. Ther. 11(4):312-316, such as iontophoresis, sonophoresis or micro-needle enhanced delivery, can also be used for the transdermal delivery of ABP described herein. Non-parenteral delivery modes are, for example, oral, such as in the form of pills, tablets, capsules, solutions or suspensions, or rectal administration, such as in the form of suppositories. The ABP of the present invention can be administered systemically or locally in a formulation containing a variety of conventional non-toxic pharmaceutically acceptable excipients or carriers, additives and excipients.

[0157] The dose of ABP administered can vary within wide limits to achieve the desired prophylactic effect or therapeutic response. For example, this will depend on the affinity of ABP for the selected target and the in vivo half-life of the complex between ABP and the ligand. In addition, the optimal dose will depend on the biodistribution of ABP or its conjugate, the mode of administration, the severity of the disease / condition being treated and the medical condition of the patient. For example, when used in an ointment for topical application, a high concentration of ABP can be used. However, if desired, ABP can also be given in the form of a sustained-release formulation, such as a liposome dispersion or a hydrogel-based polymer microsphere, such as PolyActiveTM or OctoDEXTM (see Bos et al., Business Briefing: Pharmatech 2003:1-6). Other available sustained-release formulations are, for example, PLGA-based polymers (PR pharmaceuticals), PLA-PEG-based hydrogels (Medincell) and PEA-based polymers (Medivas).

[0158] Accordingly, the ABP of the present invention can be formulated into a composition using pharmaceutically acceptable ingredients and established preparation methods (Gennaro, A.L. and Gennaro, A.R. (2000) Remington: The Science and Practice of Pharmacy, 20th Ed., Lippincott Williams & Wilkins, Philadelphia, PA). For the preparation of pharmaceutical compositions, pharmaceutically inert inorganic or organic excipients can be used. For the preparation of, for example, pills, powders, gelatin capsules or suppositories, lactose, talc, stearic acid and its salts, fats, waxes, solid or liquid polyols, natural and hardened oils can be used. Suitable excipients for the preparation of solutions, suspensions, emulsions, aerosol mixtures or powders to be reconstituted into a solution or aerosol mixture before use include water, alcohols, glycerol, polyols and their suitable mixtures, as well as vegetable oils.

[0159] The pharmaceutical composition may also contain additives such as, for example, fillers, binders, wetting agents, glidants, stabilizers, preservatives, emulsifiers, and in addition solvents or solubilizers or agents for achieving depot effects. The latter are such that the fusion protein can be incorporated into slow or sustained release or targeted delivery systems, such as liposomes and microcapsules.

[0160] The preparation can be sterilized in a variety of ways, including filtration through a filter that retains bacteria, or incorporation of a sterilizing agent in the form of a sterile solid composition that can be dissolved or dispersed in sterile water or other sterile medium just before use.

[0161] ABP can be suitable and can be used to treat or prevent diseases. Thus, in some embodiments, the ABP according to the present invention can be used in methods for treating and / or preventing medical conditions such as disorders or diseases. Similarly, the ABP of the present invention can be used to treat diseases. The disease to be treated or prevented can be a proliferative disease. Such proliferative diseases can preferably be tumors or cancers. Due to the ability of the ABP of the present invention to bind FLT3, the ABP can be used to treat cancers composed of cells expressing FLT3, wild-type or mutant FLT3. For the methods of the present invention, the cancer can be any cancer, including any of the following: acute lymphocytic carcinoma, acute myeloid leukemia (AML), alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder carcinoma), bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal cancer, anal canal cancer or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia, liquid tumor, liver cancer, lung cancer (e.g., non-small cell lung cancer and long adenocarcinoma), lymphoma, mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, B-cell chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkitt’s lymphoma, ovarian cancer, pancreatic cancer, peritoneal cancer, omental cancer and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, synovial sarcoma, stomach cancer, testicular cancer, thyroid cancer and ureteral cancer. Preferably, the cancer is characterized by the expression of FLT3. Most preferably, the cancer is FLT3-positive leukemia, such as AML or ALL.

[0162] The subject to be treated with the fusion protein can be a human or a non-human animal. Such animals are preferably mammals, such as humans, pigs, cows, rabbits, mice, rats, primates, goats, sheep, chickens or horses, and most preferably humans.

[0163] The ABP of the present invention can also be used for the diagnosis of diseases, such as the diseases described herein. For this purpose, the ABP can be labeled with a suitable detectable signal marker. Such labeled ABP can allow the detection or quantification of FLT3 levels or cancers, such as leukemia, or any of the above cancers or subjects. When designated for in vivo use, the detectable signal marker is preferably detectable in vivo.

[0164] The labeled ABP can be used in immunoimaging techniques. Then, for example, a detectable signal marker can be selected based on the immunoimaging technique for diagnosis, such as a radionuclide (or gamma emitter) that emits gamma in the case of gamma camera imaging technique / SPECT, a metal or a positron emitter in the case of MRI or PET imaging technique. In this regard, one or more detectable signal markers of the present disclosure include gamma camera imaging agents, PET imaging agents, and MRI imaging agents, such as radionuclides, fluorescers, fluorogens, chromophores, chromogens, phosphorescers, chemiluminescent agents, and bioluminescent agents.

[0165] Suitable detectable signal markers can be radionuclides. The radionuclides can be selected from 3H, 14C, 35S, 99Tc, 123I, 125I, 131I, 68Ga, 97Ru, 67Ga, 68Ga, 72As, 89Zr, and 201Tl.

[0166] Suitable detectable signal markers can also be fluorophores or fluorogens. The fluorophores or fluorogens can be selected from the group consisting of fluorescein, rhodamine, dansyl, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde, fluorescamine, fluorescein derivatives, Oregon Green, Rhodamine Green, Rhodol Green, or Texas Red.

[0167] The labeled ABP can be directly or indirectly coupled to a detectable signal label. For example, ABP can be directly (e.g., through tyrosine residues of ABP) or indirectly (e.g., through a linker - as a metal chelator) coupled to a detectable signal marker. In some other embodiments, ABP can be coupled to a molecule that can be coupled to a detectable signal marker at the time and place of use (in vitro or in vivo).

[0168] The detectable signal marker can bind to ABP through one or more diethylenetriaminepentaacetic acid (DTPA) residues coupled to ABP.

[0169] The present invention also contemplates an in vitro method for detecting or diagnosing a disease as defined herein. Such methods can include contacting a sample obtained from a subject with a preferably labeled ABP of the present invention. The sample can be a blood, urine, or cerebrospinal fluid sample, but preferably can be a liquid sample or a biopsy sample. The disease to be detected or diagnosed is preferably leukemia, such as ALL or AML.

[0170] As used herein, the terms "of the invention", "according to the invention", "in accordance with the invention", etc. refer to all aspects and embodiments of the invention described and / or claimed herein.

[0171] As used herein, the term "comprising" shall be interpreted to cover both "comprising" and "consisting of", both of which meanings are specifically intended and are thus separate disclosed embodiments according to the invention. When used herein, "and / or" shall be regarded as specifically disclosing each of the two specified features or components, with or without the other. For example, "A and / or B" shall be regarded as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein. In the context of the present invention, the terms "about" and "approximately" indicate a range of precision that a person skilled in the art will understand still ensures the technical effect of the feature being discussed. The term generally indicates a deviation of ±20%, ±15%, ±10%, for example ±5% from the indicated numerical value. As will be understood by a person of ordinary skill in the art, for a given technical effect, such a specific deviation of the numerical value will depend on the nature of the technical effect. For example, natural or biological technical effects can generally have a greater such deviation compared to artificial or engineering technical effects. As will be understood by a person of ordinary skill in the art, for a given technical effect, such a specific deviation of the numerical value will depend on the nature of the technical effect. For example, natural or biological technical effects can generally have a greater such deviation compared to artificial or engineering technical effects. When an indefinite or definite article is used in reference to a singular noun, e.g., in the case of "a" or "the", this includes a plural number of that noun unless specifically stated otherwise.

[0172] It should be understood that, in accordance with the teachings contained herein, applying the teachings of the present invention to a particular problem or environment, and including variations or other features of the present invention, such as additional aspects and embodiments, will be within the ability of a person of ordinary skill in the art.

[0173] Unless the context otherwise indicates, the descriptions and definitions of the features set forth above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

[0174] All references, patents, and publications cited herein are hereby incorporated by reference in their entirety.

[0175] In view of the foregoing, it will be understood that the present invention also relates to the following embodiments item by item:

[0176] Item 1: A bispecific antigen-binding protein (ABP) comprising a first antigen-binding domain capable of binding to the human fms-like tyrosine kinase 3 (FLT3) antigen and a second antigen-binding domain capable of binding to the human cluster of differentiation 3 (CD3) antigen.

[0177] Item 2: The bispecific ABP according to Item 1, wherein the bispecific ABP binds FLT3 with an EC50 of less than 10 nM, preferably less than 9 nM, more preferably less than 8 nM, more preferably less than 7 nM, more preferably less than 6 nM, or less than 5.5 nM.

[0178] Item 3: The bispecific ABP according to Item 1 or 2, wherein the bispecific ABP binds FLT3 with an EC50 of more than 0.5 nM, more preferably more than 1 nM, more preferably more than 1.3 nM, more preferably more than 2 nM, more preferably more than 3 nM, or 4 nM, or more than 4.5 nM.

[0179] Item 4: The bispecific ABP according to any one of Items 1 to 3, wherein the bispecific ABP binds FLT3 with an EC50 of less than 10 nM and more than 0.5 nM, more preferably less than 9 nM and more than 1 nM, preferably less than 8 nM and more than 1.3 nM, preferably less than 7 nM and more than 3 nM, preferably less than 6 nM and more than 4 nM, preferably less than 5.5 nM and more than 4.5 nM.

[0180] Item 5: The bispecific ABP according to any one of Items 2 to 4, wherein the EC50 of the bispecific ABP binding to FLT3 is determined by flow cytometry analysis of the binding of the bispecific ABP to FLT3-positive cells using a fluorescence-activated cell sorting (FACS) device; preferably wherein the FLT3-positive cells are B-cell precursor leukemia cells, preferably NALM-16 cells (such as those deposited at DSMZ under ACC 680); and / or preferably, wherein a fluorescently labeled secondary antibody is used to detect the binding; and / or wherein the bispecific ABP is incubated with the FLT3-positive cells for about 30 minutes before flow cytometry.

[0181] Item 6: The bispecific ABP according to any one of Items 1 to 5, wherein the bispecific ABP binds FLT3 with a kD of less than 50 μM, more preferably less than 20 μM, more preferably less than 10 μM, more preferably less than 5 μM, more preferably less than 1 μM.

[0182] Item 7: The bispecific ABP according to any one of Items 1 to 5, wherein the bispecific ABP binds FLT3 with a kD of more than 50 nM, more preferably more than 100 nM, more preferably more than 160 nM, more preferably more than 200 nM, more preferably more than 300 nM.

[0183] Item 8: The bispecific ABP according to any one of Items 1 to 5, wherein the bispecific ABP binds FLT3 with a kD of less than 50 μM and greater than 50 nM, more preferably less than 20 μM and greater than 100 nM, preferably less than 10 μM and greater than 160 nM, more preferably less than 5 μM and greater than 200 nM, and even more preferably less than 1 μm and greater than 300 nM.

[0184] Item 9: The bispecific ABP according to any one of Items 6 to 8, wherein the kD is measured by surface plasmon resonance, such as in a BIAcore affinity assay.

[0185] Item 10: The bispecific ABP according to any one of Items 1 to 9, wherein the bispecific ABP binds CD3 with an EC50 of less than 200 nM, preferably less than 90 nM, more preferably less than 50 nM, more preferably less than 20 nM, and even more preferably less than 15 nM.

[0186] Item 11: The bispecific ABP according to any one of Items 1 to 10, wherein the bispecific ABP binds CD3 with an EC50 of greater than 1 nM, preferably greater than 2 nM, more preferably greater than 4.1 nM, more preferably greater than 6 nM, and even more preferably greater than 8 nM.

[0187] Item 12: The bispecific ABP according to any one of Items 1 to 11, wherein the bispecific ABP binds CD3 with an EC50 of less than 200 nM and greater than 1 nM, preferably less than 200 nM and greater than 2 nM, more preferably less than 90 nM and greater than 4.1 nM, more preferably less than 20 nM and greater than 6 nM, and even more preferably less than 15 nM and greater than 8 nM.

[0188] Item 13: The bispecific ABP according to any one of Items 10 to 12, wherein the EC50 of the bispecific ABP binding to CD3 is determined by flow cytometry analysis of the binding of the bispecific ABP to CD3-positive cells using a fluorescence-activated cell sorting (FACS) device; preferably wherein the CD3-positive cells are T cell leukemia cells, preferably Jurkat cells (such as those deposited at DSMZ under ACC 282); and / or preferably, wherein a fluorescently labeled secondary antibody is used to detect the binding; and / or wherein the bispecific ABP is incubated with the CD3-positive cells for about 30 minutes before flow cytometry.

[0189] Item 14: The bispecific ABP according to any one of Items 1 to 13, wherein in an in vitro assay, the bispecific ABP inhibits the proliferation and / or viability of leukemic blood mononuclear cells of a patient with acute leukemia to equal to or less than 50%, more preferably equal to or less than 40%, more preferably equal to or less than 30%, and most preferably equal to or less than 25% compared to an untreated control.

[0190] Item 15: The bispecific ABP according to any one of Items 1 to 14, wherein the bispecific ABP comprises two first antigen-binding sites.

[0191] Item 16: The bispecific ABP according to any one of Items 1 to 15, wherein the bispecific ABP comprises two second antigen-binding sites.

[0192] Item 17: The bispecific ABP according to any one of Items 1 to 16, which is an antibody or an antibody variant.

[0193] Item 18: The bispecific ABP according to Item 17, wherein the antigen-binding domain is composed of an antibody heavy-chain variable domain and an antibody light-chain variable domain.

[0194] Item 19: The bispecific ABP according to any one of Items 1 to 18, which is a tetravalent and homodimeric bispecific antibody, and each monomer thereof comprises: (i) an N-terminal Fab fragment, which contains a variable region containing a heavy-chain variable domain and a light-chain variable domain, wherein the variable region contains a first antigen-binding site; (ii) a C-terminal scFv fragment containing a second antigen-binding site, and wherein (i) and (ii) are linked by CH2 and CH3 domains.

[0195] Item 20: The bispecific ABP according to any one of Items 17 to 19, wherein at least one of the amino acid residues in the CH2 domain of the antibody that can mediate binding to an Fc receptor is missing or mutated.

[0196] Item 21: The bispecific ABP according to any one of Items 1 to 20, wherein the second antigen-binding site comprises an antibody heavy-chain variable domain and an antibody light-chain variable domain in the C-terminal to N-terminal direction.

[0197] Item 22: An ABP capable of binding to human fms-related tyrosine kinase 3 (FLT3), comprising: (i) a heavy chain variable domain comprising a CDRH1 region shown as SEQ ID NO:01 (SYWMH), a CDRH2 region shown as SEQ ID NO:02 (EIDPSDSYKDYNQKFKD), and a CDRH3 region shown as SEQ ID NO:03 (AITTTPFDF), or wherein in each case, CDRH1, CDRH2, and / or CDRH3 independently comprises a sequence having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion compared to SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03, respectively; or comprises a CDRH1, CDRH2, or CDRH3 sequence having at least 75% sequence identity or at least 80%, preferably 90% sequence identity with SEQ ID NO:01, SEQ ID NO:02, or SEQ ID NO:03; and (ii) a light chain variable domain comprising a CDRL1 region shown as SEQ ID NO:05 (RASQSISNNLH), a CDRL2 region shown as SEQ ID NO:06 (YASQSIS), and a CDRL3 region shown as SEQ ID NO:07 (QQSNTWPYT), or wherein in each case, CDRL1, CDRL2, and / or CDRL3 independently comprises a sequence having no more than three or two, preferably no more than one amino acid substitution, deletion, or insertion compared to SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07, respectively; or comprises a CDRL1, CDRL2, or CDRL3 sequence having at least 75% sequence identity or at least 80% sequence identity with SEQ ID NO:05, SEQ ID NO:06, or SEQ ID NO:07; characterized in that the heavy chain variable domain and the light chain variable domain each comprise a human variable region framework sequence.

[0198] Item 23: The ABP according to item 22, which is a bispecific ABP according to any one of items 1 - 21, and wherein the CDR regions of (i) and (ii) in item 22 are comprised in the first antigen-binding domain.

[0199] Item 24: The ABP according to any one of items 22 or 23, wherein the human framework sequence of the heavy chain variable domain is derived from IGHV1-46, preferably IGHV1-46*3, and / or wherein the human framework sequence of the heavy chain variable domain is derived from IGKV3D-15.

[0200] Item 25: The ABP according to Item 24, wherein the heavy chain variable region comprises any one or a combination of the following mutations: K16G, V18L, K19R, V20L, K22A, M48I, K57T, N60A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A, and T108L, wherein the numbering is according to the Kabat system.

[0201] Item 26: The ABP according to Item 24 or 25, wherein the light chain variable region comprises any one or a combination of the following mutations: Y49K, Y87F, and I55A, wherein the numbering is according to the Kabat system.

[0202] Item 27: The ABP of Item 1, wherein the heavy chain variable region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 15, 17, 19, 21, or 23, or independently in each case, optionally having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2, or 1 amino acid substitutions, insertions, or deletions as compared to these sequences; and / or wherein the light chain variable region comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from SEQ ID NO: 16, 18, 20, 22, or 24, or independently in each case, optionally having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2, or 1 amino acid substitutions, insertions, or deletions as compared to these sequences.

[0203] Item 28: The ABP according to Item 27, wherein in the heavy chain variable region, amino acid positions 16, 18, 19, 20, 22, 48, 57, 60, 69, 70, 75, 76, 78, 80, 81, 87, and 108 are as in any one of SEQ ID NO: 15, 17, 19, 21, or 23; and / or wherein in the light chain variable region, amino acid positions 49, 55, and 87 are as in any one of SEQ ID NO: 16, 18, 20, 22, or 24; wherein the numbering is according to the Kabat system.

[0204] Item 29: The ABP of any one of Items 1 to 28, which comprises at least one, preferably two, second antigen-binding domains, wherein the second antigen-binding domain binds to CD3, preferably wherein the second antigen-binding domain is fused to the heavy chain of the first antibody-binding domain.

[0205] Item 30: The ABP according to Item 29, wherein the second antigen-binding domain comprises a scFv fragment, and the scFv fragment comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 14, 25, 26, and 27, or, independently in each case, optionally having no more than 10, 9, 8, 7, 6, 5, 4, preferably no more than 3, 2, or 1 amino acid substitutions, insertions, or deletions as compared with a sequence selected from SEQ ID NO: 14, 25, 26, and 27.

[0206] Item 31: The ABP according to Item 27 or Item 28, comprising at least one antibody heavy chain, which comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, and 74, or having no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, preferably no more than 1 amino acid substitution, insertion, or deletion as compared with this sequence; and / or comprising at least one antibody light chain, which comprises an amino acid sequence having at least 80% sequence identity with a sequence selected from SEQ ID NO: 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, and 75, or having no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, preferably no more than 1 amino acid substitution, insertion, or deletion as compared with this sequence

[0207] Item 32: The ABP according to Item 31, which comprises one, preferably two antibody heavy chains and one preferably two antibody light chains, each of which has at least 80% sequence identity with or has no more than 20, 15, 10, 9, 8, 7, 6, 4, preferably 3 or 2, preferably no more than 1 amino acid substitution, deletion, or insertion as compared with the following amino acid sequences:

[0208] a. A sequence selected from the following: SEQ ID NO: 28 for the heavy chain and SEQ ID NO: 29 for the light chain;

[0209] b. A sequence selected from the following: SEQ ID NO: 30 for the heavy chain and SEQ ID NO: 31 for the light chain;

[0210] c. A sequence selected from the following: SEQ ID NO: 32 for the heavy chain and SEQ ID NO: 33 for the light chain;

[0211] d. A sequence selected from the following: SEQ ID NO:34 of the heavy chain and SEQ ID NO:35 of the light chain;

[0212] e. A sequence selected from the following: SEQ ID NO:36 of the heavy chain and SEQ ID NO:37 of the light chain;

[0213] f. A sequence selected from the following: SEQ ID NO:38 of the heavy chain and SEQ ID NO:39 of the light chain;

[0214] g. A sequence selected from the following: SEQ ID NO:40 of the heavy chain and SEQ ID NO:41 of the light chain;

[0215] h. A sequence selected from the following: SEQ ID NO:42 of the heavy chain and SEQ ID NO:43 of the light chain;

[0216] i. A sequence selected from the following: SEQ ID NO:44 of the heavy chain and SEQ ID NO:45 of the light chain;

[0217] j. A sequence selected from the following: SEQ ID NO:46 of the heavy chain and SEQ ID NO:47 of the light chain;

[0218] k. A sequence selected from the following: SEQ ID NO:48 of the heavy chain and SEQ ID NO:49 of the light chain;

[0219] l. A sequence selected from the following: SEQ ID NO:50 of the heavy chain and SEQ ID NO:51 of the light chain;

[0220] m. A sequence selected from the following: SEQ ID NO:52 of the heavy chain and SEQ ID NO:53 of the light chain;

[0221] n. A sequence selected from the following: SEQ ID NO:54 of the heavy chain and SEQ ID NO:55 of the light chain;

[0222] o. A sequence selected from the following: SEQ ID NO:56 of the heavy chain and SEQ ID NO:57 of the light chain;

[0223] p. A sequence selected from the following: SEQ ID NO:58 of the heavy chain and SEQ ID NO:59 of the light chain;

[0224] q. A sequence selected from the following: SEQ ID NO:60 of the heavy chain and SEQ ID NO:61 of the light chain;

[0225] r. A sequence selected from the following: SEQ ID NO:62 of the heavy chain and SEQ ID NO:63 of the light chain;

[0226] s. A sequence selected from the following: SEQ ID NO:64 of the heavy chain and SEQ ID NO:65 of the light chain;

[0227] t. A sequence selected from the following: SEQ ID NO:66 of the heavy chain and SEQ ID NO:67 of the light chain;

[0228] u. A sequence selected from the following: SEQ ID NO:68 of the heavy chain and SEQ ID NO:69 of the light chain;

[0229] v. A sequence selected from the following: SEQ ID NO:70 of the heavy chain and SEQ ID NO:71 of the light chain;

[0230] w. A sequence selected from the following: SEQ ID NO:72 of the heavy chain and SEQ ID NO:73 of the light chain;

[0231] x. A sequence selected from the following: SEQ ID NO:74 of the heavy chain and SEQ ID NO:75 of the light chain;

[0232] wherein the heavy chain and the light chain pair with each other.

[0233] Item 33: An ABP capable of binding to human FLT3 or an antigen-binding fragment thereof, which is capable of competing with the binding of an ABP according to any one of Items 1 to 32.

[0234] Item 34: An ABP according to any one of Items 1 to 33, which has the activity of binding to T cells and tumor cells expressing FLT3, and preferably, wherein the antibody increases the recruitment of T cells to tumor cells expressing FLT3 by binding to FLT3 and CD3.

[0235] Item 35: An ABP according to any one of Items 1 to 34, wherein the heavy chain variable region and the light chain variable region of the antibody molecule containing the second binding domain are the heavy chain variable region and the light chain variable region of UCHT1.

[0236] Item 36: An ABP according to any one of Items 1 to 35, which contains heavy chain constant regions CH1 to CH3, and wherein at least one amino acid residue of the human CH2 domain capable of mediating binding to the Fc receptor is missing or mutated.

[0237] Item 37: An ABP according to any one of Items 1 to 36, wherein the ABP is a tetrameric antibody molecule or a homodimeric and tetravalent antibody molecule.

[0238] Item 38: An isolated nucleic acid encoding an ABP according to any one of Items 1 to 37, or an antigen-binding fragment or monomer of the ABP.

[0239] Item 39: A recombinant host cell comprising the nucleic acid of item 38.

[0240] Item 40: A pharmaceutical composition comprising: (i) an ABP of any one of items 1 to 37, or (ii) the nucleic acid of item 38, or (iii) a recombinant host cell according to item 39, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0241] Item 41: A component for use in medicine, wherein the component is selected from: an ABP of any one of items 1 to 37, an isolated nucleic acid of item 38, a recombinant host cell according to item 39, and a pharmaceutical composition according to item 40.

[0242] Item 42: The component for use according to item 41, wherein the use in medicine is for the treatment of a proliferative disease associated with the expression of FLT3.

[0243] Item 43: The component for use according to item 41 or 42, wherein the component is for enhancing T cell-mediated killing of FLT3-positive tumor cells and / or inhibiting their proliferation.

[0244] Item 44: The component for use according to any one of items 41 to 43, wherein the component is for the diagnosis, prevention and / or treatment of a proliferative disease, wherein the proliferative disease is preferably cancer, wherein the cancer is selected from leukemia, such as acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL), or solid tumors, selected from prostate cancer, colorectal cancer, gastric cancer, lung cancer, osteosarcoma, breast cancer, pancreatic cancer, or squamous cell carcinoma; preferably, the cancer is leukemia, such as AML or ALL.

[0245] Item 45: The component for use according to item 44, wherein the cancer is associated with tumor cells expressing FLT3.

[0246] Brief description of the drawings and sequences

[0247] The figure shows:

[0248] Figure 1 Depicts the Fc-attenuated IgGsc form used to construct the bispecific FLT3xCD3 antibody variant described in the present invention. Variants of the FLT3 antigen-binding domain (V1 to V5) were obtained by CDR grafting of the V region of the FLT3 antibody 4G8 using replacement strategies of different stringencies. To generate different CD3-antigen-binding domains (V6 to V9), the UCHT1scFv sequence was used.

[0249] Figure 2Show the amino acid sequences of the heavy and light chains of CC-2. A: Heavy chain sequence of the FLT3(4G8)X CD3 (humanized hUCHT1) bispecific IgGsc form antibody molecule (SEQ ID NO:68). The heavy chain contains the murine heavy chain (HC) variable region of 4G8, IgG1 CH1 domain, IgG1 hinge region, modified IgG1 CH2 domain, IgG1 CH3 domain, and humanized CD3 (UCHT1) single-chain Fv fragment. B: Show the amino acid sequence of the kappa light chain of FLT3(4G8)x CD3 (humanized hUCHT1) (SEQ ID NO:69). This light chain completes the heavy chain construct of SEQ ID NO:68 to form chimeric 4G8xUCHT1 IgGsc and Fabsc molecules respectively (see Figure 1 ).

[0250] Figure 3 Depict the binding of different CC-2 variants to soluble recombinant FLT3 protein. The corresponding antibody variants were immobilized on a protein A-coated Biacore chip, and the binding of His-tagged recombinant FLT3 protein (Sino Biologicals) was determined using a Biacore X instrument (GE Healthcare). Results of various mutant variants (V1 to V5) of the FLT3 antigen-binding domain are shown.

[0251] Figure 4 Show the binding of CC-2 variants to Nalm16 cells expressing FLT3. The calculated EC50 values are indicated on the corresponding bars of Figure 3 . Mutant variants (V1 to V5) of the FLT3 antigen-binding domain were tested.

[0252] Figure 5 Depict the depletion of leukemia cells from blood samples of AML patients using various different CC-2 constructs of the present invention. A comparison of mutant variants (V1 to V5) of the FLT3 antigen-binding domain is shown. Also indicated are the Figure 3 Biacore result values and the Figure 4 flow cytometry EC50.

[0253] Figure 6 Show the binding of CC-2 variants to Jurkat cells expressing CD3. The calculated EC50 values are indicated on the corresponding bars of Figure 5 . Mutant variants (V7 to V9) of the CD3 antigen-binding domain were tested.

[0254] Figure 7 Depict the depletion of leukemia cells from blood samples of AML patients using various different CC-2 constructs of the present invention. A comparison of mutant variants (V7 to V9) of the CD3 antigen-binding domain is shown. Also indicated areFigure 6 Flow cytometry EC50 value.

[0255] Figure 8 Depicts the results of T cell activation and blast reduction by different CC-2 variants in a flow cytometry assay. The CC2 variants used were different in both their FLT3 antigen-binding domain and CD3 antigen-binding domain (antibodies used were V6-V6 (V6); V6-V9 (V9); V4-V6 (V4)). A: CD4-positive cell reduction; B: CD8-positive cell reduction; C: blast count.

[0256] Figure 9 Depicts the depletion of NALM16 leukemia cells by different variants of CC-2 in a flow cytometry assay. The CC2 variants used were different in both their FLT3 antigen-binding domain and CD3 antigen-binding domain (antibodies used were V6-V6 (V6); V6-V8 (V8); V4-V6 (V4); V6-V7 (V7)).

[0257] Figure 10 Depicts the in vivo anti-leukemia activity of CC-2 variant 4 in immunodeficient NSG mice transplanted with primary AML (A) or ALL (B). As an example, the CC-2 variant with FLT3 antigen-binding domain V4 (V4-V6) is shown.

[0258] Figure 11 Depicts the antibody-dependent

[0259] cytotoxicity (antibody-dependent cellular cytotoxicity) (ADCC) of monospecific anti-FLT3 antibody variants. Specific lysis of FLT3+ leukemia cells (SEM; DSMZ No. ACC 546) by allogeneic polyclonal natural killer cells (pNKC) in the presence of the indicated FLT3 constructs or Fc control (5 μg / ml each) measured by 2hBATDA-europium cytotoxicity assay is shown.

[0260] All ABP of the present invention is described in the sequence listing and Table 1 below:

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275] Examples

[0276] Certain aspects and embodiments of the present invention will now be illustrated by way of examples and with reference to the descriptions, drawings, and tables set forth herein. Such examples of the methods, uses, and other aspects of the present invention are merely representative and should not be considered as limiting the scope of the present invention to such representative examples.

[0277] The examples show:

[0278] Example 1: Generation of Recombinant Bispecific FLT3xCD3 ABP (CC-2)

[0279] A recombinant bispecific ABP in IgGsc form was constructed using the 4G8 anti-FLT3 antibody ( Figure 1 ), that is, the variable domain of the murine FLT3 antibody 4G8 was fused to the human constant and variable regions of the CD3 antibody UCHT1 in the following order. VL-CL of the light chain and VH-CH1-CH2mod-CH3-scFv (UCHT1) of the heavy chain (see Figure 1 ). In these ABPs, the FLT3 binding site exists in the form of a Fab2 fragment, while the CD3 binding site exists in the form of a scFv fragment (see again Figure 1)。To eliminate FcR binding, the following modifications were introduced into the hinge region and CH2 domain (EU-index): E233P; L234V; L235A; ΔG236; D265G;; A327Q; A330S (see also International Patent Application WO 2013 / 092001 in this regard). As described in International Patent Application WO 2013 / 092001, the construct was cloned into an expression vector derived from pcDNA3.1 (InVitrogen, Thermo Fisher) and transiently transfected into CHO cells. The ABP was purified from the supernatant of transfected cells by affinity chromatography using protein A resin (purchased from GE Health Care Freiburg, Germany). The resulting murine FLT3xCD3 bispecific antibody was designated V6-V6.

[0280] Example 2: Generation of a humanized 4G8 antibody

[0281] Humanizing the 4G8 anti-FLT3 antibody by grafting the CDR regions of the light chain of antibody 4G8 (meaning the CDR loops of SEQ ID NOs: 5 to 7) into the (variable domain) of the human κ light sequence IGKV3-15*01, which is deposited in the IMGT / LIGM database under accession number M23090. See also Ichiyoshi Y., Zhou M., Casali P. A human anti-insulin IgG autoantibody apparently arises through clonal selection from an insulin-specific 'germ-line' natural antibody template. Analysis by V gene segment reassortment and site-directed mutagenesis. J. Immunol. 154(1):226-238 (1995). Further, the CDR regions of the heavy chain of antibody 4G8 (meaning the CDR loops of SEQ ID NOs: 01 to 03) are incorporated into the (variable domain) of the heavy chain sequence IGHV1-46*03, which is deposited in the IMGT / LIGM database under accession number L06612 (see also Watson C.T., et al. Complete haplotype sequence of the human immunoglobulin heavy-chain variable, diversity, and joining genes and characterization of allelic and copy-number variation. Am. J. Hum. Genet. 92(4):530-546 (2013). The resulting humanized 4G8 is used as the basis for the mutant analysis / variants of the present invention.

[0282] Example 3: Generation of variants of the bispecific FLT3xCD3 ABP (CC-2) of the present invention

[0283] Multiple variants (V1 to V5) of the FLT3-binding domain of humanized 4G8 are generated. In addition, multiple publicly available variants (V7 to V9) of the CD3-binding domain scFv derived from the humanized CD3 antibody UCHT1 are used in various combinations with the FLT3-binding domain variants of the present invention. The following preferred mutant variants with specific technical effects, which are desirable for the use of such humanized 4G8 variants, are identified (CDRs are underlined and mutations are indicated for the corresponding heavy and light chains present in the humanized 4G8 FLT3 antigen-binding domain designated as variant 0 or V0):

[0284] Humanized 4G8 FLT3 variable region variant 1 (V1)

[0285] Heavy chain variable region:

[0286] Mutations: K16G, V18L, K19R, V20L, K22A, K57T, N60A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A, T108L

[0287] QVQLVQSGAEVKKPGGSLRLSCAASGYTFT SYWMH WVRQAPGQGLEWI

[0288] G EIDPSDSYTDYAQKFKD RVTISRDTSKNTLYLQLSSLRAEDTAVYYCAR A ITTTPFDF WGQGTLVTVSS.

[0289] Light chain variable region:

[0290] Mutations: K49Y, I55A

[0291] EIVMTQSPATLSVSPGERATLSC RASQSISNNLH WYQQKPGQAPRLLIY YA

[0292] SQSAS GIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQSNTWPYT FGGGTK

[0293] LEIK

[0294] Humanized 4G8FLT3 variable region variant 2 (V2)

[0295] Heavy chain variable region:

[0296] Mutations: K16G, V18L, K19R, V20L, K22A, K57T, N60A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A, T108L

[0297] QVQLVQSGAEVKKPGGSLRLSCAASGYTFT SYWMH WVRQAPGQGLEWI

[0298] G EIDPSDSYTDYAQKFKD RVTISRDTSKNTLYLQLSSLRAEDTAVYYCAR A

[0299] ITTTPFDF WGQGTLVTVSS

[0300] Variable region of light chain:

[0301] EIVMTQSPATLSVSPGERATLSC RASQSISNNLH WYQQKPGQAPRLLIK YASQSIS GIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQSNTWPYT FGGGTKLEIK

[0302] Humanized 4G8 FLT3 variable region variant 3 (V3)

[0303] Variable region of heavy chain:

[0304] Mutation: 48I

[0305] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWIG EIDPSDSYKDYNQKFKD RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR AITTTPFDF WGQGTTVTVSS

[0306] Variable region of light chain:

[0307] Mutations: K49Y, I55A

[0308] EIVMTQSPATLSVSPGERATLSC RASQSISNNLH WYQQKPGQAPRLLIY YASQSAS GIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQSNTWPYT FGGGTKLEIK

[0309] Humanized 4G8 FLT3 variable region variant 4 (V4)

[0310] Heavy chain variable region:

[0311] Mutation: 48I

[0312] QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWIG EIDPSDSYKDYNQKFKD RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR AITTTPFDF WGQGTTVTVSS

[0313] Light chain variable region:

[0314] Mutation: 49K, 87F

[0315] EIVMTQSPATLSVSPGERATLSC RASQSISNNLH WYQQKPGQAPRLLIK YASQSIS GIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQSNTWPYT FGGGTKLEIK

[0316] Humanized 4G8FLT3 variable region variant 5 (V5)

[0317] Heavy chain variable region:

[0318] Mutation: K16G, V18L, K19R, V20L, K22A, M69I, T70S, T75K, S76N, V78L, M80L, E81Q, S87A, T108L

[0319] QVQLVQSGAEVKKPGGSLRLSCAASGYTFT SYWMH WVRQAPGQGLEWIG EIDPSDSYKDYNQKFKD RVTISRDTSKNTLYLQLSSLRAEDTAVYYCAR AITTTPFDF WGQGTLVTVSS

[0320] Light chain variable region:

[0321] EIVMTQSPATLSVSPGERATLSC RASQSISNNLH WYQQKPGQAPRLLIK YASQSIS GIPARFSGSGSGTEFTLTISSLQSEDFAVYFC QQSNTWPYT FGGGTKLEIK

[0322] Humanized UCHT1 CD3scFv variant 1 (V7)

[0323] Mutations: N60A, Q61D, K62S, D65G, K73D

[0324] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFG QGTKVEIK GGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYADSFKGRFTISVDDSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS Humanized UCHT1 CD3scFv variant 2 (V8)

[0325] Mutation: K73D

[0326] DIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK GGGGSGGGGSGGGGS EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDDSKNTAYLQMNSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSS Humanized UCHT1 CD3scFv variant 3 (V9)

[0327] Mutation: Light chain - heavy chain replacement (VL - VH → VH - VL):

[0328] EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRQAPGKGLEWV

[0329] ALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYYCA

[0330] RSGYYGDSDWYFDVWGQGTLVTVSS GGGGSGGGGSGGGGS DIQMTQSP

[0331] SSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGV

[0332] PSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIK

[0333] Create bispecific ABP forms using the above variants as indicated in Table 1 above.

[0334] Example 4: Binding affinity of variants of the FLT3 antigen-binding domain of humanized 4G8.

[0335] In Figure 3 , the dissociation constants of the binding of different FLT3-binding domain variants V1 to V5 as indicated above were tested using soluble recombinant FLT3 protein. The corresponding antibody variants were immobilized on a Biacore chip coated with protein A, and the binding of His-tagged recombinant FLT3 protein (Sino Biologicals) was determined using a Biacore X instrument (GE Healthcare). The results are listed in Table 2:

[0336]

[0337] In addition, the binding affinity of the antibody was also tested using the binding of the antibody to its target expressed on cells. The binding of the CC-2 variant to Nalm16 cells expressing FLT3 ( Figure 4 ) or Jurkat cells expressing CD3 ( Figure 6 ) was analyzed. After transient transfection of CHO cells with the insertion sequences listed above, variants V1 to V9 (V6 was V6-V6) were generated. The antibodies were purified by protein A affinity chromatography and size exclusion chromatography. V1 to V5 contained different variants of the FLT3 antibody as indicated above and the same CD3-binding antibody (UCHT1 variant, which binds to CD3 with an EC50 of approximately 10 nM). V6 to V9 contained the parental 4G8 antibody that binds to FLT3 with an EC50 of approximately 1 nM and different UCHT1 variants. To measure the antibodies that bind to FLT3 and CD3, NALM16 (FLT3+) and Jurkat (CD3+) cells were incubated with the respective variants for 30 minutes, washed, stained with a goat anti-human Fcγ-specific and PE-conjugated secondary antibody (Jackson Immuno Research), washed again and analyzed by flow cytometry (FACSCalibur, BD Biosciences).

[0338] Conclusion : As Figure 3 and 4 shown, as measured by surface plasmon resonance (Biacore) and flow cytometry respectively, variant 5 has the highest binding affinity for FLT3, followed by V4.

[0339] Example 5: Reduction of leukemic cells from blood samples of AML patients by different FLT3-binding or CD3-binding domain variants.

[0340] Peripheral blood mononuclear cells (PBMCs) were obtained from patients with acute leukemia by density gradient centrifugation, incubated with the indicated CC-2 variants at 1 μg / ml for 5 days, and analyzed by flow cytometry using a FACSCanto-II (BD Biosciences). Leukemic cells were identified using antibodies against CD33, CD34, or CD117. To calibrate cell numbers, a limited amount of compensation beads (BD Biosciences) was added to each sample. Results were obtained from three (V4, V5, V6, V9) and two (V3) independent experiments performed with cells from two different donors. All values were related to data obtained using a control IgGsc antibody with irrelevant specificity. Results for FLT3-binding variants are shown in Figure 5 and results for CD3-binding variants are shown in Figure 7 .

[0341] Conclusion : Anti-leukemic activity initially followed the affinity values on both sides of the bispecific molecule. V5 had the highest activity and affinity for FLT3. However, surprisingly, the less-affine V4 variant had significantly better cytotoxic potential against leukemic cells than the other variants, indicating that for certain applications, a lower affinity range of the FLT3 antibody could be selected. Similar results were observed for the CD3-binding moiety.

[0342] In Figure 8 and 9 , T cell activity and blast reduction were analyzed at different antibody concentrations. A PBMC preparation from patient MM2 was obtained and incubated with the indicated concentrations of the CC-2 variants. After 5 days, T cell activation and reduction of leukemic cells were evaluated by flow cytometry ( Figure 8 ). Nalm16 cells were incubated with the indicated concentrations of the CC-2 variants at an E:T ratio of 2:1 for 3 days and then analyzed by flow cytometry ( Figure 9 ).

[0343] Example 6: In vivo anti-leukemic activity of the ABP of the present invention

[0344] To test the in vivo application of the CC-2 variants of the present invention, the therapeutic potential of the preferred variants binding FLT3 and CD3 was tested in an immunodeficient mouse model ( Figure 10)。Immunodeficient NSG mice were implanted with primary AML (left) or ALL (right) cells. On day 7, PBMC and CC-2 (V4-V6) or control antibody were injected, and the bsAb treatment was repeated on day 10. On day 17, the leukemia burden in the bone marrow (ratio of hCD45 + / mCD45 + cells) was determined by flow cytometry.

[0345] Conclusion : This experiment demonstrated that even less-affinity CC2 variants, such as V4, also have therapeutic potential.

[0346] The inventions described by way of example herein can be practiced appropriately without any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, terms such as "comprising", "including", "containing", etc. should be read broadly and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description rather than of limitation, and are not intended to exclude any equivalents of the features shown and described or portions thereof, but it should be recognized that various modifications can be made within the scope of the invention as claimed. Accordingly, it should be understood that although the invention has been specifically disclosed by way of exemplary embodiments and optional features, those skilled in the art can make modifications and variations to the invention embodied herein, and such modifications and variations are considered to be within the scope of the invention.

[0347] Example 7: Humanized 4G8 Variant in Monospecific IgG Form

[0348] To test the superiority of the antibody variants of the present invention produced, the new variable domain sequences were cloned into a monospecific anti-FLT3 human IgG form. For this purpose, the corresponding variable heavy and light chain sequences were cloned into a monospecific human IgG form. For this purpose, the antibody genes were codon-optimized for expression in human cells, and NheI and Not restriction sites were designed at the 5' and 3' ends. The synthetic genes were then cloned into a mammalian expression vector according to standard procedures. After sequence verification, a sufficient amount of plasmid for transfection was prepared using the Plasmid Plus purification kit (Qiagen).

[0349] HEK 293 (human embryonic kidney 293) mammalian cells were passaged to the optimal stage for transient transfection. The cells were transiently transfected with the expression vector and cultured for another 6 days.

[0350] The culture was harvested by centrifugation at 4000 rpm and filtered through a 0.22 mm filter. The first step of purification was carried out by nickel affinity chromatography and elution was performed using PBS containing 400 mM imidazole. The second step of purification was carried out by size exclusion chromatography and elution was performed in PBS (phosphate buffered saline) at pH 7.2. The antibody concentration was determined by UV spectrophotometry and the antibody was concentrated as needed. The antibody purity was determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and HPLC (high performance liquid chromatography). HPLC was performed on an Agilent 1100 series instrument using a MabPac size exclusion column running at 0.2 ml / min in PBS.

[0351] To elucidate the superiority of the generated 4G8 variants, a monospecific IgG having the heavy and light chain variable domain sequences of the V4 variant (variable domain heavy and light chain sequences as shown in SEQ ID NO: 21 and 22, respectively) was compared with the parental (mouse) IgG V6 (variable domain heavy and light chain sequences as shown in SEQ ID NO: 4 and 8, respectively) and an Fc control in an ADCC assay. For this purpose, pNKC was generated by incubating non-plastic adherent PBMC with K562-41BBL-IL15 feeder cells obtained from St Jude's Children's Research Hospital as previously described (Schmiedel BJ et al. Int J Cancer 2011; 128: 2911–2922; Fujisaki H et al. Cancer Res 2009; 69: 4010–4017). BATDA-europium killing was performed as previously described (Baessler T et al. Cancer Res 2009: 69: 1037-1045). Figure 11 The results are shown in.

[0352] Figure 11 The data in show exemplary results for 1 pNKC donor at the indicated effector:target ratios. In this experimental setup, a superior lysis rate of V4 compared to the V6 FLT3 monospecific IgG antibody variant was observed, thus demonstrating the improved therapeutic potential of the variants of the present invention compared to the parental 4G8 antibody. Sequence Listing <110> Deutsches Krebsforschungszentrum Stiftung des oeffentlichen Rechts University of Tuebingen <120> Improved Anti-FLT3 Antigen-Binding Protein <130> D31605WO <150> EP18193889.5 <151> 2018-09-11 <150> EP19189566.3 <151> 2019-08-01 <160> 79 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 1 Ser Tyr Trp Met His 1 5 <210> 2 <211> 17 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 2 Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 3 <211> 9 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 3 Ala Ile Thr Thr Thr Pro Phe Asp Phe 1 5 <210> 4 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 4 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 5 <211> 11 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 5 Arg Ala Ser Gln Ser Ile Ser Asn Asn Leu His 1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 6 Tyr Ala Ser Gln Ser Ile Ser 1 5 <210> 7 <211> 9 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 7 Gln Gln Ser Asn Thr Trp Pro Tyr Thr 1 5 <210> 8 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 8 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 9 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 10 <211> 98 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 10 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 11 <211> 15 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 11 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 <210> 12 <211> 109 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 12 Ala Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 1 5 10 15 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 20 25 30 Val Gly Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 35 40 45 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 50 55 60 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 65 70 75 80 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gln 85 90 95 Leu Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105 <210> 13 <211> 109 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 13 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser Gly 100 105 <210> 14 <211> 244 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 14 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr 165 170 175 Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile 180 185 190 Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 210 215 220 Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 15 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 16 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 16 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 17 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 18 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 18 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 19 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 20 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 20 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 21 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 21 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 22 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 22 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 23 <211> 118 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 24 <211> 107 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 24 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 25 <211> 244 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 25 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr 165 170 175 Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr Ile 180 185 190 Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 210 215 220 Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 26 <211> 244 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 26 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro Tyr 165 170 175 Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr Ile 180 185 190 Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr Tyr 210 215 220 Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu Val 225 230 235 240 Thr Val Ser Ser <210> 27 <211> 244 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 27 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu Glu 180 185 190 Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys 225 230 235 240 Val Glu Ile Lys <210> 28 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 28 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 29 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 29 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 30 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 30 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 31 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 31 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 32 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 32 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 33 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 33 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 34 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 34 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 35 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 35 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 36 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 36 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 37 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 37 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 38 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 38 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 39 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 39 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 40 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 40 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 41 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 41 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 42 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 42 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 43 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 43 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 44 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 44 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 45 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 45 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 46 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 47 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 47 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 48 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 49 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 49 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 50 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 50 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 51 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 51 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 52 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 52 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 53 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 53 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 54 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 54 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 55 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 55 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 56 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 56 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 57 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 57 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 58 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 58 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 485 490 495 Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys 500 505 510 Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 515 520 525 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 530 535 540 Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 545 550 555 560 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 580 585 590 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 595 600 605 Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln 610 615 620 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 625 630 635 640 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 645 650 655 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 660 665 670 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr 675 680 685 Lys Val Glu Ile Lys 690 <210> 59 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 59 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 60 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 60 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Thr Asp Tyr Ala Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 485 490 495 Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys 500 505 510 Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 515 520 525 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 530 535 540 Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 545 550 555 560 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 580 585 590 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 595 600 605 Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln 610 615 620 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 625 630 635 640 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 645 650 655 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 660 665 670 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr 675 680 685 Lys Val Glu Ile Lys 690 <210> 61 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 61 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 62 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 485 490 495 Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys 500 505 510 Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 515 520 525 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 530 535 540 Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 545 550 555 560 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 580 585 590 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 595 600 605 Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln 610 615 620 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 625 630 635 640 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 645 650 655 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 660 665 670 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr 675 680 685 Lys Val Glu Ile Lys 690 <210> 63 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 63 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Gln Ser Ala Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 64 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 485 490 495 Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys 500 505 510 Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 515 520 525 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 530 535 540 Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 545 550 555 560 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 580 585 590 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 595 600 605 Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln 610 615 620 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 625 630 635 640 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 645 650 655 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 660 665 670 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr 675 680 685 Lys Val Glu Ile Lys 690 <210> 65 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 65 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 66 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 66 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 485 490 495 Val Ala Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys 500 505 510 Phe Lys Asp Arg Phe Thr Ile Ser Val Asp Lys Ser Lys Asn Thr Ala 515 520 525 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 530 535 540 Cys Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val 545 550 555 560 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser 565 570 575 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 580 585 590 Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr 595 600 605 Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr Leu Asn Trp Tyr Gln Gln 610 615 620 Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 625 630 635 640 Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 645 650 655 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr 660 665 670 Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp Thr Phe Gly Gln Gly Thr 675 680 685 Lys Val Glu Ile Lys 690 <210> 67 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 67 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 68 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 68 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Lys Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 69 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 69 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 70 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 70 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Ala Asp Ser Phe Lys Gly Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 71 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 71 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 72 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 72 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 450 455 460 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn 465 470 475 480 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 485 490 495 Ile Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 500 505 510 Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln 515 520 525 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro 530 535 540 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Gly 545 550 555 560 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val 565 570 575 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 580 585 590 Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val 595 600 605 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Leu Ile Asn Pro 610 615 620 Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe Lys Asp Arg Phe Thr 625 630 635 640 Ile Ser Val Asp Asp Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser 645 650 655 Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gly Tyr 660 665 670 Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Leu 675 680 685 Val Thr Val Ser Ser 690 <210> 73 <211> 214 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 73 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly 1 5 10 15 Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn 20 25 30 Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr 65 70 75 80 Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Asn Thr Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 74 <211> 693 <212> PRT <213> Artificial <220> <223> Variant antibody sequence <400> 74 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Leu Lys Leu Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met His Trp Val Arg Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asp Pro Ser Asp Ser Tyr Lys Asp Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met His Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Ile Thr Thr Thr Pro Phe Asp Phe Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Gly Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gln Leu Pro Ser Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Ser 435 440 445 Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 450 455 460 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Phe Thr Gly 465 470 475 480 Tyr Thr Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp ...

Claims

1. An antigen-binding protein (ABP) capable of binding to human fms-related tyrosine kinase 3 (FLT3), comprising: (a) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 21, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 22; or (b) two heavy chain variable regions and two light chain variable regions, wherein the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 21, and the light chain variable region consists of the amino acid sequence of SEQ ID NO:

22.

2. The ABP according to claim 1, comprising: (i) a CDRH1 region shown by SEQ ID NO: 01 (SYWMH), a CDRH2 region shown by SEQ ID NO: 02 (EIDPSDSYKDYNQKFKD), and a CDRH3 region shown by SEQ ID NO: 03 (AITTTPFDF); and (ii) a CDRL1 region shown by SEQ ID NO: 05 (RASQSISNNLH), a CDRL2 region shown by SEQ ID NO: 06 (YASQSIS), and a CDRL3 region shown by SEQ ID NO: 07 (QQSNTWPYT).

3. The ABP according to claim 1 or claim 2, which binds to the FLT3 with a k of less than 50 μM and greater than 50 nM. D 4. The ABP according to claim 1 or claim 2, which binds to human cells expressing FLT3 with an EC of less than 10 nM and higher than 0.5 nM. 50 5. The ABP according to claim 1 or claim 2, which binds to human cells expressing FLT3 with an EC of less than 5.5 nM and higher than 4.5 nM. 50 6. The ABP according to claim 1 or claim 2, which comprises an effector group and / or is labeled.

7. The ABP according to claim 1 or claim 2, which is isolated.

8. The ABP according to claim 1, which is an antibody or a fragment of an antibody.

9. The ABP according to claim 8, wherein the antibody is a monoclonal antibody, a chimeric antibody or a human chimeric antibody.

10. The ABP according to claim 8 or 9, wherein the antibody is an IgG, IgE, IgD, IgA or IgM immunoglobulin. ​​​11. The ABP according to claim 8 or claim 9, which is an antibody fragment selected from the following list: Fab, Fab’-SH, Fv, scFv, and F(ab’)2.

12. The ABP according to claim 1 or claim 2, wherein the ABP is modified or engineered to increase antibody-dependent cellular cytotoxicity (ADCC).

13. The ABP according to claim 12, wherein the ABP is α-fucosylated.

14. The ABP according to claim 1 or claim 2, which comprises one or more additional antigen-binding domains that bind an antigen other than the FLT3.

15. The ABP according to claim 14, wherein the additional antigen-binding domain binds human CD3.

16. The ABP according to claim 15, which is bispecific and comprises two binding sites that bind FLT3 and two binding sites that bind an antigen other than the FLT3.

17. The ABP according to claim 16, wherein the two binding sites that bind an antigen other than the FLT3 bind human CD3.

18. The ABP according to claim 17, wherein the two binding sites that bind human CD3 comprise a UCHT1 anti-CD3 scFv construct.

19. The ABP according to claim 18, wherein the scFv consists of the amino acid sequence of SEQ ID NO:

14.

20. The ABP according to claim 14, which comprises two antibody heavy chain sequences and two antibody light chain sequences, wherein each of the antibody heavy chain sequences and the antibody light chain sequences consists of the amino acid sequence of the following V4: 。 21. An isolated nucleic acid, which comprises a sequence encoding the ABP or an antigen-binding fragment or monomer of the ABP according to any one of claims 1 to 20.

22. A nucleic acid construct, which comprises the nucleic acid of claim 21 and one or more additional sequence features that allow the expression in a cell of the encoded ABP or bispecific ABP or a component of the ABP or bispecific ABP.

23. A recombinant host cell, which comprises the nucleic acid of claim 21 or the nucleic acid construct according to claim 22.

24. A pharmaceutical composition comprising: (i) an ABP or bispecific ABP of any one of claims 1 to 20, or (ii) a nucleic acid of claim 21 or a nucleic acid construct according to claim 22, or (iii) a recombinant host cell according to claim 23 and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

25. A component for use in medicine, wherein the component is selected from the list consisting of: an ABP or bispecific ABP of any one of claims 1 to 20, an isolated nucleic acid of claim 21 or a nucleic acid construct according to claim 22, a recombinant host cell according to claim 23 and a pharmaceutical composition according to claim 24.

26. A component according to claim 25 for use in the diagnosis and / or treatment of acute myeloid leukaemia (AML) or acute lymphoblastic leukaemia (ALL).

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