EA040387B1 - ANTIBODY CONSTRUCTS TO FLT3 AND CD3 - Google Patents

Description

The present invention relates to a bispecific antibody construct comprising a first binding domain that binds to human FLT3 on the surface of a target cell and a second binding domain that binds to human CD3 on the surface of a T cell. In addition, the present invention provides a polynucleotide encoding said antibody construct, a vector containing said polynucleotide, and a host cell transformed or transfected with said polynucleotide or vector. In addition, according to the present invention, a method for producing said antibody construct according to the present invention, a medical use of said antibody construct, and a kit containing said antibody construct are provided.

Technical field

Acute myeloid leukemia (AML) is a heterogeneous malignant hematologic disease that is the most common type of acute leukemia diagnosed in adults. AML represents approximately one third of all leukemias, with approximately 14,500 new cases reported in 2013 in the United States alone, and has poor overall survival rates. Over the past thirty years there has been little improvement in the standard of care for patients with AML. However, recent discoveries in molecular and cellular biology have radically transformed our understanding of human hematopoiesis, both in health and disease. A number of key elements involved in the pathogenesis of the disease have been identified and can be investigated as potentially relevant targets. One such activating trigger gene, typically mutated in about 30% of AML cases, is FLT3.

Fms-like tyrosine kinase 3 (FLT3), also known as fetal liver kinase 2 (FLK-2), human stem cell kinase 1 (SCK-1), or differentiation cluster antigen (CD135), is a hematopoietic receptor tyrosine kinase that has been cloned by two independent groups in the 1990s. The FLT3 gene, located on chromosome 13q12 in humans, encodes a class III receptor tyrosine kinase protein that is homologous to other members of the class III family, including the stem cell factor receptor (c-KIT), the macrophage colony-stimulating factor (FMS) receptor, and the platelet-derived growth factor receptor ( rTRF).

Upon binding to the FLT3 ligand, the FLT3 receptor undergoes homodimerization, thus allowing for autophosphorylation of specific tyrosine residues in the peri-membrane domain and downstream activation via the PI3K/Akt, MAPK, and STAT5 pathways. FLT3 therefore plays a critical role in the control of proliferation, survival, and differentiation of normal hematopoietic cells.

Human FLT3 is expressed in CD34+CD38 hematopoietic stem cells (HSCs) as well as a subgroup of dendritic progenitor cells. FLT3 expression can also be detected in multipotent progenitors such as CD34+CD38+CD45RA-CD123 ^low common myeloid progenitor (CMP), CD34+CD38+CD45RA+CD123 ^low granulocyte and monocyte progenitors (GMP) and CD34+CD38+CD10+ CD19 - common lymphoid progenitors (CLP). Interestingly, FLT3 expression is virtually absent in CD34+CD38-CD45RA-CD123 megakaryocyte and erythrocyte (MER) progenitor cells. FLT3 expression is accordingly mainly restricted to early myeloid and lymphoid progenitor cells, with some expression observed in more mature monocytic cell lines. This limited FLT3 expression pattern contrasts sharply with the FLT3 ligand expression pattern, which is expressed in most hematopoietic tissues and in the prostate, kidney, lung, colon, and heart. Due to these variable expression patterns, FLT3 expression represents a rate-determining step in determining tissue-specific FLT3 signaling pathways.

The most common FLT3 mutation in AML is the FLT3 intrinsic tandem duplication (FLT3-ITD), which is found in 20-38% of patients with cytogenetically normal AML. FLT3-ITD mutations occur when there is a duplication and insertion of a part of the coding sequence of the peri-membrane domain in a head-to-tail orientation. FLT3 mutations have not been identified in patients with chronic lymphoid leukemia (CLL), non-Hodgkin's lymphoma, and multiple myeloma, suggesting a strong specificity for AML disease. Mutant FLT3 activation is commonly observed in all FAB subtypes, however, it is significantly increased in patients with AML characterized by FAB M5 (monocytic leukemia), while the FAB M2 and M6 subtypes (granulocytic or erythroid leukemia) are significantly less frequently associated with FLT3 activation, at the level of normal FLT3 expression patterns.

A small percentage of AML patients (5-7%) have single amino acid mutations in the FLT3 tyrosine kinase domain (TKD FLT3), most commonly at position D835 or in some cases T842 or I836; even fewer patients (~1%) have mutations in the near-membrane domain of FLT3 involving residues 579, 590, 591, and 594. Patients with AML characterized by FLT3-ITD mutant have an aggressive form of the disease characterized by early relapse and poor survival, then both overall survival and event-free survival

- 1 040387 FLT3-TKD mutations are not significantly affected. In addition, patients with AML who have the mutation

FLT3-ITD and co-existing TET2 or DNMT3A mutations have an unfavorable overall risk profile compared with patients with AML characterized by FLT3-ITD mutant TET2 or

DNMT3A is wild-type, highlighting the clinical and biological heterogeneity of AML.

Both FLT3-ITD mutations and FLT3 TKD mutations induce ligand-independent FLT3 activation leading to downstream activation of the Ras/MAPK and PI3K/Akt pathways. However, the downstream signaling pathways associated with any of the mutations differ mainly in the predominant activation of STAT5 by FLT3-ITD, which leads to increased proliferation potential and aberrant regulation of DNA repair pathways.

Regardless of FLT3 mutational status, FLT3 phosphorylation appears to occur in over two-thirds of AML patients, and FLT3 is expressed in over 80% of AML blasts and ~0% of all AML patients, making it an attractive therapeutic target associated with pathogenesis. disease in a large sample size.

A number of small molecule inhibitors have been proposed as attractive therapy options for AML patients with FLT3 mutations. The first FLT3 tyrosine kinase (TKI) inhibitors obtained were characterized by a lack of selectivity, efficacy, and unfavorable pharmacokinetic properties. Newer and more selective agents have been developed to cope with these problems; however, their effectiveness has been limited as a result of secondary resistance.

Several early FLT3 TKIs included, among others, midostaurin (PKC412), lestaurtinib (CEP-701), sunitinib (SUI1248) and sorafenib (BAY 43-9006). The response rate in phase I and phase II studies to these multikinase targeting agents in patients with relapsed or refractory AML is limited, presumably due to their inability to provide effective FLT3 inhibition without dose-limiting toxicity. Quisartinib (AC220) has been developed as a second-generation FLT3 TKI with high selectivity for wild-type FLT3 and FLT3-ITD and has been shown to be beneficial, particularly in peri-transplant settings in an earlier cohort of patients. However, secondary FLT3 mutations identified in patients with relapsing disease treated with quisartinib highlight the need to develop better therapeutic strategies for patients with AML, while confirming the validity of FLT3 as a therapeutic target.

A number of targeted agents have been tested in AML patients with de novo, relapsed/refractory, or secondary disease.

Epigenetic silencing of tumor suppressor genes plays an important role in the pathogenesis of AML, and DNA methyltransferase (DNMT) inhibitors such as azacitidine and decitabine have provided some clinical success. In addition, the recent identification of mutations affecting post-translational histone modifications (eg, EZH2 and ASXL1 mutations) or DNA methylation (eg, DNMT3A, TET2, IDH1/2) in a subgroup of patients with AML has led to the development of various therapeutic options, including EZH2, DOT1L, IDH1/2 inhibitors along with HDAC and proteasome inhibitors. However, the results of preclinical studies of many of these compounds in AML cells suggest that these inhibitors may alter the phenotype and gene expression characteristic of hematopoietic differentiation, rather than cause direct cytotoxicity for AML blasts. Accordingly, there remains a strong unmet medical need to identify new targets/methods to combat AML and provide targeted lysis of AML blast cells. Other candidate therapeutic agents for AML include Aurora kinase inhibitors, including AMG 900, and inhibitors of polo-like kinases, which play an important role in cell cycle progression.

The standard of care for patients with AML remains chemotherapy with stem cell transplantation when possible. However, the occurrence of relapse/refractory in the vast majority of treated patients is a reason to look for additional therapies. The identification and characterization of several leukemia-specific antigens, along with a clearer understanding of the immune-mediated effects of graft against leukemia, have paved the way for the development of immunomodulatory strategies to combat hematological malignancies, which have been reviewed in several articles.

Gemtuzumab ozogamicin (GO) is an antibody/drug conjugate directed against CD33, a universal myeloid cell surface marker. GO was withdrawn from the market after randomized trials showed no improvement in outcomes with GO therapy. However, there is a need to re-evaluate GO in AML and several trials have been initiated to carefully evaluate the efficacy and toxicity of GO. Other anti-AML biological agents include lintuzumab (SGN-33), a humanized anti-CD33 monoclonal antibody in unconjugated form or conjugated with radioactive bismuth, and SL-401, consisting of human IL-3 coupled to diphtheria toxin, to load the IL-3 receptor , which is overexpressed in most AML blasts. Next-generation monoclonal antibodies targeting both tumor-associated antigen and effector cytolytic T cells include AMG 330 (a bispecific T cell activator or BiTE molecule targeting CD33) and

MGD006, a dual affinity reorienting molecule that binds to CD123 and CD3.

Recent success with chimeric antigen receptor-bearing T cells in refractory CLL and acute lymphoblastic leukemia (ALL) has paved the way for the development of myeloid-specific CAR-T cells, including CD123 CAR-T and CD33 CAR- T. A number of efforts have also been made to develop dendritic cell-based vaccines, as well as combinations with checkpoint blockade inhibitors, to improve treatment outcomes.

Therapeutic antibodies against FLT3 have also been obtained. Antibody therapy is considered more effective, with a low likelihood of developing secondary resistance mechanisms, since the antibody is directed against the extracellular domain of FLT3, which is less prone to mutations compared to the intracellular kinase domain. The Imclone antibody, IMC-EB10, was evaluated in patients with relapsing AML in a Phase I study, but the study was discontinued due to lack of efficacy (ClinicalTrials.gov, ID: NCT00887926). Accordingly, there remains a strong need to evaluate the use of second-generation monoclonal antibodies, including bispecific antibodies, in the treatment of AML.

Since there is still a need for additional available options for the treatment of hematological diseases associated with the expression of FLT3, methods and means are provided herein to solve this problem in the form of a bispecific antibody construct containing a binding domain directed to FLT3 on the surface of tumor target cells, and a second binding domain directed to CD3 on the surface of T cells.

Accordingly, according to a first aspect, the present invention provides a bispecific antibody construct comprising a first binding domain that binds to human FLT3 on the surface of a target cell and a second binding domain that binds to human CD3 on the surface of a T cell, said first binding domain binding with an FLT3 epitope that is located in the extracellular region of FLT3, according to the sequences of SEQ ID NO: 801-804.

It should be noted that in this document, the terms in the singular, including the definition of the specified(th, oe) include the appropriate forms of the plural, unless the context clearly implies otherwise. Accordingly, for example, when referring to a reagent, one or more different such reagents are included, and when referring to a (specified) method, equivalent steps and methods known to those skilled in the art are included, which may be modified methods or replace methods as described herein. .

Unless otherwise indicated, the term at least preceding the series of elements is intended to refer to each element in the series. Many equivalents of specific embodiments of the present invention as described herein are known to those skilled in the art, or such equivalents can be determined using no more than routine experimentation. It is assumed that such equivalents are covered by the present invention.

The term and / or in any sections of this document includes the meanings of and, or and all or any other combinations of elements combined by the specified term.

The term about or about herein means values within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a specified value or range.

In the present description and the appended claims, except where the context otherwise implies, the term contain and its variants, such as contains and containing, is intended to include the specified integer value or step, or group of integer values or steps, but does not exclude any other integer value. a value or step, or a group of integer values or steps. In this document, the term containing may be replaced by the term encompassing or including, or sometimes by the term having.

As used herein, consisting of excludes any elements, steps, or ingredients not listed in the claim element. In this document, the term consisting essentially of does not exclude materials or steps that do not significantly affect the main and new features claimed in the claims.

In all cases in this document, any of the terms containing, essentially consisting of, and consisting of may be replaced by any of the other two terms.

The term antibody construct refers to a molecule whose structure and/or function is(s) based on the structure and/or function of an antibody, such as a full-length or whole immunoglobulin molecule. Thus, the antibody construct is capable of binding to a specific target or antigen. In addition, the antibody construct according to the present invention satisfies

- 3 040387 minimum structural requirements for an antibody that ensures target binding. This minimum requirement may, for example, be defined as the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 from the VL region) and/or three heavy chain CDRs (i.e., CDR1, CDR2 and CDR3 from the VH region), preferably all six CDR regions. Antibodies on which the constructs of the present invention are based include, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.

The definition of antibody constructs in accordance with the present invention includes full-length or whole antibodies, including also camelid antibodies and other immunoglobulin antibodies obtained using biotechnological methods or processes, or methods or processes for constructing proteins. Said full length antibodies may be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies. Also included in the definition of antibody constructs are fragments of full length antibodies such as VH, VHH, VL, (s)dAb, Fv, Fd, Fab, Fab', F(ab') ₂ or r-IgG (half-body). The antibody constructs of the present invention may also be modified antibody fragments, also referred to as antibody variants, such as scFv, di-scFv or 6u(c)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies , single chain diabodies, tandem diabodies (Tandab), tandem di-scFvs, tandem tri-scFvs, minibodies exemplified by the structure below: (VH-VL-CH3)2, (scFv-CH3) ₂ , ((scFv) 2-CH3+CH3), ((scFv)2-CH3) or (scFvCH3-scFv)2, multibodies such as triabodies or tetrabodies, and single domain antibodies such as nanobodies or single domain antibodies with only one variable domain, which can be represented by a VHH, VH, or VL domain that specifically binds an antigen or epitope, independent of other variable regions or domains.

The binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it must not contain both. Fd fragments, for example, contain two VH regions and often retain the antigen-binding function of the intact antigen-binding domain to some extent. Additional examples of antibody fragment formats, antibody variants, or binding domains include (1) Fab fragment, a monovalent fragment containing the VL, VH, CL, and CH1 domains; (2) F(ab') ₂ fragment, bivalent fragment containing two Fab-fragment connected by a disulfide bridge in the hinge region; (3) Fd fragment containing two VH and CH1 domains; (4) an Fv fragment containing the VL and VH domains of one antibody arm, (5) a dAb fragment (Ward et al., (1989) Nature 341:544-546) which contains a VH domain; (6) isolated complementarity determining region (CDR); and (7) single chain Fv (scFv), the latter being preferred (eg, derived from the scFV library). Examples of embodiments of antibody constructs according to the present invention are described, for example, in WO 00/006605, WO 2005/040220, WO 2008/119567, WO 2010/037838, WO 2013/026837, WO 2013/026833, US 2014/0308285, US 2014/0302037, WO 2014/144722, WO 2014/151910 and WO 2015/048272.

In addition, the definition of the term antibody construct includes monovalent, bivalent and polyvalent/multivalent constructs and, respectively, monospecific constructs that specifically bind to only one antigenic structure, as well as bispecific and polyspecific/multispecific constructs that specifically bind more than one antigenic structure, for example two, three or more, via separate binding domains. In addition, the definition of the term antibody construct includes molecules consisting of a single polypeptide chain, as well as molecules consisting of more than one polypeptide chain, while these chains can be either identical (homodimers, homotrimers or homooligomers) or different (heterodimer, heterotrimer or heterooligomer). Examples of the antibodies identified above and variants or derivatives thereof are described, inter alia, in: Harlow and Lane, Antibodies a laboratory manual, CSHL Press (1988); Using Antibodies: a laboratory manual, CSHL Press (1999); Kontermann and Dubel, Antibody Engineering, Springer, 2nd ed. 2010; Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

The antibody constructs of the present invention are preferably prepared in vitro antibody constructs. The term refers to an antibody construct as defined above, the variable region of which is wholly or partly (e.g., at least one CDR region) obtained without the use of immune cells, e.g., using in vitro phage display, protein chip, or any other method. to test the ability of candidate sequences to bind to an antigen. Said term accordingly preferably excludes sequences obtained solely by genomic rearrangement in an animal's immune cell. A recombinant antibody is an antibody obtained through the use of recombinant DNA technology or genetic engineering.

The term monoclonal antibody (mAb) or monoclonal antibody construct as used herein refers to an antibody derived from a population of substantially homogeneous antibodies, ie.

- 4 040387 the individual antibodies that make up the population are identical except for possible naturally occurring mutations and/or post-translational modifications (eg isomerizations, amidations) which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site or determinant on an antigen, in contrast to standard (polyclonal) antibody formulations, which typically include different antibodies directed against different determinants (or epitopes). In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and therefore do not contain contaminants from other immunoglobulins. The modifier monoclonal indicates the nature of the antibody, essentially derived from a homogeneous population of antibodies, and should not be construed as determining the production of antibodies in any particular way.

To obtain monoclonal antibodies, any technique that ensures the production of antibodies by continuous cultures of cell lines can be used. For example, monoclonal antibodies for use may be prepared by the hybridoma method first described in: Koehler et al., Nature, 256: 495 (1975), or may be prepared using recombinant DNA techniques (see, for example, US Patent No. 4,816,567 ). Examples of additional techniques for generating human monoclonal antibodies include the trioma technique, the human B cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72), and the EBV hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

Hybridomas can then be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) to identify one or more hybridomas that produce an antibody that specifically binds to a given antigen. The immunogen may be any form of the relevant antigen, such as a recombinant antigen, naturally occurring forms, any variants or fragments thereof, and the corresponding antigenic peptide. Surface plasmon resonance used in the BIAcore system can be used to increase the efficiency of phage antibodies that bind to an epitope of a target antigen such as FLT3 or CD3epsilon (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

Another example of a method for producing monoclonal antibodies is provided by screening a protein expression library, such as phage display or ribosome display libraries. Phage display is described, for example, in the sources: Ladner et al., US patent No. 5223409; Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991).

In addition to using display libraries, the relevant antigen can be used to immunize a non-human animal, such as a rodent (such as a mouse, hamster, rabbit, or rat). In one embodiment, said non-human animal contains at least a portion of a human immunoglobulin gene. For example, it is possible to construct mouse strains deficient in the production of mouse antibodies with large fragments of human Ig (immunoglobulin) loci. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with the desired specificity can be generated and selected. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US 20030070185, WO 96/34096 and WO 96/33735.

The monoclonal antibody can also be derived from a non-human animal and then modified, eg, humanized, deimmunized, chimerized, and the like, using recombinant DNA techniques known in the art. Examples of modified antibody constructs include humanized variants of non-human antibodies, affinity matured antibodies (see, e.g., Hawkins et al., J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)) and mutant antibodies with altered effector function(s) (see, for example, US Pat. No. 5,648,260, Kontermann and Dubel (2010), supra, and Little (2009), supra).

In immunology, affinity maturation is known as the process by which B cells produce antibodies with increased affinity for an antigen during an immune response. Repeated exposures to the same antigen in the host produce antibodies with successively increasing affinities. As with the natural prototype, in vitro affinity maturation is based on the principles of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody constructs, and antibody fragments. Random mutations in the CDR region are introduced using radiation, chemical mutagens, or error-prone PCR. In addition, genetic diversity can be increased through strand shuffling. Two or three rounds of mutation and selection using display techniques such as phage display typically produce antibody fragments with affinities in the lower nanomolar range.

- 5 040387

A preferred type of variation in antibody constructs resulting from amino acid substitutions involves substitution of one or more hypervariable region residues of the parent antibody (eg, a humanized or human antibody). Typically, the final variant/variants selected for further development have improved biological properties relative to the original antibody from which they are derived. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, multiple hypervariable region sites (eg, 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus obtained are presented on filamentous phage particles in monovalent form and fused with the gene III product of the M13 phage, packaged in each particle. The biological activity of the phage display variants (eg, binding affinity) is then screened as described herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. In an alternative or additional embodiment, it may be advantageous to perform a crystal structure analysis of the antigen-antibody complex to identify points of contact between the binding domain and, for example, human FLT3. Such contact residues and adjacent residues are candidates for replacement according to the techniques of the present invention. Once such variants are obtained, a panel of variants is screened as described herein, and antibodies with the best performance as assessed in one or more relevant assays can be selected for further development.

The monoclonal antibodies and antibody constructs of the present invention particularly include chimeric antibodies (immunoglobulins) in which part of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, and the remainder part of the chain(s) is (are) identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they demonstrate the required biological activity (US patent No. 4816567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). As used herein, chimeric antibodies of interest include primatized antibodies comprising the variable domain of antigen-binding sequences derived from a non-human primate (eg, Old World monkeys, great apes, etc.) and human constant region sequences. Various methods for producing chimeric antibodies have been described. See, for example, Morrison et al., Proc. Natl. Acad. ScL U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., US Pat. No. 4,816,567; Boss et al., US patent No. 4816397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

An antibody, antibody construct, antibody fragment, or antibody variant may also be modified by specifically removing human T cell epitopes (a technique referred to as deimmunization) using the methods described, for example, in WO 98/52976 or WO 00/34317. Briefly, the variable domains of the heavy and light chains of an antibody can be analyzed for the presence of peptides that bind to MHC class II; these peptides are potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). To detect potential T-cell epitopes, a computer modeling technique called peptide stringing can be used; in addition, a database of human MHC class II binding peptides can be searched for motifs present in VH and VL sequences as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus represent potential T-cell epitopes. Detected potential T-cell epitopes can be eliminated by substitution of a small number of amino acid residues in the variable domains, or, preferably, by single amino acid substitutions. As a rule, conservative substitutions are made. Often, but not exclusively, an amino acid representative of a position in human germline antibody sequences can be used. Human germline sequences are described, for example, in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook, G.P. et al. (1995) Immunol. Today Vol. 16(5): 237-242; and Tomlinson et al. (1995) EMBO J. 14:14:4628-4638. The VBASE catalog is an extensive catalog of human immunoglobulin variable region sequences (compiled by: Tomlinson, LA. et al., MRC Center for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, such as framework regions and CDR regions. Human consensus frameworks can also be used, for example as described in US Pat. No. 6,300,064.

Humanized antibodies, antibody constructs, variants or fragments thereof (such as Fv, Fab, Fab', F(ab') ₂ or other antibody antigen-binding subsequences) are antibodies or immunoglobulins composed primarily of human sequences that contain (a) the minimum sequence(s) deriving(s) from a non-human

- 6 040387 immunoglobulin. In general, humanized antibodies are human immunoglobulins (recipient antibody) in which the hypervariable region (also CDR) residues of the recipient are replaced with residues from the hypervariable region (donor antibody) of a non-human species (e.g., rodent) such as mouse, rat, hamster or rabbit with the required specificity, affinity and potential. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibodies herein may also contain residues that are not found in either the recipient antibody or the donor antibody. These modifications are made to further refine and optimize the performance of the antibodies. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically a human immunoglobulin. More information can be found in: Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

Humanized antibodies or fragments thereof can be prepared by replacing Fv variable domain sequences that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Examples of methods for producing humanized antibodies or fragments thereof are provided in: Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214; and US 5585089; US 5693761; US 5693762; US 5859205; and US 6,407,213. These methods include the isolation, manipulation, and expression of nucleic acid sequences that encode, in whole or in part, immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids can be obtained from a hybridoma producing an antibody to a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into a suitable expression vector.

Humanized antibodies can also be generated using transgenic animals such as mice that express human heavy and light chain genes but are unable to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an example of a CDR region grafting method that can be used to produce humanized antibodies as described herein (US Patent No. 5,225,539). Any of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of said CDRs may be replaced with non-human CDRs. It is only necessary to change a certain number of CDR regions to ensure that the humanized antibody binds to the predetermined antigen.

A humanized antibody can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline sequence substitutions, and/or backmutations. Such altered immunoglobulin molecules can be prepared using any of a number of techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982, and EP 239400).

The term human antibody, human antibody construct, and human binding domain includes antibodies, antibody constructs, and binding domains containing antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including , such as those described by Kabat et al. (1991) (see above). Antibodies, antibody constructs, or human binding domains of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis, or by in vivo somatic mutation), for example, in CDR regions, and in particular in CDR3. In antibodies, antibody constructs, or human binding domains, at least one, two, three, four, five, or more positions may be substituted with an amino acid residue that is not encoded in the human germline immunoglobulin sequence. The definition of antibodies, antibody constructs, and human binding domains herein also encompasses wholly human antibodies, which include only non-artificially and/or genetically altered human antibody sequences, as such sequences can be generated using technologies or systems such as Xenomouse. .

In some embodiments, the antibody constructs of the present invention are isolated or substantially pure antibody constructs. Isolated or substantially pure, in relation to the description of antibody constructs as described herein, means an antibody construct that has been identified, separated from, and/or isolated from a component of its production environment. Preferably, said antibody construct is free or substantially free of bonds to all other components of its production environment. Contaminant components of the environment for its production, originating, for example, from recombinant transfections

- 7 040387 cells, are materials that generally interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones and other protein-like or non-protein-like solutes. The antibody constructs may, for example, comprise at least about 5%, or at least about 50%, by weight of the total protein content of a given sample. It is contemplated that the isolated protein may comprise from 5% to 99.9% by weight of the total protein content, as the case may be. Significantly higher concentrations of the polypeptide can be achieved by using an inducible promoter or a highly expressed promoter, so as to provide increased levels of said polypeptide upon production. The definition includes the production of an antibody construct in a wide range of organisms and/or host cells known in the art. In preferred embodiments, the antibody construct is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary beaker sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or preferably silver staining. Typically, however, an isolated antibody construct is obtained by at least one purification step.

The term binding domain in the context of the present invention characterizes a domain that (in particular) binds/interacts with a given target epitope or a given target site/recognizes a given target epitope or a given target site on target molecules (antigens), in this case: FLT3 and CD3, respectively. The structure and function of said first binding domain (FLT3 recognition) and preferably also the structure and/or function of the second binding domain (CD3 recognition) are based on the structure and/or function of an antibody, such as a full length or whole immunoglobulin molecule. In accordance with the present invention, the first binding domain is characterized by the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 from the VL region) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 from the VH region). The second binding domain preferably also satisfies the minimum structural requirements for an antibody that allows target binding. More preferably, the second binding domain comprises at least three light chain CDR regions (i.e. CDR1, CDR2 and CDR3 from the VL region) and/or three heavy chain CDR regions (i.e. CDR1, CDR2 and CDR3 from the VH- areas). It is envisaged to obtain or be able to obtain said first and/or second binding domains using phage display or library screening methods, rather than by grafting CDR sequences from an already existing (monoclonal) antibody onto a scaffold.

In accordance with the present invention, binding domains are in the form of one or more polypeptides. Such polypeptides may include protein-like portions and non-protein-like portions (eg, chemical linkers or chemical crosslinkers such as glutaraldehyde). Proteins (including their fragments, preferably biologically active fragments, and peptides, usually containing less than 30 amino acids) contain two or more amino acids linked together by a covalent peptide bond (to form a chain of amino acids). The term polypeptide as used herein describes a group of molecules that typically consists of more than 30 amino acids. Polypeptides can also form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, and the like may or may not be identical. The corresponding higher order structures of such multimers are therefore referred to as homo- or hetero-dimers, homo- or hetero-trimers, and the like. An example of a heteromultimer is an antibody molecule which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms peptide, polypeptide and protein also refer to naturally modified peptides/polypeptides/proteins, said modification being carried out, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, and the like. The peptide, polypeptide or protein herein may also be chemically modified, such as pegylated. Such modifications are well known in the art and are described herein below.

Preferably, the binding domain that binds to FLT3 and/or the binding domain that binds to CD3 is(are) human binding domains. Antibodies and antibody constructs containing at least one human binding domain avoid some of the problems associated with antibodies or antibody constructs containing non-human variable and/or constant regions, e.g., rodent (e.g., mouse, rat, hamster, or a rabbit). The presence of such rodent-derived proteins may result in the rapid clearance of antibodies or antibody constructs, or may result in the generation of an immune response against said antibody or antibody construct in a patient. To avoid the use of rodent-derived antibodies or antibody constructs, human or wholly human antibodies/antibody constructs can be generated by introducing human antibody function elements to a rodent such that said rodent produces wholly human antibodies.

The ability to clone and reverse engineer human loci several megabases in YAC chromosomes and introduce them into the mouse germ line provides an efficient way to determine the functional components of very large or roughly mapped loci and to generate useful models of human disease. In addition, the use of this technology to replace mouse loci with their human equivalents may provide unique information for understanding the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

An important practical application of such a strategy is represented by the humanization of the mouse humoral immune system. Administration of human immunoglobulin (Ig) loci to mice in which endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying programmed antibody expression and assembly, as well as their role in B cell development. In addition, such a strategy may provide an ideal source for the production of wholly human monoclonal antibodies (mAbs), an important milestone towards realizing the promise of antibody therapy in human diseases. Fully human antibodies or antibody constructs are expected to minimize the immunogenic and allergic reactions associated with mouse mAbs or derivatized mouse mAbs and thus increase the efficacy and safety of administered antibodies/antibody constructs. The use of wholly human antibodies or antibody constructs can be expected to provide a significant advantage in the treatment of chronic and recurrent human diseases such as inflammation, autoimmunity, and cancers that require repeated administration of the compounds.

One way to achieve this goal was to construct lines of mice deficient in the production of mouse antibodies with large fragments of human Ig loci; presumably such mice will produce a large repertoire of human antibodies in the absence of mouse antibodies. Large fragments of human Ig ensure the maintenance of a significant diversity of variable genes, as well as proper regulation of the production and expression of antibodies. Using the mechanisms of diversity and selection of antibodies in mice and lack of immunological tolerance to human proteins, the repertoire of human antibodies reproduced by these lines of mice should produce high affinity antibodies against any antigen of interest, including against human antigens. Using hybridoma technology, antigen-specific human monoclonal antibodies (mAbs) with the desired specificity can be easily generated and selected. This general strategy has been demonstrated in the production of the first strains of XenoMouse mice (see Green et al. Nature Genetics 7:13-21 (1994)). XenoMouse lines were constructed using yeast artificial chromosomes (YACs) containing fragments in the germline configuration of 245 kb and 190 kb of the human heavy chain locus and the human kappa light chain locus, respectively, which contained core variable and constant region sequences. These human Ig-containing YACs were found to be compatible with both the antibody rearrangement and expression system in mice, and were able to replace inactivated mouse Ig genes, as indicated by their ability to induce B cell development to produce a fully human antibody repertoire similar to that of an adult. human and the formation of antigen-specific human mAbs. These results also suggested that the introduction of larger portions of human Ig loci containing more V genes, additional regulatory elements, and human Ig constant regions could reproduce a substantially complete repertoire characteristic of the human humoral response to infection and immunization. Recently, Green et al's study was expanded to examine the introduction of more than about 80% of the human antibody repertoire by introducing, germline-configured YAC fragments with a few Mb of human heavy chain and kappa light chain loci, respectively. See Mendez et al. Nature Genetics 15:146-156 (1997) and US Patent Application Ser. No. 08/759620.

The production of XenoMouse mice is further discussed and described in US Patent Applications Ser. No. 07/466008; ser. No. 07/610515; ser. No. 07/919297; ser. No. 07/922649; ser. No. 08/031801; ser. No. 08/112848; ser. No. 08/234145; ser. No. 08/376279; ser. No. 08/430938; ser. No. 08/464584; ser. No. 08/464582; serial no. 08/463191; ser. No. 08/462837; ser. No. 08/486853; ser. No. 08/486857; ser. No. 08/486859; ser. No. 08/462513; ser. No. 08/724752; and ser. No. 08/759620; U.S. Patent Nos. 6,162,963; 6150584; 6114598; 6075181 and 5939598, and Japanese Patent No. 3068180 B2, No. 3068506 B2 and No. 3068507 B2. See also sources: Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and WO 03/47336.

In an alternative approach, others, including GenPharm International, Inc., have used the minilocus approach. In the miniloci method, an exogenous Ig locus is mimicked by incorporating fragments (individual genes) from the Ig locus. Accordingly, one or more VH genes, one or more DH genes, one or more JH genes, a mu chain constant region, and a second constant region (preferably a gamma chain constant region) form a construct for insertion into an animal body. This approach is described in U.S. Pat. No. 5,545,807 issued to Surani et al.

- 9 040387 US tax no. 5545806; 5625825; 5625126; 5633425; 5661016; 5770429; 5789650; 5814318; 5877397; 5,874,299 and 6,255,458 issued to Lonberg and Kay in US Patent Nos. 5,591,669 and 6,023,010 to Krimpenfort; and US Pat. No. 5,612,205; No. 5721367 and No. 5789215 issued by Berns et al.; U.S. Patent No. 5,643,763 to Choi and Dunn and GenPharm International U.S. Patent Application Ser. No. 07/574748, Ser. No. 07/575962, Ser. No. 07/810279, Ser. No. 07/853408, Ser. No. 07/904068, Ser. No. 07/990860, Ser. No. 08/053131, Ser. No. 08/096762, Ser. No. 08/155301, Ser. No. 08/161739, Ser. No. 08/165699, Ser. No. 08/209741. See also EP 0546073 B1, WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/ 13852 and WO 98/24884 and US Pat. No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994) and Tuaillon et al. (1995), Fishwild et al. (1996).

Kirin has also been shown to generate human antibodies from mice injected with microcell-mediated fusion of large fragments of chromosomes or entire chromosomes. See European Patent Applications No. 773288 and No. 843961. Xenerex Biosciences is developing technology that has the potential to generate human antibodies. In this technology, SCID mice are injected with human lymphatic cells, such as B and/or T cells, for replenishment. Mice are then immunized with an antigen and may develop an immune response against said antigen. See US Pat. Nos. 5,476,996; No. 5698767 and No. 5958765.

Human anti-mouse (HAMA) mediated responses have led to industry-leading chimeric or otherwise humanized antibodies. It is expected, however, that certain human anti-chimeric antibodies (HACA) mediated responses will be observed, particularly with chronic or repeated dosing of said antibody. Accordingly, it is desirable to provide antibody constructs containing a human anti-FLT3 binding domain and a human anti-CD3 binding domain in order to overcome the problems and/or effects of a HAMA or HACA mediated response.

The terms (particularly) binds to, (particularly) recognizes, (particularly) targets and (particularly) reacts with mean, in accordance with the present invention, that the binding domain interacts or interacts specifically with a given epitope or a given target site on target molecules (antigens), in this case, FLT3 and CD3, respectively.

The term epitope refers to a site on an antigen to which a binding domain specifically binds, such as an antibody or immunoglobulin, or a derivative, fragment or variant of an antibody or immunoglobulin. An epitope is antigenic and, accordingly, an epitope is sometimes also referred to herein as an antigenic structure or an antigenic determinant. Accordingly, the binding domain is the site of interaction with the antigen. It is also believed that the specified binding/interaction determines the specific recognition.

Epitopes can be formed either by a continuous sequence of amino acids or by a non-continuous sequence of amino acids brought together by the tertiary folding of the protein. A linear epitope is an epitope whose primary amino acid sequence contains a recognizable epitope. A linear epitope typically includes at least 3 or at least 4 amino acids, and more often at least 5 or at least 6 or at least 7 amino acids, for example, from about 8 to about 10 amino acids arranged in unique sequence.

A conformational epitope, as opposed to a linear epitope, is an epitope characterized in that the primary amino acid sequence that makes up said epitope is not the only defining recognizable component of the epitope (e.g., an epitope characterized in that its primary amino acid sequence is not necessarily recognized by the binding domain ). Typically, a conformational epitope contains more amino acids than a linear epitope. When recognizing conformational epitopes, the binding domain recognizes the three-dimensional structure of the antigen, preferably a peptide or protein, or fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is located in the FLT3 protein). For example, when a protein molecule is folded into a three-dimensional structure, certain amino acids and/or the polypeptide backbone forming a conformational epitope are brought close together, allowing the antibody to recognize said epitope. Methods for determining the conformation of epitopes include, but are not limited to, X-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and site-directed spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

The epitope mapping method is described below. In the event that a region (contiguous amino acid stretch) of the human FLT3 protein is/replaced with a corresponding region of a non-human, non-primate FLT3 antigen (e.g., mouse FLT3, but other FLT3s, e.g., chicken, rat, hamster, rabbit, etc.), it is expected that there will be a decrease in the binding domain, unless said binding domain is cross-reactive with the non-human, non-primate FLT3 used. Said reduction is preferably at least

- 10 040387 re 10, 20, 30, 40 or 50%; more preferably at least 60%, 70% or 80% and most preferably 90%, 95% or even 100% compared to binding to the corresponding region in the human FLT3 protein, assuming 100% binding to the corresponding region in the human FLT3 protein. Expression of the aforementioned human/non-human FLT3 FLT3 chimeras in CHO cells is contemplated. It is also contemplated to fuse human/non-human FLT3 FLT3 chimeras to the transmembrane domain and/or cytoplasmic domain of another membrane-bound protein such as EpCAM, see Examples 1 and 2.

In an alternative or additional epitope mapping method, multiple truncated versions of the human FLT3 extracellular domain can be generated to determine the specific region that is recognized by the binding domain. In these truncated versions, different extracellular domains/subdomains or regions of FLT3 are removed stepwise, starting from the N-terminus. Truncated versions of FLT3 are shown, which were obtained and used in the context of the present invention. It is possible to express truncated versions of FLT3 in CHO cells. It is also contemplated that truncated versions of FLT3 can be fused to the transmembrane domain and/or cytoplasmic domain of another membrane-bound protein, such as EpCAM. It is also contemplated that truncated versions of FLT3 may include a signal peptide domain at the N-terminus, for example, a signal peptide derived from a mouse IgG heavy chain signal peptide. It is also possible to include in the truncated versions of FLT3 the v5 domain at the N-terminus (after the signal peptide), which allows to verify their correct expression on the cell surface, it is expected that in this case there will be a decrease or loss of binding of those truncated versions of FLT3 that no longer include the FLT3 region recognized by the binding domain. Said reduction in binding is preferably at least 10%, 20%, 30%, 40%, 50%; more preferably at least 60%, 70%, 80% and most preferably 90%, 95% or even 100%, assuming that the binding of the total human FLT3 protein (or its extracellular region or domain) is 100%, see Example 3.

An additional method for determining the contribution of a specific target antigen residue to recognition by an antibody construct or binding domain is by alanine scanning (see, for example, Morrison KL & Weiss GA. Cur Opin Chem Biol. 2001 Jun; 5(3):302-7), in which each residue to be analyzed is replaced with an alanine, for example by site-directed mutagenesis. Alanine is used because of the presence of a small, chemically inert methyl functional group, which nevertheless mimics the secondary structure template found in many other amino acids. Occasionally, bulky amino acids, such as valine or leucine, may be used where size conservation of mutated residues is required. Alanine scanning is an established technology that has been used for a long period of time.

The interaction of a binding domain and an epitope or region containing an epitope implies that the binding domain exhibits measurable affinity for the specified epitope/region containing the specified epitope on a particular protein or antigen (in this case, FLT3 and CD3, respectively) and usually does not show significant reactivity for proteins or antigens other than FLT3 or CD3. Measurable affinity includes binding with an affinity of approximately 10-6 M (kDa) or greater. Preferably, a binding ^is considered specific if the ^binding affinity is about ^10-12-10-8 M, ^10-12-10-9 M, ^{10-12-10-10 M} , ^10-11-10-8 M, preferably about 10- ^11-10-9 M. Testing for specific binding or reaction of a binding domain to a target can easily be carried out, including by comparing the reaction of said binding domain with a target protein or antigen, and the reaction of said binding domain with proteins or antigens other than FLT3 or CD3. Preferably, the binding domain of the present invention does not substantially bind to proteins or antigens other than FLT3 or CD3 (i.e., the first binding domain is unable to bind to proteins other than FLT3 and the second binding domain is unable to bind to proteins other than from CD3).

The term substantially does not bind or is unable to bind means that the binding domain of the present invention does not bind a protein or antigen other than FLT3 or CD3, i. does not show a reactivity of more than 30%, and preferably shows a reactivity of no more than 20%, more preferably no more than 10%, in particular preferably no more than 9%, 8%, 7%, 6% or 5% with respect to proteins or antigens other than FLT3 or CD3, assuming that the binding of FLT3 or CD3, respectively, is 100%.

It is believed that specific binding is mediated by specific motifs in the amino acid sequence of the binding domain and antigen. Accordingly, the achievement of binding is due to their primary, secondary and/or tertiary structure, as well as secondary modifications of these structures. The specific interaction of an antigen interaction site with its corresponding specific antigen may result in a simple binding of said site to said antigen. In addition, the specific interaction of the site of interaction with the antigen with

- 11 040387 corresponding specific antigen can, according to an alternative or additional option, lead to the initiation of a signal, for example, due to the induction of a change in the conformation of the antigen, oligomerization of the antigen, and the like.

According to another aspect of the present invention, a bispecific antibody construct is provided comprising a first binding domain that binds to human FLT3 on the surface of a target cell and a second binding domain that binds to human CD3 on the surface of a T cell, said first binding domain binding to an FLT3 epitope, which is located in the human FLT3 region having the sequence shown in SEQ ID NO: 814 (cluster 1) or SEQ ID NO: 816 (cluster 3).

Preferably, the first binding domain of said construct of the bispecific antibody of the present invention comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from group consisting of the sequences SEQ ID NO: 151-156, SEQ ID NO: 161-166, SEQ ID NO: 171-176, SEQ ID NO: 181-186, SEQ ID NO: 191-196, SEQ ID NO: 201 -206, SEQ ID NO: 211-216, SEQ ID NO: 221-226, SEQ ID NO: 231-236, SEQ ID NO: 241-246, SEQ ID NO: 251-256, SEQ ID NO: 261-266 , SEQ ID NO: 271-276, SEQ ID NO: 281-286, SEQ ID NO: 291-296, SEQ ID NO: 301-306, SEQ ID NO: 311-316, SEQ ID NO: 321-326, SEQ ID NO: 331336, SEQ ID NO: 341-346, SEQ ID NO: 351-356, SEQ ID NO: 361-366, SEQ ID NO: 371-376, SEQ ID NO: 381-386, SEQ ID NO: 391 -396, SEQ ID NO: 401-406, SEQ ID NO: 411-416, SEQ ID NO: 421-426, SEQ ID NO: 431-436, SEQ ID NO: 441-446, SEQ ID NO: 451-456 , SEQ ID NO: 461-466, SEQ ID NO: 471-476, SEQ ID NO: 481-486, SEQ ID NO: 491-496, SEQ ID NO: 501-506 , SEQ ID NO: 511-516, SEQ ID NO: 521-526, SEQ ID NO: 531-536, SEQ ID NO: 541-546, SEQ ID NO: 551-556, SEQ ID NO: 561-566, SEQ ID NO: 571576, SEQ ID NO: 581-586, SEQ ID NO: 591-596, SEQ ID NO: 601-606, SEQ ID NO: 611-616, SEQ ID NO: 621-626, SEQ ID NO: 631 -636, SEQ ID NO: 641-646, SEQ ID NO: 651-656, SEQ ID NO: 661-666, SEQ ID NO: 671-676, SEQ ID NO: 681-686, SEQ ID NO: 691-696 , SEQ ID NO: 701-706, SEQ ID NO: 711-716, SEQ ID NO: 721-726, SEQ ID NO: 731-736, SEQ ID NO: 741-746, SEQ ID NO: 751-756, SEQ ID NO: 761-766, SEQ ID NO: 771-776, SEQ ID NO: 781-786, SEQ ID NO: 791-796.

The term variable refers to portions of the domains of an antibody or immunoglobulin that exhibit sequence variability and are involved in determining the specificity and binding affinity (affinity) of a particular antibody (i.e., the variable domain(s)). When the heavy chain variable region (VH) and the light chain variable region (VL) are paired, a single antigen-binding site is formed.

Variability is unevenly distributed across the variable domains of antibodies; it is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are referred to as hypervariable regions or complementarity determining regions (CDRs). The more conserved (ie, non-hypervariable) portions of variable domains are called framework regions (FRMs, or FRs); they provide a three-dimensional scaffold for the six CDRs to form an antigen-binding surface. Each of the variable domains of naturally occurring heavy and light chains contains four FRM regions (FR1, FR2, FR3, and FR4) primarily adopting a β-sheet configuration connected by three hypervariable regions that form loops connecting β-sheet structures, and in in some cases included in them. The hypervariable regions of each chain are held in close proximity to each other by the FRM region and, together with the hypervariable regions of the other chain, contribute to the formation of an antigen binding site (see Kabat et al., supra).

The terms CDR (CDR region) and, in the plural, CDR regions, refer to complementarity determining regions, of which three determine the binding pattern of the light chain variable region (CDR-L1, CDR-L2 and CDR-L3), and three more determine the binding pattern of the light chain variable region. heavy chain variable region binding (CDR-H1, CDR-H2 and CDR-H3). The CDR regions contain most of the residues responsible for specific antibody-antigen interactions and thus contribute to the functional activity of the antibody molecule: they are the main determinant of antigen specificity.

Different classification and numbering systems can be used to accurately define the boundaries and length of a CDR. CDRs may accordingly be named according to definitions by Kabat, Chothia, contact or any other boundary, including according to the numbering system as described herein. Despite the differences in the definition of boundaries, all of these systems to some extent agree with respect to the so-called hypervariable regions within the variable sequences. The CDRs defined in accordance with said systems may, respectively, differ in length and area of boundaries with an adjacent frame region. See, for example, Kabat (cross-species sequence variability approach), Chothia (antigen-antibody complex crystallographic approach), and/or MacCallum (Kabat et al., supra; Chothia et al., J. Mol. Biol, 1987, 196:901-917 and MacCallum et al., J. Mol Biol, 1996, 262:732). Another standard for characterizing antigen binding sites is the AbM definition used in the AbM antibody modeling software from Oxford Molecular. See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Rontermann, R., Springer-Verlag, Heidelberg). Where overlapping but not identical regions are identified by the two residue identification techniques, they can be combined to determine a hybrid CDR. However, numbering according to the so-called Kabat system is preferred.

Typically, the CDRs form a loop-like structure, which can be classified as a canonical structure. The term canonical structure refers to the basic chain conformation adopted by the antigen-binding (CDR) loops. Through comparative structural studies, five of the six antigen-binding loops were found to have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Corresponding loops between antibodies can therefore have very similar three-dimensional structures despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989 , 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800). In addition, there is a relationship between the adopted loop structure and its surrounding amino acid sequences.

The conformation for a particular canonical class is determined by the length of the loop and the amino acid residues found at key positions within the loop as well as within the conserved framework (ie, outside the loop). Membership in a particular canonical class can, accordingly, be established based on the presence of said key amino acid residues.

The term canonical structure may also take into account features such as the linear sequence of an antibody, eg as cataloged by Kabat (Kabat et al., supra). The Kabat numbering scheme (system) is a widely accepted standard for uniform numbering of amino acid residues in the variable domain of an antibody and is the preferred scheme for use in the present invention, as further indicated elsewhere in this document. In determining the canonical structure of an antibody, additional structural features may also be taken into account. For example, differences not fully reflected in the Kabat numbering can be described in the numbering system of Chothia et al. and/or identified using other techniques such as crystallography and 2D or 3D numerical simulations. Accordingly, a given antibody sequence can be assigned to a canonical class, which allows, inter alia, the identification of suitable reference sequences (eg, based on the desire to include a plurality of canonical structures in the library). The amino acid sequence numbering of antibodies according to Kabat and structural features as described by Chothia et al., see above, as well as their significance for the interpretation of the canonical aspects of the structure of antibodies, are described in published sources. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known in the art. See Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. for an overview of the structure of antibodies. Harlow et al., 1988.

The light chain CDR3 region, and in particular the heavy chain CDR3 region, may be the most important determinants in antigen binding within the light and heavy chain variable regions. In some antibody constructs, the heavy chain CDR3 appears to be the primary site of contact between antigen and antibody. In vitro selection schemes in which variations are present only in CDR3 can be used to obtain varying antibody binding properties, or to determine which residues contribute to antigen binding. Therefore, as a rule, CDR3 is the most significant source of molecular diversity within the binding site of an antibody. For example, the length of H3 may be as little as two amino acid residues, or may be greater than 26 amino acids.

In a classic full length antibody or immunoglobulin, each light (L) chain is linked to the heavy (H) chain by a single covalent disulfide bond, and the two H chains are linked together by one or more disulfide bonds, depending on the isotype of the H chains. The CH domain closest to the VH is usually referred to as CH1. The constant (C) domains are not directly involved in antigen binding, but exhibit various effector functions such as antibody-dependent cell-mediated cytotoxicity and complement activation. The Fc region of an antibody is located in the heavy chain constant domains and is capable, for example, of interacting with Fc receptors located on the cell surface.

The sequence of antibody genes after assembly and somatic mutation varies greatly, and these variable genes encode, according to estimates, 10 ¹⁰ different antibody molecules (Immunoglobulin Genes, ^2nd ed., eds. Jonio et al., Academic Press, San Diego, CA , 1995). Accordingly, the immune system provides a repertoire of immunoglobulins. The term repertoire refers to at least one nucleotide sequence derived in whole or in part from at least one sequence encoding at least one immunoglobulin. Specified

- 13 040387 sequence or sequences can be obtained by in vivo rearrangement of V-, D- and J-segments of heavy chains, and V- and J-segments of light chains. Alternatively, the specified sequence or sequences can be obtained from the cell in response to which the rearrangement occurs, for example, during in vitro stimulation. Alternatively, a portion of said sequence or sequences, or the entirety of said sequence or sequences, may be obtained using DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, for example, US Pat. No. 5,565,332. sequences, including in the form of a genetically heterogeneous set.

A preferred antibody construct according to the present invention can also be defined as a bispecific antibody construct comprising a first (preferably human) binding domain that binds to human FLT3 on the surface of the target cell and a second binding domain that binds to human CD3 on the surface of the T cell. , wherein said first binding domain binds to the same FLT3 epitope as an antibody selected from the group consisting of FL-1 to FL-65, ie. an antibody containing a VH region containing CDR-H1, CDR-H2 and CDR-H3, and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of the sequences of SEQ ID NO: 151 -156, SEQ ID NO: 161-166, SEQ ID NO: 171-176, SEQ ID NO: 181-186, SEQ ID NO: 191-196, SEQ ID NO: 201-206, SEQ ID NO: 211-216 , SEQ ID NO: 221-226, SEQ ID NO: 231-236, SEQ ID NO: 241-246, SEQ ID NO: 251-256, SEQ ID NO: 261266, SEQ ID NO: 271-276, SEQ ID NO : 281-286, SEQ ID NO: 291-296, SEQ ID NO: 301-306, SEQ ID NO: 311-316, SEQ ID NO: 321-326, SEQ ID NO: 331-336, SEQ ID NO: 341 -346, SEQ ID NO: 351-356, SEQ ID NO: 361-366, SEQ ID NO: 371-376, SEQ ID NO: 381-386, SEQ ID NO: 391-396, SEQ ID NO: 401-406 , SEQ ID NO: 411-416, SEQ ID NO: 421-426, SEQ ID NO: 431-436, SEQ ID NO: 441-446, SEQ ID NO: 451-456, SEQ ID NO: 461-466, SEQ ID NO: 471-476, SEQ ID NO: 481-486, SEQ ID NO: 491-496, SEQ ID NO: 501506, SEQ ID NO: 511-516, SEQ ID NO: 521-526, SEQ ID NO: 531 -536, SEQ ID NO: 541-546, SEQ ID NO: 551-556, SEQ ID NO : 561-566, SEQ ID NO: 571-576, SEQ ID NO: 581-586, SEQ ID NO: 591-596, SEQ ID NO: 601-606, SEQ ID NO: 611-616, SEQ ID NO: 621 -626, SEQ ID NO: 631-636, SEQ ID NO: 641-646, SEQ ID NO: 651-656, SEQ ID NO: 661-666, SEQ ID NO: 671-676, SEQ ID NO: 681-686 , SEQ ID NO: 691-696, SEQ ID NO: 701-706, SEQ ID NO: 711-716, SEQ ID NO: 721-726, SEQ ID NO: 731-736, SEQ ID NO: 741746, SEQ ID NO : 751-756, SEQ ID NO: 761-766, SEQ ID NO: 771-776, SEQ ID NO: 781-786, SEQ ID NO: 791-796.

Assessing whether an antibody construct binds to the same epitope on FLT3 as another given antibody construct can be done, for example, by mapping epitopes to chimeric or truncated target molecules, for example, as described herein above and in the accompanying examples.

A preferred antibody construct according to the present invention may also be defined as a bispecific antibody construct comprising a first binding domain (preferably a human domain) that binds to human FLT3 on the surface of the target cell and a second binding domain that binds to human CD3 on the surface. T cells, wherein said first binding domain competes for binding with an antibody selected from the group consisting of FL-1 - FL-65, ie. an antibody containing a VH region containing CDR-H1, CDRH2 and CDR-H3, and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of the sequences described above.

An assessment of whether an antibody construct competes for binding with another given antibody construct may be performed in a competitive assay, such as a competitive ELISA or cellular competition assay. Avidinized microparticles (granules) may also be used. Similar to the avidin-coated ELISA plate, when reacted with a biotinylated protein, each of these beads can be used as a substrate for the assay. The antigen is applied to the bead and then precoated with the first antibody. A second antibody is added and any additional binding determined. Possible recording methods include flow cytometry.

In one embodiment, the first binding domain of said antibody construct of the present invention comprises a VH region selected from the group consisting of the sequences shown in SEQ ID NO: 157, SEQ ID NO: 167, SEQ ID

NO: 177, SEQ ID NO

NO: 237, SEQ ID NO

NO: 297, SEQ ID NO

NO: 357, SEQ ID NO

NO: 417, SEQ ID NO

NO: 477, SEQ ID NO

NO: 537, SEQ ID NO

NO: 597, SEQ ID NO

NO: 657, SEQ ID NO

187 SEQ ID NO

247 SEQ ID NO

307 SEQ ID NO

367 SEQ ID NO

427 SEQ ID NO

487 SEQ ID NO

547 SEQ ID NO

607 SEQ ID NO

667 SEQ ID NO

197, SEQ ID NO 257, SEQ ID NO 317, SEQ ID NO 377, SEQ ID NO 437, SEQ ID NO 497, SEQ ID NO 557, SEQ ID NO 617, SEQ ID NO 677, SEQ ID NO

207 SEQ ID NO

267 SEQ ID NO

327 SEQ ID NO

387 SEQ ID NO

447 SEQ ID NO

507 SEQ ID NO

567 SEQ ID NO

627 SEQ ID NO

687 SEQ ID NO

217 SEQ ID NO

277 SEQ ID NO

337 SEQ ID NO

397 SEQ ID NO

457 SEQ ID NO

517 SEQ ID NO

577 SEQ ID NO

637 SEQ ID NO

697 SEQ ID NO

227 SEQ ID

287 SEQ ID

347 SEQ ID

407 SEQ ID

467 SEQ ID

527 SEQ ID

587 SEQ ID

647 SEQ ID

707 SEQ ID

- 14 040387

NO: 717, SEQ ID NO: 727, SEQ ID NO: 737, SEQ ID NO: 747, SEQ ID NO: 757, SEQ ID NO: 767, SEQ ID

NO: 777, SEQ ID NO: 787 and SEQ ID NO: 797.

In a further embodiment, the first binding domain of said antibody construct of the present invention comprises a VL region selected from the group consisting of the sequences shown in SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 178, SEQ ID NO : 188, SEQ ID NO: 198, SEQ ID NO: 208, SEQ ID NO: 218, SEQ ID NO: 228,

SEQ ID NO: 238, SEQ ID NO: 248, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 278, SEQ ID NO: 288,

SEQ ID NO: 298, SEQ ID NO: 308, SEQ ID NO: 318, SEQ ID NO: 328, SEQ ID NO: 338, SEQ ID NO: 348,

SEQ ID NO: 358, SEQ ID NO: 368, SEQ ID NO: 378, SEQ ID NO: 388, SEQ ID NO: 398, SEQ ID NO: 408,

SEQ ID NO: 418, SEQ ID NO: 428, SEQ ID NO: 438, SEQ ID NO: 448, SEQ ID NO: 458, SEQ ID NO: 468,

SEQ ID NO: 478, SEQ ID NO: 488, SEQ ID NO: 498, SEQ ID NO: 508, SEQ ID NO: 518, SEQ ID NO: 528,

SEQ ID NO: 538, SEQ ID NO: 548, SEQ ID NO: 558, SEQ ID NO: 568, SEQ ID NO: 578, SEQ ID NO: 588,

SEQ ID NO: 598, SEQ ID NO: 608, SEQ ID NO: 618, SEQ ID NO: 628, SEQ ID NO: 638, SEQ ID NO: 648,

SEQ ID NO: 658, SEQ ID NO: 668, SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 698, SEQ ID NO: 708,

SEQ ID NO: 718, SEQ ID NO: 728, SEQ ID NO: 738, SEQ ID NO: 748, SEQ ID NO: 758, SEQ ID NO: 768,

SEQ ID NO: 778, SEQ ID NO: 788 and SEQ ID NO: 798.

In another embodiment, the first binding domain of said antibody construct of the present invention comprises a VH region and a VL region selected from the group consisting of pairs of VH regions and VL regions shown in SEQ ID NOs: 157+158, SEQ ID NO: 167+168, SEQ ID NO: 177+178, SEQ ID NO: 187+188, SEQ ID NO: 197+198, SEQ ID NO: 207+208, SEQ ID NO: 217+218, SEQ ID NO: 227+228, SEQ ID NO: 237+238, SEQ ID NO: 247+248, SEQ ID NO: 257+258, SEQ ID NO: 267+268, SEQ ID NO: 277+278, SEQ ID NO: 287+ 288, SEQ ID NO: 297+298, SEQ ID NO: 307+308, SEQ ID NO: 317+318, SEQ ID NO: 327+328, SEQ ID NO: 337+338, SEQ ID NO: 347+348, SEQ ID NO: 357+358, SEQ ID NO: 367+368, SEQ ID NO: 377+378, SEQ ID NO: 387+388, SEQ ID NO: 397+398, SEQ ID NO: 407+408, SEQ ID NO: 417+418, SEQ ID NO: 427+428, SEQ ID NO: 437+438, SEQ ID NO: 447+448, SEQ ID NO: 457+458, SEQ ID NO: 467+468, SEQ ID NO: 477+478, SEQ ID NO: 487+488, SEQ ID NO: 497+498, SEQ ID NO: 507+508, SEQ ID NO: 517+518, SEQ ID NO: 527+528, SEQ ID NO: 537+538, SEQ ID NO: 547+548, SEQ ID NO: 557+558, SEQ ID NO: 567+568, SEQ ID NO: 577+578, SEQ ID NO: 587+588, SEQ ID NO: 597+598, SEQ ID NO: 607+608, SEQ ID NO: 617+618, SEQ ID NO: 627+628, SEQ ID NO: 637+638, SEQ ID NO: 647+648, SEQ ID NO: 657+658, SEQ ID NO: 667+668, SEQ ID NO: 677+678, SEQ ID NO: 687+688, SEQ ID NO: 697+698, SEQ ID NO: 707+ 708, SEQ ID NO: 717+718, SEQ ID NO: 727+728, SEQ ID NO: 737+738, SEQ ID NO: 747+748, SEQ ID NO: 757+758, SEQ ID NO: 767+768, SEQ ID NO: 777+778, SEQ ID NO: 787+788 and SEQ ID NO: 797+798.

In another further embodiment, the first binding domain of said antibody construct of the present invention comprises a polypeptide selected from the group consisting of the sequences shown in SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO : 189, SEQ ID NO: 199, SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269 , SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO : 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789 and SEQ ID NO: 799.

The first binding domains described above (defined by their CDR, VH and VL regions, and combinations thereof) are characterized as binding domains that bind to an FLT3 epitope that is located in the region shown in SEQ ID NO: 819.

The term bispecific as used herein refers to an antibody construct that is at least bispecific, ie. contains at least a first binding domain and a second binding domain, characterized in that said first binding domain binds to one antigen or target (in this case, FLT3), and the second binding domain binds to another antigen or target (in this case, CD3 ). Accordingly, the antibody constructs of the present invention differ in specificity for at least two different antigens or targets. The term bispecific antibody construct of the present invention also encompasses multispecific antibody constructs, such as trispecific antibody constructs, the latter comprising three binding domains, or constructs differing in specificity for more than three (e.g., four, five...) targets.

Considering that the antibody constructs in accordance with the present invention are (according to

- 15 040387 least) bispecific, they do not occur in nature and differ markedly from naturally occurring products. A bispecific antibody or immunoglobulin construct, respectively, is an artificial hybrid antibody or immunoglobulin containing at least two separate binding sites with different specificities. The bispecific antibody constructs can be generated using a variety of methods, including hybridoma fusion or Fab' fragment fusion. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990).

The at least two binding domains and variable domains of an antibody construct of the present invention may or may not contain peptidic linkers (spacer peptides). The term peptide linker according to the present invention comprises an amino acid sequence by which the amino acid sequences of one (variable and/or binding) domain and another (variable and/or binding) domain of the indicated antibody construct according to the present invention are connected to each other. An essential technical feature of such a peptide linker is that it does not exhibit any polymerization activity. Suitable peptide linkers include those described in US Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. Peptide linkers can also be used to attach other domains or modules or regions (such as half-life increasing domains) to an antibody construct of the present invention.

If a linker is used, the length and sequence of said linker is preferably sufficient to ensure that each of said first and second domains can, independently of the other, retain its differential binding specificity. Peptide linkers connecting at least two binding domains (or two variable domains) in an antibody construct of the present invention preferably contain only a small number of amino acid residues, such as 12 amino acid residues or less. Accordingly, peptide linkers containing 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. Provided peptide linker containing less than 5 amino acids, contains 4, 3, 2 amino acids or one amino acid, while Gly-rich linkers are preferred. In particular, the preferred single amino acid in the context of said peptide linker is Gly. Suitably, said peptide linker may consist of a single Gly amino acid. Another preferred embodiment of the peptide linker is the amino acid sequence Gly-Gly-Gly-Gly-Ser, i. Gly ₄ Ser (SEQ ID NO: 1) or polymers thereof, i.e. (Gly ₄ Ser)x, where x is an integer of 1 or more (eg, 2 or 3). Preferred linkers are shown in SEQ ID NOs: 1-9. The characteristics of said peptide linker, including the lack of support for secondary structures, are known in the art and are described, for example, in Dall'Acqua et al. (Biochem. (1998) 37, 9266-9273), Cheadle et al. (Mol Immunol (1992) 29, 21-30), and Raag and Whitlow (FASEB (1995) 9(1), 73-80). Preferred are peptide linkers which, among other things, do not support the formation of any secondary structures. Linking these domains to each other can be achieved, for example, using genetic engineering, as described in the examples. Methods for preparing fused and operably linked bispecific single chain constructs and expressing them in mammalian or bacterial cells are well known in the art (see, for example, sources: WO 99/54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

As described herein above, the present invention provides a preferred embodiment wherein the format of said antibody construct is selected from the group consisting of (scFv) ₂ , scFv/single domain mAb, diabodies, and oligomers of any of the listed formats.

According to a particular preferred embodiment and as described in the accompanying examples, said antibody construct of the present invention is a bispecific single chain antibody construct, more preferably a bispecific single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH, encode separate genes, they can be combined using recombinant methods, through a synthetic linker - as described above in this document - which allows them to be produced as a single protein chain in which the VL and VH are paired to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). These antibody fragments are prepared using standard techniques known to those skilled in the art and the function of the fragments is evaluated in the same manner as the function of whole or full length antibodies. A single chain variable fragment (scFv) is thus a fusion protein comprising the immunoglobulin heavy chain (VH) and light chain (VL) variable regions, typically joined by a short linker peptide that is about 10 to about 25 amino acids in length, preferably about 15 to 20 amino acids. Said linker is typically rich in glycine for flexibility and serine or threonine for solubility, and may connect the N-terminus of the VH to the C-terminus of the VL, or vice versa. The specified protein retains the specificity of the original immunoglobulin, despite the removal of constant regions and the introduction of the linker.

Bispecific single chain molecules are known in the art and are described in WO 99/54440, Mack, J. Immunol. (1997), 158, 3965-3970, Mack, PNAS, (1995), 92, 7021-7025, Kufer, Cancer Immunol. Immunother., (1997), 45, 193-197, Loffler, Blood, (2000), 95, 6, 2098-2103, Bruhl, Immunol., (2001), 166, 2420-2426, Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. The described techniques for making single chain antibodies (see, inter alia, US Pat. (s) target(s).

Bivalent (also referred to as divalent) or bispecific single chain variable fragments (bi-scFv or di-scFv having the (scFv) ₂ format can be constructed by linking two scFv molecules (eg, with linkers as described above herein). In that case, if the two scFv molecules have the same binding specificity, the resulting (scFv) ₂ molecule is preferably said to be bivalent (i.e. has two valences for the same target epitope.) In the event that the two scFv molecules have different binding specificity, the final molecule (scFv) ₂ is preferably referred to as bispecific.This connection can be made by making a single peptide chain with two VH and two VL regions, producing tandem scFvs (see, for example, Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244) Another possibility is to generate scFv molecules with linker peptides too short to folding two variable regions (eg, approximately five amino acids long), which causes the scFv to dimerize. This type is known as a diabody (see, for example, Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90 (14): 6444-8.).

According to a further preferred embodiment of the antibody design of the present invention, the heavy chain (VH) and light chain (VL) of the binding domain that binds to the target FLT3 or CD3 antigen are not connected directly via the above-described peptide linker, but the binding domain is formed due to the formation of a bispecific molecules as described for the diabody. Accordingly, the VH chain of the CD3 binding domain can be fused to the VL chain of the FLT3 binding domain through such a peptide linker, and the VH chain of the FLT3 binding domain can be fused to the VL chain of the CD3 binding domain through such a peptide linker.

Single domain antibodies contain only one (monomeric) antibody variable domain that is capable of selectively binding to a specific antigen, independent of other V regions or domains. The first single domain antibodies were constructed from heavy chain antibodies found in camelids and are referred to as VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR) from which single domain antibodies called VNAR fragments can be generated. An alternative approach is to cleave dimeric variable domains from common immunoglobulins, eg human or rodent, into monomers, thus producing VH or VL as a single domain antibody. Although currently most studies of single domain antibodies are based on heavy chain variable domains, light chain-derived nanobodies have also been shown to specifically bind to target epitopes. Examples of single domain antibodies are referred to as sdAbs, nanobodies, or single variable domain single domain antibodies.

(Single domain mAb)2, respectively, is a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibodies that are individually selected from the group consisting of VH, VL, VHH and V _NAR . The linker is preferably in the form of a peptide linker. Similarly, a scFv/single domain mAb is a monoclonal antibody construct consisting of at least one single domain antibody as described above and one scFv molecule as described above. In this case, too, the linker is preferably in the form of a peptide linker.

In addition, according to the present invention, a bispecific antibody construct is provided, comprising a first binding domain that binds to human FLT3 on the surface of a target cell, and a second binding domain that binds to human CD3 on the surface of a T cell, characterized in that said first binding domain binds to an FLT3 epitope that is located in the region shown in SEQ ID NO: 819 (cluster 1).

Accordingly, according to a further aspect of the present invention, the first binding domain of said bispecific antibody construct comprises a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3, selected from the group consisting of the sequences SEQ ID NO: 151-156, SEQ ID NO: 161-166, SEQ ID NO: 171-176, SEQ ID NO: 181-186, SEQ ID NO: 191-196, SEQ ID NO : 201-206, SEQ ID NO: 211-216, SEQ ID NO: 221-226, SEQ ID NO: 231-236, SEQ ID NO: 241-246, SEQ ID NO: 251-256, SEQ ID NO: 261 -266, SEQ ID NO: 271-276, SEQ ID NO: 281-286, SEQ ID NO: 291-296, SEQ ID NO: 301-306, SEQ ID NO: 311-316, SEQ ID NO: 321-17 040387

326, SEQ ID NO: 331-336, SEQ ID NO: 341-346, SEQ ID NO: 351-356, SEQ ID NO: 361-366, SEQ ID NO: 371-376, SEQ ID NO: 381-386, SEQ ID NO: 391-396, SEQ ID NO: 401-406, SEQ ID NO: 411-416, SEQ ID NO: 421-426, SEQ ID NO: 431-436, SEQ ID NO: 441-446, SEQ ID NO: 451-456, SEQ ID NO: 461-466, SEQ ID NO: 471-476, SEQ ID NO: 481-486, SEQ ID NO: 491-496, SEQ ID NO: 501-506, SEQ ID NO: 511-516, SEQ ID NO: 521-526, SEQ ID NO: 531-536, SEQ ID NO: 541-546, SEQ ID NO: 551-556, SEQ ID NO: 561566, SEQ ID NO: 571-576, SEQ ID NO: 581-586, SEQ ID NO: 591-596, SEQ ID NO: 601-606, SEQ ID NO: 611-616, SEQ ID NO: 621-626, SEQ ID NO: 631-636, SEQ ID NO: 641-646, SEQ ID NO: 651-656, SEQ ID NO: 661-666, SEQ ID NO: 671-676, SEQ ID NO: 681-686, SEQ ID NO: 691-696, SEQ ID NO: 701-706, SEQ ID NO: 711-716, SEQ ID NO: 721-726, SEQ ID NO: 731-736, SEQ ID NO: 741-746, SEQ ID NO: 791-796.

In one embodiment, the first binding domain of said antibody construct of the present invention comprises a VH region selected from the group consisting of the sequences shown in the sequences of SEQ ID NO:

NO NO NO NO NO NO NO NO NO NO

177 SEQ ID NO

237 SEQ ID NO

297 SEQ ID NO

357 SEQ ID NO

417 SEQ ID NO

477 SEQ ID NO

537 SEQ ID NO

597 SEQ ID NO

657 SEQ ID NO

187 SEQ ID NO

247 SEQ ID NO

307 SEQ ID NO

367 SEQ ID NO

427 SEQ ID NO

487 SEQ ID NO

547 SEQ ID NO

607 SEQ ID NO

667 SEQ ID NO

197 SEQ ID NO

257 SEQ ID NO

317 SEQ ID NO

377 SEQ ID NO

437 SEQ ID NO

497 SEQ ID NO

557 SEQ ID NO

617 SEQ ID NO

697 SEQ ID NO

207 SEQ ID NO

267 SEQ ID NO

327 SEQ ID NO

387 SEQ ID NO

447 SEQ ID NO

507 SEQ ID NO

567 SEQ ID NO

627 SEQ ID NO

707 SEQ ID NO

157 SEQ ID NO: 167 SEQ ID

217 SEQ ID NO

277 SEQ ID NO

337 SEQ ID NO

397 SEQ ID NO

457 SEQ ID NO

517 SEQ ID NO

577 SEQ ID NO

637 SEQ ID NO

717 SEQ ID NO

227 SEQ ID

287 SEQ ID

347 SEQ ID

407 SEQ ID

467 SEQ ID

527 SEQ ID

587 SEQ ID

647 SEQ ID

727, SEQ ID

737, SEQ ID NO: 747 and SEQ ID NO: 797.

In a further embodiment, the first binding domain of said construct of the present invention comprises a VL region selected from the group consisting of the sequences shown in SEQ ID NO

SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO:

178 SEQ ID NO

238 SEQ ID NO

298 SEQ ID NO

358 SEQ ID NO

418 SEQ ID NO

478 SEQ ID NO

538 SEQ ID NO

598 SEQ ID NO

658 SEQ ID NO

188 SEQ ID NO

248 SEQ ID NO

308 SEQ ID NO

368 SEQ ID NO

428 SEQ ID NO

488 SEQ ID NO

548 SEQ ID NO

608 SEQ ID NO

668 SEQ ID NO

738, SEQ ID NO: 748 and SEQ ID NO

198 SEQ ID NO

258 SEQ ID NO

318 SEQ ID NO

378 SEQ ID NO

438 SEQ ID NO

498 SEQ ID NO

558 SEQ ID NO

618 SEQ ID NO

698 SEQ ID NO

798.

208 SEQ ID NO

268 SEQ ID NO

328 SEQ ID NO

388 SEQ ID NO

448 SEQ ID NO

508 SEQID NO

568 SEQ ID NO

628 SEQ ID NO

708 SEQ ID NO

158 SEQ ID NO

218 SEQ ID NO

278 SEQ ID NO

338 SEQ ID NO

398 SEQ ID NO

458 SEQ ID NO

518 SEQ ID NO

578 SEQ ID NO

638 SEQ ID NO

718 SEQ ID NO

168, 228, 288, 348, 408, 468, 528, 588, 648, 728,

In another embodiment, the first binding domain of said antibody construct of the present invention comprises a VH region and a VL region selected from the group consisting of pairs of VH regions and VL regions shown in SEQ ID NOs: 157+158, SEQ ID NO: 167+168, SEQ ID NO: 177+178, SEQ ID NO: 187+188, SEQ ID NO: 197+198, SEQ ID NO: 207+208, SEQ ID NO: 217+218, SEQ ID NO: 227+228, SEQ ID NO: 237+238, SEQ ID NO: 247+248, SEQ ID NO: 257+258, SEQ ID NO: 267+268, SEQ ID NO: 277+278, SEQ ID NO: 287+ 288, SEQ ID NO: 297+298, SEQ ID NO: 307+308, SEQ ID NO: 317+318, SEQ ID NO: 327+328, SEQ ID NO: 337+338, SEQ ID NO: 347+348, SEQ ID NO: 357+358, SEQ ID NO: 367+368, SEQ ID NO: 377+378, SEQ ID NO: 387+388, SEQ ID NO: 397+398, SEQ ID NO: 407+408, SEQ ID NO: 417+418, SEQ ID NO: 427+428, SEQ ID NO: 437+438, SEQ ID NO: 447+448, SEQ ID NO: 457+458, SEQ ID NO: 467+468, SEQ ID NO: 477+478, SEQ ID NO: 487+488, SEQ ID NO: 497+498, SEQ ID NO: 507+508, SEQ ID NO: 517+518, SEQ ID NO: 527+528, SEQ ID NO: 537+538, SEQ ID NO: 547+548, SEQ ID NO: 557+558, SEQ ID NO: 567+568, SEQ ID NO: 577+578, SEQ ID NO: 587+588, SEQ ID NO: 597+598, SEQ ID NO: 607+608, SEQ ID NO: 617+618, SEQ ID NO: 627+628, SEQ ID NO: 637+638, SEQ ID NO: 647+648, SEQ ID NO: 657+658, SEQ ID NO: 667+668, SEQ ID NO: 697+698, SEQ ID NO: 707+708, SEQ ID NO: 717+718, SEQ ID NO: 727+ 728, SEQ ID NO: 737+738, SEQ ID NO: 747+748 and SEQ ID NO: 797+798.

In a further embodiment, the first binding domain of said antibody construct of the present invention comprises a polypeptide selected from the group consisting of the sequences shown in SEQ ID NO: 159, SEQ ID NO

SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO: SEQ ID NO SEQ ID NO

179, SEQ ID NO: 189, SEQ ID NO: 199, SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO 239, SEQ ID NO: 249, SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319, SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449, SEQ ID NO: 459, SEQ ID NO 479 and SEQ ID NO : 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 539, SEQ ID NO: 549, SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO

169

229,

289,

349,

409,

469,

529,

589,

649,

- 18 040387

SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729,

SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 799.

The present invention also provides a bispecific antibody construct comprising a first binding domain that binds to human FLT3 on the surface of a target cell and a second binding domain that binds to human CD3 on the surface of a T cell, characterized in that said first binding domain binds to an FLT3 epitope that is located in the region shown in SEQ ID NO: 821 (cluster 3).

Accordingly, according to a further aspect of the present invention, the first binding domain of said bispecific antibody construct comprises a VH region containing CDR-H1, CDR-H2 and CDR-H3 and a VL region containing CDR-L1, CDR-L2 and CDR-L3, as follows: SEQ ID NO: 671-676, SEQ ID NO: 681-686, SEQ ID NO: 751-756, SEQ ID NO: 761-766, SEQ ID NO: 771-776 and SEQ ID NO: 781-786 .

In one embodiment, the first binding domain of said construct of an antibody of the present invention comprises the VH region shown in the sequences SEQ ID NO: 677, SEQ ID NO: 687, SEQ ID NO: 757, SEQ ID NO: 767, SEQ ID NO: 777 and SEQ ID NO: 787.

In a further embodiment, the first binding domain of said antibody construct of the present invention comprises the VL region shown in SEQ ID NO: 678, SEQ ID NO: 688, SEQ ID NO: 758, SEQ ID NO: 768, SEQ ID NO: 778 and SEQ ID NO: 788.

In another embodiment, the first binding domain of said antibody construct of the present invention comprises a VH region and a VL region selected from the group consisting of pairs of VH regions and VL regions shown in SEQ ID NOs: 677+678, SEQ ID NO: 687+688, SEQ ID NO: 757+758, SEQ ID NO: 767+768, SEQ ID NO: 777+778 and SEQ ID NO: 787+788.

In a further embodiment, the first binding domain of said antibody construct of the present invention comprises a polypeptide selected from the group consisting of the sequences shown in SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 759, SEQ ID NO: 769 , SEQ ID NO: 779 and SEQ ID NO: 789.

Another preferred antibody construct according to the present invention can also be defined as a bispecific antibody construct comprising a first binding domain (preferably a human domain) that binds to human FLT3 on the surface of the target cell and a second binding domain that binds to human CD3 on the surface. T cells, wherein said first binding domain competes for binding with an antibody selected from the group consisting of FL-1 - FL-53, FL-55 - FL-60 and FL-65, i. an antibody containing a VH region containing CDR-H1, CDR-H2 and CDR-H3, and a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of the sequences described above.

T cells or T lymphocytes are a type of lymphocyte (which is itself a type of white blood cell) that play a central role in cell-mediated immunity. There are several subgroups of T cells, each with its own function. T cells can be distinguished from other lymphocytes such as B cells and NK cells by the presence of the T cell receptor (TCR) on the cell surface. The TCR is responsible for the recognition of antigens associated with major histocompatibility complex (MHC) molecules and is composed of two different protein chains. In 95% of T cells, the TCR consists of alpha (α) and beta (β) chains. When the TCR interacts with the antigenic peptide and the MHC (peptide/MHC complex), the T-lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

The CD3 receptor complex is a protein complex and consists of four chains. In mammals, this complex contains a CD3y chain (gamma), a CD3δ chain (delta), and two CD3ε chains (epsilon). These chains bind to the T cell receptor (TCR) and the so-called ζ (zeta) chain to form the CD3 receptor T cell complex and generate an activation signal in T lymphocytes. The CD3y (gamma), CD3δ (delta), and CD3ε (epsilon) chains are closely related cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. The intracellular terminal fragments of CD3 molecules contain one conserved motif, known as the immunoreceptor tyrosine activating motif, or ITAM for short, which is essential for TCR signaling. The CD3epsilon molecule is a polypeptide that in humans encodes the CD3E gene located on chromosome 11. The most preferred epitope of CD3 epsilon is located in the region of amino acid residues 1-27 of the extracellular domain of human CD3 epsilon.

Redirected lysis of target cells by recruiting T cells with a multispecific, at least bispecific, antibody construct includes formation of a cytolytic synapse and delivery of perforin and granzymes. Activated T cells are capable of serially killing target cells and are not affected by immunological escaping mechanisms that interact with peptide antigen processing and presentation, or T cell clonal differentiation; see, for example, WO 2007/042261.

Cytotoxicity mediated by anti-FLT3xCD3 bispecific antibody constructs can be measured in various ways. See examples section 10. Effector cells can be, for example, stimulated CD8-positive enriched T cells (human) or unstimulated peripheral blood mononuclear cells (PBMCs) (human). In the event that the target cells are macaque-derived, either express macaque FLT3 or are transfected with macaque FLT3, said effector cells must also be macaque-derived, eg a macaque T-cell line, eg 4119LnPx. Target cells must express (at least the extracellular domain of) FLT3, eg human or macaque FLT3. The target cells can be a cell line (such as CHO) stably or transiently transfected with FLT3, eg human or macaque FLT3. Alternatively, the target cells can be a FLT3 positive, naturally FLT3 expressing cell line, such as the FLT3 positive human AML cell lines EOL-1, MOLM-13 and MV4-11. Generally, EC50 values are expected to be lower for target cell lines expressing higher levels of FLT3 on the cell surface. The ratio of effector to target cells (E:T) is usually about 10:1, but can also vary. The cytotoxic activity of anti-FLT3xCD3 bispecific antibody constructs can be measured in a chromium-51 release assay (incubation time approximately 18 hours) or in a FACS-based cytotoxicity assay (incubation time approximately 48 hours). It is also possible to modify the time of incubation (cytotoxic reaction) in the analysis. Other methods for measuring cytotoxicity are well known to those skilled in the art and include MTT or MTS assays, ATP based assays including bioluminescent assays, sulforhodamine B (SRB) assay, WST assay, clonogenic assay, and ECIS technology.

Cytotoxic activity mediated by the anti-FLT3xCD3 bispecific antibody constructs of the present invention is preferably measured in a cellular cytotoxicity assay. It can also be measured in a chromium-51 release assay. It reflects the EC ₅₀ value, which corresponds to the half-maximal effective concentration (the concentration of the antibody construct that induces a cytotoxic response corresponding to the average between the initial and maximum values). Preferably, the EC ₅₀ value of the anti-FLT3xCD3 bispecific antibody construct is <5000 pM or <4000 pM, more preferably <3000 pM or <2000 pM, even more preferably <1000 pM or <500 pM, even more preferably <400 pM or < 300 pM, even more preferably -<200 pM, even more preferably -<100 pM, even more preferably -<50 pM, even more preferably -<20 pM or <10 pM, and most preferably -<5 pM.

The EC ₅₀ targets presented above can be measured in various assays. One skilled in the art will recognize that the EC ₅₀ value can be expected to be lower when stimulated/enriched for CD8+ T cells are used as effector cells compared to unstimulated PBMCs. It can also be expected that EC ₅₀ values will be lower if the target cells express a significant amount of the target antigen, compared to values at a low target expression rate. For example, when using stimulated/enriched for CD8+ human T cells as effector cells (and using any FLT3-transfected cells as target cells, such as CHO cells or FLT3-positive human AML lines EOL-1, MOLM-13 and MV4-11), the EC ₅₀ value of the anti-FLT3xCD3 bispecific antibody construct is preferably <1000 pM, more preferably -<500 pM, even more preferably -<250 pM, even more preferably -<100 pM, even more preferably -<50 pM, even more preferably -<10 pM and most preferably -<5 pM. When using human PBMCs as effector cells, the EC ₅₀ value of the anti-FLT3xCD3 bispecific antibody construct is preferably <5000 pM or <4000 pM (in particular, in cases where the target cells are FLT3-positive human AML lines EOL-1, MOLM- 13 and MV4-11), more preferably -<2000 pM (particularly when the target cells are FLT3 transfected cells such as CHO cells), more preferably -<1000 pM or <500 pM, even more preferably - <200 pM, even more preferably -<150 pM, even more preferably -<100 pM, and most preferably -<50 pM or less. When using a macaque T cell line such as LnPx4119 as effector cells and a FLT3 transfected macaque cell line such as CHO cells as the target cell line, the EC ₅₀ value of the anti-FLT3xCD3 bispecific antibody construct is preferably <2000 pM or < 1500 pM, more preferably <1000 pM or <500 pM, even more preferably <300 pM or <250 pM, even more preferably <100 pM and most preferably <50 pM.

Preferably, the anti-FLT3xCD3 bispecific antibody constructs of the present invention do not induce/mediate lysis or substantially do not induce/mediate lysis

- 20,040,387 FLT3 negative cells such as CHO cells. The term do not induce lysis, do not substantially induce lysis, do not mediate lysis, or do not substantially mediate lysis means that the antibody construct of the present invention does not induce or mediate more than 30% lysis, preferably not more than 20%, more preferably not more than 10%, particularly preferably no more than 9, 8, 7, 6 or 5% FLT3 negative cells, assuming 100% cell lysis of the FLT3 positive human lung carcinoma line SHP-77 (see above). This generally applies to antibody construct concentrations up to 500 nM.

The skilled artisan will know how cell lysis can be measured without additional complexity. In addition, specific recommendations for measuring cell lysis are given in the present description.

The difference in the cytotoxic activity of monomeric and dimeric isoforms of individual constructs of a bispecific antibody to FLT3xCD3 is called the efficiency difference. This efficiency difference can, for example, be calculated as the ratio of the EC ₅₀ values for the monomeric and dimeric form of the molecule, see example 17. The efficiency difference between the FLT3xCD3 bispecific antibody constructs of the present invention is preferred, more preferably -<4, even more preferably -< 3, even more preferably -<2 and most preferably -<1.

The first and/or second (or any additional) binding domain or domains of the antibody construct of the present invention are preferably cross-species specific for mammals of the primate order. Cross-species specific CD3 binding domains are described, for example, in WO 2008/119567. In one embodiment, the first and/or second binding domain, in addition to binding to human FLT3 and human CD3, respectively, also binds to FLT3/CD3 of primates, including (but not limited to) New World primates (such as Callithrix jacchus , Saguinus Oedipus or Saimiri sciureus), Old World primates (such as baboons and macaques), gibbons, orangutans, and non-human hominins. It is provided that the first binding domain of the antibody construct of the present invention that binds to human FLT3 on the surface of the target cell also binds to at least macaque FLT3 and/or the second binding domain that binds to human CD3 on the surface of the T cell also binds to at least macaque CD3. The preferred macaque is Masaca fascicularis. Also included is Masaca mulatta (rhesus monkey).

In one aspect of the present invention, the first binding domain binds to human FLT3 and further binds to macaque FLT3, such as Macaca fascicularis FLT3, and more preferably macaque FLT3 expressed on the surface of macaque cells. A preferred Macaca fascicularis FLT3 is shown in SEQ ID NO: 802. The affinity of said first binding domain for macaque FLT3 is preferably <15 nM, more preferably <10 nM, even more preferably <5 nM, even more preferably <1 nM , even more preferably -<0.5 nM, even more preferably -<0.1 nM, and most preferably -<0.05 nM or even <0.01 nM.

Preferably, the difference in affinity between macaque FLT3 binding and human FLT3 binding [macaque FLT3:human FLT3] (assessed using e.g. BiaCore or Scatchard assay) is <100, preferably <20, more preferably <15, also preferably <10, even more preferably <8, more preferably <6 and most preferably <2. Preferred ranges of difference in affinity of antibody constructs of the present invention for macaque FLT3 binding and human FLT3 binding are 0.1 to 20, more preferably 0.2 to 10, even more preferably 0.3 to 6, still more preferably 0.5 to 3 or 0.5 to 2.5, and most preferably 0.5 to 2 or 0.6 to 2. See examples section 5.

In one embodiment of said antibody construct of the present invention, the second binding domain binds to human CD3 epsilon and Callithrix jacchus, Saguinus Oedipus, or Saimiri sciureus CD3 epsilon. Preferably, said second binding domain binds to an extracellular epitope of said CD3-epsilon chains. It is also contemplated that said second binding domain can bind to a human extracellular epitope and Macas' CD3-epsilon chain. The most preferred epitope of CD3 epsilon is located in the region of amino acid residues 1-27 of the extracellular domain of human CD3 epsilon. Even more specifically, said epitope contains at least the amino acid sequence Gln-Asp-Gly-Asn-Glu. Both Callithrix jacchus and Saguinus oedipus are New World primates of the family Callitrichidae, and Saimiri sciureus is a New World primate of the family Cebidae.

In particular, it is preferred that, in said antibody construct of the present invention, the second binding domain that binds to human CD3 on the surface of the T cell contains a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from:

(a) CDR-L1 according to the sequence of SEQ ID NO: 27 of WO 2008/119567, CDR-L2 according to the sequence of SEQ ID NO: 28 of WO 2008/119567 and CDR-L3 according to the sequence of SEQ ID

- 21 040387

NO: 29 from WO 2008/119567;

(b) CDR-L1 according to SEQ ID NO: 117 from WO 2008/119567, CDR-L2 according to SEQ ID NO: 118 from WO 2008/119567 and CDR-L3 according to SEQ ID

NO: 119 from WO 2008/119567 and (c) CDR-L1 according to SEQ ID NO: 153 from WO 2008/119567, CDR-L2 according to SEQ ID NO: 154 from WO 2008/119567 and CDR-L3 according to SEQ ID NO: 155 from WO 2008/119567.

In an alternative preferred embodiment of said antibody construct of the present invention, said second binding domain that binds to human CD3 on the surface of a T cell comprises a VH region comprising CDR-H1, CDR-H2 and CDRH3 selected from:

(a) CDR-H1 according to SEQ ID NO: 12 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 13 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 14 from WO 2008/119567 ;

(b) CDR-H1 according to SEQ ID NO: 30 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 31 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 32 from WO 2008/119567 ;

(c) CDR-H1 according to SEQ ID NO: 48 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 49 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 50 from WO 2008/119567 ;

(d) CDR-H1 according to SEQ ID NO: 66 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 67 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 68 from WO 2008/119567 ;

(e) CDR-H1 according to SEQ ID NO: 84 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 85 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 86 from WO 2008/119567 ;

(f) CDR-H1 according to SEQ ID NO: 102 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 103 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 104 from WO 2008/119567 ;

(g) CDR-H1 according to SEQ ID NO: 120 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 121 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 122 from WO 2008/119567 ;

(h) CDR-H1 according to SEQ ID NO: 138 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 139 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 140 from WO 2008/119567 ;

(i) CDR-H1 according to SEQ ID NO: 156 from WO 2008/119567, CDR-H2 according to SEQ ID NO: 157 from WO 2008/119567 and CDR-H3 according to SEQ ID NO: 158 from WO 2008/119567 and (j) CDR-H1 according to the sequence of SEQ ID NO: 174 of WO 2008/119567, CDR-H2 according to the sequence of SEQ ID NO: 175 of WO 2008/119567 and CDR-H3 according to the sequence of SEQ ID NO: 176 of WO 2008/ 119567.

It is also preferred that the second binding domain of said antibody construct of the present invention that binds to human CD3 on the surface of a T cell contains a VL region selected from the group consisting of the VL region shown in SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO : 99 and SEQ ID NO: 102 (see also SEQ ID NO: 35, 39, 125, 129, 161 or 165 of WO 2008/119567).

Alternatively, it is preferred that said second binding domain that binds to human CD3 on the surface of the T cell contains a VH region selected from the group consisting of the VH region shown in SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98 and SEQ ID NO: 101 (See also SEQ ID NO: 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105, 109, 123, 127, 141, 145, 159, 163, 177 or 181 of WO 2008/119567).

More preferably, said antibody construct of the present invention is characterized by a second binding domain that binds to human CD3 on the surface of a T cell, comprising a VL region and a VH region selected from the group consisting of:

(a) the VL region shown in SEQ ID NO: 17 or 21 of WO 2008/119567 and the VH region shown in SEQ ID NO: 15 or 19 of WO 2008/119567;

(b) the VL region shown in SEQ ID NO: 35 or 39 of WO 2008/119567 and the VH region shown in SEQ ID NO: 33 or 37 of WO 2008/119567;

(c) the VL region shown in SEQ ID NO: 53 or 57 of WO 2008/119567 and the VH region shown in SEQ ID NO: 51 or 55 of WO 2008/119567;

- 22 040387 (d) the VL region shown in the sequence of SEQ ID NO: 71 or 75 of WO 2008/119567, and

the VH region shown in SEQ ID NO: 69 or 73 of WO 2008/119567;

(e) the VL region shown in SEQ ID NO: 89 or 93 of WO 2008/119567, and

the VH region shown in SEQ ID NO: 87 or 91 of WO 2008/119567;

(f) the VL region shown in SEQ ID NO: 107 or 111 of WO 2008/119567 and the VH region shown in SEQ ID NO: 105 or 109 of WO 2008/119567;

(g) the VL region shown in SEQ ID NO: 125 or 129 of WO 2008/119567 and the VH region shown in SEQ ID NO: 123 or 127 of WO 2008/119567;

(h) the VL region shown in SEQ ID NO: 143 or 147 of WO 2008/119567 and the VH region shown in SEQ ID NO: 141 or 145 of WO 2008/119567;

(i) the VL region shown in SEQ ID NO: 161 or 165 of WO 2008/119567 and the VH region shown in SEQ ID NO: 159 or 163 of WO 2008/119567; and (j) a VL region shown in SEQ ID NO: 179 or 183 of WO 2008/119567 and a VH region shown in SEQ ID NO: 177 or 181 of WO 2008/119567.

Also preferred for said antibody construct of the present invention is a second binding domain that binds to human CD3 on the surface of a T cell, comprising a VL region as shown in SEQ ID NO: 102 and a VH region as shown in SEQ ID NO. : 101.

According to a preferred embodiment of said construct, the antibody of the present invention has the binding domains, and in particular the second binding domain (which binds to human CD3 on the surface of the T cell), have the following format: the pairs of VH regions and VL regions are in single chain antibody format ( scFv). These VH and VL regions are arranged in the order VH-VL or VL-VH. Preferably, the VH region is located N-terminally from the linker sequence and the VL region is located C-terminally from the linker sequence.

A preferred embodiment of the above antibody construct of the present invention is characterized by a second binding domain that binds to human CD3 on the surface of a T cell comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103 (See also SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185, or 187 of WO 2008/119567).

An additional function of the antibody construct of the present invention, in addition to the function of binding to target FLT3 and CD3 molecules, is also contemplated. In the indicated format, said antibody construct is a trifunctional or multifunctional antibody construct that targets target cells by binding to FLT3, mediating cytotoxic T cell activity by binding to CD3, and providing an additional function such as a fully functional Fc constant domain mediating antibody- dependent cellular cytotoxicity by recruiting effector cells such as NK cells, a label (fluorescent, etc.), a therapeutic agent such as a toxin or radionuclide, and/or a method for increasing serum half-life, and the like.

Examples of methods for increasing the serum half-life of antibody constructs of the present invention include peptides, proteins, or protein domains that are fused or otherwise attached to said antibody construct. The group of peptides, proteins or protein domains includes the binding of peptides to other proteins with a preferred pharmacokinetic profile in the human body, such as serum albumin (see WO 2009/127691). In an alternative concept, such half-life increasing peptides include peptides that bind to the neonatal Fc receptor (FcRn, see WO 2007/098420), which can also be used in the constructs of the present invention. The concept of attaching domains of larger proteins or complete proteins includes, for example, fusion with human serum albumin, variants or mutants of human serum albumin (see WO 2011/051489, WO 2012/059486, WO 2012/150319, WO 2013/135896, WO 2014/ 072481, WO 2013/075066) or its domains, as well as fusion with the constant region of immunoglobulins (Fc domains) and their variants. Such Fc domain variants can be optimized/modified to provide the desired pairing of dimers or multimers, to abolish Fc receptor (eg, Fcγ receptor) binding, or for other reasons. An additional art-known concept used to increase the half-life of small protein compounds in the human body is to pegylate said compounds, for example, the antibody construct of the present invention.

In a preferred embodiment, said antibody construct of the present invention is described as follows:

(a) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179 , SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9; and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103; and optionally, a histidine (His) tag such as that shown in SEQ ID NO 10;

(b) a polypeptide containing, in the following order, starting from the N-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQ ID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103;

optionally, a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9;

a polypeptide having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 104-134; and optionally, a histidine (His) tag such as that shown in SEQ ID NO 10;

(c) a polypeptide containing, in the following order, starting from the N-terminus:

a polypeptide with the amino acid sequence QRFVTGHFGGLX1PANG (SEQ ID NO: 135), where X1 is Y or H; and a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9;

- 24 040387 a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ

ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103;

a polypeptide with the amino acid sequence QRFVTGHFGGLHPANG (SEQ ID NO: 137) or

QRFCTGHFGGLHPCNG (SEQ ID NO: 139); and optionally, a histidine (His) tag such as that shown in SEQ ID NO 10;

(d) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO : 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98 and SEQ ID NO: 101;

a peptide linker with the amino acid sequence shown in the sequence of SEQ ID NO: 8;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 178, SEQ ID NO: 188, SEQ ID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

208 SEQ ID NO

268 SEQ ID NO

328 SEQ ID NO

388 SEQ ID NO

448 SEQ ID NO

508 SEQID NO

568 SEQ ID NO

628 SEQ ID NO

688 SEQ ID NO

218 SEQ ID NO

278 SEQ ID NO

338 SEQ ID NO

398 SEQ ID NO

458 SEQ ID NO

518 SEQ ID NO

578 SEQ ID NO

638 SEQ ID NO

698 SEQ ID NO

228 SEQ ID NO

288 SEQ ID NO

348 SEQ ID NO

408 SEQ ID NO

468 SEQ ID NO

528 SEQ ID NO

588 SEQ ID NO

648 SEQ ID NO

708 SEQ ID NO

238 SEQ ID NO

298 SEQ ID NO

358 SEQ ID NO

418 SEQ ID NO

478 SEQ ID NO

538 SEQ ID NO

598 SEQ ID NO

658 SEQ ID NO

718 SEQ ID NO

248 SEQ ID NO

308 SEQ ID NO

368 SEQ ID NO

428 SEQ ID NO

488 SEQ ID NO

548 SEQ ID NO

608 SEQ ID NO

668 SEQ ID NO

728 SEQ ID NO

748, SEQ ID NO: 758, SEQ ID NO: 768, SEQ ID NO: 778, SEQ ID NO: 788 and SEQ ID NO

198, 258, 318, 378, 438, 498, 558, 618, 678, 738, 798;

and a serine residue at the C-terminus;

a polypeptide with the amino acid sequence shown in SEQ ID NO: 140; and a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 157, SEQ ID NO: 167, SEQ ID NO: 177, SEQ ID NO: 187 , SEQ ID NO: 197,

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

207 SEQ ID NO

267 SEQ ID NO

327 SEQ ID NO

387 SEQ ID NO

447 SEQ ID NO

507 SEQ ID NO

567 SEQ ID NO

627 SEQ ID NO

687 SEQ ID NO

217 SEQ ID NO

277 SEQ ID NO

337 SEQ ID NO

397 SEQ ID NO

457 SEQ ID NO

517 SEQ ID NO

577 SEQ ID NO

637 SEQ ID NO

697 SEQ ID NO

227 SEQ ID NO

287 SEQ ID NO

347 SEQ ID NO

407 SEQ ID NO

467 SEQ ID NO

527 SEQ ID NO

587 SEQ ID NO

647 SEQ ID NO

707 SEQ ID NO

237 SEQ ID NO

297 SEQ ID NO

357 SEQ ID NO

417 SEQ ID NO

477 SEQ ID NO

537 SEQ ID NO

597 SEQ ID NO

657 SEQ ID NO

717 SEQ ID NO

247 SEQ ID NO

307 SEQ ID NO

367 SEQ ID NO

427 SEQ ID NO

487 SEQ ID NO

547 SEQ ID NO

607 SEQ ID NO

667 SEQ ID NO

727 SEQ ID NO

257, 317, 377,

437, 497, 557, 617,

677, 737,

SEQ ID NO: 747, SEQ ID NO: 757, SEQ ID NO: 767, SEQ ID NO: 777, SEQ ID NO: 787 and SEQ ID NO: 797;

a peptide linker with the amino acid sequence shown in SEQ ID NO: 8;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99 and SEQ ID NO: 102; and a serine residue at the C-terminus;

a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 141;

(e) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 62, SEQ ID NO: 71, SEQ ID NO: 80, SEQ ID NO: 89, SEQ ID NO: 98 and SEQ ID NO: 101;

a peptide linker with the amino acid sequence shown in SEQ ID NO: 8;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 158, SEQ ID NO: 168, SEQ ID NO: 178, SEQ ID NO: 188, SEQ ID NO: 198, SEQ ID NO: 208, SEQ ID NO: 218, SEQ ID NO: 228, SEQ ID NO: 238, SEQ ID NO: 248, SEQ ID NO: 258, SEQ ID NO: 268, SEQ ID NO: 278, SEQ ID NO: 288, SEQ ID NO : 298, SEQ ID NO: 308, SEQ ID NO: 318, SEQ ID NO: 328, SEQ ID NO: 338, SEQ ID NO: 348, SEQ ID NO: 358, SEQ ID NO: 368, SEQ ID NO: 378 , SEQ ID NO: 388, SEQ ID NO: 398, SEQ ID NO: 408, SEQ ID NO: 418, SEQ ID NO: 428, SEQ ID NO: 438, SEQ ID NO: 448, SEQ ID NO: 458, SEQ ID NO: 468, SEQ ID NO: 478, SEQ ID NO: 488, SEQ ID NO: 498,

- 25 040387

SEQ ID NO: 508, SEQ ID NO: 518, SEQ ID NO: 528, SEQ ID NO: 538, SEQ ID NO: 548, SEQ ID NO: 558,

SEQ ID NO: 568, SEQ ID NO: 578, SEQ ID NO: 588, SEQ ID NO: 598, SEQ ID NO: 608, SEQ ID NO: 618,

SEQ ID NO: 628, SEQ ID NO: 638, SEQ ID NO: 648, SEQ ID NO: 658, SEQ ID NO: 668, SEQ ID NO: 678,

SEQ ID NO: 688, SEQ ID NO: 698, SEQ ID NO: 708, SEQ ID NO: 718, SEQ ID NO: 728, SEQ ID NO: 738,

SEQ ID NO: 748, SEQ ID NO: 758, SEQ ID NO: 768, SEQ ID NO: 778, SEQ ID NO: 788 and SEQ ID NO: 798;

a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 142; and a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 157, SEQ ID NO: 167, SEQ ID NO: 177, SEQ ID NO: 187, SEQ ID NO: 197,

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

SEQID NO

207 SEQ ID NO

267 SEQ ID NO

327 SEQ ID NO

387 SEQ ID NO

447 SEQ ID NO

507 SEQ ID NO

567 SEQ ID NO

627 SEQ ID NO

687 SEQ ID NO

217 SEQ ID NO

277 SEQ ID NO

337 SEQ ID NO

397 SEQ ID NO

457 SEQ ID NO

517 SEQ ID NO

577 SEQ ID NO

637 SEQ ID NO

697 SEQ ID NO

227 SEQ ID NO

287 SEQ ID NO

347 SEQ ID NO

407 SEQ ID NO

467 SEQ ID NO

527 SEQ ID NO

587 SEQ ID NO

647 SEQ ID NO

707 SEQ ID NO

237 SEQ ID NO

297 SEQ ID NO

357 SEQ ID NO

417 SEQ ID NO

477 SEQ ID NO

537 SEQ ID NO

597 SEQ ID NO

657 SEQ ID NO

717 SEQ ID NO

247 SEQ ID NO

307 SEQ ID NO

367 SEQ ID NO

427 SEQ ID NO

487 SEQ ID NO

547 SEQ ID NO

607 SEQ ID NO

667 SEQ ID NO

727 SEQ ID NO

257, 317, 377, 437,

497, 557, 617, 677, 737,

SEQ ID NO: 747, SEQ ID NO: 757, SEQ ID NO: 767, SEQ ID NO: 777, SEQ ID NO: 787 and SEQ ID NO: 797;

a peptide linker having the amino acid sequence shown in the sequence of SEQ ID NO: 8;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 27, SEQ ID NO: 36, SEQ ID NO: 45, SEQ ID NO: 54, SEQ ID NO: 63, SEQ ID NO: 72, SEQ ID NO: 81, SEQ ID NO: 90, SEQ ID NO: 99 and SEQ ID NO: 102; and a serine residue at the C-terminus;

[CD 3 VL] polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 143;

(f) [V5 Hetero-Fc] polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9; and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 144; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 145;

(g) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and

- 26 040387

SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739,

SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 146; and a polypeptide containing, in the following order, starting from the N-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 147;

(h) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199, SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO : 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO : 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599 , SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SE Q ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 148; and a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100, and SEQ ID NO: 103; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 149; or (i) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9; and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103; and a polypeptide with the amino acid sequence shown in the sequence of SEQ ID NO: 150.

(j) a polypeptide comprising, in the following order, starting from the N-terminus, a polypeptide having an amino acid sequence selected from the group consisting of the sequences SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189, SEQ ID NO: 199,

SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and SEQ ID NO: 259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO: 309, SEQ ID NO: 319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 349 and SEQ ID NO: 359, SEQ ID NO: 369, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO: 409, SEQ ID NO: 419 and SEQ ID NO: 429, SEQ ID NO: 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO: 469, SEQ ID NO: 479 and SEQ ID NO: 489, SEQ ID NO: 499,

- 27 040387

SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO: 529, SEQ ID NO: 539, SEQ ID NO: 549 and SEQ ID NO: 559,

SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO: 609, SEQ ID NO: 619 and

SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 669, SEQ ID NO: 679 and

SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 729, SEQ ID NO: 739,

SEQ ID NO: 749 and SEQ ID NO: 759, SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO: 789; and SEQ ID NO:

799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9; and a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9; and a third domain having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 843-850.

According to a preferred aspect of the present invention, the binding of said first binding domain to human FLT3 is reduced by the FLT3 ligand by 25% or less, preferably 20% or less, more preferably 15% or less, also preferably 10% or less, even more preferably 8% or less, more preferably 6% or less, and most preferably 2% or less.

As detailed in Example 18, it was surprisingly found that constructs containing FL-53, FL-54, FL-61, F-62, FL-63, and FL-64, all of which bind to FLT3 epitope cassette 3, all still show a binding signal that exceeds the threshold described in Example 18, although the epitope for these binding elements is in the region described for the interaction of the FLT3 ligand with FLT3.

In addition, given the interaction of the FLT3 ligand with the region of epitope cluster 3, it was also suggested that the binding element for a more distant epitope cluster, such as FLT3 cluster 1, would not be affected by competition with the FLT3 ligand. There were, however, a significant number of connecting elements that did not meet the 75% threshold condition. Binding elements FL-1-FL-53, FL-55-FL-60 and FL-65 were included in the group of binding elements insensitive to competition with the FLT3 ligand.

As described above, several preferred antibody constructs of the present invention are modified by fusion with another moiety, such as albumin or albumin variants. As one skilled in the art will appreciate, if said fusion constructs are characterized in terms of their properties, in particular target affinity or cytotoxic activity, similar fusion constructs or unmodified bispecific antibody constructs can be expected to have similar (or even better) properties. For example, if the albumin-fused bispecific antibody construct has measurable or desirable cytotoxic activity or target affinity, the same/similar or even higher cytotoxic activity/target affinity can be expected to be observed for constructs without albumin.

According to another preferred embodiment, the bispecific antibody construct of the present invention comprises (in addition to the two binding domains) a third domain that contains two polypeptide monomers each containing a hinge, a CH2 domain, and a CH3 domain, said two polypeptides (or polypeptide monomers) fused to each other via a peptide linker. Preferably, said third domain comprises, N-terminally to C-terminally: hinge-CH2-CH3-linker-hinge-CH2-CH3. Preferred amino acid sequences of said third domain are shown in SEQ ID NO: 843850. Each of said two polypeptide monomers preferably has an amino acid sequence that is selected from the group consisting of SEQ ID NO: 835-842, or a sequence of at least 90 % identical to the specified sequences. In another preferred embodiment, said first and second binding domains of a bispecific antibody construct of the present invention are fused to said third domain via a peptide linker, which is, for example, selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5 , 6, 7, 8 and 9.

In accordance with the present invention, the hinge is an IgG hinge region. The specified area can be identified in a similar way using Kabat numbering, see provisions 223-243 of Kabat. In accordance with the above, the minimum requirement for a hinge is satisfied by amino acid residues corresponding to the segment of the IgG1 sequence from D231 to P243 in accordance with Kabat numbering. The terms CH2 and CH3 refer to constant regions 2 and 3 of immunoglobulin heavy chains. These areas can also be identified in a similar way using Kabat numbering, see Kabat provisions 244-360 for

- 28 040387

CH2 and provisions according to Kabat 361-478 for CH3. Immunoglobulins are expected to contain some variation affecting the Fc IgG1 region, the Fc IgG ₂ region, the Fc IgG ₃ region, the Fc IgG4 region, the Fc IgM region, the Fc IgA region, the Fc IgD region, and the Fc IgE region (see, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). The term Fc monomer refers to the last two IgA heavy chain constant regions, IgD and IgG, and the last three IgE and IgM heavy chain constant regions. The Fc monomer may also include a flexible hinge located N-terminally from said domains. In the case of IgA and IgM, the Fc monomer may include a J chain. In the case of IgG, the Fc portion contains the CH2 and CH3 immunoglobulin domains, as well as a hinge between the first two domains and CH2. Although the boundaries of the Fc portion of an immunoglobulin may vary, an example of a human IgG heavy chain Fc portion containing a functional hinge, CH2 and CH3 domains can be defined, for example, such that it contains residues D231 (in the hinge domain) to P476 ( at the C-terminus of the CH3 domain), or residues D231 to L476, respectively, in the case of IgG ₄ ; the indicated numbering corresponds to the Kabat system.

An antibody construct of the present invention may suitably comprise, in the N-terminal to C-terminal direction:

(a) a first binding domain;

(b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: SEQ ID NOs: 1-9;

(c) a second binding domain;

(d) a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9;

(e) a first third domain polypeptide monomer (comprising a hinge, a CH2 domain, and a CH3 domain);

(f) a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 852, 853, 854 and 855; and (g) a second third domain polypeptide monomer (comprising a hinge, a CH2 domain, and a CH3 domain).

It is also preferred that said antibody construct of the present invention comprise, in N-terminal to C-terminal direction:

a first binding domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 159, SEQ ID NO: 169, SEQ ID NO: 179, SEQ ID NO: 189,

SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO SEQ ID NO

199, SEQ ID NO: 209, SEQ ID NO: 219 and SEQ ID NO: 229, SEQ ID NO: 239, SEQ ID NO: 249 and

259, SEQ ID NO: 269, SEQ ID NO: 279 and SEQ ID NO: 289, SEQ ID NO: 299, SEQ ID NO

319 and SEQ ID NO: 329, SEQ ID NO: 339, SEQ ID NO: 379 and SEQ ID NO: 389, SEQ ID NO: 399, SEQ ID NO 439, SEQ ID NO: 449 and SEQ ID NO: 459, SEQ ID NO 499, SEQ ID NO: 509, SEQ ID NO: 519 and SEQ ID NO

309, 369, 429, 489,

349 and SEQ ID NO: 359, SEQ ID NO

409, SEQ ID NO: 419 and SEQ ID NO

469, SEQ ID NO: 479 and SEQ ID NO

529, SEQ ID NO: 539, SEQ ID NO: 549 and

559, SEQ ID NO: 569, SEQ ID NO: 579 and SEQ ID NO: 589, SEQ ID NO: 599, SEQ ID NO 619 and SEQ ID NO: 629, SEQ ID NO: 639, SEQ ID NO: 649 and SEQ ID NO: 659, SEQ ID NO: 679 and SEQ ID NO: 689, SEQ ID NO: 699, SEQ ID NO: 709, SEQ ID NO: 719 and SEQ ID NO: 739, SEQ ID NO: 749 and SEQ ID NO: 759 , SEQ ID NO: 769, SEQ ID NO: 779 and SEQ ID NO

609, 669, 729, 789;

and SEQ ID NO: 799;

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1-9;

a second binding domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64 , SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103 (see also SEQ ID NOs: 23, 25, 41, 43, 59, 61 , 77, 79, 95, 97, 113, 115, 131, 133, 149, 151, 167, 169, 185 or 187 from WO 2008/119567);

a peptide linker having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NOs: 1, 2, 4, 5, 6, 8 and 9; and a third domain having an amino acid sequence selected from the group consisting of the sequences of SEQ ID NO: 843-850.

In a preferred embodiment, an antibody construct of the present invention comprises or consists of a polypeptide shown in SEQ ID NOs: 856-871.

According to one preferred embodiment of an antibody construct according to the present invention, said antibody construct comprises or consists of a polypeptide shown in the sequence of SEQ ID NOS: 856, 858, 860, 862, 864, 866, 868 and 870.

According to one alternative preferred embodiment of an antibody construct according to the present invention, said antibody construct comprises or consists of a polypeptide shown in SEQ ID NOS: 857, 859, 861, 863, 865, 867, 869, and 871.

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According to a preferred embodiment of the present invention, the antibody construct of the present invention comprises or consists of a polypeptide shown in SEQ ID NOs: 858, 859, 862, 863, 864 and 865.

Covalent modifications of antibody constructs are also included within the scope of the present invention and are usually, but not always, carried out post-translationally. For example, several types of covalent modifications to an antibody construct are introduced into a molecule by reacting specific amino acid residues from said antibody construct with an organic derivatizing agent that is capable of reacting with selected side chains, or N- or C-terminal residues.

For cysteinyl residues, α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide are most commonly reacted to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues are also derivatized by reacting with bromotrifluoroacetone, a-bromo-b-(5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, pchloromercury benzoate, 2-chloromercury- 4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reacting with diethyl pyrocarbonate at pH 5.5-7.0, since this agent is relatively specific for the histidyl side chain. Also suitable for use is para-bromphenacyl bromide; this reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. The reaction of lysinyl and amino-terminal residues is carried out with anhydrides of succinic acid or other carboxylic acids. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and a transaminase-catalyzed reaction with glyoxylate.

Arginyl residues are modified by reaction with one or more standard reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be carried out under alkaline conditions due to the high pKa value of the guanidine functional group. In addition, these reagents can react with lysine groups as well as the arginine epsilon-amino group.

Specific modification of tyrosyl residues can be carried out, with particular emphasis on the introduction of spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most often, N-acetylimidazole and tetranitromethane are used to obtain O-acetyltyrosyl compounds and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵ I or ¹³¹ I to obtain labeled proteins for use in radioimmunoassay, for which the method described above with chloramine T is suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N--R'), where R and R' are optionally different alkyl groups such as 1-cyclohexyl-3- (2-morpholinyl-4-ethyl)carbodiimide or 1ethyl-3-(4-azonium-4,4-dimethylpentyl)carbodiimide. In addition, aspartyl or glutamyl residues are converted to asparaginyl and glutaminyl residues by reacting with ammonium ions.

Derivatization with bifunctional agents is suitable for crosslinking antibody constructs of the present invention to a water insoluble support matrix or surface for use in a variety of methods. Commonly used crosslinkers include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g. as 3,3'dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate produce photoactivated intermediates that are capable of crosslinking in the presence of light. Alternatively, reactive, water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates are used to immobilize the protein, as described in US Pat. Nos. 3,969,287; 3691016; 4195128; 4247642; 4229537 and 4330440.

Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, said residues are deamidated under moderately acidic conditions. Any form of these residues is included in the scope of the present invention.

Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of the side chains of lysine, arginine, and histidine (T. E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

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Another type of covalent modification of antibody constructs within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, glycosylation patterns can depend both on the sequence of the protein (eg, on the presence or absence of specific glycosylated amino acid residues, as discussed below) and on the host cell or organism in which said protein is produced. Specific expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a 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 recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Accordingly, the presence of any of these tripeptide sequences in a polypeptide results in a site of potential glycosylation. 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 may also be used.

Glycosylation sites may conveniently be added to an antibody construct by changing the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Said change may also be effected by adding one or more serine or threonine residues to the start sequence, or substituting one or more serine or threonine residues in the start sequence (for O-linked glycosylation sites). For simplicity, the amino acid sequence of antibody constructs is preferably changed by substitutions at the DNA level, in particular by introducing mutations of preselected bases in the DNA encoding said polypeptide so as to obtain codons that translate into the desired amino acids.

Another way to increase the number of carbohydrate moieties on an antibody construct is to chemically or enzymatically attach glycosides to the protein. The advantage of these procedures is that the production in the host cell of a protein capable of undergoing N- and O-linked glycosylation is not required. Depending on the method of attachment used, the sugar(s) may be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as cysteine sulfhydryl groups, (d) free hydroxyl groups such as serine, threonine or hydroxyproline hydroxyl groups, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan aromatic residues, or (f) glutamine amide group. These methods are described in WO 87/05330, and in the source: Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

Removal of carbohydrate moieties present on the original antibody construct may be carried out chemically or enzymatically. Chemical deglycosylation requires exposing the protein to a trifluoromethanesulfonic acid compound, or an equivalent compound. This treatment results in cleavage of most or all of the sugars except for the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) and the polypeptide remains intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using various endo- and exoglycosidases as described in Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by using the tunicamycin compound as described in Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunikamicin blocks the formation of protein bonds with N-glycosides.

Other modifications of this antibody design are also contemplated by the present invention. For example, another type of covalent modification of said antibody construct includes coupling said antibody construct to various non-protein-like polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, according to the method described in patents. USA No. 4640835; 4496689; 4301144; 4670417; 4,791,192 or 4,179,337. In addition, as is known in the art, amino acid substitutions can be made at various positions within an antibody construct, for example, to facilitate the addition of polymers such as PEG.

In some embodiments, covalent modification of antibody constructs of the present invention includes the addition of one or more labels. The labeling group can be attached to the antibody construct via spacer fragments of various lengths to reduce potential steric hindrance. In the art known and can be used in the implementation of the present invention, various methods of labeling proteins. The term label or labeling group refers to any detectable label. In general, labels fall into different classes depending on the assay in which they are intended to be detected - examples below include, but are not limited to:

a) isotopic labels, which may be radioactive isotopes or heavy isotopes such as radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁸⁹ Zr, ⁹⁰ Y, ⁹⁹ Tc, ⁿ¹ In, ¹²⁵ I, ^131I );

b) magnetic marks (eg magnetic particles);

c) redox active fragments;

d) optical dyes (including, but not limited to, chromophores, phosphorescent substances and fluorophores) such as fluorescent groups (for example, FITC, rhodamine, lanthanide phosphors), chemiluminescent groups and fluorophores, which can be either low molecular weight fluorescent substances, either protein-like fluorescent substances;

e) enzymatic groups (eg horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase);

f) biotinylated groups;

g) predetermined polypeptide epitopes recognized by the secondary reporter (eg, leucine zipper pairing sequences, secondary antibody binding sites, metal binding domains, epitope tags, etc.).

By fluorescent label is meant any molecule that can be detected due to its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl coumarins, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red ( Texas Red), IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy5, Cy5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, Cascade Blue, Cascade Yellow, and Phycoerythrin (PE) (Molecular Probes, Eugene, OR) , FITC, rhodamine, and Texas Red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook, Richard P. Haugland.

Suitable protein-like fluorescent labels also include, but are not limited to, green fluorescent protein, including the GFP protein of Renilla, Ptilosarcus or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number: U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801, 8th floor West Boulevard de Maisonneuve, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462- 471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408 -5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98 /26277, WO 99/49019, US Pat.

Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were first identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759) and have since been found in various other proteins. Known leucine zippers include naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for the production of soluble oligomeric proteins are described in PCT application WO 94/10308, and a leucine zipper derived from lung surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191. The use of a modified leucine zipper that provides stable trimerization of a heterologous protein fused to it is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteins containing a FLT3 antibody fragment or derivative fused to a peptide leucine zipper are expressed in suitable host cells and the resulting soluble FLT3 antibody oligomeric fragments or derivatives are recovered from the culture supernatant.

The antibody construct of the present invention may also contain additional domains which, for example, facilitate the isolation of said molecule or are associated with tailoring the pharmacokinetic profile of said molecule. Domains that facilitate the isolation of the antibody construct can be selected from peptide motifs or reintroduced fragments that can be captured by the isolation method, eg on a isolation column. Non-limiting embodiments of such additional domains include peptide motifs known as Myc tag, HAT tag, HA tag, TAP tag, GST tag, chitin binding domain (CBD tag), maltose binding protein (MBP tag), Flag-tag, Strep-tag, and their variants (for example, StrepII- 32 040387 tag), as well as His-tag. All antibody constructs disclosed herein, characterized on the basis of identified CDR regions, preferably contain a histidine (His) tag domain, generally known as a repeat of consecutive His residues in the amino acid sequence of the molecule, preferably containing five, more preferably six His residues (hexa -histidine). The His-tag can be located, for example, at the N- or C-terminus of said antibody construct; preferably, it is located at the C-terminus. Most preferably, a hexahistidine tag (HHHHHH) (SEQ ID NO: 10) is linked to the C-terminus of the antibody construct of the present invention via a peptide bond.

The first binding domain of the antibody construct of the present invention binds to human FLT3 on the surface of the target cell. The preferred amino acid sequence of human FLT3 is shown at sequences 801, 803, 804 and 805. It is to be understood that the term on the surface in the context of the present invention means that the binding domain specifically binds to an epitope contained in the extracellular domain of FLT3 (FLT3 ECD, see SEQ ID NO:813). The first binding domain according to the invention suitably preferably binds to FLT3 when expressed by cells or cell lines naturally expressing it and/or cells or cell lines transformed or (stably/transiently) transfected with FLT3. In a preferred embodiment, said first binding domain also binds to FLT3 when FLT3 is used as a target molecule or ligand in an in vitro binding assay such as BIAcore or Scatchard assay. The target cell can be any prokaryotic or eukaryotic cell that expresses FLT3 on the surface; preferably, the target cell is a cell that is resident in a human or animal body, such as an ovarian cancer cell, a pancreatic cancer cell, a mesothelioma cell, a lung cancer cell, a gastric cancer cell, and a triple-negative breast cancer cell.

The term FLT3 EVA refers to a form of FLT3 that is substantially free of the transmembrane and cytoplasmic domains of FLT3. One skilled in the art will appreciate that the transmembrane domain identified for the FLT3 polypeptide of the present invention is identified based on criteria commonly used in the art to identify this type of hydrophobic domain. The exact boundaries of the transmembrane domain may vary, but most likely by no more than about 5 amino acids on either side of the domain specifically described herein. The preferred EVA of human FLT3 is shown in SEQ ID NO: 813.

The affinity of said first binding domain for human FLT3 is preferably <20 nM, more preferably -<10 nM, even more preferably -<5 nM, even more preferably -<2 nM, even more preferably -<1 nM, even more preferably - <0.6 nM, even more preferably <0.5 nM and most preferably <0.4 nM. Affinity can be measured, for example, in the analysis of Biacore or in the analysis of Scatchard, for example, as described in the examples. Other methods for determining affinity are also well known to those skilled in the art.

Also contemplated are modifications to the amino acid sequences of antibody constructs as described herein. For example, it may be desirable to improve the binding affinity (affinity) and/or other biological properties of said antibody construct. Amino acid sequence variants of antibody constructs are obtained by introducing suitable nucleotide changes into the nucleic acid of said antibody constructs, or by peptide synthesis. All amino acid sequence modifications described below should provide an antibody construct that retains the desired biological activity (binding to FLT3 and to CD3) of the unmodified parent molecule.

The term amino acid, or amino acid residue, generally refers to an amino acid as defined in the art, for example, an amino acid selected from the group consisting of: alanine (Ala, or A); arginine (Arg, or R); asparagine (Asn, or N); aspartic acid (Asp, or D); cysteine (Cys, or C); glutamine (Gln, or Q); glutamic acid (Glu, or E); glycine (Gly, or G); histidine (His, or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys, or K); methionine (Met, or M); phenylalanine (Phe, or F); proline (Pro, or P); serine (Ser, or S); threonine (Thr, or T); tryptophan (Trp, or W); tyrosine (Tyr, or Y) and valine (Val, or V), although modified, synthetic or rare amino acids may be used if necessary. Typically, amino acids are classified into groups having a non-polar side chain (eg, Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (eg Asp, Glu); a positively charged side chain (eg Arg, His, Lys); or uncharged polar side chain (eg Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp and Tyr) amino acids.

Amino acid modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequences of antibody constructs. To obtain the final design, any combination of deletions, insertions and replacements is carried out, provided that the specified

- 33 040387 the final design has the required characteristics. Changes in amino acids can also cause changes in post-translational processes in antibody constructs, such as changes in the number or position of glycosylation sites.

For example, 1, 2, 3, 4, 5 or 6 amino acids can be inserted or deleted in each of the CDR regions (of course, given their length), and 1, 2, 3, 4, 5, 6, 7, 8, 9, 1o, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be inserted or deleted in each of the FR regions. Preferably insertion of amino acid sequences includes fusion to the amino and/or carboxyl terminus of amino acid sequences ranging from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues up to polypeptides containing one hundred or more residues, as well as insertion inside sequences of one or more amino acid residues. An embodiment of an insertable antibody construct of the present invention comprises fusion to the N-terminus or C-terminus of said enzyme antibody construct, or fusion to a polypeptide that increases the serum half-life of said antibody construct.

Sites of greatest interest for substitution mutagenesis include the heavy and/or light chain CDR regions, in particular the hypervariable regions, but alterations to the heavy and/or light chain FRs are also contemplated. Said substitutions are preferably conservative substitutions as described herein. Preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids may be substituted in the CDR region, and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or 25 amino acids may be substituted in the framework regions (FR regions), depending on the length of said CDR or FR region. For example, if the sequence of a CDR region spans 6 amino acids, one, two, or three of these amino acids are substitutions. Similarly, if the sequence of the CDR region spans 15 amino acids, one, two, three, four, five, or six of these amino acids are substitutions.

A suitable method for identifying specific residues or regions of antibody constructs located at a mutagenesis-preferred location is called alanine-scanning mutagenesis, as described in Cunningham and Wells, Science, 244: 1081-1085 (1989). The method identifies a residue or group of target residues within an antibody construct (e.g., charged residues such as arg, asp, his, lys, and glu) and replaces it with a neutral or negatively charged amino acid (most preferably, alanine or polyalanine) to affect the interaction of amino acids with the epitope.

Amino acid positions demonstrating functional sensitivity to substitutions are then refined by introducing additional or different variants at or in place of substitution sites. Thus, while the site or region of introduction of the amino acid sequence variation is predetermined, as such the nature of the mutation need not be predetermined. For example, to analyze or optimize the expression of a mutation at a given site, alanine scanning or random mutagenesis at the target codon or region can be performed and the expressed antibody construct variants screened for the optimal combination of desired activity. Techniques for introducing substitution mutations at predetermined sites in DNA of known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Mutant screening is performed using antigen-binding activity assays, such as FLT3 or CD3 binding assays.

In general, when substituting amino acids in one or more or all regions of the heavy and/or light chain CDRs, it is preferred that the substitution sequence thus obtained be at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and most preferably 90% or 95% identical to the original CDR sequence. This means that the degree of sequence identity with substitutions depends on the length of the CDR region. For example, a CDR region containing 5 amino acids is preferably 80% identical to the corresponding substitution sequence so that at least one amino acid can be substituted. Accordingly, the CDR regions of a given antibody construct may differ in varying degrees of identity to the corresponding sequences with substitutions, for example, CDRL1 may be 80% identical, and CDRL3 may be 90% identical.

Preferred substitutions (or substitutions) are conservative substitutions. However, any substitution (including non-conservative substitution, or one or more of the examples of substitutions shown in Table 1 below) is contemplated, so long as the antibody construct retains the ability to bind to FLT3 via the first binding domain and to CD3 or CD3 epsilon via a second binding domain; and/or its CDR regions are identical to the resulting corresponding substitution sequence (at least 60 or 65%, more preferably 70 or 75%, even more preferably 80 or 85%, and most preferably 90 or 95% identical to the original region sequence CDR).

Conservative substitutions are given in table. 1 in the column headed preferred substitutions. In cases where such substitutions lead to a change in biological activity, more significant changes can be introduced, called examples of substitutions in table. 1 or additionally provided below when describing classes of amino acids, and products can be screened for the desired characteristic.

Table 1. Amino acid substitutions

Source amino acid Substitution examples Preferred substitutions Ala (A) val, leu, ile val Arg(R) lys, gin, asn lys Asn(N) gin, his, asp, lys, arg gin Asp(D) glu, asn glu Cys (C) ser, ala ser Gin (Q) asn, glu asn Glu(E) asp, gin asp Gly (G) Ala ala His(H) asn, gin, lys, arg arg He(I) leu, val, met, ala, phe leu Leu(L) norleucine, ile, val, met, ala ile Lys (K) arg, gin, asn arg Met(M) leu, phe, ile leu Phe(F) leu, val, ile, ala, tyr tyr Pro (P) Ala ala Ser(S) Thr thr Thr(T) Ser ser TRP(W) tyr, phe tyr Tyr (Y) trp, phe, thr, ser phe Val(V) ile, leu, met, phe, ala leu

Substantial modifications to the biological properties of an antibody construct of the present invention are made by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of substitution, e.g., in a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the main part of the side chain. Naturally occurring residues are divided into groups based on the general properties of the side chains: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

Non-conservative substitutions entail the replacement of a representative of one of the indicated classes by a representative of another class. Any cysteine residue not involved in maintaining the proper conformation of the antibody construct can usually be replaced by serine to increase the oxidative stability of the molecule and prevent aberrant crosslinking. At the same time, a cysteine bond or bonds may be added to the antibody to improve stability (particularly if the antibody is an antibody fragment, such as an Fv fragment).

Amino acid sequence identity and/or similarity is determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (Smith and Waterman, 1981, Adv. Appl. Math. 2: 482), Needleman and Wunsch's sequence identity alignment algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443), Pearson and Lipman's similarity search method (Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444), computerized versions of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin), Best Fit sequence alignment programs as described in the source: Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using default settings or by inspection. Preferably, percent identity is calculated using FastDB based on the following parameters: mismatch penalty=1; penalty for skipping in sequence^; pass penalty=0.33; pooling penalty=30, Current Methods in Sequence Comparison and Analysis, Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

An example of a suitable algorithm is PILEUP. PILEUP creates multiple alignment sequences from a group of related sequences using progressive pairwise alignment. It also allows you to build a tree that reflects the cluster relationships used for alignment. PILEUP uses the simplified progressive alignment method of Feng & Doolittle, 1987, J. Mol. Evol. 35:351-360; this method is similar to that described in the source: Higgins and Sharp, 1989, CABIOS 5:151-153. Suitable PILEUP parameters include default gap insertion penalty=3.00, default gap lengthening penalty=0.10, and trailing gap penalty.

Another example of a suitable algorithm is the BLAST algorithm described in Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402 and Karin et al., 1993, Proc. Natl. Acad. sci. U.S.A. 90:5873-5787. In particular, a suitable BLAST algorithm program is the WU-BLAST-2 program derived from: Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search options, most of which are set to default. The following values of the adjustable parameters are set: overlap length=1, overlap fraction=0.125, threshold word length (T)=L. Parameters HSP S and HSP S2 take dynamic values set by the program itself depending on the composition of a particular sequence and the composition of a particular database searched for the sequence of interest; however, these values may be adjusted to increase sensitivity.

An additional suitable algorithm is the Gapped BLAST algorithm according to the source: Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses the substitution scores from BLOSUM-62; threshold T=9; two-match method for launching extensions without gaps, price for extensions of gaps of length k=10+k; Xu=16; Xg=40 for the database search stage and up to 67 for the data derivation stage of the specified algorithms. Gap alignments are triggered based on an estimate corresponding to approximately 22 bits.

In general, the homology, similarity, or amino acid identity of the individual variants of the CDR regions is at least 60% relative to the sequences provided herein, more typically, the homology or identity is preferably increased to at least 65% or 70%, more preferably at least up to 75% or 80%, even more preferably at least up to 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and up to almost 100%. Similarly, percent (%) nucleic acid sequence identity relative to the nucleic acid sequence of the binding proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to nucleotide residues in the coding sequence of a specified antibody construct. The specific method uses the BLASTN module of the WU-BLAST-2 package with default parameter settings, with the overlap length and overlap ratio set to 1 and 0.125, respectively.

In general, the homology, similarity, or identity of nucleic acid sequences encoding individual variants of the CDR regions and nucleotide sequences provided herein is at least 60%, and more typically, the homology or identity is preferably increased to at least 65, 70 , 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% and almost up to 100%. Accordingly, a CDR variant is a variant with a given homology, similarity, or identity relative to the original CDR region of the present invention, and retains biological function, including, but not limited to, retains at least 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% specificity and/or activity of the original CDR region.

In one embodiment, the percent identity of human germline and antibody constructs of the present invention is >70% or >75%, more preferably >80% or >85%, even more preferably >90%, and most preferably >91 %, >92%, >93%, >94%, >95% or even >96%. See Example 8. Identity to antibodies that are products of human germline genes is believed to be an important feature to reduce the risk of therapeutic proteins stimulating an anti-drug immune response in a patient during treatment. Hwang and Foote (Hwang & Foote, Immunogenicity of engineered antibodies; Methods 36 (2005) 3-10) demonstrate that reduction of non-human portions of antibody drug constructs results in a reduced risk of anti-drug antibody induction in patients during treatment. Comparison of an exhaustive number of clinically evaluated antibody-based drugs and corresponding immunogenicity data revealed a trend of proteins becoming less immunogenic when antibody V-regions were humanized (mean 5.1% of patients) compared to antibodies carrying g unaltered unaltered human V-regions (mean 23.59% of patients). Thus, it is desirable to provide V-region-based protein therapeutics in the form of antibody constructs having a higher degree of identity to human sequences. For this purpose of determining germline identity, the V-region of the VL can be aligned with the amino acid sequences of the human V-segments and J-segments of the human germline.

- 36 040387 (http://vbase.mrc-cpe.cam.ac.uk/) using the Vector NTI software, and amino acid sequence calculated by dividing identical amino acid residues by the total number of VL amino acid residues in percent. The same can be done for the VH segments (http://vbase.mrc-cpe.cam.ac.uk/) except that the VH CDR3 may be excluded due to significant diversity and lack of existing partners to align VH CDR3 germline human lines. Thereafter, recombinant techniques can be used to increase sequence identity to the germline genes of human antibodies.

In a further embodiment, the bispecific antibody constructs of the present invention exhibit high levels of monomer yields under standard assay conditions, eg, a standard two-step purification process. Preferably, the monomer yield of the antibody constructs of the present invention is >0.25 mg/l supernatant, more preferably >0.5 mg/l, even more preferably >1 mg/l, and most preferably >3 mg/l l supernatant.

Similarly, the yield of the dimeric isoform of the antibody construct and, accordingly, the percentage of monomers (ie, monomer: (monomer+dimer)) of the antibody constructs can be determined. Evaluation of the productivity of the monomeric and dimeric antibody constructs and the calculated percentage of monomers can, for example, be obtained from the purification step by size exclusion chromatography (SEC) of the culture supernatant on a standardized assay scale in roller bottles. In one embodiment, the percentage of antibody monomeric constructs is >80%, more preferably >85%, even more preferably >90%, and most preferably >95%.

In one embodiment, the antibody design has a preferred plasma stability (ratio of EC ₅₀ with plasma to EC ₅₀ without plasma) of <5 or <4, more preferably -<3.5 or <3, even more preferably -<2.5 or <2, and most preferably -<1.5 or <1. Plasma stability of an antibody construct can be tested by incubating said construct in human plasma at 37° C. for 24 hours, followed by determination of the EC ₅₀ in a chromium-51 release cytotoxicity assay. The effector cells in the cytotoxicity assay can be stimulated CD8-positive enriched human T cells. Target cells may, for example, be CHO cells transfected with human FLT3. The selected ratio of effector to target cells (E:T) may be 10:1. The pool of human plasma used for this purpose comes from the blood of healthy donors collected using EDTA-coated syringes. Cellular components are removed by centrifugation and the upper plasma phase is collected and then pooled. As a control, immediately prior to construct cytotoxicity analysis, antibodies are diluted in RPMI-1640 medium. Plasma stability is calculated as the ratio of EC ₅₀ (after incubation with plasma) to EC ₅₀ (control). See example 13.

It is also preferred that the conversion of the antibody constructs of the present invention from monomers to dimers is negligible. Said conversion can be measured under various conditions and analyzed using high performance size exclusion chromatography. See example 11. For example, incubation of monomeric isoforms of antibody constructs can be carried out for 7 days at 37° C. and concentrations of, for example, 100 μg/ml or 250 μg/ml in an incubator. Preferably, under these conditions, the percentage of dimers of the antibody constructs of the present invention is <5%, more preferably -<4%, even more preferably <3%, even more preferably -<2.5%, even more preferably -<2%, even more preferably <1.5% and most preferably <1% or <0.5% or even 0%.

It is also preferred that very little conversion to form dimers of the bispecific antibody constructs of the present invention be observed after several freeze/thaw cycles. For example, the monomer concentration of the indicated antibody construct is adjusted to 250 μg/ml, for example, in a conventional receptor buffer, and subjected to three freeze/thaw cycles (freeze at -80°C for 30 min followed by thawing for 30 min at room temperature), followed by high performance SEC to determine the initial percentage of antibody construct monomers that have converted to dimers. Preferably, the percentage of dimers of the bispecific antibody constructs is <%, more preferably -<4%, even more preferably -<3%, even more preferably -<2.5%, even more preferably -<2%, even more preferably -<1, 5% and most preferably <1% or even <0.5%, eg after three freeze/thaw cycles.

The bispecific antibody constructs of the present invention preferably exhibit favorable thermal stability with aggregation temperatures of >45°C or >50°C, more preferably ->52°C or >54°C, even more preferably ->56°C or >57° C, and most preferably ->58°C or >59°C. The thermal stability index can be determined from the aggregation temperature of the antibody as described below: The antibody solution at a concentration of 250 μg/ml is transferred into a disposable cuvette and placed in a dynamic light scattering (DLS) device.

- 37 040387

The sample is heated from 40 to 70°C at a rate of 0.5°C/min with constant registration of the measured radius. The increase in radius, which indicates protein melting and aggregation, is used to calculate the aggregation temperature of the antibody. See example 12.

Alternatively, melting point curves can be determined using differential scanning calorimetry (DSC) to determine the inherent biophysical stability of antibody construct proteins. These experiments were carried out using a MicroCal LLC (Northampton, Massachusetts, USA) VP-DSC device. The energy absorption of the sample containing the antibody construct is recorded at 20 to 90° C. and compared with the sample containing the formulation buffer alone. The antibody constructs are brought to a final concentration of 250 μg/ml, for example, in running SEC buffer. To record the appropriate melting curve, the total temperature of the sample is gradually increased. At each temperature T, the energy absorption of the sample and the reference formulation buffer is recorded. Plot the difference in energy absorption Cp (kcal/mol/°C) between the sample and the reference buffer versus the corresponding temperature. The melting point is defined as the temperature at the first energy absorption maximum.

It is also contemplated that the anti-FLT3xCD3 bispecific antibody constructs of the present invention do not cross-react (i.e., do not substantially bind) with human FLT3 paralogs KIT v1 (SEQ ID NO: 805), CSF1R v1 (SEQ ID NO: 806) , PDGFRA (SEQ ID NO: 807) and/or NTM v3 (SEQ ID NO: 808). In addition, it is contemplated that the anti-FLT3xCD3 bispecific antibody constructs of the present invention do not cross-react (i.e., do not substantially bind) with macaque/cynomolgus monkey FLT3 paralogs KIT v1, CSF1R v1, PDGFRA and/or NTM v3. See example 7.

It is also contemplated that the anti-FLT3xCD3 bispecific antibody constructs of the present invention have a turbidity (estimated by OD340 after concentration of the purified monomeric antibody construct to 2.5 mg/mL and overnight incubation) of <0.2, preferably -<0.15 , more preferably -<0.12, even more preferably -<0.1, and most preferably -<0.08. See example 14.

In a further embodiment, an antibody construct of the present invention is stable at acidic pH values. The more tolerant said antibody construct behaves at non-physiological pH values, such as pH 5.5 (the pH value required to perform, for example, cation exchange chromatography), the higher the recovery rate of said antibody construct when eluted from an ion exchange column relative to the total amount of protein loaded . The recovery rate of said antibody construct on an ion exchange (e.g. cation exchange) column at pH 5.5 is preferably >30%, more preferably >40%, more preferably >50%, even more preferably >60%, even more preferably - >70%, even more preferably ->80%, even more preferably ->90%, even more preferably ->95% and most preferably ->99%. See example 15.

It is also contemplated that the bispecific antibody constructs of the present invention exhibit therapeutic efficacy or antitumor activity. Its evaluation can, for example, be carried out during a study according to the following example of a xenograft model of a common human tumor:

On study day 1, 5x106 cells of a FLT3 positive human cancer cell line (eg, OVCAR-8) are injected subcutaneously into the dorsal right flank of female NOD/SCID mice. After the average tumor size reaches approximately 100 mm ³ , mice are transplanted with in vitro expanded human CD3 positive T cells by injecting approximately 2x107 cells into the abdominal cavity of the animals. Mice in the base-treated control group 1 do not receive effector cells and are used as controls without transplantation to compare with the base-treated control group 2 (which receive effector cells) to monitor the effect on tumor growth of T cells alone. Antibody treatment is started when the mean tumor size reaches approximately 200 mm ³ . The value of the mean tumor size in each treatment group on the day of treatment initiation should not be statistically different from the value in any other group (analysis of variance). Mice are treated with the FLT3xCD3 bispecific antibody construct at 0.5 mg/kg/day by intravenous bolus injection for approximately 15-20 days. Tumors are measured with a caliper during the course of the study and progression is assessed by intergroup comparison of tumor volumes (TV). Tumor growth inhibition T/C [%] is determined by calculating TV as T/C%=100χ(average TV in test group)/(average TV in control group 2).

The skilled artisan knows how to modify or correct certain parameters of a given study, such as the number of tumor cells injected, the site of injection, the number of transplanted human T cells, the number of bispecific antibody constructs to be administered, and the time frame, while still being able to obtain a meaningful and reproducible

- 38 040387 results. Preferably, tumor growth inhibition T/C [%] is <70 or <60, more preferably <50 or <40, even more preferably <30 or <20 and most preferably

-<10 or <5, or even <2.5.

The present invention also provides a polynucleotide/nucleic acid molecule encoding an antibody construct of the present invention.

A polynucleotide is a biopolymer consisting of 13 or more nucleotide monomers connected in a chain by covalent bonds. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides with separate biological functions. Nucleotides are organic molecules that serve as monomers or subunits of nucleic acid molecules such as DNA or RNA. The nucleic acid molecule or polynucleotide can be double-stranded and single-stranded, linear and circular. It is preferably in a vector, which is preferably in a host cell. Said host cell, for example, after transformation or transfection with a vector or polynucleotide of the present invention, is capable of expressing an antibody construct. For this purpose, a polynucleotide or nucleic acid molecule is operably linked to control sequences.

The genetic code is a set of rules according to which the information encoded in the genetic material (nucleic acids) is translated into proteins. Biological decoding in living cells is carried out by the ribosome, which combines the amino acids in the order specified by the mRNA, using tRNA molecules to transport the amino acids and to read three nucleotides of the mRNA at a time. The specified code determines, given by the sequences of the specified nucleotide triplets, which are called codons, the order in which each successive amino acid is added during protein synthesis. With few exceptions, a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid mutation. Since the vast majority of genes code for a completely identical code, said particular code is often referred to as the canonical or standard genetic code. The genetic code determines the protein sequence for a given coding region, while other genomic regions may influence when and where said proteins are produced.

In addition, according to the present invention, a vector containing a polynucleotide/nucleic acid molecule according to the present invention is provided.

A vector is a nucleic acid molecule used as a basis for transferring (foreign) genetic material into a cell. The term vector includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. In general, the constructed vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is usually a sequence of nucleotides, often a DNA sequence, which contains an insert (transgene) and a larger sequence which serves as the backbone of said vector. Modern vectors may have additional features besides the transgene insert and backbone: a promoter, a genetic marker, antibiotic resistance, a reporter gene, a targeting sequence, a tag for protein purification. Vectors, referred to as expression vectors (expression constructs), in particular, are designed to express the transgene in the target cell, and usually contain control sequences.

The term control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to use promoters, polyadenylation signals, and enhancers.

A nucleic acid is operably linked if it is in a state of functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to a polypeptide's DNA if it is expressed as a precursor protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of said sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In general, operably linked means that the DNA sequences to be combined are contiguous and, in the case of a secretory leader sequence, contiguous and in-frame. However, enhancers do not have to be continuous. The association is carried out by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with standard practice.

Transfection is the process of intentionally introducing nucleic acid molecules or polynucleotides (including vectors) into target cells. This term is basically

- 39 040387 is used to refer to virus-free methods for eukaryotic cells. The term transduction is often used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening temporary pores or holes in the cell membrane to allow uptake of the material. Transfection can be carried out using calcium phosphate, by electroporation, cell compression, or by mixing a cationic lipid with material to produce liposomes that fuse with the cell membrane to deliver their contents.

The term transformation is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, as well as into non-animal eukaryotic cells, including plant cells. Transformation is thus a genetic change in a bacterial or non-animal eukaryotic cell resulting from direct uptake through the cell membrane(s) from the environment and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be realized using artificial methods. To effect transformation, cells or bacteria must be in a state of competence, which may be a time-limited response to environmental conditions such as starvation and cell density.

In addition, the present invention provides a host cell transformed or transfected with a polynucleotide/nucleic acid molecule or a vector of the present invention.

As used herein, the terms host cell or recipient cell include any individual cells or cell culture that can be or have been recipients of vectors, exogenous nucleic acid molecules, and polynucleotides encoding an antibody construct of the present invention; and/or recipients of said antibody construct itself. The introduction of the appropriate material into the cell is carried out by transformation, transfection, and the like. The term host cell is also intended to include the progeny or potential progeny of a single cell. Since certain modifications may occur in subsequent generations due to natural, random or intentional mutation or environmental influences, such progeny may not actually be completely identical (in morphology or in genomic or total DNA set) to the original cell, but are also included in the scope term according to this document. Suitable host cells include prokaryotic or eukaryotic cells, and include, but are not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells such as insect cells and mammalian cells, such as mice, rats, macaques, or humans.

The antibody construct of the present invention may be produced by bacteria. After expression, the antibody construct of the present invention is isolated from the E. coli cell paste as a soluble fraction; it can be purified by, for example, affinity chromatography and/or size exclusion. The final purification may be carried out in a manner analogous to the purification of an antibody expressed, for example, in CHO cells.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable hosts for cloning or expression of an antibody construct of the present invention. Of the lower eukaryotic microorganisms, Saccharomyces cerevisiae, or ordinary baker's yeast, is most often used as hosts. However, a number of other genera, species and strains are publicly available and suitable for the purposes of the present invention, such as Schizosaccharomyces pombe, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans and K. marxianus; Yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, e.g. Schwanniomyces occidentalis; filamentous fungi such as Neurospora, Penicillium, Tolypocladium; and Aspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for expression of the glycosylated antibody construct of the present invention are from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants have been identified, and corresponding permissive insect cell hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori. Various viral strains are available for transfection, such as Autographa californica NPV variant L-1 and Bombyx mori nuclear polyhedrosis virus (NPV) strain Bm-5, and such viruses can be used in accordance with the present invention as described herein, in in particular, as a virus for transfection of Spodoptera frugiperda cells.

Plant cell cultures such as cotton, corn, potato, soybean, petunia, tomato, Arabidopsis and tobacco can also be used as hosts. Cloning and

- 40 040387 expression vectors suitable for use in the production of proteins in plant cell culture are known to those skilled in the art. See, for example, sources: Hiatt et al., Nature (1989) 342: 76-78, Owen et al. (1992) Bio/Technology 10: 790-794, Artsaenko et al. (1995) The Plant J 8: 745750 and Fecker et al. (1996) Plant Mol Biol 32: 979-986.

However, vertebrate cells were of maximum interest, and propagation of vertebrate cells in culture (tissue culture) became a routine procedure. Examples of suitable mammalian host lines are the CV1 monkey kidney cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); newborn hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, 1413 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL5 1); TRI cells (Mather et al., Annals N. Y Acad. Sci. (1982) 383: 44-68); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

In a further embodiment, the present invention provides a method for producing an antibody construct of the present invention, comprising culturing a host cell of the present invention under conditions that allow expression of said antibody construct of the present invention and isolating the resulting antibody construct from the culture.

As used herein, the term culture refers to the in vitro maintenance, differentiation, growth, proliferation and/or propagation of cells in a medium under suitable conditions. The term expression includes any step involved in the production of antibody constructs of the present invention, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

Using recombinant techniques, said antibody construct can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. In the event that said antibody construct is produced intracellularly, the first step is to remove debris particles originating from host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Source: Carter et al., Bio/Technology 10: 163167 (1992) describes a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cellular debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatants in such expression systems are typically first concentrated using a commercially available protein concentration filter, such as an ultrafiltration unit from Amicon or Millipore Pellicon. Any of the above steps may include the use of a protease inhibitor such as PMSF to inhibit proteolysis; antibiotics may be included to prevent the growth of incidental contaminants.

An antibody construct of the present invention derived from host cells can be isolated or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Other protein purification techniques are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin sepharose (SEPHAROSE™) chromatography, anion exchange or cation exchange resin chromatography (e.g., polyaspartic acid column). ), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation, depending on the antibody to be isolated. When an antibody construct of the present invention includes a CH3 domain, Bakerbond ABX resin (J.T. Baker, Phillipsburg, NJ) is suitable for purification.

Affinity chromatography is the preferred purification technique. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrendivinyl)benzene provide higher flow rates and shorter processing times than are achievable with agarose.

In addition, the present invention provides a pharmaceutical composition comprising an antibody construct of the present invention or an antibody construct produced according to the method of the present invention. For the pharmaceutical composition of the present invention, it is preferred that the homogeneity of said antibody construct be >80%, more preferably >81%, >82%, >83%, >84% or >85%, more preferably

- 41 040387 >86%, >87%, >88%, >89% or >90%, even more preferably >91%, >92%, >93%, >94% or >95% and most preferably >96 %, >97%, >98% or >99%.

As used herein, the term pharmaceutical composition refers to a composition that is suitable for administration to a patient, preferably a human patient. In particular, the preferred pharmaceutical composition according to the present invention contains one or a combination of these constructs of antibodies(a) according to the present invention, preferably in a therapeutically effective amount. Preferably, one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and/or adjuvants are additionally included in the formulation with said pharmaceutical composition. Acceptable constituents of said composition are preferably non-toxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions according to the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

Proposed according to the present invention the composition may contain a pharmaceutically acceptable carrier. In general, as used herein, a pharmaceutically acceptable carrier means any and all aqueous and non-aqueous solutions, sterile solutions, diluents, buffers such as phosphate buffered saline (PBS), water, suspensions, emulsions such as oil/water emulsions, various types wetting agents, liposomes, dispersion media and coatings that are compatible with pharmaceutical administration, in particular parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be prepared using well known standard methods.

In certain embodiments, pharmaceutical compositions are provided comprising an antibody construct of the present invention and further comprising one or more excipients, such as those illustratively described in this section and elsewhere herein. However, according to the present invention, excipients can be used for a variety of purposes, for example, to correct the physical, chemical or biological properties of the compositions, for example, to correct the viscosity, and / or methods according to the present invention, to improve the efficiency and / or stabilization of such compositions and methods with protection against degradation and deterioration resulting, for example, from stresses during production, transport, storage, preparation before use and administration, and subsequently.

In some embodiments, said pharmaceutical composition may contain formulation materials designed to modify, maintain, or retain, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, aroma, sterility, stability, rate of dissolution or release, adsorption or permeation. compositions (see REMINGTON'S PHARMACEUTICAL SCIENCES, ^18th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company). In such embodiments, suitable formulation materials may include, but are not limited to:

amino acids such as glycine, alanine, glutamine, asparagine, threonine, proline, 2-phenylalanine, including charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine antimicrobial agents such as antibacterial and fungicidal agents antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium hydrosulfite;

buffers, buffer systems and buffering agents that are used to maintain physiological or slightly lower pH values in the composition, typically in the pH range from about 5 to about 8 or 9; examples of buffers are borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids, succinate, phosphate, histidine and acetate; for example, a Tris buffer with a pH of about 7.0-8.5, or an acetate buffer with a pH of about 4.0-5.5;

non-aqueous solvents such as propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate;

aqueous vehicles, including water, alcoholic/aqueous solutions, emulsions or suspensions, including saline solutions and buffered media;

biodegradable polymers such as polyesters;

bulking agents such as mannitol or glycine;

chelating agents such as ethylenediaminetetraacetic acid (EDTA);

isotonic and absorption delaying agents;

complexing agents such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin) excipients;

monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); the carbonic waters may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol;

(low molecular weight) proteins, polypeptides or protein-like carriers such as human serum albumin or bovine, gelatin or immunoglobulins, preferably human-derived;

coloring and flavoring agents;

sulfur-containing reducing agents such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol and sodium thiosulfate diluting agents;

emulsifying agents;

hydrophilic polymers such as polyvinylpyrrolidone, salt-forming counterions such as sodium;

preservatives such as antimicrobials, antioxidants, chelating agents, inert gases, and the like;

examples are benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);

metal complexes, such as complexes of proteins with Zn;

solvents and co-solvents (such as glycerol, propylene glycol or polyethylene glycol);

sugars and sugar alcohols such as trehalose, sucrose, octasulfate, mannitol, sorbitol or xylitol, stachyose, mannose, sorbose, xylose, ribose, myoinositose, galactose, lactitol, ribitol, myoinositol, galactitol, glycerol, cyclitols (e.g. inositol), polyethylene glycol; and polyhydric sugar alcohols;

suspending agents;

surfactants or wetting agents such as pluronics, PEGs, sorbitol esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol; surfactants can be detergents, preferably having a molecular weight >1.2 kDa, and/or a polyether, preferably having a molecular weight >3 kDa;

non-limiting examples of preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples of preferred polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000;

stability enhancing agents such as sucrose or sorbitol;

tonicity enhancing agents such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol;

parenteral delivery vehicles, including sodium chloride solution, dextrose Ringer's solution, dextrose and sodium chloride solution, lactated Ringer's solution, or fixed oils;

bases for intravenous delivery, including replenishers of fluid and nutrients, replenishers of electrolytes (for example, based on Ringer's solution with dextrose).

Those of skill in the art will appreciate that different constituents of a pharmaceutical composition (eg, those listed above) may have different effects, eg, an amino acid may act as a buffer, stabilizer, and/or antioxidant; mannitol can function as a bulking agent and/or tonicity agent; sodium chloride can function as a delivery base and/or tonicity enhancing agent, and the like.

It is envisaged that the composition according to the present invention may contain, in addition to the polypeptide according to the present invention as defined herein, additional biologically active agents, depending on the intended use of said composition. Such agents may be drugs that act on the gastrointestinal tract, drugs that act as cytostatics, drugs that prevent hyperuricemia, drugs that inhibit immune responses (for example, corticosteroids), drugs that modulate the inflammatory response, drugs that act on the circulatory system, and/or agents such as cytokines known in the art. Also contemplated is the use of the antibody construct of the present invention in co-therapy, i. in combination with another anti-cancer drug.

In some embodiments, the optimal pharmaceutical composition is determined by one of skill in the art based on, for example, the intended route of administration, delivery format, and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, supra. In some embodiments, such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of an antibody construct of the present invention. In some embodiments, the nature of the primary base or carrier in the pharmaceutical composition may be aqueous or non-aqueous. For example, a suitable vehicle or carrier may be water for injection, physiological saline, or artificial cerebrospinal fluid, with the possible addition of other materials commonly used in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are further examples of bases. In some embodiments, compositions containing an antibody construct of the present invention can be prepared for storage by mixing the selected composition, having the desired purity, with optional prescription agents (REMINGTON'S PHARMACEUTICAL SCIENCES, supra) in the form of a compacted lyophilisate or aqueous solution. In addition, in some embodiments, the antibody construct of the present invention may be formulated as a lyophilisate using suitable excipients such as sucrose.

Where parenteral administration is contemplated, therapeutic compositions for use according to the present invention may be presented in the form of a pyrogen-free parenteral acceptable aqueous solution containing the desired antibody construct of the present invention in a pharmaceutically acceptable vehicle. In particular, a suitable base for parenteral injection is sterile distilled water; in this case, the formulation containing the antibody construct of the present invention is a sterile isotonic solution, properly protected. In some embodiments, upon formulation, the desired molecule may be formulated with an agent, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules, or liposomes, which may provide a controlled or sustained release of the product, which can be delivered by depot injection. In some embodiments, hyaluronic acid may also be used, the effect of which is to increase the duration of stay in the bloodstream. In some embodiments, drug delivery via implantable devices may be used to introduce the desired antibody construct.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations containing an antibody construct of the present invention as sustained or controlled delivery/release formulations. Techniques for preparing a variety of other sustained or controlled delivery formulations, such as liposomal vehicles, biodegradable microparticles or porous granules, and injectable depots, are also known to those skilled in the art. See, for example, International Patent Application No. PCT/US93/00829, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions. Sustained release formulations may include semi-permeable polymeric matrices in the form of shaped objects such as films or microcapsules. Extended release matrices may include polyesters, hydrogels, polylactides (as described in US Pat. No. 3,773,919 and EP 058481 published), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman et al., 1983, Biopolymers 2:547-556), poly(2-hydroxyethyl methacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech. 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra), or poly-D(-)-3-hydroxybutyric acid (EP Published Application No. EP 133988). Sustained release formulations may also include liposomes, which can be prepared using any of a number of methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. sci. U.S.A. 82: 3688-3692; published European Patent Application No. EP 036676; EP 088046 and EP 143949.

Said antibody construct may also be encapsulated in microcapsules, for example, using coacervation or interfacial polymerization techniques (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., , liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in: Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

Pharmaceutical compositions used for in vivo administration are typically sterile formulations. Sterilization can be carried out by filtration through sterilizing filtration membranes. If lyophilization of the composition is carried out, sterilization using said method may be carried out either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Compositions for parenteral administration are usually placed in a container with a sterile access device, such as an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic needle.

According to another aspect of the present invention, self-buffering formulations containing an antibody construct of the present invention are provided that can be used as pharmaceutical compositions as described in international patent application WO 06138181A2 (PCT/US 2006/022599). Descriptions of protein stabilization, formulation materials, and methods suitable for use in these instances are provided in various sources such as Arakawa et al.: Solvent interactions in pharmaceutical formulations, Pharm Res. 8(3): 285-91 (1991); Ken

- 44 040387 drick et al., Physical stabilization of proteins in aqueous solution in Rational Design of Stable Protein Formulations: Theory and Practice; Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al., Surfactant-protein interactions, Pharm Biotechnol. 13:159-75 (2002); see in particular the passages relating to excipients and related methods for the preparation of self-buffering protein formulations of the present invention, in particular proteinaceous pharmaceutical products and methods, for veterinary and/or human medical use.

Salts can be used in accordance with certain embodiments of the present invention, for example, to correct the ionic strength and/or isotonicity of the composition and/or to improve the solubility and/or physical stability of the protein or other ingredient of the composition according to the present invention. As is well known, ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein, screening charged and polar groups in the protein, and reducing the strength of their electrostatic, attractive, and repulsive interactions. The ions can also stabilize the protein in a denatured state by binding, in particular, to the denatured peptide bonds (--CONH) of said protein. In addition, ionic interaction with charged and polar groups in a protein can also reduce intermolecular electrostatic interactions and thus prevent or reduce protein aggregation and insolubility.

The effects of ionic forms on proteins differ significantly. A multi-category classification of ions and their effects on proteins has been developed that can be used in formulating the pharmaceutical compositions of the present invention. One example is the Hofmeister series, which arranges ionic and polar non-ionic solutes based on their effect on the conformational stability of proteins in solution. Stabilizing solutes are called cosmotropic.

Destabilizing solutes are called chaotropic. Cosmotropes are widely used at high concentrations (eg, >1 M ammonium sulfate) to precipitate proteins from solution (salting out). Chaotropes are widely used for protein denaturation and/or solubilization (salting-in). The relative effectiveness of ions for salting in and salting out determines their position in the Hofmeister series.

In accordance with various embodiments of the present invention, free amino acids can be used in formulations containing an antibody construct of the present invention as bulking agents, stabilizers, and antioxidants, as well as other standard uses. Lysine, proline, serine and alanine can be used to stabilize the proteins in the formulation. Glycine is suitable for use in lyophilization to ensure the proper structure and properties of the compacted lyophilisate. Arginine may be suitable for inhibiting protein aggregation in both liquid and lyophilized formulations. Methionine is suitable for use as an antioxidant.

Polyols include sugars such as mannitol, sucrose and sorbitol, and polyhydric alcohols such as, for example, glycerol and propylene glycol, and as used herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotropes. They are suitable for use as stabilizing agents in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are also suitable for correcting the tonicity of formulations. Suitable polyols for use in some embodiments of the present invention are, among others, mannitol, widely used to provide structural stability of compacted lyophilisate in lyophilized formulations. It guarantees the structural stability of the compacted lyophilisate. As a rule, it is used together with a lyoprotectant, such as sucrose. Sorbitol and sucrose are preferred tonicity correcting agents and stabilizers for protection against freeze-thaw stress during shipping or bulk products during manufacture. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate the surface of lysine and arginine residues. Accordingly, in general, they are not among the preferred polyols for use in the present invention. In addition, sugars that form such reactive groups, such as sucrose, which hydrolyzes to form fructose and glucose under acidic conditions, and thus causes glycation, are also therefore not among the preferred polyols of the present invention. PEG is suitable for stabilizing proteins and as a cryoprotectant, and therefore can be used according to the present invention.

In embodiments, formulations containing an antibody construct of the present invention further comprise surfactants. Protein molecules can be prone to adsorption to surfaces as well as to denaturation and subsequent aggregation at air/liquid, solid/liquid, and liquid/liquid interfaces. These effects are usually inversely proportional to protein concentration. Specified destructive interactions

- 45 040387 are inversely proportional to protein concentration and are generally exacerbated by physical agitation, such as occurs during transport and handling of the product. Surfactants are often used to prevent, minimize or reduce surface adsorption. Suitable surfactants for this purpose include polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan polyethoxylates, and poloxamer 188. Surfactants are also widely used to control the conformational stability of proteins. The use of surfactants in this regard is protein-specific, as any given surfactant will generally stabilize some proteins and destabilize others.

Polysorbates are susceptible to oxidative degradation and, when supplied, often contain sufficient amounts of peroxides to oxidize the side chains of protein residues, in particular methionine. Accordingly, polysorbates must be used with caution and, if used, the lowest effective concentrations should be used. In this regard, polysorbates are an example of the general rule that excipients should be used at the lowest effective concentration.

In embodiments, formulations containing an antibody construct of the present invention additionally contain one or more antioxidants. Destructive oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining proper oxygen levels and ambient temperature and avoiding exposure to light. Antioxidant adjuvants may also be used to prevent oxidative degradation of proteins. Suitable antioxidants in this respect include reducing agents, oxygen/free radical scavengers, and chelating agents. Antioxidants for use in therapeutic protein formulations according to the present invention are preferably water soluble and remain active throughout the shelf life of the product. In this regard, EDTA is the preferred antioxidant according to the present invention. Antioxidants can damage proteins. For example, reducing agents such as glutathione in particular can break intramolecular disulfide bonds. Accordingly, the antioxidants for use according to the present invention are chosen, among other things, so as to eliminate or sufficiently reduce the possibility of damage to the proteins in the composition by themselves.

The compositions according to the present invention may include metal ions, which are protein cofactors necessary for the formation of protein coordination complexes, such as zinc, necessary for the formation of certain suspensions of insulin. Metal ions can also inhibit some processes that break down proteins. However, metal ions also catalyze the physical and chemical processes that break down proteins. Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to form isoaspartic acid. Ca ⁺² ions (at concentrations up to 100 mM) can increase the stability of human deoxyribonuclease. Mg ⁺² , Mn ⁺² and Zn ⁺² , however, can destabilize rhDNase. Similarly, Ca ⁺² and Sr ⁺² can stabilize factor VIII; it can be destabilized by Mg ⁺² , Mn ⁺² and Zn ⁺² , Cu ⁺² and Fe ⁺² , and its aggregation can be increased by Al ⁺³ ions.

Embodiments of formulations containing an antibody construct of the present invention further contain one or more preservatives. Preservatives are needed in the development of multi-dose parenteral formulations involving more than one extraction from a single container. Their primary function is to inhibit the growth of microorganisms and ensure the sterility of the product throughout the shelf life or duration of use of the medicinal product. Commonly used preservatives include benzyl alcohol, phenol and m-cresol. Although preservatives have a long history of use with small molecule parenterals, the development of protein formulations that include preservatives can be challenging.

Preservatives almost always have a destabilizing effect on proteins (cause aggregation), which has become the main factor limiting their use in multi-dose protein formulations. Until now, most protein drug formulations have been designed for single use only. However, where multi-dose formulations are possible, this provides additional benefits in terms of patient convenience and increased marketability. A good example is human growth hormone (hGH), the development of preservative formulations with which has led to the commercialization of more convenient reusable pen systems. There are currently at least four versions of such pen devices with preservative-containing hGH formulations on the market. Norditropin (Liquid, Novo Nordisk), Nutropin AQ (Liquid, Genentech), and Genotropin (lyophilized dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope (Eli Lilly) contains m-cresol. Several aspects must be taken into account in the production and development of preservative-containing dosage forms. The effective concentration of the preservative in the drug product must be optimized. This requires testing a given preservative in the medicinal product.

- 46 040387 form in the range of concentrations that provide antimicrobial efficacy, but do not violate the stability of the protein.

As one might expect, the development of liquid formulations containing preservatives is more difficult than the development of lyophilized formulations. Freeze-dried products can be lyophilized without preservative and reconstituted with preservative-containing diluent at the time of use. In this way, the period of time during which the preservative is in contact with the protein is shortened, which significantly minimizes the associated stability risks. In the case of liquid formulations, preservative effectiveness and stability must be maintained throughout the product's shelf life (approximately 18-24 months). It is important to note that the effectiveness of the preservative must be demonstrated in the final formulation containing the active drug and all excipients.

The antibody constructs as described herein may also be incorporated into immunoliposomes. A liposome is a small vesicle formed by various types of lipids, phospholipids and/or surfactants, which is suitable for drug delivery to a mammal. The components of a liposome are usually arranged in a bilayer similar to the arrangement of lipids in biological membranes. Liposomes containing said antibody construct are prepared using methods known in the art, for example as described in Epstein et al., Proc. Natl. Acad. sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl Acad. sci. USA 77: 4030 (1980); U.S. Patent Nos. 4,485,045 and 4,544,545 and WO 97/38731. Liposomes with extended circulation time are described in US Pat. No. 5,013,556. In particular, suitable liposomes can be obtained by reverse phase evaporation of a lipid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters with pores of a certain size to obtain liposomes of the desired diameter. Fab' fragments of the antibody construct of the present invention can be conjugated to liposomes as described in Martin et al. J Biol. Chem. 257: 286-288 (1982), via a disulfide exchange reaction. The liposome optionally contains a chemotherapeutic agent. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

Once formulated, the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystalline, or dehydrated or lyophilized powder. Such formulations may be stored either in ready-to-use form or in a form (eg, lyophilized) intended to be reconstituted prior to administration.

The biological activity of a pharmaceutical composition described herein can be determined, for example, by performing cytotoxicity assays as described in the examples below in WO 99/54440 or in Schlereth et al., Cancer Immunol. Immunother. 20 (2005), 1-12. Efficacy or in vivo efficacy as used herein refers to response to therapy with a pharmaceutical composition of the present invention as assessed using, for example, National Cancer Institute (NCI) standardized response criteria. The success or in vivo efficacy of a therapy using a pharmaceutical composition of the present invention refers to the efficacy of the composition for its intended purpose, i.e. the ability of said composition to produce the desired effect, i. e. depletion of pathological cells, such as tumor cells. Monitoring of in vivo efficacy can be performed using generally accepted standard methods for assessing the relevant nosological units, including, but not limited to, counting leukocytes, counting formed elements, sorting cells with activated fluorescence, bone marrow aspiration. In addition, various disease-specific clinical biochemical parameters and other conventional standard methods can be used. In addition, computed tomography, X-ray, nuclear magnetic resonance imaging (for example, to evaluate based on the response criteria adopted by the National Cancer Institute [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher RI, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP Report of an international workshop to standardize response criteria for non- Hodgkin's lymphomas NCI Sponsored International Working Group J Clin Oncol 1999 Apr;17(4):1244]), positron emission tomography, leukocyte count, formed element count, fluorescence-activated cell sorting, bone marrow aspiration, biopsy/ histological examination of the lymph nodes, as well as various clinical biochemical parameters specific for lymphoma (for example, the level of lactate dehydrogenase), and other generally accepted standard measures. soby.

Another major difficulty in the development of drugs, such as the pharmaceutical composition of the present invention, is to ensure the predictability of the modulation of pharmacokinetic properties. To do this, the pharmacokinetic profile of the drug candidate can be established, i.e. a pharmacokinetic profile that affects the ability of a particular drug to provide treatment for a given condition.

- 47 040387

Pharmacokinetic parameters of a drug that affect the ability of a drug to provide treatment for a particular disease entity include, but are not limited to: half-life, volume of distribution, first pass metabolism in the liver, and degree of binding in blood serum. Each of the factors mentioned above can affect the effectiveness of a particular drug.

Half-life refers to the time required for 50% of an administered drug to be eliminated by biological processes such as metabolism, excretion, and the like. By first-pass metabolism in the liver is understood the predisposition of the drug to be metabolized upon first contact with the liver, i.e. during the first passage through the liver. The volume of distribution means the degree of retention of the drug in various compartments of the body, such as, for example, intracellular and extracellular space, tissues and organs, and the like, and the distribution of the drug within these compartments. The degree of binding in blood serum means the predisposition of the drug to interact and bind to blood serum proteins, such as albumin, resulting in a decrease or loss of the biological activity of said drug.

Pharmacokinetic parameters also include bioavailability, latency period (Tlag), T _max , rate of absorption, onset of action, and/or C _max for a given amount of drug administered. Bioavailability refers to the amount of drug in the blood compartment. Latent period means the period of delay between the administration of a drug and its detection and the ability to measure in blood or plasma. T _max is the time period after which the maximum blood concentration of the drug is reached, and C _max is the maximum blood concentration achieved for a given drug. For the period of time until the concentration of the drug in the blood or tissue, which is necessary to ensure its biological effect, is affected by all indicators. Pharmacokinetic performance of bispecific antibody constructs demonstrating cross-species specificity, which can be determined during preclinical testing in non-chimpanzee primate animals as described above, is also given, for example, in Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1-12).

In one embodiment, an antibody construct of the present invention or an antibody construct prepared according to the method of the present invention is provided for use in the prevention, treatment, or amelioration of hematologic cancer or metastatic cancer.

The compositions described herein are suitable as pharmaceutical compositions for the treatment, alleviation and/or prevention of a medical condition as described herein in a patient in need thereof. The term treatment refers to both therapeutic treatment and prophylactic or preventive measures. Treatment includes the use or administration of said composition into the body, isolated tissue or cell of a patient suffering from a disease/disorder, having a symptom of the disease/disorder, or a predisposition to the disease/disorder, to cure, heal, alleviate, alleviate, alter, eliminate, alleviate, improve or effect on said disease, symptom of said disease, or predisposition to said disease.

The term alleviation as used herein refers to any improvement in the disease state of a patient suffering from a tumor or cancer, or metastatic cancer as described hereinafter, by administering to a subject in need thereof the antibody constructs of the present invention. Such an improvement may also be expressed as slowing or stopping the progression of a patient's tumor or cancer or metastatic cancer. The term "prevention" as used herein means avoiding the occurrence or recurrence of a tumor or cancer or metastatic cancer in a patient, as described hereinafter, by administering to a subject in need thereof the antibody constructs of the present invention.

The term disease refers to any condition in which treatment with a specified antibody construct or pharmaceutical composition as described herein provides an advantage. This term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the disease in question.

A neoplasm is an abnormal growth of tissue, usually but not always forming a mass. In this case, the formation subsequently formed is usually called a tumor. Neoplasms or tumors can be benign, potentially malignant (precancerous), or malignant. Malignant neoplasms are generally referred to as cancer. They usually invade and destroy the surrounding tissue and may form metastases, i.e. spread to other parts, tissues or organs of the body. Thus, the term metastatic cancer encompasses metastases to other tissues or organs other than those of the original tumor. Lymphomas and leukemias are lymphoid neoplasms. For the purposes of the present invention, they are also encompassed by the terms tumor or cancer.

According to a preferred embodiment of the present invention, the hematological cancer is AML and the metastatic cancer may be derived from it.

Preferred neoplastic or cancerous diseases in the context of the present invention are selected from the group consisting of breast cancer, carcinoid tumor, cervical cancer, colon and rectal cancer, endometrial cancer, gastric cancer, head and neck cancer, mesothelioma, liver cancer, lung cancer , ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, kidney cancer and stomach cancer. More preferably, the neoplastic or cancerous disease, preferably a solid tumor, may be selected from the group consisting of ovarian cancer, pancreatic cancer, mesothelioma, lung cancer, gastric cancer and triple negative breast cancer. Metastatic cancer may be from any of the above diseases.

The present invention also provides a method for treating or ameliorating a hematologic cancer or a metastatic cancer comprising the step of administering to a subject in need thereof an antibody construct of the present invention or an antibody construct obtained in accordance with the method of the present invention.

The terms subject in need or subjects in need of treatment include subjects already suffering from the disorder as well as subjects in whom said disorder is intended to be prevented. A subject or patient in need includes a human and other mammalian subjects receiving prophylactic or therapeutic treatment.

Said antibody construct of the present invention is typically designed to take into account specific routes and routes of administration, specific dosages and frequencies of administration, specific treatment options for specific diseases, including bioavailability and resistance ranges. The materials of the composition are preferably formulated at concentrations suitable for the site of administration.

Accordingly, according to the present invention, formulations and compositions can be developed for delivery by any suitable route of administration. In the context of the present invention, routes of administration include, but are not limited to, the following:

topical (eg, dermal, inhalation, nasal, ocular, auricular/ear, vaginal, mucosal);

enteral (eg, oral, gastrointestinal, sublingual, sublabial, buccal, rectal); and parenteral (eg, intravenous, intraarterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, epidural, intrathecal, subcutaneous, intraperitoneal, extraamniotic, intraarticular, intracardiac, intradermal, intralesional, intrauterine, intravesical, intravitreal, percutaneous, intranasal, transmucosal, intraarticular, intraluminal ).

Pharmaceutical compositions and antibody constructs of the present invention are particularly suitable for parenteral administration, eg, subcutaneous or intravenous delivery, eg, by injection, such as a bolus injection, or by infusion, such as continuous infusion. Pharmaceutical compositions may be administered using a medical device. Examples of medical devices for the administration of pharmaceutical compositions are described in US Pat. Nos. 4,475,196; 4439196; 4447224; 4447233; 4486194; 4487603; 4596556; 4790824; 941880; 5064413; 5312335; 5312335; 5383851 and 5399163.

In particular, the present invention provides for the uninterrupted administration of a suitable composition. According to a non-limiting example, uninterrupted or substantially uninterrupted, i. e. continuous administration can be implemented using a small pump system worn by the patient to metered influx of the therapeutic agent into the patient's body. A pharmaceutical composition containing an antibody construct of the present invention may be administered using said pumping systems. Such pumping systems are well known in the art and typically involve the periodic replacement of cartridges containing the therapeutic agent for infusion. Replacing the cartridge in such a pumping system may entail a temporary interruption in the otherwise uninterrupted supply of the therapeutic agent to the patient's body. In such a case, it is still considered that the administration phase before cartridge replacement and the administration phase after cartridge replacement, within the meaning of the pharmaceutical methods and methods of the present invention, together provide uninterrupted administration of such a therapeutic agent.

Continuous or uninterrupted administration of the antibody constructs of the present invention may be administered intravenously or subcutaneously using a fluid delivery device.

- 49 040387 environments or pumping system of small size, including a mechanism for bringing the fluid into motion to ensure the movement of the fluid from the tank, and a trigger to start the moving mechanism. Pump systems for subcutaneous administration may include a needle or cannula to pierce the skin of a patient and deliver the appropriate composition to the patient's body. Said pumping systems can be fixed directly to the skin or attached to the patient's skin independently of a vein, artery or blood vessel, which provides direct contact between the pumping system and the patient's skin. The pump system may be attached to the patient's skin over a period of 24 hours to several days. The pumping system may be of small size and contain a small volume reservoir. According to a non-limiting example, the volume of the reservoir for a pharmaceutical composition suitable for administration can be from 0.1 to 50 ml.

Continuous administration may also be carried out transdermally by means of a patch that is worn on the skin and is to be replaced at regular intervals. One skilled in the art will be aware of patch-based drug delivery systems suitable for this purpose. It should be noted that transdermal administration is particularly suitable for uninterrupted administration, since the replacement of the first expended patch can advantageously be carried out simultaneously with the placement of a new second patch, for example, on the surface of the skin in the immediate vicinity of the first expended patch and immediately prior to the removal of the first expended patch. . There are no problems with intermittent supply or disconnection of the power supply.

In the event that the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in a suitable liquid prior to administration. The lyophilized material can be reconstituted, for example, in bacteriostatic water for injection (BWFI), physiological saline, phosphate buffered saline (PBS), or the same formulation as the protein prior to lyophilization.

Compositions of the present invention may be administered to a subject at a suitable dose, which may be determined, for example, in dose escalation studies with increasing doses demonstrating the cross-species specificity of the antibody constructs of the present invention described herein to non-chimpanzee primates, such as macaques. As described above, the cross-species specific antibody construct of the invention described herein can be advantageously used in an identical form in preclinical testing in non-chimpanzee primates and as a drug in humans. The dosage regimen is determined by the attending physician, taking into account clinical factors. As is well known in the medical arts, dosages for each patient depend on many factors, including patient size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and others. drugs that are administered at the same time.

The term effective dose or effective dosage is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term therapeutically effective dose is defined as an amount sufficient to cure or at least partially stop a disease and its complications in a patient already suffering from said disease. Amounts or doses effective for the indicated use depend on the condition (intention) to be treated, the antibody construct being delivered, the therapeutic conditions and goals, the severity of the disease, prior therapy, the patient's clinical history and response to the therapeutic agent, route of administration, size (body weight, body surface area or organ size) and/or the condition (age and general health) of the patient and the general condition of the patient's own immune system. The appropriate dose may be adjusted at the discretion of the attending physician so that it can be administered to the patient in a single dose or as a series of injections and provide optimal therapeutic effect.

A typical dosage may range from about 0.1 μg/kg to about 30 mg/kg or more, depending on the factors mentioned above. In specific embodiments, the dosage may range from 1.0 µg/kg to about 20 mg/kg, optionally from 10 µg/kg to about 10 mg/kg, or from 100 µg/kg to about 5 mg/kg.

A therapeutically effective amount of an antibody construct of the present invention preferably results in a reduction in the severity of symptoms of a disease, an increase in the frequency or duration of periods without symptoms of a disease, or the prevention of impairment or disability due to disease involvement. For the treatment of FLT3-expressing tumors, a therapeutically effective amount of an antibody construct of the present invention, e.g., an anti-FLT3/anti-CD3 antibody construct, preferably inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% relative to untreated patients. The ability of a compound to inhibit

- 50 040387 tumor growth can be assessed in an animal model to predict efficacy against human tumors.

The pharmaceutical composition may be administered as the sole therapeutic agent or, as necessary, in combination with additional therapies such as anti-cancer therapy, for example, with other protein-like and non-protein-like drugs. Said medicinal products may be administered simultaneously with a composition containing an antibody construct according to the invention described herein, or separately, before or after administration of said antibody construct, at specified times and at prescribed doses.

The term effective non-toxic dose as used herein refers to a tolerated dose of an antibody construct of the present invention that is sufficiently high to result in abnormal cell depletion, tumor elimination, tumor downsizing, or disease stabilization without or substantially without significant toxic effects. Such effective non-toxic doses can be determined, for example, during dose escalation studies described in the art, and should be below the dose that induces serious adverse events (dose-limiting toxicity, DLT).

The term toxicity as used herein refers to the toxic effects of a drug that appear as adverse events or serious adverse events. These side effects may relate to the lack of tolerance of the specified drug in general and/or the lack of local tolerance after administration. Toxicity may also include teratogenic or carcinogenic effects caused by the drug.

The term safety, in vivo safety, or tolerability as used herein refers to administration of a drug without inducing serious adverse events immediately after administration (local tolerance) and over a longer period of use of said drug. Safety, in vivo safety or tolerability can be assessed, for example, at regular intervals during treatment and during follow-up. Measurements include clinical assessment of, for example, organ manifestations and screening for laboratory abnormalities. Clinical evaluation may be performed and abnormalities recorded/coded according to NCICTC and/or MedDRA standards. Assessment of organ manifestations may include evaluation for criteria such as allergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulation, etc., as presented, for example, in the Common terminological criteria for adverse events, v3.0 (CTCAE). Laboratory values that may be tested include, for example, hematological, clinical chemistry, coagulogram, and urinalysis, as well as studies of other body fluids such as serum, plasma, lymphatic or cerebrospinal fluid, cerebrospinal fluid, and the like. The safety assessment can accordingly be carried out, for example, by physical examination, imaging techniques (i.e. ultrasound, x-ray, CT scan, magnetic resonance imaging (MRI), other measurements using technical devices (i.e. electrocardiogram) , by vital signs, changes in laboratory parameters and reporting of adverse events.For example, adverse events in non-chimpanzee primates using and in the methods of the present invention can be investigated by histopathological and/or histochemical methods.

The above terms are also mentioned, for example, in S6: Preclinical safety evaluation of biotechnology-derived Pharmaceuticals; Harmonized Tripartite ICH Guidelines; ICH Steering Committee meeting 16 July 1997

According to a further embodiment of the present invention, there is provided a kit comprising an antibody construct of the present invention, an antibody construct produced according to the method of the present invention, a polynucleotide of the present invention, a vector of the present invention, and/or a host cell of the present invention.

In the context of the present invention, the term kit means two or more components - one of which corresponds to the antibody construct, pharmaceutical composition, vector or host cell of the present invention - co-packaged in a container, recipient or otherwise. Thus, a kit can be described as a collection of products and/or accessories sufficient to achieve a particular purpose, which can be sold as a single kit.

The kit may contain one or more recipients (such as vials, ampoules, containers, syringes, vials, pouches) of any suitable shape, size, and any suitable material (preferably waterproof, such as plastic or glass) containing the antibody construct or pharmaceutical composition. according to the present invention in a suitable dosage for administration (see above). Said kit may further comprise instructions for use (for example, in the form of a booklet or instructions for use), means for administering the antibody construct of the present invention, such as a syringe, pump, infusion device, or the like, means for reconstituting an antibody construct of the present invention and/or a means for diluting an antibody construct of the present invention.

The present invention also provides kits containing single dose forms. The kit according to the present invention may also contain a first recipient containing a dried/lyophilized antibody construct and a second recipient containing an aqueous formulation. According to certain embodiments of the present invention, kits are provided containing single-chamber and multi-chamber pre-filled syringes (eg, liquid syringes and lyophilisate syringes).

Brief description of the drawings

Fig. 1: (a) Schematic representation of the structure of the monomeric FLT3 protein and (b) crystal structure of the FLT3 homodimer interacting with the FLT3LG homodimer.

Fig. 2: Schematic representation of the molecular structure of human/mouse chimeric FLT3 used to characterize the epitope cluster binding element.

Fig. 3: Schematic representation of the structure of the truncated FLT3 constructs used to characterize the epitope cluster-binding element.

Fig. 4: Analysis of the competition of the FLT3 binding element according to the present invention with the FLT3 ligand for FLT3 binding.

CHO cells transfected with human FLT3 were incubated in the presence and absence of 10 μg/ml FLT3 ligand for 30 min (no wash step). ScFv periplasmic preforms were added and incubated for 30 min. Detection was performed using mouse anti-FLAG + PE-conjugated goat anti-mouse and measuring mean fluorescence intensity by FACS (mean level (+ ligand)/mean level (no ligand) x 100%).

Fig. 5: FACS analysis of binding of selected cross-species specific bispecific single chain constructs to human FLT3 (hu) transfected CHO cells, HPBaLL human CD3+ T cell line, cynomolgus monkey FLT3 (cyno) transfected CHO cells, and LnPx macaque CD3+ T cell line 4119. Red line indicates cells incubated with 2 μg/ml of purified monomeric protein, which were then incubated with mouse anti-I2C and PE-labeled goat anti-mouse IgG for detection. The black line in the histogram corresponds to the negative control: cells incubated with anti-I2C antibody only, as well as PE-labeled detection antibody (see example 6).

Fig. 6: Cytotoxic activity induced by selected cross-species specific single chain constructs directed to CD56 depleted unstimulated human PBMCs as effector cells and human FLT3 transfected CHO cells as target cells (Example 9).

Fig. 7: Cross-reactive binding to CD3, FLT3 and its isoforms). 5 µg/ml protein BiTE: 4°C 60 min; 2 μg/ml anti-I2C antibody 3E5.A5: 4°C, 30 min; PE-conjugated goat anti-mouse antibody 1:100: 4°C, 30 min.

Fig. 8: No binding to paralogs and non-transfected CHOs. 5 µg/ml protein BiTE: 4°C 60 min; 2 μg/ml anti-I2C antibody 3E5.A5: 4°C, 30 min; PE-conjugated goat anti-mouse antibody 1:100: 4°C, 30 min.

Fig. 9: Mapping of epitope clusters - Cluster E1.

Fig. 10: Anti-FLT3 scFc constructs are active when unstimulated human PBMCs are used against human FLT3-transfected CHO cells in the absence and presence of FLT3LG (FLT3 ligand).

Examples

The present invention is illustrated by the following examples. These examples should not be construed as limiting the scope of the present invention. The present invention is limited solely by the claims.

Example 1 Generation of CHO Cells Expressing Wild Type FLT3 and Chimeric FLT3

The FLT3 antigen can be subdivided into six different subdomains or regions as defined for Examples 1 and 2. The AK sequence of these five subdomains is shown in SEQ ID NOs: 814-818.

Received the following molecules; see also FIG. 1:

Human LSP - V5 x Flt3-ElmuxEpC-pEFDHFR SEQ ID NO: 827

Human LSP - V5 x Flt3-E2muxEpC-pEFDHFR SEQ ID NO: 828

Human LSP - V5 x Flt3-E3muxEpC-pEFDHFR SEQ ID NO: 829

Human LSP - V5 x Flt3-E3AmuxEpC-pEFDHFR SEQ ID NO: 830

Human LSP - V5 x Flt3-E3BmuxEpC-pEFDHFR SEQ ID NO: 831

Human LSP - V5 x Flt3-E4muxEpC-pEFDHFR SEQ ID NO: 832

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Human LSP - V5 x Flt3-E5muxEpC-pEFDHFR SEQ ID NO: 833

Human LSP - V5 x Flt3-E6muxEpC-pEFDHFR SEQ ID NO: 834

Human LSP - FLT3 - full mouse ECP

Human LSP - FLT3- human full-length EVA full-length human FLT3 SEQ ID NO: 801 full-length cynomolgus monkey FLT3 SEQ ID NO: 802

To generate CHO/HEK cells expressing human, mouse extracellular domain and FLT3 chimeric extracellular domain (ECD), the respective coding sequences for human FLT3, mouse FLT3, and eight versions of human/mouse chimeric FLT3 (see above) were cloned into a plasmid referred to as pEF -DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). The original signal peptide was used for expression on the surface of human and mouse FLT3 cells. All cloning procedures were performed according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). For each construct, dihydrofolate reductase (DHFR) deficient CHO cells were transfected with the appropriate plasmid for eukaryotic expression as described by Kaufman R.J. (1990) Methods Enzymol. 185, 537-566.

Expression of human FLT3, chimeric FLT3 and mouse FLT3 on CHO cells was verified using FACS analysis.

Example 2 Epitope Mapping of Anti-FLT3 Antibody Construct

Cells transfected with human, mouse FLT3 and chimeric human FLT3 molecules (See Example 1) were stained with a crude undiluted periplasmic extract containing anti-FLT3xCD3 bispecific antibody constructs (CD3 binding domain designated as I2C) fused to human albumin (version 1) in PBS /1.5% FCS. Bound molecules were detected with in-house made anti-CD3 binding domain mouse monoclonal antibody (50 µl) and then PE-conjugated anti-mouse Fc-gamma IgG (1:100, 50 µl; Jackson Immunoresearch # 115-116-071) All antibodies were diluted in the FSB / 1.5% FCS. As a negative control, cells incubated with PBS/2% FCS were used instead of the periplasmic extract. Samples were evaluated by flow cytometry.

The regions that were recognized by the respective FLT3 binding domains are shown in the sequence table (Table 2). The disappearance of the FACS signal in the respective FLT3 chimeric constructs containing the mouse epitope cassette was recorded as confirmation of the relevance of the respective cassette for binding. The corresponding results in table. 2 are consistent with the results in example 3.

Example 3 Generation of CHO Cells Expressing Wild Type FLT3 and Truncated FLT3

The extracellular domain of the FLT3 antigen can be subdivided into different subdomains or regions, epitope clusters E1-E6, respectively, defined by the following amino acid positions:

El AK 27-79 SEQ ID NO: 819

E2 DI AK 79-167 SEQ ID NO: 820

E3 D2 AK 168-244 SEQ ID NO: 821

ESA D2A AK 168-206 SEQ ID NO: 822

E3B D2B AK 207-244 SEQ ID NO: 823

E4 D3 AK 245-345 SEQ ID NO:824

E5 D4 AK 346-434 SEQ ID NO:825

E6 D5 AK 435-543 SEQ ID NO:826

To construct truncated FLT3 molecules used for epitope mapping (see FIG. 3), the sequences of the corresponding seven human regions as well as five combinations of two adjacent human regions (see above) were replaced with the corresponding regions from mouse FLT3. In addition, a V5 tag (GKPIPNPLLGLDST) was fused to the C-terminus of the chimeric molecules via a GGGGS linker. The final sequences of the chimeric molecules are shown in SEQ ID NO: 827834. In addition, full length human FLT3 (SEQ ID NO: 801) and full length cynomolgus FLT3 (SEQ ID NO: 802) were constructed, both tagged with V5 (GKPIPNPLLGLDST) fused to C-terminally via a GGGGS linker.

To obtain CHO dhfr cells expressing the above constructs, the respective coding sequences were cloned into a plasmid designated pEF-DHFR (pEFDHFR is described in: Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). CHO cells transfected with human FLT3 were also produced, but without the V5 label. All cloning procedures were performed according to standard protocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001)). To obtain each construct, DHFR-deficient CHO cells were transfected with the appropriate plasmid for eukaryotic expression, as described in the source: Kaufman R.J. (1990)

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Methods Enzymol. 185, 537-566.

Expression of these constructs on CHO cells was verified using anti-v5 mouse IgG2a monoclonal antibody (1 μg/ml; AbD Serotec, #MCA 1360). Bound monoclonal antibody was detected using PE-conjugated anti-mouse Fc-gamma IgG. As a negative control, cells were incubated with an isotype control antibody instead of the first antibody. Samples were evaluated by flow cytometry.

The results of this analysis for the disclosed FLT3 binding element are shown in table. 2. These results are consistent with the epitope mapping analysis of Example 2.

Table 2. Epitope mapping

epitope cluster Connecting element epitope cluster Connecting element epitope cluster Connecting element FL-1 E1 FL-23 E1 FL-45 E1 FL-2 E1 FL-24 E1 FL-46 E1 FL-3 E1 FL-25 E1 FL-47 E1 FL-4 E1 FL-26 E1 FL-48 E1 FL-5 E1 FL-27 E1 FL-49 E1 FL-6 E1 FL-28 E1 FL-50 E1 FL-7 E1 FL-29 E1 FL-51 E1 FL-8 E1 FL-30 E1 FL-52 E1 FL-9 E1 FL-31 E1 FL-53 E3 FL-10 E1 FL-32 E1 FL-54 E3 FL-11 E1 FL-33 E1 FL-55 E1 FL-12 E1 FL-34 E1 FL-56 E1 FL-13 E1 FL-35 E1 FL-57 E1 FL-14 E1 FL-36 E1 FL-58 E1 FL-15 E1 FL-37 E1 FL-59 E1 FL-16 E1 FL-38 E1 FL-60 E1 FL-17 E1 FL-39 E1 FL-61 E3 FL-18 E1 FL-40 E1 FL-62 E3 FL-19 E1 FL-41 E1 FL-63 E3 FL-20 E1 FL-42 E1 FL-64 E3 FL-21 E1 FL-43 E1 FL-65 E1 FL-22 E1 FL-44 E1

Example 4 Biacore Assay-Based Determination of Antibody Affinity for Human and Cynomolgus FLT3

Biacore assay experiments were performed using recombinant human/cynomolgus monkey FLT3-ECD fusion proteins with albumin to determine target binding of antibody constructs of the present invention.

More CM5 sensor chips (GE Healthcare) immobilized approximately 600-800 RU (response units) of the respective recombinant antigen using acetate buffer, pH 4.5, according to the manufacturer's instructions. The anti-FLT3xCD3 bispecific antibody construct samples were loaded as serial dilutions at the following concentrations: 50, 25, 12.5, 6.25, and 3.13 nM in HBS-EP running buffer (GE Healthcare). The flow rate was 30 μl/min for 3 minutes, then running buffer HBS-EP was reapplied for 8-20 minutes at a flow rate of 30 μl/ml. Chip regeneration was performed using a solution of 10 mM glycine, 10 mM NaCl, pH 1.5. Datasets were analyzed using BiaEval Software. A total of two independent experiments were performed.

In addition, the binding of anti-human CD3 bispecific antibody and macaque CD3 constructs was confirmed in the Biacore assay.

Example 5 Scatchard distribution-based analysis of the affinity of an anti-FLT3xCD3 bispecific antibody construct for human and macaque FLT3 on target antigen-positive cells and determination of the interspecies difference in affinity

The affinity values of the anti-FLT3xCD3 bispecific antibody constructs for human or macaque FLT3-transfected CHO cells were also determined by Scatchard analysis, as the most reliable way to measure the potential difference in affinity for human and macaque FLT3. To perform the Scatchard assay, saturation binding experiments are performed using a monovalent detection system to accurately determine the monovalent binding of the anti-FLT3xCD3 bispecific antibody constructs to the appropriate cell line.

2x104 cells of each respective cell line (recombinant human FLT3-expressing CHO cell line, recombinant macaque FLT3-expressing CHO cell line) were incubated with 50 µl of triplicate serial dilutions (12 1:2 dilutions) of the respective anti-FLT3xCD3 bispecific antibody construct (until saturation was reached), starting with 10-20 nm, followed by incubation for 16 h at 4°C with stirring and one stage of residual washing. The cells were then incubated for an additional hour with 30 µl of CD3xALEXA488 conjugate solution. After one washing step, the cells were resuspended in 150 µl of FACS buffer containing 3.5% formaldehyde, incubated for an additional 15 min, centrifuged, resuspended

- 54 040387 in FACS buffer and analyzed using the FACS CantoII apparatus and FACS Diva software. The data were obtained during two independent series of experiments, each of which included three repetitions. An appropriate Scatchard analysis was performed to extrapolate maximum binding (Bmax). The half-maximal binding concentrations of the anti-FLT3xCD3 bispecific antibody constructs were determined, reflecting the respective KDs. Triple measurements were plotted as hyperbolic curves and S-curves to demonstrate appropriate concentration ranges from minimal to optimal binding.

Example 6 Bispecific Binding and Cross-Species Cross-Reactivity

To confirm binding to human FLT3 and CD3 and to cynomolgus FLT3 and CD3, the bispecific antibody constructs of the present invention were tested by flow cytometry using CHO cells transfected with human FLT3 (SEQ ID NO: 801) using human FLT3 isoforms (FLT3 isoform human (T227M), see SEQ ID NO: 803 and human FLT3-ITD isoform, see SEQ ID NO: 804), and macaque FLT3 (SEQ ID NO: 802), respectively, of FLT3 positive human AML cell lines EOL- 1, MOLM-13 and MV4-11 (other human FLT3 positive cell lines are also acceptable), CD3-expressing human HPB-ALL leukemia T-cell lines (DSMZ, Braunschweig, ACC483), and CD3-expressing T cell lines LnPx 4119 cynomolgus monkey.

For flow cytometry, 200,000 cells of the respective lines were incubated for 60 min at 4°C with 50 µl of the purified bispecific antibody construct at a concentration of 5 µg/ml. Cells were washed twice with PBS/2% FCS and then incubated with homemade mouse antibody (2 μg/mL) specific for the CD3 binding portion of the bispecific antibody constructs for 30 min at 4°C. After washing, bound mouse antibodies were detected using goat anti-mouse Fcy antibodies conjugated to PE (1:100) for 30 min at 4°C. Samples were evaluated by flow cytometry. Untransfected CHO cells were used as a negative control.

Table 3a. Affinity values of FLT3 binding domains

FLT3-HLE BITE epitope cluster Human FLT3* affinity as determined by Octet [nM] Macaque FLT3* affinity as determined by Octet [nM] Affinity difference for FLT3 KDmac/KDhu FL 39 X I2C-scFc ⁰ El 0.52 ± 0.03 1.65±0.10 3.2 FL 16 X I2C-scFc El 0.57** 1.38** 2.4 FL 42 x I2C-scFc El 0.36 ± 0.11 1.11 ± 0.13 3.1 FL 36 X I2C scFc El 5.30±0.08 4.44 ± 0.72 0.8 FL 52 x I2C scFc El 4.80±0.25 4.14 ± 0.08 0.9 FL 23 x I2C-scFc El 0.97 ± 0.19 0.68 ± 0.01 0.7 FL 46 X I2C-scFc El 1.67 ± 0.08 7.9 ± 1.81 4.7 FL 61 X I2C-scFc E3 26.50±0.57 3.77 ± 3.74 0.14

Table 3b. Affinity values for CD3 binding domains

FLT3-HLE BiTE epitope cluster Affinity according to Biacore analysis for human FLT3* [nM] Affinity according to Biacore analysis for FLT3* macaque [nM] Affinity difference for FLT3 , KDmac/KDhu FL 39 X I2C-scFc ⁰ El 8.24±0.15 6.84 ± 0.09 0.8 FL 16 X I2C-scFc El 6.08±0.12 4.91 ± 0.10 0.8 FL 42 X I2C-scFc El 9.13 ± 1.03 7.31 ± 1.13 0.8 FL 36 X I2C scFc El 7.73 ± 014 6.06±0.42 0.8 FL 52 X I2C-scFc El 7.40±0.80 5.99±0.78 0.8 FL 23 X I2C scFc El 9.64±0.11 7.87 ± 0.15 0.8 FL 46 X !2C-scFc El 6.06 ± 0.47 4.86±0.35 0.8 FL 61 X I2C-scFc E3 11.65 ± 1.48 9.37 ± 1.46 0.8

Example 7 Confirmation of No Binding to Human Paralogs

Human FLT3 paralogs KIT v1 (SEQ ID NO: 805), CSF1R v1 (SEQ ID NO: 806), PDGFRA (SEQ ID NO: 807), and NTM v3 (SEQ ID NO: 808) were stably transfected into CHO cells. The sequence of the paralog used in this example is as identified in the sequence listing.

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Table 4a. Identity of FLT3 sequence paralogs within the full-length protein sequence

Protein % identity Request ID (%) C-KIT 29 28 CSF1R 29 28 PDGFRA 27 thirty

Table 4b. Identity of Paralogs of the FLT3 Sequence within the ECD of the Protein Sequence

Protein % Identity Request ID (%) NTM 25 28

Protein expression was confirmed by FACS analysis with specific antibodies. Flow cytometric analysis was performed as described in Example 6.

Example 8 Human Germline Identity

To analyze the identity/sequence similarity of antibody constructs to human germline antibody genes, the FLT3 binding domains of the present invention were aligned as described below: full VL alignment including all CDR regions; full VH alignment, including CDR 1 and 2 regions, but not including CDR3, relative to human germline antibody genes (Vbase). More detailed information is given in the description of the present application.

Example 9 Cytotoxic Activity

The efficacy of the anti-FLT3xCD3 bispecific antibody constructs of the present invention to redirect effector T cells against FLT3-expressing target cells was analyzed in five in vitro cytotoxicity assays:

The efficacy of anti-FLT3xCD3 bispecific antibody constructs to redirect stimulated human CD8+ effector T cells against human FLT3-transfected CHO cells was measured in an 18 hour chromium-51 release assay.

The efficacy of anti-FLT3xCD3 bispecific antibody constructs to redirect stimulated human CD8+ effector T cells against FLT3-positive human AML lines EOL-1, MOLM-13, and MV4-11 (other human FLT3-positive cell lines are also acceptable) was measured in 18 -hour analysis of the release of chromium-51.

The efficacy of anti-FLT3xCD3 bispecific antibody constructs to redirect T cells in unstimulated human PBMCs against human FLT3-transfected CHO cells was measured in a 48-hour FACS-based cytotoxicity assay. Effector cells: unstimulated human PBMC (CD14-/CD56-). Target cells: EOL-1. The ratio of effector cells to target cells (E:T): 10:1. BiTE protein: as specified________________

Anti-FLT3XCD3 antibody constructs FL 39 FL 16 FL42 FL 36 FL52 FL 23 FL 4 6 FL61 EC50 [pM] 7.5 13 34 35 91 20 25 745

Effector cells: unstimulated human PBMC (CD14-/CD56-). Target cells: MV411. The ratio of effector cells to target cells (E:T): 10:1. Protein BiTE: as indicated.

Anti-FLT3XCD3 antibody constructs FL 39 FL 16 FL42 FL 36 FL52 FL 23 FL46 FL61 EC50 [pM] 4.8 8.1 13 15 14 13 12 270

The efficacy of anti-FLT3xCD3 bispecific antibody constructs to redirect T cells in unstimulated human PBMCs against FLT3-positive human AML cell lines EOL-1, MOLM-13, and MV4-11 (other human FLT3-positive cell lines are also acceptable) was measured over a 48-hour period. FACS-based cytotoxicity assay.

To confirm that cross-reactive anti-FLT3xCD3 bispecific antibody constructs are able to redirect macaque T cells against macaque FLT3 transfected CHO cells, a 48-hour FACS-based cytotoxicity assay was performed on a macaque T cell line as effector T cells. Effector cells: unstimulated human PBMC (CD14-/CD56-). Target cells: FLT3-transfected macaque CHO cells. The ratio of effector cells to target cells (E:T): 10:1. Protein BiTE: as indicated.______________________

Anti-FLT3XCD3 antibody constructs FL 39 FL 16 FL42 FL 36 FL52 FL 23 FL46 FL61 EC50 [pM] 0.2 0.5 1.0 2.5 0.7 1.9 eleven 0.9

The efficacy of anti-FLT3xCD3 bispecific antibody constructs to redirect T cells in unstimulated human PBMCs to human FLT3-transfected CHO cells in the absence and presence of FLT3 ligand was measured in a 48-hour FACS-based cytotoxicity assay. Effector cells: unstimulated human PBMC (CD14-/CD56-). Target Cells:

- 56 040387 human FLT3 transfected CHO cells. The ratio of effector cells to target cells (E:T): 10:1. Protein BiTE: according to . with the specified.__________________________________

Anti-FLT3XCD3 antibody constructs EC50 [pM] Without FLT3 ligand with 10 ng/ml FLT3 ligand with 1 μg/ml FLT3 ligand FL 39 X I2C-scFc® 0.7 13 29 FL 16 X I2C-scFc 0.5 eleven 23 FL 42 X I2C-scFc 1.0 19 e FL 36 X I2C scFc 1.9 24 73 FL 52 X I2C-scFc 2.4 24 71 FL 23 X I2C scFc 0.9 15 49 FL 46 X I2C-scFc 1.2 18 43 FL 61 X I2C-scFc 32 662 608

Example 10.1. Chromium release assay on stimulated human T cells

Stimulated T cells enriched for CD8+ T cells were obtained as described below. A Petri dish (145 mm diameter, Greiner Bio-One GmbH, Kremsmuenster, Austria) was coated with a commercially available anti-CD3 specific antibody (OKT3, Orthoclone) at a final concentration of 1 μg/ml for 1 h at 37°C. Unbound protein was removed by a one-step PBS wash. 3-5 x 10 ⁷ human PBMC in 120 ml RPMI 1640 with stabilized glutamine/10% FCS/20 U/ml IL-2 (Proleukin®, Chiron) was added to a precoated petri dish and stimulated for two days. On the third day, the cells were harvested and washed once with RPMI 1640. IL-2 was added to a final concentration of 20 U/ml and the cells were cultured again for 1 day in the same cell culture medium described above. The culture was enriched in cytotoxic CD8+ T lymphocytes (CTL) by depleting CD4+ T cells and CD56+ NK cells using Dynal beads according to the manufacturer's protocol.

Cynomolgus FLT3 or human FLT3 transfected target CHO cells were washed twice with PBS and labeled with 11.1 mBq ⁵¹ Cr in a final volume of 100 μl RPMI with 50% FBS for 60 min at 37°C. The labeled target cells were then washed 3 times with 5 ml RPMI and used for cytotoxicity assay. The assay was performed in a 96-well plate in a total volume of 200 μl supplemented with 10:1 E:T ratio of RPMI medium. An initial concentration of 0.01-1 μg/ml of the purified bispecific antibody construct and its three-fold dilutions were used. The incubation time for this assay was 18 hours. Cytotoxicity was defined as the relative amount of chromium released in the supernatant, as applied to the difference between maximum lysis (when Triton-X was added) and spontaneous lysis (no effector cells). All measurements were carried out in quadruplicate. Chromium activity in supernatants was measured using a Wizard 3 gamma counter (Perkin Elmer Life Sciences GmbH, Cologne, Germany). Results were analyzed using Prism 5 for Windows (version 5.0, GraphPad Software Inc., San Diego, CA, USA). For comparison of cytotoxic activity, EC ₅₀ values were used, calculated using an analytical program based on sigmoidal curves of dose-effect dependence.

Example 10.2. Efficiency of redirection of stimulated human effector T cells against human FLT3-transfected CHO cells

The cytotoxic activity of the anti-FLT3xCD3 bispecific antibody construct of the present invention was analyzed in a chromium-51 ( ⁵¹ Cr) releasing cytotoxicity assay using human FLT3-transfected CHO cells as target cells and stimulated human CD8+ T cells as effector cells. . The experiment was carried out as described in example 10.1.

Example 10.3. Redirection Efficiency of Stimulated Human Effector T Cells Against FLT3 Positive Human Cell Line

The cytotoxic activity of the anti-FLT3xCD3 bispecific antibody construct was analyzed in a chromium-51 ( ⁵¹ Cr) releasing cytotoxicity assay using FLT3-positive human AML cell lines EOL-1, MOLM-13 and MV4-11 as source of target cells, and stimulated CD8+ T human α-cells as effector cells. The specified analysis was carried out as described in example 10.1.

Example 10.4. FACS-based cytotoxicity assay with unstimulated human PBMC Isolation of effector cells

Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation from lymphocyte-enriched formulations (buffy coat), a by-product in blood banks collecting blood for transfusion. The buffy coat provided a local blood bank and PBMCs were obtained on the same day as

- 57 040387 blood collection. After Ficoll density gradient centrifugation and extensive Dulbecco's (Gibco) PBS washes, remaining erythrocytes were removed from PBMCs by incubation with erythrocyte lysis buffer (155 mM NH4Cl, 10 mM KHCO3, 100 μM EDTA). Platelets were removed with supernatant after PBMC centrifugation at 100xg. The remaining lymphocytes mainly include B and T lymphocytes, NK cells and monocytes. PBMCs were maintained in culture at 37°C/5% CO2 in RPMI medium (Gibco) with 10% FCS (Gibco).

Depletion of CD14+ and CD56+ cells

CD14 MicroBeads (Milteny Biotec, MACS, #130-050-201) were used for human CD14+ cell depletion, CD56 MicroBeads (MACS, #130-050-401) were used for human NK cell depletion. PBMCs were counted and centrifuged for 10 min at room temperature and 300xg. The supernatant was discarded and the cell pellet was resuspended in MACS isolation buffer [80 μl/10 ⁷ cells; PBS (Invitrogen, #20012-043), 0.5% (v/v) PBS (Gibco, #10270106), 2 mM EDTA (Sigma-Aldrich, #E-6511)]. Added microbeads CD14 MicroBeads and CD56 MicroBeads (20 μl/10 ⁷ cells) and incubated for 15 min at 4-8°C. Cells were washed with MACS isolation buffer (1-2 ml/10 ⁷ cells). After centrifugation (see above), the supernatant was discarded and the cells were resuspended in MACS isolation buffer (500 μl/10 ⁸ cells). CD14/CD56 negative cells were then isolated using LS columns (Miltenyi Biotec, #130-042-401). PBMCs without CD14+/CD56+ cells were cultured in complete RPMI medium, i.e. RPMI1640 (Biochrom AG, #FG1215) supplemented with 10% PBS (Biochrom AG, #S0115), 1x non-essential amino acids (Biochrom AG, #K0293), 10 mM Hepes buffer (Biochrom AG, #L1613), 1 mM sodium pyruvate (Biochrom AG, #L0473) and 100 U/ml penicillin/streptomycin (Biochrom AG, #A2213) at 37° C. in an incubator until administered.

Labeling of target cells

For flow cytometric analysis of cell lysis, the fluorescent membrane dye DiOC ₁₈ (DiO) (Molecular Probes, #V22886) was used to label FLT3 transfected human or macaque FLT3 CHO cells as target cells and distinguish from effector cells. Briefly, cells were harvested, washed once with PBS, and the cell density was adjusted to 10 ⁶ cells/ml in PBS with 2% (v/v) PBS and DiO membrane dye (5 μl/10 ⁶ cells). After incubation for 3 min at 37°C, the cells were washed twice in complete RPMI medium and the cell count was adjusted to 1.25x105 cells/ml. Cell viability was determined using a 0.5% (v/v) isotonic solution of Eosin G (Roth, #45380).

Flow cytometric analysis

This assay was designed to quantify the lysis of FLT3-transfected cynomolgus or human CHO cells in the presence of serial dilutions of the anti-FLT3 bispecific antibody construct. Equal volumes of DiO-labeled target cells and effector cells (ie, PBMCs without CD14+ cells) were mixed in an E:T cell ratio of 10:1. 160 μl of this suspension was transferred to each well of a 96-well plate. 40 μl serial dilutions of anti-FLT3xCD3 bispecific antibody constructs and a bispecific negative control (a CD3-based bispecific antibody construct recognizing an irrelevant target antigen) or complete RPMI medium were added as an additional negative control. The bispecific antibody-mediated cytotoxic reaction was continued for 48 hours in a humidified incubator at 7% CO ₂ . The cells were then transferred to a new 96-well plate and the loss of target cell membrane integrity was monitored by adding propidium iodide (PI) at a final concentration of 1 μg/ml. PI is a non-membrane-permeable dye that is normally excluded from entering viable cells, while dead cells absorb it, which makes it possible to identify them by fluorescent radiation.

Samples were evaluated by flow cytometry on a FACSCanto II apparatus and analyzed using FACSDiva software (both from Becton Dickinson). Target cells were identified as DiO-positive cells. PI-negative target cells were classified as live target cells. The percentage of cytotoxicity was calculated using the following formula:

CYTOTOXICITY [%]= ^P dead target cells _χ1 θθ ^target cells n=number of events.

Using GraphPad Prism 5 software (Graph Pad Software, San Diego), percent cytotoxicity was plotted against respective concentrations of the bispecific antibody construct. Dose-response curves were analyzed using four-parameter regression logistic models to estimate sigmoid dose-response curves with a fixed slope factor, and EC ₅₀ values were calculated.

Example 10.5. Redirection Efficacy of Unstimulated Human PBMCs Against Human FLT3 Transfected CHO Cells

The cytotoxic activity of anti-FLT3xCD3 bispecific antibody constructs was analyzed in a FACS-based cytotoxicity assay using human FLT3-transfected cells.

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CHO as target cells and unstimulated human PBMC as effector cells.

This analysis was carried out as described in example 8.4 above.

Example 10.6. Efficacy of redirecting unstimulated human PBMCs against FLT3-positive human ovarian carcinoma cells

The cytotoxic activity of the anti-FLT3xCD3 bispecific antibody constructs was further analyzed in a FACS-based cytotoxicity assay using FLT3-positive human AML cell lines EOL-1, MOLM-13 and MV4-11 as source of target cells and unstimulated human PBMC as effector cells. This analysis was carried out as described in example 8.4 above.

Example 10.7.

Efficiency of redirecting macaque T cells against macaque FLT3-expressing CHO cells

Finally, the cytotoxic activity of anti-FLT3xCD3 bispecific antibody constructs was analyzed in a FACS-based cytotoxicity assay using macaque (cynomolgus macaque) FLT3-transfected CHO cells as target cells and the 4119LnPx macaque T cell line (Knappe et al. Blood 95:3256- 61 (2000)) as a source of effector cells. Labeling of target cells, macaque FLT3-transfected CHO cells, and analysis of cytotoxic activity based on flow cytometry were performed as described above.

Example 11 Conversion of Monomers to Dimers after (i) Three Freeze/Thaw Cycles and (ii) 7 Days of Incubation at 250 µg/mL

The anti-FLT3xCD3 bispecific antibody monomer construct was subjected to various stresses followed by high resolution SEC to determine the initial percentage of the antibody monomer construct converted to a dimer antibody construct.

(i) 25 μg of the monomeric antibody construct was diluted with total formulation buffer to a concentration of 250 μg/ml and then frozen at -80°C for 30 minutes followed by thawing for 30 minutes at room temperature. After three freeze/thaw cycles, the content of dimers was determined using HPEA.

(ii) 25 μg of the monomeric antibody construct was diluted with total formulation buffer to a concentration of 250 μg/ml, followed by incubation at 37° C. for 7 days. The content of dimers was determined using HPEA.

A TSK Gel G3000 SWXL high resolution SEC column (Tosoh, Tokyo, Japan) was attached to an Akta Purifier 10 FPLC (GE Lifesciences) fast liquid chromatography apparatus equipped with an A905 autosampler. Column equilibration buffer and running buffer consisted of 100 mM KH2PO4-200 mM Na ₂ SO ₄ pH adjusted to 6.6. An antibody solution (25 μg of protein) was added to a balanced column and eluted at a flow rate of 0.75 ml/min with a maximum pressure of 7 MPa. Throughout the analysis, optical absorption was monitored at wavelengths of 280, 254, and 210 nm. The analysis was performed by integrating the signal peaks at 210 nm entered into the Akta Unicorn software analysis evaluation form. The content of dimers was calculated by dividing the area of the peaks for dimers by the total area of the peaks for monomers and dimers.

Example 12 Thermal Stability

The antibody aggregation temperature was determined as described below: 40 μl of the antibody construct solution at a concentration of 250 μg/ml was transferred into a disposable cuvette and placed in a DynaPro Nanostar dynamic light scattering device (Wyatt). The sample was heated from 40 to 70°C at a rate of 0.5°C/min with constant registration of the measured radius. The increase in radius, which is indicative of protein melting and aggregation, is used to calculate the aggregation temperature of the antibody construct using the software package supplied with the DLS device.

Example 13 Stability after incubation for 24 hours in human plasma

Purified bispecific antibody constructs were incubated 1:5 in pooled human plasma at 37° C. for 96 hours at a final concentration of 2-20 μg/mL. After incubation in plasma of the construct, antibodies were compared in a chromium-51 release assay with stimulated human CD8+ enriched T cells and human FLT3-transfected CHO cells at an initial concentration of 0.01-0.1 μg/mL at a ratio of effector cells to target (E:T), which is 10:1 (analysis as described in example 8.1). Unincubated freshly thawed bispecific antibody constructs were included as controls.

Example 14 Turbidity at an antibody concentration of 2500 μg/ml ml of a solution of a purified antibody construct at a concentration of 250 μg/ml was concentrated in a centrifuge concentrator to 2500 μg/ml. After 16 hours of storage at 5°C, the turbidity of the antibody solution was determined by measuring the optical absorbance OD340 nm with the total prescription buffer in

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Example 15 Protein Homogeneity Assessed Using High Performance Cation Exchange Chromatography

Protein homogeneity of the antibody constructs of the present invention was analyzed using high performance cation exchange chromatography (CIEX).

μg of antibody construct monomer was diluted with 50 ml of binding buffer A (20 mM sodium dihydrogen phosphate, 30 mM NaCl, 0.01% sodium octanate, pH 5.5), and 40 ml of this solution was applied to a 1 ml BioPro SP-F column ( YMC, Germany) connected to an Akta Micro Fast Liquid Chromatography (FPLC) system (GE Healthcare, Germany). After binding the sample, a washing step was performed with additional binding buffer. A linearly increasing gradient of salt in buffer B (20 mM sodium dihydrogen phosphate, 1000 mM NaCl, 0.01% sodium octanate, pH 5.5) to 50% buffer B in 10 column volumes was used to elute the protein. Throughout the analysis, optical absorption was monitored at wavelengths of 280, 254, and 210 nm. The analysis was performed by integrating the signal peaks at 280 nm entered into the Akta Unicorn analysis software analysis form.

Example 16 Surface Hydrophobicity Assessed Using Butyl-HIC

The surface hydrophobicity of the bispecific antibody constructs of the present invention was tested by hydrophobic interaction chromatography (HIC) in flow mode.

μg of antibody construct monomer was diluted with total formulation buffer to a final volume of 500 μl (10 mM citric acid, 75 mM lysine HCl, 4% trehalose, pH 7.0) and applied to a 1 ml fast-flow (FF) column with butyl- sepharose (Butyl Sepharose) (GE Healthcare, Germany), connected to the system for fast liquid chromatography (FPLC) Akta Purifier (GE Healthcare, Germany). Throughout the analysis, optical absorption was monitored at wavelengths of 280, 254, and 210 nm. The analysis was performed by integrating the signal peaks at 280 nm entered into the Akta Unicorn analysis software analysis form. The elution characteristics were evaluated by comparing the area and the linear rate of rise and fall of the protein signal, thus determining the strength of the interaction of the albumin-fused BiTE with the matrix.

Example 17 Difference in Efficacy of Monomeric and Dimeric Isoforms of Bispecific Antibody Constructs

To determine differences in cytotoxic activity between the monomeric and dimeric isoforms of the individual anti-FLT3xCD3 bispecific antibody constructs (referred to as potency margin), an 18 hour chromium-51 releasing cytotoxicity assay as described above (Example 10.1) of the monomer and dimer of the purified bispecific antibody construct was performed. The effector cells were stimulated human CD8+ enriched T cells. Target cells were human FLT3 transfected CHO cells. The ratio of effector to target cells (E:T) was 10:1. The efficiency difference was calculated as the ratio of the EC ₅₀ values._________________________________________________________

FLT3XCD3 antibody constructs EC50 [pM] Ratio of monomers to dimers (Monomer EC _S0 / Dimer EC _B0 ) Monomer Dimer FL 39 x I2C-scFc® 0.37 0.23 0.5 FL 16 x I2C-scFc 0.52 0.16 2.0 FL 42 x I2C-scFc 0.53 0.76 1.2 FL 36 x I2C-scFc 2.70 0.88 1.1 FL 52 x I2C-scFc 1.45 1.69 9.9 FL 23 x I2C-scFc 0.20 0.20 0.2 FL 46 x I2C-scFc 0.34 0.15 0.9 FL 61 x I2C-scFc 18 24 6.3

Example 18 FLT3 Binding Element Competition with FLT3 Ligand for Binding to the Appropriate Target

This assay was performed to test the possibility of a soluble FLT3 ligand disrupting FLT3 binding of anti-FLT3 binding domains in accordance with the present invention.

To verify binding of human FLT3 ligand to human FLT3 transfected CHO cells, cells were incubated with human FLT3 ligand for 30 min at 4°C. Bound FLT3 ligand was detected with an anti-HIS antibody (5 μg/ml; AbD Serotec) followed by a PE-conjugated anti-mouse Fc-gamma IgG antibody (1:100; Jackson Immunoresearch # 115-116-071). As a negative control, cells were incubated with PBS/2% FBS instead of CD27.

To test competition/replacement of FLT3 binding element with FLT3 ligand, human FLT3 transfected CHO cells were incubated in the presence or absence of FLT3 ligand for 30 min at 4°C (10 μg/ml FLT3 ligand). After that, the cells were not washed, but directly stained with the FLT3 binding element (scFv). Bound scFv was detected with mouse anti-FLAG M2 monoclonal antibody (1 μg/mL; Sigma F1804) followed by PE-conjugated antibody

- 60 040387 vs Fc-gamma mouse IgG (1:100; Jackson Immunoresearch # 115-116-071). As a negative control, cells were incubated with non-specific scFv instead of anti-FLT3 scFv. FLT3 ligand and all antibodies were diluted in PBS with 2% FCS.

To evaluate the data, the average level of fluorescence was detected using FACS. The loss of more than 25% of the signal as a result of competition with the FLT3 ligand was considered a significant effect on binding, which can be understood as a significant steric interaction with the same FLT3 domain. All binding elements that score above 75% of the threshold (mean level [+ligand]/mean level [no ligand] x100>75%) (which is true for FL-1 to FL-65) are identified as insensitive to ligand competition. FLT3.

The interaction of the FLT3 ligand with its receptor, as described in published sources, occurs in the region corresponding to epitope cluster 3. Accordingly, it was expected that the signal corresponding to the average fluorescence level of all binding elements identified during the screening carried out by the inventors as specific for the epitope cluster 3, as well as binding elements identified as specific for neighboring clusters 2 and 4, will be significantly affected by competition with the FLT3 ligand. In general, these expectations were justified. Surprisingly, FL-53, FL-54, FL-61, F-62, FL-63, and FL-64, all of which bind to epitope cluster 3, still showed a signal above this threshold.

In addition, given the interaction of the FLT3 ligand with the region of epitope cluster 3, it was also suggested that the binding element of a more distant epitope cluster, such as FLT3 cluster 1, would not be affected by competition with the FLT3 ligand. However, a significant number of connecting elements did not satisfy the 75% threshold condition. Binding elements FL-1-FL-53, FL-55-FL60 and FL-65 were included in the group of binding elements that are not sensitive to competition with the FLT3 ligand.

Table 5. Sequence listing

SEQID NO: Description Source Subsequence 1. Peptide linker Artificial sequence GGGG 2. Peptide linker Artificial sequence GGGGS 3. Peptide linker Artificial sequence GGGGQ 4 . Peptide linker Artificial sequence PGGGGS 5. Peptide linker Artificial sequence PGGDGS 6. Peptide linker Artificial sequence SGGGGS 7. Peptide linker Artificial sequence GGGGSGGGS 8 . Peptide linker Artificial sequence GGGGSGGGGS 9. Peptide linker Artificial sequence GGGGSGGGGSGGGGS 10. Hexahistidine Artificial sequence NNNN eleven. CDR-L1 from F6A Artificial sequence GSSTGAVTSGYYPN 12 . CDR-L2 from F6A Artificial sequence GTKFLAP 13. CDR-L3 from F6A Artificial sequence ALWYSNRWV 14 . CDR-H1 of F6A Artificial sequence IYAMN 15. CDR-H2 of F6A Artificial sequence RIRSKYNNYATYYADSVKS 16. CDR-H3 from F6A Artificial sequence HGNFGNSYVSFFAY 17 . VH of F6A Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY VS FFAYWGQGTLVTVS S

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18 . VL of F6A Artificial sequence L 19. VH-VL out F6A Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY VSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 20. CDR-L1 from H2C Artificial sequence GSSTGAVTSGYYPN 21. CDR-L2 from H2C Artificial sequence GTKFLAP 22. CDR-L3 from H2C Artificial sequence ALWYSNRWV 23. CDR-H1 from H2C Artificial sequence KYAMN 24. CDR-H2 from H2C Artificial sequence RIRSKYNNYATYYADSVKD 25. CDR-H3 from H2C Artificial sequence HGNFGNSYISYWAY 26. VH from H2C Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY IS YWAYWGQGT LVTVS S 27 . VL from H2C Artificial sequence L 28. VH-VL out H2C Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 29. CDR-L1 from H1E Artificial sequence GSSTGAVTSGYYPN thirty. CDR-L2 from H1E Artificial sequence GTKFLAP 31. CDR-L3 from H1E Artificial sequence ALWYSNRWV 32 . CDR-H1 from H1E Artificial sequence SYAMN 33. CDR-H2 from H1E Artificial sequence RIRSKYNNYATYYADSVKG 34 . CDR-H3 from H1E Artificial sequence HGNFGNSYLSFWAY 35. VH from H1E Artificial sequence EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY L S FWAYWGQ GT LVTVS S 36. VL from H1E Artificial sequence L

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37. VH-VL from HIE Artificial sequence EVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY LSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 38. CDR-L1 from G4H Artificial sequence GSSTGAVTSGYYPN 39. CDR-L2 from G4H Artificial sequence GTKFLAP 40. CDR-L3 from G4H Artificial sequence ALWYSNRWV 41. CDR-H1 of G4H Artificial sequence RYAMN 42. CDR-H2 of G4H Artificial sequence RIRSKYNNYATYYADSVKG 43. CDR-H3 of G4H Artificial sequence HGNFGNSYLSYFAY 44 . VH from G4H Artificial sequence FAYWGQ GT LVTVS S 45. VL from G4H Artificial sequence L 46. VH-VL from G4H Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY LSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 47. CDR-L1 from A2J Artificial sequence RSSTGAVTSGYYPN 48. CDR-L2 from A2 J Artificial sequence ATDMRPS 49. CDR-L3 from A2J Artificial sequence ALWYSNRWV 50. CDR-H1 of A2J Artificial sequence VYAMN 51. CDR-H2 from A2J Artificial sequence RIRSKYNNYATYYADSVKK 52 . CDR-H3 from A2J Artificial sequence HGNFGNSYLSWWAY 53. VH from A2J Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY LSWWAYWGQGTLVTVSS 54. VL from A2J Artificial sequence L 55. VH-VL out A2J Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY LSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQ KPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 56. CDR-L1 of E1L Artificial sequence GSSTGAVTSGYYPN

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57 . CDR-L2 from EIL Artificial sequence GTKFLAP 58 . CDR-L3 from EIL Artificial sequence ALWYSNRWV 59. CDR-H1 from EIL Artificial sequence KYAMN 60. CDR-H2 from EIL Artificial sequence RIRSKYNNYATYYADSVKS 61. CDR-H3 from EIL Artificial sequence HGNFGNSYTSYYAY EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW 62 Artificial VRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTI subsequence SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY T S YYAYWGQ GT LVTVS S QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPN 62 Artificial WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK subsequence AALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTI Artificial sequence SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY 64 . EIL TSYYAYWGQGTLVTVSSGGGGSGGGGSGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 65. CDR-L1 from E2M Artificial sequence RSSTGAVTSGYYPN 66. CDR-L2 from E2M Artificial sequence ATDMRPS 67 . CDR-L3 from E2M Artificial sequence ALWYSNRWV 68 . CDR-H1 from E2M Artificial sequence GYAMN 69 . CDR-H2 from E2M Artificial sequence RIRSKYNNYATYYADSVKE 70. CDR-H3 from E2M Artificial sequence HRNFGNSYLSWFAY EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNW 71. Artificial VRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTI subsequence SRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSY LSWFAYWGQGTLVTVSS QTWTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPN 72 Artificial WVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGK subsequence AALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTV L EVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTI Artificial sequence SRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSY 73 . E2M LSWFAYWGQGTLVTVSSGGGGSGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQ KPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 74 . CDR-L1 of F7O Artificial sequence GSSTGAVTSGYYPN 75. CDR-L2 of F7O Artificial sequence GTKFLAP 76. CDR-L3 of F7O Artificial sequence ALWYSNRWV 77 . CDR-H1 of F7O Artificial sequence VYAMN 78 . CDR-H2 of F7O Artificial sequence RIRSKYNNYATYYADSVKK

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79. CDR-H3 of F7O Artificial sequence HGNFGNSYISWWAY 80. VH of F7O Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISWWAYWGQGTLVTVS S 81. VL of F7O Artificial sequence L 82 . VH-VL of F7O Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 83. CDR-L1 of F12Q Artificial sequence GSSTGAVTSGNYPN 84 . CDR-L2 of F12Q Artificial sequence GTKFLAP 85. CDR-L3 from F12Q Artificial sequence VLWYSNRWV 86. CDR-H1 of F12Q Artificial sequence SYAMN 87. CDR-H2 of F12Q Artificial sequence RIRSKYNNYATYYADSVKG 88. CDR-H3 of F12Q Artificial sequence HGNFGNSYVSWWAY 89. VH of F12Q Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY VSWWAYWGQGTLVTVS S 90. VL of F12Q Artificial sequence QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L 91. VH-VL of F12Q Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY VSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 92. CDR-L1 from I2C Artificial sequence GSSTGAVTSGNYPN 93. CDR-L2 from I2C Artificial sequence GTKFLAP 94. CDR-L3 from I2C Artificial sequence VLWYSNRWV 95. CDR-H1 from I2C Artificial sequence KYAMN 96. CDR-H2 from I2C Artificial sequence RIRSKYNNYATYYADSVKD 97. CDR-H3 from I2C Artificial sequence HGNFGNSYISYWAY 98 . VH from I2C Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY IS YWAYWGQGT LVTVS S

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99. VL from I2C Artificial sequence QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L 100. VH-VL from I2C Artificial sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 101. VH from F12q Artificial sequence EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWWAYWGQGTLVTVS S 102 . VL of F12q Artificial sequence QTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPN WVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGK AALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTV L 103. F12q scFv EVQLVESGGGLVQPGGSLRLSCAASGFTFNSYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTI SRDDSKNTAYLQMNSLKTEDTAVYYCVRHGNFGNSY VSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 104 . HAL B Human Sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL 105. HAL B 7 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA GTFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

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106. HALB098 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHРYFYAPЕLLFFAKRYKAAFTЕСCQAADКААСLLР KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 107. HALB114 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL 108 . HALB254 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL

- 67 040387

109. HALB253 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 110. HALB131 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLV AASQAALGL 111. HALB135 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL

- 68 040387

112 . HALB133 Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL Artificial sequence Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH 113. HALB234 CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH 114 . HALB C34S CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL

- 69 040387

115. HALB7C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA GTFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL 116. HALB098 C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 117 . HALB114 C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL

- 70 040387

118 . HALB254 C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 119. HALB253C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 120. HALB131C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLV AASQAALGL

- 71 040387

121. HALB135C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL 122. HALB133C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL 123. HALB234C34S Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQSPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL

- 72 040387

124 . HALB C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL 125. HALB7C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA GTFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAAMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL 126. HALB098 C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL

- 73 040387

127 . HALB114 C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL 128. HALB254C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALGVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL 129. HALB253C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASQAALGL

- 74 040387

130. HALB131C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHРYFYAPЕLLFFAKRYKAAFTЕСCQAADКААСLLР KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPHLV AASQAALGL 131. HALB135C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPHLV AASKAALGL 132 . HALB133C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH CIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNA ETFTFHADICTLSEKERQIKKQTALVELVKHKPKAT KEQLKAVMDKFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL

- 75 040387

133. HALB234C34A Artificial sequence DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQAPF EDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGD KLCTVATLRETYGEMADCCAKQEPERNECFLQHKDD NPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIAR RHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLE CADDRADLAKYICENQDSISSKLKECCEKPLLEKSH СIАЕVENDEMPADL Р S LAAD FVE S KDVC KNYAEAKD VFLGMFLYEYARRHPDYSWLLLRLAKTYETTLEKC CAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFE QLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSWLNQLCVLHEKTP VSDRVTKCCTESLVNRRPCFSALDVDETYVPKEFNA ET FT FHADICTLSEKERQIKKQТALVELVKHКР КАТ KEQLKAVMDDFAAFVEKCCKADDKETCFAEEGPKLV AASKAALGL 134 . AI56 Artificial sequence RDWD FDVFGGGT PVGG 135. Linear FcRn binding peptide Artificial sequence QRFVTGHFGGLXPANG 136. Linear FcRn-binding peptide Y Artificial sequence QRFVTGHFGGLYPANG 137 . Linear FcRn binding peptide H Artificial sequence QRFVTGHFGGLHPANG 138 . Core FcRn binding peptide H Artificial sequence TGHFGGLHP 139. Cyclic FcRn binding peptide H Artificial sequence QRFCTGHFGGLHPCNG 140. Crossbody 1 HC ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVP SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VWD VS Η Е D Р Е VK FNW YVD GVE VHNAKT KPCEEQYG STYRCVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFF LYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 141. Crossbody 1 LC GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG AVTVAWKADSSРVKAGVETТТРSKQSNNКYAASSYL SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VT С VWD VS Η Е D Р Е VK FNW YVD GVE VHNAKT К Р С Е Е QYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKALPA РIЕКТIS KAKGQ PREP QVYT L Р Р S RKEMT KNQVS LT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

- 76 040387

142 . Crossantbody 2 NS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWN S GALT S GVHT FPAVLQ S S GL YS L S SWT VP SSNFGTQTYTCNVDHKPSNTKVDKTVEPKSSDKTHT CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTC VWD VS Η E D P E VK FNWYVD GVE VHNAKT К P RE EQ YN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTIS KAKGQPREPQVYTLP P S REEMTKNQVS LTCLV KGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFF LYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK 143. Crossbody 2 LC GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPG AVTVAWKADSSPVKAGVETTTPSKQSNNКYAASSYL SLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSEP KSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTISKAKGQ PREPQVYT L P P S RKEMT KN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 144 . Hetero-Fc linking Fc element DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTIS KAKGQ P RE PQVYT L P P S RKEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 145. Hetero-Fc, partner Fc DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDS DGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 146. Maxitelo 1 Fc to target EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 147 . Maxitelo 1 Fc to CD3 EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTP PVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 148 . Maxitelo 2 Fc to target EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 149. Maxitelo 2 Fc to CD3 EPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYDTTP PVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK

- 77 040387

150. Mono-Fc APELLGGPSVFLFPPKPKDTLM1SRTPEVTCVWDV S Η E D P EVKFNWYVDGVEVHNAKT КP REEQYN S TYRV VSVLTVLHQDWLNGKEYKCKVSNKALPAPiEKHSK AKGQ P RE PQVTT L P P S REEMT KNQVS LT CLVKGFYP SD1AVEWESNGQPENNYDTTPPVLDSDGSFFLYSDL TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG К 151. FL1 VH CDR ¹ NARMGVS 152 . FL1 VH CDR2 H1FSNAEKSYRTSLKS 153. FL1 VH CDR3 1PGYGGNGDYHYYGMDV 154 . FL1 VL CDR! RASQG1RNDLG 155. FL1 VL CDR2 ASSTLQS 156. FL1 VL CDR3 LQHNNFPWT 157 . F ^L_ ! vh QVTLKESGPVLVKPTETLTLTCTVSGFSL1NARMGV SW1RQPPGKALEWLAH1FSNAEKSYRTSLKSRLT1S KDTSKSQWLTMTNMDPVDTATYYCAR1PGYGGNGD YHYYGMDVWGQGTTVTVS S 158 . FL. ¹ VL D1QMTQSPSSLSASLGDRVTITCRASQG1RNDLGWY QQKPGKAPKRL1YASSTLQSGVPSRFSGSGSGTEFT LT1SSLQPEDFATYYCLQHNNFPWTFGQGTKVE1K 159. ^FL_1 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSL1NARMGV SW1RQPPGKALEWLAH1FSNAEKSYRTSLKSRLT1S KDTSKSQWLTMTNMDPVDTATYYCAR1PGYGGNGD YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSD1 QMTQSPSSLSASLGDRVT1TCRASQG1RNDLGWYQQ KPGKAPKRLTYASSTLQSGVPSRFSGSGSGTEFTLT 1SSLQPEDFATYYCLQHNNFPWTFGQGTKVEIK 160. ^FL_1 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSL1NARMGV SW1RQPPGKALEWLAH1FSNAEKSYRTSLKSRLT1S KDTSKSQWLTMTNMDPVDTATYYCAR1PGYGGNGD YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSD1 QMTQSPSSLSASLGDRVT1TCRASQG1RNDLGWYQQ KPGKAPKRL1YASSTLQSGVPSRFSGSGSGTEFTLT 1SSLQPEDFATYYCLQHNNFPWTFGQGTKVEIKSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVAR1RSKYNNYATYYADSVKDR FT1SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSY1SYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGL1GGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 161. FL2 VH CDRi NARMGVS 162 . FL2 VH CDR2 H1FSNDEKTYSTSLKS 163. FL2 VH CDR3 1PYYGSGSHNYGMDV 164 . FL2 VL CDRi RASQD1RNDFG 165. FL2 VL CDR2 AASTLQS 166. FL2 VL CDR3 LQYNTYPWT 167 . FL_2 vh QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SW1RQPPGKALEWLAH1FSNDEKTYSTSLKSRLT1S RDTSKGQWLTMTKMDPVDTATYYCAR1PYYGSGSH NYGMDVWGQGTTVTVSS 168 . FL_2 VL D1QMTQSPSSLSASVGDRVTITCRASQD1RNDFGWY QQKPGKAPQRLLYAASTLQSGVPSRFSGSGSGTEFT LT1SSLQPEDFATYYCLQYNTYPWTFGQGTKVE1K 169. FL_2 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SW1RQPPGKALEWLAH1FSNDEKTYSTSLKSRLT1S RDTSKGQWLTMTKMDPVDTATYYCAR1PYYGSGSH NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSD1QM TQSPSSLSASVGDRVT1TCRASQD1RNDFGWYQQKP GKAPQRLLYAASTLQSGVPSRFSGSGSGTEFTLT1S SLQPEDFATYYCLQYNTYPWTFGQGTKVE1K

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170. FL_2 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SWIRQPPGKALEWLAHIFSNDEKTYSTSLKSRLTIS RDTSKGQWLTMTKMDPVDTATYYCARIPYYGSGSH NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQDIRNDFGWYQQKP GKAPQRLLYAASTLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQYNTYPWTFGQGTKVEIKSGGGG SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 171. FL3 VH CDR1 NARMAVS 172 . FL3 VH CDR2 HIFSNGEKSYSTSLKS 173. FL3 VH CDR3 IVGYSDWLLPFDH 174 . FL3 VL CDR1 RASQNINRFLN 175. FL3 VL CDR2 AASSLQS 176. FL3 VL CDR3 LQHNSYPWT 177 . FL_3 vh QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMAV SWIRQPPGKALEWLAHIFSNGEKSYSTSLKSRLTIS KDTSKTQWLTMTNTDPVDTATYFCARIVGYSDWLL PFDHWGQGIMVTVSS 178 . FL_3 VL DIQMTQSPSSLSASVGDRVTITCRASQNINRFLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCLQHNSYPWTFGQGTKVDIK 179 . FL_3 s cFv QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMAV SWIRQPPGKALEWLAHIFSNGEKSYSTSLKSRLTIS KDTSKTQWLTMTNTDPVDTATYFCARIVGYSDWLL PFDHWGQGIMVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQNINRFLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCLQHNSYPWTFGQGTKVDIK 180 . FL_3 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMAV SWIRQPPGKALEWLAHIFSNGEKSYSTSLKSRLTIS KDTSKTQWLTMTNTDPVDTATYFCARIVGYSDWLL PFDHWGQGIMVTVSSGGGGSGGGGSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQNINRFLNWYQQKPGK APKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCLQHNSYPWTFGQGTKVDIKSGGGGSE VQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWV RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTIS RDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYI SYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTWT QEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQK PGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTL SGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 181. FL4 VH CDR1 NAKMGVS 182 . FL4 VH CDR2 HIFSNDEKSYSTSLKS 183. FL4 VH CDR3 IVGYGSGWYGYFDY 184 . FL4 VL CDR1 RASQDIRDDLG 185. FL4 VL CDR2 GASTLQS 186. FL4 VL CDR3 LQHNSYPLT 187 . FL_4 vh QVTLKESGPALVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 188 . FL_4 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLGWY QQKPGNAPKRLIYGASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK

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189 . FL_4 scFv QVTLKESGPALVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KGQWLTMTNMD P VDTAT YYCARIVGYG S GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIK 190 . FL_4 Bispecific molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KGQWLTMTNMD P VDTAT YYCARI VGYG S GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 191. FL5 VH CDR1 NARMGVS 192 . FL5 VH CDR2 HIFWNDEKSYSTSLKS 193 . FL5 VH CDR3 IPYYGSGSYNYGMDV 194 . FL5 VL CDR1 RASQGIRNDLG 195 . FL5 VL CDR2 AASSLQS 196. FL5 VL CDR3 LQHNTYPLT 197 . FL_5 vh QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVS S 198 . FL_5 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNTYPLTFGGGTKVDIK 199 . FL_5 scFv QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNTYPLTFGGGTKVDIK 200 . FL_5 Bispecific molecule QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNTYPLTFGGGTKVDIKSGGGG SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 201. FL6 VH CDR1 NARMAVS 202 . FL6 VH CDR2 HIFSNDEKSYSPSLKS 203 . FL6 VH CDR3 IVGYGTGWYGFFDY 204 . FL6 VL CDR1 RASQGIRNDLG 205 . FL6 VL CDR2 AASVLQS 206. FL6 VL CDR3 LQHNSYPLT

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207 . FL_6 vh QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSPSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVS S 208 . FL_6 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWF QQKPGKAPKRLIYAASVLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 209 . FL_6 s cFv QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSPSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPG KAPKRLIYAASVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIK 210 . FL_6 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSPSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWFQQKPG KAPKRLIYAASVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 211. FL7 VH CDR1 NARMGVS 212 . FL7 VH CDR2 HIFSNDEKSYSTSLKN 213. FL7 VH CDR3 IVGYGTGWFGYFDY 214 . FL7 VL CDR1 RASQDIRTDLA 215. FL7 VL CDR2 AASSLQS 216. FL7 VL CDR3 LQHNRYPLT 217 . FL_7 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVS S 218 . FL_7 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRTDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNRYPLTFGGGTKVDIK 219. FL_7 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRTDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNRYPLTFGGGTKVDIK 220. FL_7 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRTDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNRYPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 221. FL8 VH CDR1 YARMGVS

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222 . FL8 VH CDR2 QIFSNDEKSYSTSLKS 223. FL8 VH CDR3 IVGYGTGWYGFFDY 224 . FL8 VL CDR1 RASQGIRNDLG 225. FL8 VL CDR2 AASSLQS 226. FL8 VL CDR3 LQHNSYPLT 227 . FL_8 vh QVTLKESGPALVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KGT SN S QWLTMTNMD P EDTAT YYCARIVGYGT GWY GFFDYWGQGILVTVS S 22Θ . FL_8 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 229. FL_8 s cFv QVTLKESGPALVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KGT SN S QWLTMTNMD P EDTAT YYCARI VGYGT GWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIK 230. FL_8 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KGT SN S QWLTMTNMD P EDTAT YYCARI VGYGT GWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 231. FL9 VH CDR1 NARMGVS 232 . FL9 VH CDR2 HIFSNDEKSYSTSLKS 233. FL9 VH CDR3 IPGYGGNFYYHYYGMDV 234 . FL9 VL CDR1 RASQGIRNDLA 235. FL9 VL CDR2 AASTVQS 236. FL9 VL CDR3 LQHNSFPWT 237 . FL_9 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVS S 238 . FL_9 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLAWY QQKPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFA LTISSLQPEDFATYYCLQHNSFPWTFGQGTKVDIK 239. FL_9 s cFv QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQ KPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFALT ISSLQPEDFATYYCLQHNSFPWTFGQGTKVDIK

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240. FL_9 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQ KPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFALT ISSLQPEDFATYYCLQHNSFPWTFGQGTKVDIKSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 241. FL10 VH CDR1 NARMGVS 242. FL10 VH CDR2 HIFSNDEKSYSTSLKS 243. FL10 VH CDR3 MPEYSSGWSGAFDI 244 . FL10 VL CDR1 RASQDIRDDLG 245. FL10 VL CDR2 GASTLQS 246. FL10 VL CDR3 LQHNSYPLT 247. FL_10 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 248 . FL_10 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLGWY QQKPGNAPKRLIYGASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 249. FL_10 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIK 250. FL_10 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 251. FL11 VH CDR1 NAKMGVS 252. FL11 VH CDR2 QIFSNGEKSYSTSLKS 253. FL 11 VH CDR3 IVGYGSGWYGYFDY 254 . FL11 VL CDR1 RASQGIRNDLG 255. FL11 VL CDR2 GASTLQS 256. FL 11 VL CDR3 LQHNSYPLT 257 . FL_11 vh QVTLKESGPVLVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAQIFSNGEKSYSTSLKSRLTIS KDT S KGQWLTMTNMDPVDTAT YYCS RI VGYGS GWY GYFDYWGQGTLVTVSS 258 . FL_11 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYGASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK

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259 . FL_11 scFv QVTLKESGPVLVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAQIFSNGEKSYSTSLKSRLTIS KDT S KGQ WLTMTNMD P VDTAT YYC S RI VGYG S GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 260 . FL_11 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTLSGFSLNNAKMGV SWIRQPPGKALEWLAQIFSNGEKSYSTSLKSRLTIS KDT S KGQ WLTMTNMD P VDTAT YYC S RI VGYG S GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 261. FL12 VH CDR1 NARMGVS 262 . FL12 VH CDR2 NIFSNDEKSYSTSLKS 263 . FL12 VH CDR3 IVGYGSGWYGYFDY 264 . FL12 VL CDR1 RASQGIRNDLG 265 . FL12 VL CDR2 AASSLQS 266. FL12 VL CDR3 LQHNSYPLT 267 . FL_12 vh QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNMDPEDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 268 . FL_12 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPQRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 269 . FL_12 scFv QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNMDPEDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPQRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 270 . FL_12 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNMDPEDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPQRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 271. FL 13 VH CDR1 NARMGVS 272 . FL 13 VH CDR2 HIFSNDEKSYSTSLKN 273 . FL 13 VH CDR3 IVGYGSGWYGFFDY 274 . FL 13 VL CDR1 RASQGIRNDLG 275 . FL 13 VL CDR2 AASTLQS 276. FL 13 VL CDR3 LQHNSYPLT

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277 . FL_13 vh QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSS 278 . FL_13 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 279 . FL_13 s cFv QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 280 . FL_13 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 281. FL14 VH CDR1 NARMAVS 282 . FL14 VH CDR2 HIFSNDEKSYSTSLKS 283. FL14 VH CDR3 IVGYGSGWYGYFDY 284 . FL14 VL CDR1 RASQDIRNDLG 285. FL14 VL CDR2 AASTLQS 286. FL14 VL CDR3 LQHNSYPLT 287 . FL_14 vh QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 288 . FL_14 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 289. FL_14 scFv QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 290. FL_14 Bispecific molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 291. FL 15 VH CDR1 NARMAVS

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292 . FL 15 VH CDR2 HIFSNDEKSYSTSLKS 293. FL 15 VH CDR3 IVGYGSGWYGYFDY 294 . FL 15 VL CDR1 RASQDIGNDLG 295. FL 15 VL CDR2 AASSLQS 296. FL 15 VL CDR3 LQHNSYPLT 297 . FL_15 vh QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KGQWLTMTNMDPVDTAT YYCARI VGYGS GWY GYFDYWGQGTLVTVSS 29Θ. FL_15 VL DIQMTQSPSSLSASVGDRVTITCRASQDIGNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 299. FL_15 scFv QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KGQWLTMTNMDPVDTAT YYCARI VGYGS GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 300. FL_15 Bispecific molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KGQWLTMTNMDPVDTAT YYCARI VGYGS GWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 301. FL 16 VH CDR1 NARMAVS 302 . FL 16 VH CDR2 HIFSNDEKSYSTSLKS 303. FL 16 VH CDR3 IVGYGSGWYGYFDY 304 . FL 16 VL CDR1 RASQDIRYDLA 305. FL 16 VL CDR2 AASSLQS 306. FL 16 VL CDR3 LQHNFYPLT 307 . FL_16 vh QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 308 . FL_16 VL DIQMTQSPSSVSASVGDRVTITCRASQDIRYDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNFYPLTFGGGTKVEIK 309. FL_16 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQDIRYDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNFYPLTFGGGTKVEIK

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310. FL_16 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQDIRYDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNFYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 311. FL 17 VH CDR1 NARMGVS 312 . FL 17 VH CDR2 NIFSNDEKSYSTSLKS 313. FL 17 VH CDR3 IVGYGSGWYGYFDY 314 . FL 17 VL CDR1 RASQDIRNDLG 315. FL 17 VL CDR2 ATSIRQS 316. FL 17 VL CDR3 LQHNSFPLT 317 . FL_17 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 318 . FL_17 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWY QQKPGKAPKRLIYATSIRQSGVPSRFTGSGSGTEFT LTISGLQPEDFATYFCLQHNSFPLTFGGGTKVEIK 319 . FL_17 s cFv QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYATSIRQSGVPSRFTGSGSGTEFTLTISG LQPEDFATYFCLQHNSFPLTFGGGTKVEIK 320 . FL_17 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKALEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYATSIRQSGVPSRFTGSGSGTEFTLTISG LQPEDFATYFCLQHNSFPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 321. FL 18 VH CDR1 NARMGVS 322 . FL 18 VH CDR2 HIFSNDEKSFSTSLKN 323. FL 18 VH CDR3 MVGYGSGWYAYFDY 324 . FL 18 VL CDR1 RASQSISSYLN 325. FL 18 VL CDR2 AASSLQS 326. FL 18 VL CDR3 LQHNSYPLT 327 . FL_18 vh QVTLKESGPVLVKPTQTLLTTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVS S 328 . FL_18 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK

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329. FL_18 scFv QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 330. FL_18 Bispecific molecule QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 331. FL 19 VH CDR1 YARMGVS 332 . FL 19 VH CDR2 QIFSNDEKSYSTSLKS 333. FL 19 VH CDR3 IVGYGTGWYGYFDY 334 . FL 19 VL CDR1 RASQDIGDDLG 335. FL 19 VL CDR2 AASTLQS 336. FL 19 VL CDR3 LQHNSYPLT 337 . FL_19 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVSS 338 . FL_19 VL DIQMTQSPSSLSASVGDRVTITCRASQDIGDDLGWY QQKPGKAPKRLIYAASTLQSGVPFRFSGSGSGTDFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 339. FL_19 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGDDLGWYQQKPG KAPKRLIYAASTLQSGVPFRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 340. FL_19 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLSYARMGV SWIRQPPGKALEWLAQIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGDDLGWYQQKPG KAPKRLIYAASTLQSGVPFRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 341. FL20 VH CDR1 NARMAVS 342 . FL20 VH CDR2 HIFSNDEKSYSTSLKS 343. FL20 VH CDR3 IVGYGTGWYGFFDY 344 . FL20 VL CDR1 RASQGIRNDLA 345. FL20 VL CDR2 AASSLQS 346. FL20 VL CDR3 LQHNSYPLT

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347 . FL_2 0 vh QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVS S 348 . FL_2 0 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 349 . FL_2 0 s cFv QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 350 . FL_2 0 Bispecific molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 351. FL 21 VH CDR1 NARMGVS 352 . FL 21 VH CDR2 HIFSNDEKSYSTSLKS 353. FL 21 VH CDR3 IPGYGGNFYYHYYGMDV 354 . FL 21 VL CDR1 RTSRGIRNDLG 355. FL 21 VL CDR2 AASTLQS 356. FL 21 VL CDR3 LQHNNFPWT 357 . FL_21 vh QVTLKESGPALVKPTETLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KS QWLTMTNMD P VDTAT YYCARI P GYGGN FY YHYYGMDVWGQGTTVTVS S 358 . FL_21 VL DIQMTQSPSSLSASVGDRVTITCRTSRGIRNDLGWY QQIPGRAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNNFPWTFGQGTKVEIK 359. FL_21 scFv QVTLKESGPALVKPTETLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KS QWLTMTNMD P VDTAT YYCARI P GYGGN FY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRTSRGIRNDLGWYQQ IPGRAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCLQHNNFPWTFGQGTKVEIK 360. FL_21 Bispecific molecule QVTLKESGPALVKPTETLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KS QWLTMTNMD P VDTAT YYCARI P GYGGN FY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRTSRGIRNDLGWYQQ IPGRAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLT ISSLQPEDFATYYCLQHNNFPWTFGQGTKVEIKSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 361. FL22 VH CDR1 NARMGVS

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362 . FL22 VH CDR2 HIFSNDEKSYSTSLKS 363. FL22 VH CDR3 MPEYSSGWSGAFDI 364 . FL22 VL CDR1 RASQGISNYLA 365 . FL22 VL CDR2 AASTLQS 366. FL22 VL CDR3 LQHNTYPWT 367 . FL_22 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 36Θ . FL_22 VL DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNTYPWTFGQGTKVEIK 369. FL_22 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNTYPWTFGQGTKVEIK 370 . FL_22 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNTYPWTFGQGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 371. FL 23 VH CDR1 NARMGVS 372 . FL 23 VH CDR2 HIFSNDEKSYSTSLKS 373. FL 23 VH CDR3 MPEYSSGWSGAFDI 374 . FL 23 VL CDR1 RASQDIGYDLG 375. FL 23 VL CDR2 AASTLQS 376. FL 23 VL CDR3 LQHNSFPWT 377 . FL_2 3 vh QVTLKESGPALVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 378 . FL_2 3 VL DIQMTQSPSSLSASVGDRVTITCRASQDIGYDLGWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LIISSLQPEDFATYYCLQHNSFPWTFGQGTKVEIK 379. FL_2 3 scFv QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGYDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLIISS LQPEDFATYYCLQHNSFPWTFGQGTKVEIK

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380. FL_2 3 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGYDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLIISS LQPEDFATYYCLQHNSFPWTFGQGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ kpgqaprgliggtkflapgtparfsgsllggkaa.lt LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 381. FL 2 4 VH CDR1 NVRMGVS 382 . FL24 VH CDR2 HIFSNDEKSYSTSLKS 383. FL 2 4 VH CDR3 MPEYSSGWSGAFDI 384 . FL 2 4 VL CDR1 RASQDIRDDLV 385. FL 2 4 VL CDR2 AASTLQS 386. FL 2 4 VL CDR3 LQHHSYPWT 387 . FL_2 4 vh QVTLKESGPALVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLILTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 388 . FL_2 4 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLVWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHHSYPWTFGQGTKVEIK 3Θ9 . FL_2 4 s cFv QVTLKESGPALVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLILTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHHSYPWTFGQGTKVEIK 390 . FL_2 4 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLILTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHHSYPWTFGQGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 391. FL 2 5 VH CDR1 NARMGVS 392 . FL 2 5 VH CDR2 HIFSNDEKSYSTSLKS 393. FL 2 5 VH CDR3 MPEYSSGWSGAFDI 394 . FL 2 5 VL CDR1 RASQDIRDDLG 395. FL 25 VL CDR2 AASTLQS 396. FL 2 5 VL CDR3 LQHNSFPFT 397 . FL_25 vh QVTLKESGPALVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 398 . FL_25 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLGWY QQKPGKAPKRLIYAASTLQSGVPSRFTGGGSGTEFT LTISSLQPEDFATYYCLQHNSFPFTFGGGTKVEIK

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399. FL_2 5 scFv QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFTGGGSGTEFTLTISS LQPEDFATYYCLQHNSFPFTFGGGTKVEIK 400. FL_2 5 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFTGGGSGTEFTLTISS LQPEDFATYYCLQHNSFPFTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ kpgqaprgliggtkflapgtparfsgsllggkaalt LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 401. FL26 VH CDR1 NARMGVS 402 . FL26 VH CDR2 HIFSNDEKSYSTSLKS 403. FL26 VH CDR3 MPEYSSGWSGAFDI 404 . FL26 VL CDR1 RASQGIRNDLV 405. FL26 VL CDR2 GTTLQS 406. FL26 VL CDR3 LQHNSYPLT 407. FL_2 6 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 408 . FL_2 6 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLVWY QQKPGKAPKRLIYGTSTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 409. FL_2 6 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 410. FL_2 6 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 411. FL27 VH CDR1 NARMGVS 412 . FL27 VH CDR2 HIFSNDEKSYSTSLKS 413. FL27 VH CDR3 MPEYSSGWSGAFDI 414 . FL27 VL CDR1 RTSQGIRNDLV 415. FL27 VL CDR2 AASTLQS 416. FL27 VL CDR3 LQHYSYPLT

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417 . FL_2 7 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 418 . FL_27 VL DIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLVWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYFCLQHYSYPLTFGGGTKVEIK 419 . FL_2 7 s cFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLVWYQQKPG KAPKRLIYAASTFCARMPEYSSGFT 420 . FL_2 7 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYFCLQHYSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 421. FL2 8 VH CDR1 NARMGVS 422 . FL2 8 VH CDR2 HIFSNDEKSYSTSLKS 423. FL2 8 VH CDR3 MPEYSSGWSGAFDI 424 . FL2 8 VL CDR1 RASQGIGDDLG 425. FL2 8 VL CDR2 ATSVLQS 426. FL2 8 VL CDR3 LQHNSYPLT 427 . FL_2 8 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 428 . FL_2 8 VL DIQMTQSPSSLSASVGDRVTITCRASQGIGDDLGWY QQIPGKAPKRLIYATSVLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 429. FL_2 8 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIGDDLGWYQQIPG KAPKRLIYATSVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 430. FL_2 8 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIGDDLGWYQQIPG KAPKRLIYATSVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 431. FL29 VH CDR1 NARMGVS

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432. FL29 VH CDR2 HIFSNDEKSYRTSLKS 433. FL29 VH CDR3 IVGYGSGWYAYFDY 434 . FL29 VL CDR1 RASQGIRNDLG 435. FL29 VL CDR2 AASSLQS 436. FL29 VL CDR3 LQHNSYPLT 437. FL_2 9 vh QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSS 43Θ . FL_2 9 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 439. FL_2 9 scFv QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 440. FL_2 9 Bispecific molecule QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 441. FL 30 VH CDR1 NARMGVS 442 . FL 30 VH CDR2 LIYWNDDKRYSPSLKS 443. FL 30 VH CDR3 MVGYGSGWYAYFDY 444 . FL 30 VL CDR1 RASQGIRNDLG 445. FL 30 VL CDR2 AASSLQS 446. FL 30 VL CDR3 LQHNSYPLT 447 . FL_3 0 vh QVTLKESGPVLVKPTQTLLTTCTFSGFSLSNARMGV SWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQWLTMTNMD PVDTATYYCARMVGYGS GWY AYFDYWGQGTLVTVSS 448. FL_3 0 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 449. FL_3 0 scFv QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQWLTMTNMD P VDTAT YYCARMVGYGS GWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK

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450. FL_3 0 Bispecific molecule QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTIT KDTSKNQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 451. FL 31 VH CDR1 NARMGVS 452. FL 31 VH CDR2 HIFSNDEKSYSTSLKS 453. FL 31 VH CDR3 IVGYGTGWYGFFDY 454 . FL 31 VL CDR1 RTSQGIRNDLG 455. FL 31 VL CDR2 AASSLQS 456. FL 31 VL CDR3 LQHNSYPLT 457. FL_31 vh QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVS S 458. FL_31 VL DIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 459. FL_31 scFv QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 460. FL_31 Bispecific molecule QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 461. FL 32 VH CDR1 NARMGVS 462 . FL 32 VH CDR2 HIFSNDEKSYSTSLKS 463. FL 32 VH CDR3 IVGYGSGWYGYFDY 464. FL 32 VL CDR1 RASQGIRNDLV 465. FL 32 VL CDR2 AASTLQS 466. FL 32 VL CDR3 LQHYSYPLT 467. FL_32 vh QVTLKESGPTLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 468 . FL_32 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLVWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFI LTISSLQPEDFATYFCLQHYSYPLTFGGGTKLEIK

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469. FL_32 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFILTISS LQPEDFATYFCLQHYSYPLTFGGGTKLEIK 470. FL_32 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFILTISS LQPEDFATYFCLQHYSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 471. FL 33 VH CDR1 NARMGVS 472. FL 33 VH CDR2 HIFSNDEKSYSTSLKS 473. FL 33 VH CDR3 IVGYGSGWYGYFDY 474 . FL 33 VL CDR1 RASQGIGDDLG 475. FL 33 VL CDR2 ATSVLQS 476. FL 33 VL CDR3 LQHNSYPLT 477. FL_3 3 vh QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 478. FL_3 3 VL DIQMTQSPSSLSASVGDRVTITCRASQGIGDDLGWY QQIPGKAPKRLIYATSVLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKLEIK 479. FL_33 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIGDDLGWYQQIPG KAPKRLIYATSVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIK 480. FL_33 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMGV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKGQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIGDDLGWYQQIPG KAPKRLIYATSVLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 481. FL 3 4 VH CDR1 NARMGVS 482 . FL 3 4 VH CDR2 HIFWNDEKSYSTSLKS 483. FL 3 4 VH CDR3 IPYYGSGSYNYGMDV 484. FL 34 VL CDR1 RASQGIRNDLG 485. FL 3 4 VL CDR2 AASSLQS 486. FL 3 4 VL CDR3 LQHNSYPLT

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487 . FL_34 vh QVTLKESGPALVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVS S 488 . FL_34 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKLEIK 489 . FL_34 s cFv QVTLKESGPALVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNSYPLTFGGGTKLEIK 490 . FL_34 Bispecific molecule QVTLKESGPALVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKALEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGG SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 491. FL 35 VH CDR1 NARMGVS 492 . FL 35 VH CDR2 HIFSNDEKSYSTSLKN 493. FL 35 VH CDR3 IVGYGTGWYGFFDY 494 . FL 35 VL CDR1 RASQDIRDDLV 495. FL 35 VL CDR2 GTTLQS 496. FL 35 VL CDR3 LQHHSYPLT 497 . FL_35 vh QVTLKESGPALVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWY GFFDYWGQGTQVTVS S 498 . FL_35 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLVWY QQKPGKAPKRLIYGTSTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHHSYPLTFGGGTKLEIK 499. FL_35 scFv QVTLKESGPALVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWY GFFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHHSYPLTFGGGTKLEIK 500. FL_35 Bispecific molecule QVTLKESGPALVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWY GFFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHHSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 501. FL 3 6 VH CDR1 YARMGVS

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502 . FL 3 6 VH CDR2 HIFSNDEKSYSTSLKS 503. FL 36 VH CDR3 MPEYSSGWSGAFDI 504. FL 36 VL CDR1 RASQDIRNLA 505. FL 36 VL CDR2 AASSLQS 506. FL 3 6 VL CDR3 LQHNSYPLT 507 . FL_3 6 vh QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 508 . FL_3 6 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKLEIK 509. FL_3 6 scFv QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSYTKGSGSPGGLEPLTYPLG 510. FL_3 6 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 511. FL 37 VH CDR1 NIKMGVS 512. FL 37 VH CDR2 HIFSNDEKSYSTSLKS 513. FL 37 VH CDR3 MPEYSSGWSGAFDI 514. FL 37 VL CDR1 RASQDISNYLA 515. FL 37 VL CDR2 AASSLQS 516. FL 37 VL CDR3 LQHNSFPLT 517. FL_37 vh QVTLKESGPVLVKPTETLTLTCTVSGFSLRNIKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 518. FL_37 VL DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSFPLTFGGGTKLEIK 519. FL_37 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSLRNIKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDISNYLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSFPLTFGGGTKLEIK

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520. FL_37 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNIKMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDISNYLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSFPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 521. FL 38 VH CDR1 NARMGVS 522. FL 38 VH CDR2 HIFSNDEKSYSTSLKS 523. FL 38 VH CDR3 MPEYSSGWSGAFDI 524 . FL 38 VL CDR1 RASQGIRNDLG 525. FL 38 VL CDR2 AASSLQS 526. FL 38 VL CDR3 LQHNSYPLT 527 . FL_3 8 vh QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTLVTVSS 528. FL_3 8 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKLEIK 529. FL_3 8 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIK 530. FL_3 8 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 531. FL 39 VH CDR1 NARMGVS 532. FL 39 VH CDR2 HIFSNDEKSYSTSLKN 533. FL 39 VH CDR3 IVGYGSGWYGFFDY 534 . FL 39 VL CDR1 RASQGIRNDLG 535. FL 39 VL CDR2 AASTLQS 536. FL 39 VL CDR3 LQHNSYPLT 537 . FL_3 9 vh QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSS 538. FL_3 9 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK

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539. FL_3 9 scFv QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIK 540. FL_3 9 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 541. FL40 VH CDR1 NARMGVS 542. FL40 VH CDR2 NIFSNDEKSYSTSLKS 543. FL40 VH CDR3 IVGYGSGWYGYFDY 544 . FL40 VL CDR1 RASQDIRNDLG 545. FL40 VL CDR2 ATSIRQS 546. FL40 VL CDR3 LQHNSFPLT 547 . FL_4 0 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSS 548. FL_4 0 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLGWY QQKPGKAPKRLIYATSIRQSGVPSRFTGSGSGTEFT LTISGLQPEDFATYFCLQHNSFPLTFGCGTKVEIK 549. FL_4 0 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYATSIRQSGVPSRFTGSGSGTEFTLTISG LQPEDFATYFCLQHNSFPLTFGGGTKVEIK 550. FL_4 0 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLANIFSNDEKSYSTSLKSRLTIS KGTSKSQWLTMTNVNPVDTGTYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLGWYQQKPG KAPKRLIYATSIRQSGVPSRFTGSGSGTEFTLTISG LQPEDFATYFCLQHNSFPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 551. FL41 VH CDR1 NARMGVS 552 . FL41 VH CDR2 HIFSNDEKSFSTSLKN 553. FL41 VH CDR3 MVGYGSGWYAYFDY 554. FL41 VL CDR1 RASQSISSYLN 555. FL41 VL CDR2 AASSLQS 556. FL41 VL CDR3 LQHNSYPLT

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557 . FL_41 vh QVTLKESGPVLVKPTQTLLTTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVS S 558 . FL_41 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK 559 . FL_4 1 s cFv QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 560 . FL_4 1 Bispecific molecule QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSFSTSLKNRLTIS KDTSKSQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPG KAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 561. FL42 VH CDR1 NARMAVS 562 . FL42 VH CDR2 HIFSNDEKSYSTSLKS 563. FL42 VH CDR3 IVGYGTGWYGFFDY 564 . FL42 VL CDR1 RASQGIRNDLA 565. FL42 VL CDR2 AASSLQS 566. FL42 VL CDR3 LQHNSYPLT 567 . FL_42 vh QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVS S 568 . FL_42 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK 569. FL_42 scFv QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK 570. FL_42 Bispecific molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 571. FL 4 3 VH CDR1 NARMGVS

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572. FL 43 VH CDR2 HIFSNDEKSYSTSLKS 573. FL 43 VH CDR3 MPEYSSGWSGAFDI 574. FL 43 VL CDR1 RASQGIRNDLV 575. FL 4 3 VL CDR2 GTTLQS 576. FL 43 VL CDR3 LQHNSYPLT QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV 577 SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLVWY 57Θ. FL 4 3 VL QQKPGKAPKRLIYGTSTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS 579. FL 4 3 scFv GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIK QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLVWYQQKPG KAPKRLIYGTSTLQSGVPSRFSGSGSGTEFTLTISS S i η Bispecific LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS molecule EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 581. FL44 VH CDR1 NARMGVS 582. FL44 VH CDR2 HIFSNDEKSYSTSLKS 583. FL 4 4 VH CDR3 MPEYSSGWSGAFDI 584 . FL 4 4 VL CDR1 RTSQGIRNDLV 585. FL 4 4 VL CDR2 AASTLQS 586. FL44 VL CDR3 LQHYSYPLT QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV 587 SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS DIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLVWY 588. FL44 VL QQKPGKAPKRLIYAASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYFCLQHYSYPLTFGCGTKVEIK QVTLKESGPVLVKPTETLTLTCTVSGFSFNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS 589. FL 4 4 scFv GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYFCLQHYSYPLTFGCGTKVEIK

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590. FL_4 4 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLVWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYFCLQHYSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ kpgqaprgliggtkflapgtparfsgsllggkaalt LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 591. FL45 VH CDR1 NARMGVS 592. FL45 VH CDR2 HIFSNDEKSYRTSLKS 593. FL45 VH CDR3 IVGYGSGWYAYFDY 594 . FL 4 5 VL CDR1 RASQGIRNDLG 595. FL45 VL CDR2 AASSLQS 596. FL 4 5 VL CDR3 LQHNSYPLT 597. FL_4 5 vh QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSS 598 . FL_4 5 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK 599. FL_4 5 scFv QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIK 600. FL_4 5 Bispecific molecule QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYRTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 601. FL 4 6 VH CDR1 NARMGVS 602. FL 4 6 VH CDR2 LIYWNDDKRYSPSLKS 603. FL 4 6 VH CDR3 MVGYGSGWYAYFDY 604 . FL 4 6 VL CDR1 RASQGIRNDLG 605. FL 4 6 VL CDR2 AASSLQS 606. FL 4 6 VL CDR3 LQHNSYPLT 607. FL_4 6 vh QVTLKESGPVLVKPTQTLLTTCTFSGFSLSNARMGV SWIRQPPGKCLEWLALIYWNDDKRYSPSLKSRLTIT KDTSKNQWLTMTNMDPVDTATYYCARMVGYGSGWY AYFDYWGQGTLVTVSS 608 . FL_4 6 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK

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609 . FL_4 6 scFv QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQWLTMTNMD PVDTATYYCARMVGYG S GWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIK 610 . FL_4 6 Bispecific molecule QVTLKESGPVLVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQWLTMTNMD P VDTAT YYCARMVGYG S GWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 611. FL 4 7 VH CDR1 NARMGVS 612 . FL 4 7 VH CDR2 HIFSNDEKSYSTSLKS 613 . FL 4 7 VH CDR3 IVGYGTGWYGFFDY 614 . FL 4 7 VL CDR1 RTSQGIRNDLG 615 . FL 47 VL CDR2 AASSLQS 616. FL 4 7 VL CDR3 LQHNSYPLT 617 . FL_4 7 vh QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVS S 618 . FL_4 7 VL DIQMTQSPSSLSASVGDRVTITCRTSQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK 619 . FL_4 7 scFv QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIK 620 . FL_4 7 Bispecific molecule QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTDMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRTSQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 621. FL 4 8 VH CDR1 NARMGVS 622 . FL 4 8 VH CDR2 HIFWNDEKSYSTSLKS 623 . FL 4 8 VH CDR3 IPYYGSGSYNYGMDV 624 . FL 4 8 VL CDR1 RASQGIRNDLG 625 . FL 4 8 VL CDR2 AASSLQS 626. FL 4 8 VL CDR3 LQHNTYPLT

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627 . FL_4 8 vh QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKCLEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVS S 628 . FL_4 8 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNTYPLTFGCGTKVDIK 629 . FL_4 8 s cFv QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKCLEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNTYPLTFGCGTKVDIK 630 . FL_4 8 Bispecific molecule QVTLKESGPMLVKPTETLTLTCTFSGFSLRNARMGV SWIRQPPGKCLEWLAHIFWNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPYYGSGSY NYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM TQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTIS SLQPEDFATYYCLQHNTYPLTFGCGTKVDIKSGGGG SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 631. FL 4 9 VH CDR1 NARMGVS 632 . FL 4 9 VH CDR2 HIFSNDEKSYSTSLKN 633. FL 4 9 VH CDR3 IVGYGTGWFGYFDY 634 . FL 4 9 VL CDR1 RASQDIRTDLA 635. FL 4 9 VL CDR2 AASSLQS 636. FL 4 9 VL CDR3 LQHNRYPLT 637 . FL_4 9 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVS S 638 . FL_4 9 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRTDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNRYPLTFGCGTKVDIK 639. FL_4 9 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRTDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNRYPLTFGCGTKVDIK 640. FL_4 9 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGTGWF GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRTDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNRYPLTFGCGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 641. FL50 VH CDR1 NARMGVS

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642 . FL50 VH CDR2 HIFSNDEKSYSTSLKS 643. FL50 VH CDR3 IPGYGGNFYYHYYGMDV 644 . FL50 VL CDR1 RASQGIRNDLA 645. FL50 VL CDR2 AASTVQS 646. FL50 VL CDR3 LQHNSFPWT 647 . FL_5 0 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVS S 64Θ. FL_5 0 VL DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLAWY QQKPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFA LTISSLQPEDFATYYCLQHNSFPWTFGCGTKVDIK 649. FL_5 0 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQ KPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFALT ISSLQPEDFATYYCLQHNSFPWTFGCGTKVDIK 650. FL_5 0 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLSNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIPGYGGNFY YHYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSDI QMTQSPSSLSASVGDRVTITCRASQGIRNDLAWYQQ KPGKAPKRLIYAASTVQSGVPSRFSGSGSGTEFALT ISSLQPEDFATYYCLQHNSFPWTFGCGTKVDIKSGG GGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYA MNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFG NSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQ TWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNW VQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKA ALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 651. FL51 VH CDR1 NVRMGVS 652 . FL51 VH CDR2 HISSNDEKSYSTSLRS 653. FL51 VH CDR3 MPGDSNTWRGFFDY 654 . FL51 VL CDR1 RTSQSVNNLA 655. FL51 VL CDR2 GAST RAT 656. FL51 VL CDR3 LQHNSYPLT 657 . FL_51 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKCLEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSS 658 . FL_51 VL EIVMTQSPATLSVSPGERATLSCRTSQSVNNNLAWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVEIK 659. FL_51 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKCLEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMT QSPATLSVSPGERATLSCRTSQSVNNNLAWYQQKPG QAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIK

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660. FL_51 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKCLEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMT QSPATLSVSPGERATLSCRTSQSVNNNLAWYQQKPG QAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 661. FL52 VH CDR1 YARMGVS 662. FL52 VH CDR2 HIFSNDEKSYSTSLKS 663. FL52 VH CDR3 MPEYSSGWSGAFDI 664. FL52 VL CDR1 RASQDIRNLA 665. FL52 VL CDR2 AASSLQS 666. FL52 VL CDR3 LQHNSYPLT 667 . FL_52 vh QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 668. FL_52 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRNDLAWY QQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKLEIK 669. FL_52 scFv QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSYTKNGSPTED 670. FL_52 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 671. FL53 VH CDR1 SYGMH 672 . FL53 VH CDR2 VISYDGSNEFYADSVKG 673. FL53 VH CDR3 GGEITMVRGVIGYYYYGMDV 674. FL53 VL CDR1 RTSQSISSYLN 675. FL53 VL CDR2 AASSLQS 676. FL53 VL CDR3 LQHNSYPLT 677. FL_53 vh S 678. FL_53 VL DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVDIK

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679. scFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVDIK FL53 Bispecific QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGCGTKVDIKS 680. FL53 GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK molecule YAMNWVRQAPGKGLEWVARIRS KYNNYATYYAD SVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 681. FL54 VH CDR1 SYGMH 682 . FL54 VH CDR2 VISYDGSNEFYADSVKG 683. FL54 VH CDR3 GGEITMVRGVIGYYYYGMDV 684 . FL54 VL CDR1 RASQGVRNNLV 685. FL54 VL CDR2 GAST RAT 686. 687. FL54 VL CDR3 vh LQHNSYPLT QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVS S EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY 688. FL_54 VL QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGCGTKVEIK QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV 689. FL_54 scFv IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGCGTKVEIK QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGCGTKVEIKS 690. FL54 GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK molecule YAMNWVRQAPGKGLEWVARIRS KYNNYATYYAD SVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 691. FL55 VH CDR1 SYGMH 692. FL55 VH CDR2 VISYDGSNKKYYADSVKG 693. FL55 VH CDR3 SYGMDV 694. FL55 VL CDR1 RASQGISSWLA 695. FL55 VL CDR2 AASSLQS 696. FL55 VL CDR3 QQANSFPWT

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697. FL_55 vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSS 698. FL_55 VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQANSFPWTFGCGTKLEIK 699. FL_55 s cFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQANSFPWTFGCGTKLEIK 700. FL_55 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKCLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQANSFPWTFGCGTKLEIKSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRS KYNNYATYYAD SVKDRFTIS RDD S KNTA YLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQ GTLVTVSSGGGGSGGGGSGGGGSQTWTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL IGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 701. FL56 VH CDR1 NVRMGVS 702. FL56 VH CDR2 HIFSNDEKLYTTSLKS 703. FL56 VH CDR3 IVGYGTGWYGYFDY 704 . FL 5 6 VL CDR1 RASQDIRDDLG 705. FL 5 6 VL CDR2 ATSIRQS 706. FL56 VL CDR3 LQHHSFPLT 707. FL_5 6 vh QVTLKESGPVLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKLYTTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVS S 708. FL_56 VL DIQMTQSPSSLSASIGDRVTITCRASQDIRDDLGWY QREPGKAPKRLIYATSIRQSGVPSRFSGSGSGTEFT LTISGLQPEDFATYFCLQHHSFPLTFGGGTKVDIK 709. FL_56 scFv QVTLKESGPVLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKLYTTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASIGDRVTITCRASQDIRDDLGWYQREPG KAPKRLIYATSIRQSGVPSRFSGSGSGTEFTLTISG LQPEDFATYFCLQHHSFPLTFGGGTKVDIK 710. FL_5 6 Bispecific molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKLYTTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GYFDYWGQGTQVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASIGDRVTITCRASQDIRDDLGWYQREPG KAPKRLIYATSIRQSGVPSRFSGSGSGTEFTLTISG LQPEDFATYFCLQHHSFPLTFGGGTKVDIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 711. FL57 VH CDR1 YARMGVS

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712 . FL57 VH CDR2 HISSNDEKSFSTALES 713. FL57 VH CDR3 MPGDSNTWRGFFDY 714 . FL57 VL CDR1 RTSQTVTNSYIA 715. FL57 VL CDR2 GTSTRAT 716. FL57 VL CDR3 QKYGSSPLT 717 . FL_57 vh QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHISSNDEKSFSTALESRLTIS TDTSKSQMVLTMTNVDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSS 71Θ . FL_57 VL EIVMTQSPGTLSLSPGERATLSCRTSQTVTNSYIAW YQQRPGQAPRLLIYGTSTRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQKYGSSPLTTFGGGTKLEIK 719. FL_57 scFv QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHISSNDEKSFSTALESRLTIS TDTSKSQMVLTMTNVDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMT QSPGTLSLSPGERATLSCRTSQTVTNSYIAWYQQRP GQAPRLLIYGTSTRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQKYGSSPLTFGGGTKLEIK 720. FL_57 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHISSNDEKSFSTALESRLTIS TDTSKSQMVLTMTNVDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMT QSPGTLSLSPGERATLSCRTSQTVTNSYIAWYQQRP GQAPRLLIYGTSTRATGIPDRFSGSGSGTDFTLTIS RLEPEDFAVYYCQKYGSSPLTFGGGTKLEIKSGGGG SEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMN WVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFT ISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNS YISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTV VTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQ QKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAAL TLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 721. FL 5 8 VH CDR1 NVRMGVS 722 . FL58 VH CDR2 HISSNDEKSYSTSLRS 723. FL 5 8 VH CDR3 MPGDSNTWRGFFDY 724 . FL 5 8 VL CDR1 RTSQSVNNLA 725. FL 5 8 VL CDR2 GAST RAT 726. FL 5 8 VL CDR3 LQHNSYPLT 727 . FL_5 8 vh QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSS 728 . FL_5 8 VL EIVMTQSPATLSVSPGERATLSCRTSQSVNNNLAWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 729. FL_5 8 scFv QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGGSGGGGSEIVMT QSPATLSVSPGERATLSCRTSQSVNNNLAWYQQKPG QAPRLLIYGASTRATGISYPARGSGSPGQVECLTYL

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730. FL_5 8 Bispecific molecule QVTLKESGPTLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHISSNDEKSYSTSLRSRLTIS TDTSKSQWLTMTNMDPVDTATYYCARMPGDSNTWR GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMT QSPATLSVSPGERATLSCRTSQSVNNNLAWYQQKPG QAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 731. FL59 VH CDR1 NVRMGVS 732. FL59 VH CDR2 HIFSNDEKSYSTSLKS 733. FL59 VH CDR3 MPEYSSGWSGAFDI 734 . FL 5 9 VL CDR1 RASQDIRDDLG 735. FL59 VL CDR2 GASTLQS 736. FL 5 9 VL CDR3 LQHNSYPLT 737. FL_5 9 vh QVTLKESGPMLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSS 738 . FL_5 9 VL DIQMTQSPSSLSASVGDRVTITCRASQDIRDDLGWY QQKPGNAPKRLIYGASTLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTRLEIK 739. FL_5 9 scFv QVTLKESGPMLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTRLEIK 740. FL_5 9 Bispecific molecule QVTLKESGPMLVKPTETLTLTCTVSGFSLRNVRMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRDDLGWYQQKPG NAPKRLIYGASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTRLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 741. FL60 VH CDR1 NARMGVS 742 . FL60 VH CDR2 HIFSNDEKSYSTSLKS 743. FL60 VH CDR3 MVGYGSGWYAYFDY 744 . FL60 VL CDR1 RSSQSLLHSNGYNYLY 745. FL60 VL CDR2 EVSNRFS 746. FL60 VL CDR3 MQALQTPLT 747. FL_60 vh QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KRQWLTMTNMD PVDTATYYCARMVGYGS GWY AYFDYWGQGTLVTVSS 748 . FL_60 VL DIVMTQTPLSLSVTPGQPASISCRSSQSLLHSNGYN YLYWYLQKPGQPPQLLIYEVSNRFSGVPDRFSGSGS GTDFTLKISRVEAEDVGVYYCMQALQTPLTFGGGTK VEIK

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749. FL_60 scFv QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KRQWLTMTNMD PVDTATYYCARMVGYGS GWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMT QTPLSLSVTPGQPASISCRSSQSLLHSNGYNYLYWY LQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFT LKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIK 750. FL_60 Bispecific molecule QVTLKESGPALVKPTQTLTLTCTFSGFSLSNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDT S KRQWLTMTNMD P VDTAT YYCARMVGYGS GWY AYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMT QTPLSLSVTPGQPASISCRSSQSLLHSNGYNYLYWY LQKPGQPPQLLIYEVSNRFSGVPDRFSGSGSGTDFT LKISRVEAEDVGVYYCMQALQTPLTFGGGTKVEIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 751. FL61 VH CDR1 SYGMH 752 . FL61 VH CDR2 VISYDGSNEFYADSVKG 753. FL61 VH CDR3 GGEITMVRGVIGYYYYGMDV 754 . FL61 VL CDR1 RASQSISSYLN 755. FL61 VL CDR2 AASSLQS 756. FL61 VL CDR3 LQHNSYPLT 757 . FL_61 vh S 758 . FL_61 VL DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 759. FL_61 scFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIK 760. FL_61 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 761. FL62 VH CDR1 SYGMH 762 . FL62 VH CDR2 VISYDGSNEFYADSVKG 763. FL62 VH CDR3 GGEITMVRGVIGYYYYGMDV 764 . FL62 VL CDR1 RASQGVRNNLV 765. FL62 VL CDR2 GAST RAT 766. FL62 VL CDR3 LQHNSYPLT

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767 . FL_62 vh S 768 . FL_62 VL EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGGGTKVEIK 769. FL_62 scFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGGGTKVEIK 770. FL_62 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS EIVMTQSPATLSVSPGERATLSCRASQGVRNNLVWY QQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFT LTISSLQSEDFATYYCLQHNSYPLTFGGGTKVEIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 771. FL63 VH CDR1 SYGMH 772 . FL63 VH CDR2 VISYDGSNEFYADSVKG 773. FL63 VH CDR3 GGEITMVRGVIGYYYYGMDV 774 . FL63 VL CDR1 RTSQSISSYLN 775. FL63 VL CDR2 AASSLQS 776. FL63 VL CDR3 LQHNSYPLT 777 . FL_63 vh S 778 . FL_63 VL DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 779. FL_63 scFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK

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780 . FL_63 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRTSQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 781. FL64 VH CDR1 SYGMH 782 . FL64 VH CDR2 VISYDGSNEFYADSVKG 783 . FL64 VH CDR3 GGEITMVRGVIGYYYYGMDV 784 . FL64 VL CDR1 RASQSISSYLN 785 . FL64 VL CDR2 AASSLQS 786. FL64 VL CDR3 LQHNSYPLT 787 . FL_64 vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVS 788 . FL_64 VL DIQMTQSPSSLSASVGNRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 789 . FL_64 s cFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGNRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIK 790 . FL_64 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNEFYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGNRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVDIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VL 791. FL65 VH CDR1 SYGMH 792 . FL65 VH CDR2 VISYDGSNKKYYADSVKG 793 . FL65 VH CDR3 SYGMDV 794 . FL65 VL CDR1 RASQGISSWLA 795 . FL65 VL CDR2 AASSLQS 796. FL65 VL CDR3 QQANSFPWT 797 . FL_65 vh QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSS 798 . FL_65 VL DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT LTISSLQPEDFATYYCQQANSFPWTFGQGTKLEIK

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799. FL_65 scFv QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQANSFPWTFGQGTKLEIK 800. FL_65 Bispecific molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYDGSNKYYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARSYGMDVWGQG TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSVSAS VGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYA ASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATY YCQQANSFPWTFGQGTKLEIKSGGGGSEVQLVESGG GLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGL EWVARIRS KYNNYATYYAD SVKDRFTIS RDD S KNTA YLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQ GTLVTVSSGGGGSGGGGSGGGGSQTWTQEPSLTVS PGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGL IGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDE AEYYCVLWYSNRWVFGGGTKLTVL 801. FLT3 human vl NM_004119 Human Sequence MPALARDGGQLPLLWFSAMIFGTITNQDLPVIKCV LINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSS GTVYEAAAVEVDVSASIT LQVLVDAP GNIS CLWVFK HSSLNCQPHFDLQNRGWSMVILKMTETQAGEYLLF IQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQD ALVCISESVPEPIVEWVLCDSQGESCKEESPAWKK EEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQT PQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELE NKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARND TGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKG LDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFT LNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCS DKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMS EAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD NISFYATIGVCLLFIWLTLLICHKYKKQFRYESQL QMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEF GKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKE KADSSEREALMSELKMMTQLGSHENIVNLLGACTLS GPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKE HNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLH GNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFA YQVAKGMEFLEFKSCVHRDLAARNVLVTHGKWKIC D FGLARD IMS D SN YWRGNARL P VKWMAP E S L FEGI YTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYK LIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSF PNLTSFLGCQLADAEEAMYQNVDGRVSECP HTYQNR RPFSREMDLGLLSPQAQVEDS

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802. FLT3 cynomolgus monkey xl ХМ_00558554 4 Macaque sequence MPALARGGGRLPLLWFSAMIFGTITNQDLPVIKCV LINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSA GTVYEAATVEVDVSASIТ LQVLVDT Р GNIS CLWVFK HSSLNCQPHFDVQNRGWSMVILKMTETQAGEYLLF IQSEATNYTILFTVSIRNTLLFTLRRPYFRKMENQD ALVCISESVPEPIVEWVLCDSQGESCKEESPAWKK EEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQT PQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELE NKALEEGSYFEMSTYSTNRTMIRILFAFVSSVARND TGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKG LDDGYSVSKFCNHKHQPGEYIFHAENGDAQFTKMFT LNIRRKPQVLAEASASQASCSSDGYPLPSWTWKKCS DKSPNCTEDIPEGVWNRKANRKVFGQWVSSSTLNMS EAMKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD SISFYATIGVCLLFIWLTMLICHKYKKQFRYESQL QMVQVTGSSDNEYFYVDFRDCDYDLKWEFPRENLEF GKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKE KADSSEREALMSELKMMTQLGSHENIVNLLGACTLS GPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKE HNFSFYPTFQSHPNSSMPGSRDVQIHPHSDPISGLH GNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFA YQVAKGMEFLEFKSCVHRDLAARNVLVTHGKWKIC D FGLARD IMS D SN YWRGNARL РVKWMAP Е S L FEGI YTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYK LIRNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSF PHLTSFLGCQLEDAEEAMYQNVDGRVSERP HIYQNR RPFSREMDSGPLSPKAQVEDS 803. Human FLT3 (T227M) isoform Human Sequence MPALARDGGQLPLLWFSAMIFGTITNQDLPVIKCV LINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSS GTVYEAAAVEVDVSASIT LQVLVDAP GNIS CLWVFK HSSLNCQPHFDLQNRGWSMVILKMTETQAGEYLLF IQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQD ALVCISESVPEPIVEWVLCDSQGESCKEESPAWKK EEKVLHELFGMDIRCCARNELGRECTRLFTIDLNQT PQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELE NKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARND TGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKG LDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFT LNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCS DKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMS EAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD NISFYATIGVCLLFIWLTLLICHKYKKQFRYESQL QMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEF GKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKE KADSSEREALMSELKMMTQLGSHENIVNLLGACTLS GPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKE HNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLH GNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFA YQVAKGMEFLEFKSCVHRDLAARNVLVTHGKWKIC D FGLARD IMS D SN YWRGNARL P VKWMAP E S L FEGI YTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYK LIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSF PNLTSFLGCQLADAEEAMYQNVDGRVSECP HTYQNR RPFSREMDLGLLSPQAQVEDS

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804. Human FLT3ITD isoform Human Sequence MPALARDGGQLPLLWFSAMIFGTITNQDLPVIKCV LINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSS GTVYEAAAVEVDVSASIТ LQVLVDAP GNIS CLWVFK HSSLNCQPHFDLQNRGWSMVILKMTETQAGEYLLF IQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQD ALVCISESVPEPIVEWVLCDSQGESCKEESPAWKK EEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQT PQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELE NKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARND TGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKG LDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFT LNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCS DKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMS EAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD NISFYATIGVCLLFIWLTLLICHKYKKQFRYESQL QMVQVTGSSDNEYFYVDFREYEYSSSDNEYFYVDFR EYEYDLKDLKWEFPRENLEFGKVLGSGAFGKVMNAT AYGISKTGVSIQVAVKML KE KAD S S ЕREALMS ЕLКМ MTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLL NYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSS MPGSREVQIHPDSDQISGLHGNSFHSEDEIEYENQK RLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCV HRDLAARNVLVTHGKWKICDFGLARDIMSDSNYW RGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWE IFSLGVNPYPGIPVDANFYKLIQNGFKMDQPFYATE EIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAE E AMYQNVDGRVSECPHTYQNRRPFSREMDLGLLSPQA QVEDS 805. Paralog KIT vl human Human Sequence MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSP PSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILD ETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIY VFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVT NYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYH RLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPWSV SKASYLLREGEEFTVTCTIKDVSSSVYSTWKRENSQ TKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVF MCYANNTFGSANVTTTLEWDKGFINIFPMINTTVF VNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWE DYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSN SDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAG FPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPP FGKLWQS SI DS SAFKHNGTVECKAYNDVGKT SAYF NFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIV MILTYKYLQKPMYEVQWKWEEINGNNYVYIDPTQL PYDHKWEFPRNRLSFGKTLGAGAFGKWEATAYGLI К S DAAMTVAVKML К Р SАН LT Е REALMS Е L KVL S YL G NHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRR KRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNE YMDMKPGVSYWPTKADKRRSVRIGSYIERDVTPAI MEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRD LAARNILLTHGRITKICDFGLARDIKNDSNYWKGN ARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFS LGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMY DIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIY SNLANCSPNRQKPWDHSVRINSVGSTA SSSQPLLV HDDV

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806. Paralog CSF1R vl human NM_005211 Human Sequence MGPGVLLLLLVATAWHGQGIPVIEPSVPELWKPGA TVTLRCVGNGSVEWDGPPSPHWTLYSDGSSSILSTN NATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPW N VL AQ Е VWF Е DQDALLPCLLTDPVLEAGVSLVRVR GRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQCSAL MGGRKVMSISIRLKVQKVIPGP PALTLVPAELVRIR GEAAQIVCSASSVDVNFDVFLQHNNTKLAIPQQSDF HNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHS TSMFFRWESAYLNLSSEQNLIQEVTVGEGLNLKVM VEAYPGLQGFNWTYLGPFSDHQPEPKLANATTKDTY RHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFEL TLRYPPEVSVIWTFINGSGTLLCAASGYPQPNVTWL QCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTV QSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAG AHTHPPDEFLFTPVWACMSIMALLLLLLLLLLYKY KQKPKYQVRWKIIESYEGNSYTFIDPTQLPYNEKWE FPRNNLQFGKTLGAGAFGKWEATAFGLGKEDAVLK VAVKMLKSTAHADEKEALMSELKIMSHLGQHENIVN LLGACTHGGPVLVITEYCCYGDLLNFLRRKAEAMLG PSLSPGQDPEGGVDYKNIHLEKKYVRRDSGFSSQGV DTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRDLL Н F S S QVAQ GMAFLAS KN СIНRDVAARNVLLTNGHVA KIGDFGLARDIMNDSNYIVKGNARLPVKWMAPESIF DCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSK FYKLVKD GYQMAQ РAFAP KNIYSIMQACWALЕ Р Т Н R PTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSG SSSSELEEESSSEHLTCCEQGD IAQPLLQPNNYQFC 807 . Human PDGFRA paralog NM_006206 Human Sequence MGTSHPAFLVLGCLLTGLSLILCQLSLPSILPNENE KWQLNSSFSLRCFGESEVSWQYPMSEEESSDVEIR NEENNSGLFVTVLEVSSASAAHTGLYTCYYNHTQTE ENELEGRHIYIYVPDPDVAFVPLGMTDYLVIVEDDD SAIIPCRTTDPETPVTLHNSEGWPASYDSRQGFNG TFTVGPYICEATVKGKKFQTIPFNVYALKATSELDL EMEALKTVYKSGETIWTCAVFNNEWDLQWTYPGE VKGKGITMLEEIKVPSIKLVYTLTVPEATVKDSGDY ECAARQATREVKEMKKVTISVHEKGFIEIKPTFSQL EAVNLHEVKHFWEVRAYPPPRISWLKNNLTLIENL TEITTDVEKIQEIRYRSKLKLIRAKEEDSGHYTIVA QNEDAVKSYTFELLTQVPSSILDLVDDHHGSTGGQT VRCTAEGTPLPDIEWMICKDIKKCNNETSWTILANN VSNIITEIHSRDRSTVEGRVTFAKVEETIAVRCLAK NLLGAENRELKLVAPТLRSЕLTVAAAVLVLLVIVII SLIVLWIWKQKPRYEIRWRVIESISPDGHEYIYVD PMQLPYDSRWEFPRDGLVLGRVLGSGAFGKWEGTA YGLSRSQPVMKVAVKMLKPTARSSEKQALMSELKIM THLGPHLNIVNLLGACTKSGPIYIITEYCFYGDLVN YLHKNRDSFLSHHPEKPKKELDIFGLNPADESTRSY VILSFENNGDYMDMKQADTTQYVPMLERKEVSKYSD IQRSLYDRPASYKKKSMLDSEVKNLLSDDNSEGLTL L D L L S FT YQVARGME FLAS KN СVH RD LAARNVL LAQ GKIVKICDFGLARDIMHDSNYVSKGSTFLPVKWMAP ESIFDNLYTTLSDVWSYGILLWEIFSLGGTPYPGMM VDSTFYNKIKSGYRMAKPDHATSEVYEIMVKCWNSE PEKRPSFYHLSEIVENLLPGQYKKSYEKIHLDF LKS DHPAVARMRVDSDNAYIGVTYKNEEDKLKDWEGGLD EQRLSADSGYIIPLPDIDPVPEEEDLGKRNRHSSQT SEESAIETGSSSSTFIKREDETIEDIDMMDDIGIDS SDLVEDSFL

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808 . Paralog NTM v3 human Human Sequence MGVCGYLFLPWKCLVWSLRLLFLVPTGVPVRSGDA TFPKAMDNVTVRQGESATLRCTIDNRVTRVAWLNRS TILYAGNDKWCLDPRWLLSNTQTQYSIEIQNVDVY DEGPYTCSVQTDNHPKTSRVHLIVQVSPKIVEISSD ISINEGNNISLTCIATGRPEPTVTWRHIS PKAVGFV SEDEYLEIQGITREQSGDYECSASNDVAAPWRRVK VTVNYPPYISEAKGTGVPVGQKGTLQCEASAVPSAE FQWYKDDKRLIEGKKGVKVENRPFLSKLIFFNVSEH DYGNYTCVASNKLGHTNASIMLFEVKTTALTPWKGP GAVSEVSNGTSRRAGCVWLLPLLVLHLLLKF 809. FLT3D2xEpCAM human labeled V5 Artificial sequence MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGT LRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQG ESCKEESPAWKKEEKVLHELFGTDIRCCARNELGR ECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAV HVNHGFGLTWELENKALEEGNYFEMSTYSTNRTMIR IL FAFVS SVARNDT GYYT CSSSKHPSQ SALVTIVEК GFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTW TFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFH AEN D DAQ FT KMFT LNIRRKPQVLAEASAS QAS C FS D GYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKV FGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETI LLNSPGPFPFIQDNISSGGGGSGAGVIAVIWWIA IVAGIWLVISRKKRMAKYEKAEIKEMGEMHRELNA 810. FLT3D3xEpCAM human labeled V5 Artificial sequence MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGF TIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGF GLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFV SSVARNDTGYYTCSSSKHPSQSALVTIVEKGFINAT NSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKS FPCEQKGLDNGYSISKFCNHKHQPGEYIFHAENDDA Q FT KMFT LNIRRKPQVLAEASAS QASCFSDGYPLPS WTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQWVS SSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPG PFPFIQDNISSGGGGSGAGVIAVIWWIAIVAGIV VLVISRKKRMAKYEKAEIKEMGEMHRELNA 811. FLT3D4xEpCAM human labeled V5 Artificial sequence MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGG FINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWT FSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHA END DAQ FTKMFTLNIRRKP QVLAEASAS QAS C F S D G YPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKVF GQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETIL LNSPGPFPFIQDNISSGGGGSGAGVIAVIWWIAI VAGIWLVISRKKRMAKYEKAEIKEMGEMHRELNA 812 . FLT3D5xEpCAM human labeled V5 Artificial sequence MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGI RRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKS PNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEAI KGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNIS SGGGGSGAGVIAVIWWIAIVAGIWLVISRKKRM AKYEKAEIKEMGEMHRELNA 813. EFTS-VKD human Human Sequence MPALARDGGQLPLLWFSAMIFGTITNQDLPVIKCV LINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSS GTVYEAAAVEVDVSASIT LQVLVDAP GNIS CLWVFK HSSLNCQPHFDLQNRGWSMVILKMTETQAGEYLLF IQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQD ALVCISESVPEPIVEWVLCDSQGESCKEESPAWKK EEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQT PQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELE NKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARND TGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYE IDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKG LDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFT LNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCS DKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMS EAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQD NIS

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814 . FLT3-D1 human Human Sequence AAVEVDVSASIT LQVLVDAP GNIS CLWVFKH S S LNC QPHFDLQNRGWSMVILKMTETQAGEYLLFIQSEAT NYTILFTVSIR 815. FLT3-D2 human Human Sequence TLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQ GESCKEESPAWKKEEKVLHELFGTDIRCCARNELG RECTRL 816. FLT3-D3 human Human Sequence FTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHG FGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAF VSSVARNDTGYYTCSSSKHPSQSALVTIV 817 . FLT3-D4 human Human Sequence GFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTW TFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFH AENDDAQFTKMFTLN 818 . FLT3-D5 human Human Sequence IRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDK SPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEA IKGFLVKCCAYNSLGTSCETILLNSPG 819. Human LSP V5 x Flt3 ElmuxEpCpEF DHFR Artificial sequence NQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPED LGCALRPQSSGTVYEA 820. Human LSP V5 x Flt3 E2muxEpCpEF DHFR Artificial sequence AAVEVDVSASIT LQVLVDAP GNIS CLWVFKH S S LNC QPHFDLQNRGWSMVILKMTETQAGEYLLFIQSEAT NYTILFTVSIRNTLLYT 821. Human LSP V5 x Flt3 EXTIHERCpEF DHFR Artificial sequence LRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQG ESCKEESPAWKKEEKVLHELFGTDIRCCARNELGR ECTRL 822. Human LSP V5 x Flt3 ESATICHECpEF DHFR Artificial sequence LRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQG ESC 823. Human LSP V5 x Flt3 E3BtiXerSpEF DHFR Artificial sequence KEESPAWKKEEKVLHELFGTDIRCCARNELGRECT RL 824. Human LSP V5 x Flt3 E4muxEpCpEF DHFR Artificial sequence FTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHG FGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAF VSSVARNDTGYYTCSSSKHPSQSALVTIV 825. Human LSP V5 x Flt3 E5muxEpCpEF DHFR Artificial sequence EKGFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRC TWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYI FHAEN D DAQ FT KMFT LN 826. Human LSP V5 x Flt3 EbtiXerSpEF DHFR Artificial sequence IRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDK SPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEA IKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNI S

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827. Human LSP V5 x Flt3ElmuxEpCpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLISHENNGSS AGKPSSYRMVRGSPEDLQCAPRRQSEGTVYEAAAVE VDVSASIТ LQVLVDAP GNIS СLWVFKH SSLNCQPHF DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVIAVIWWIAIVAGIWLVISRKKRMAKYEKAE IKEMGEMHRELNA 828 . Human LSP V5 x Flt3E2muxEpCpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAATVE VAESGSITLQVQLATPGDLSCLWVFKHSSLGCQPHF DLQNRGIVSMAILNVTETQAGEYLLHIQSEAANYTV LFTVNVRDTQLYVLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVIAVIWWIAIVAGIWLVISRKKRMAKYEKAE IKEMGEMHRELNA 829. Human LSP V5 x Flt3EZTIHERСpEFDHFR Artificial sequence MGWSCIILFLVATATGVHSGKPIPNPLLGLDSTSGN QDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDL GCALRPQ S S GTVYEAAAVEVDVSASIT LQVLVDAP G NISCLWVFKHSSLNCQPHFDLQNRGWSMVILKMTE TQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRP YFRKMENQDALLCISEGVPEPTVEWVLCSSHRESCK EEGPAWRKEEKVLHELFGTDIRCCARNALGRECTK LFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNH GFGLTWELENKALEEGNYFEMSTYSTNRTMIRILFA FVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFIN ATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSR KSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHAEND DAQ FTKMFTLNIRRKP QVLAEASAS QASCFSDGYPL PSWTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQW VSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNS PGPFPFIQDNISSGGGGSGAGVIAVIWWIAIVAG IWLVISRKKRMAKYEKAEIKEMGEMHRELNA

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830. Human LSP V5 x Flt3EZATIHerSpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVE VDVSASIТ LQVLVDAP GNIS СLWVFKH SSLNCQPHF DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALLCISEGVP EPTVEWVLCSSHRESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVI AVI WWI AI VAGI WLVIS RKKRMAKYEKAE IKEMGEMHRELNA 831. Human LSP V5 x Flt3EZVtichErSpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVE VDVSASIT LQVLVDAP GNIS CLWVFKH SSLNCQPHF DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEEGPAWRKEEKVLHELFG TDIRCCARNALGRECTKLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVI AVI WWI AI VAGI WLVI S RKKRMAKYEKAE IKEMGEMHRELNA 832 . Human LSP V5 x Flt3E4muxEpCpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVE VDVSASIT LQVLVDAP GNIS CLWVFKH SSLNCQPHF DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQAPQSTLPQLFL KVGEPLWIRCKAIHVNHGFGLTWELEDKALEEGSYF EMSTYSTNRTMIRILLAFVSSVGRNDTGYYTCSSSK HPSQSALVTILEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVI AVI WWI AI VAGI WLVI S RKKRMAKYEKAE IKEMGEMHRELNA

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833. Human LSP V5 x Flt3E5muxEpCpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVE VDVSASIТ LQVLVDAP GNIS СLWVFKH S S LNCQ РН F DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATSSQEEYEIDPYEKFCFS VRFKAYPRIRCTWIFSQASFPCEQRGLEDGYSISKF CDHKNKPGEYIFYAENDDAQFTKMFTLNIRRKPQVL AEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEI TEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKC CAYNSLGTSCETILLNSPGPFPFIQDNISSGGGGSG AGVIAVIWWIAIVAGIWLVISRKKRMAKYEKAE IKEMGEMHRELNA 834 . Human LSP V5 x Flt3E6muxEpCpEFDHFR Artificial sequence GKPIPNPLLGLDSTSGNQDLPVIKCVLINHKNNDSS VGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVE VDVSASIT LQVLVDAP GNIS CLWVFKH SSLNCQPHF DLQNRGWSMVILKMTETQAGEYLLFIQSEATNYTI LFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVP EPIVEWVLCDSQGESCKEESPAWKKEEKVLHELFG TDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFL KVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYF EMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSK HPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFS VRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKF CNHKHQPGEYIFHAENDDAQFTKMFTLNIRKKPQVL ANASASQASCSSDGYPLPSWTWKKCSDKSPNCTEEI PEGVWNKKANRKVFGQWVSSSTLNMSEAGKGLLVKC CAYNSMGTSCETIFLNSPGPFPFIQDNISSGGGGSG AGVIAVIVVWIAIVAGIWLVISRKKRMAKYEKAE IKEMGEMHRELNA 835. Monomer Fc -1 +c/-g Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT CVWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 836. Monomer Fc -2 +c/-g/delGK Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP 837 . Monomer Fc -3 -c/+g Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P R EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 838 . Monomer Fc -4 -c/+g/delGK Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P R EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP

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839. Monomer Fc -5 -c/-g Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVE VHNAKT К Р R EEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТIS KAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 840. Monomer Fc -6 -c/-g/delGK Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVE VHNAKT К Р R EEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТISKAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP 841. Monomer Fc -7 +c/+g Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT CVWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK 842 . Monomer Fc -8 +c/+g/delGK Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSP 843. scFc-1 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 844 . scFc-2 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P C EEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT KPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSP

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845. scFc-3 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVEVHNAKT К Р R EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТIS KAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT Р Е VT С VWD VS Н Е D Р Е VK FNWYVD GVE VHNA KTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 846. scFc-4 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVE VHNAKT К Р R EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТIS KAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT KPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSP 847 . scFc-5 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P R EEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNA KTKPREEQYGSTYRWSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 848 . scFc-6 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P R EEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQ PREPQVYT L P P S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT KPREEQYGSTYRWSVLTVLHQDWLNGKEYKCKVSN KAL PAPIEKTIS KAKGQ PREPQVYT L P P S REEMT KN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSP

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849. scFc-7 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVEVHNAKT К Р С EEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТISKAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGGS GGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MI S RT Р Е VT С VWD VS Н Е D Р Е VK FNWYVD GVE VHNA KTKPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 850. scFc-8 Artificial sequence DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRT Р Е VT С VWD VS Η Е D Р Е VK FNWYVD GVE VHNAKT К Р С EEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSNKAL РАРIЕКТISKAKGQ PREPQVYT L Р Р S REEMT KNQVS LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI S RT P E VT CVWD VS Η E D P E VK FNW YVD GVE VHNAKT KPCEEQYNSTYRCVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSP 851. Peptide linker Linker (G ₄ S ) 4 GGGGSGGGGSGGGGSGGGGS 852 . Peptide linker Linker (G ₄ S ) 5 GGGGSGGGGSGGGGSGGGGSGGGGS 853. Peptide linker Linker (G ₄ S ) ₆ GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 854 . Peptide linker Linker (G ₄ S ) 7 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 855. Peptide linker Linker (G ₄ S ) ₈ GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSG GGGS

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856. FL_16xCD3scFc Bispecific HLE molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQDIRYDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNFYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNWYVD GVEVHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQ PREP QVYT L P P S RE EMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHN AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 857 . FL_16xCD3scFc delGK Bispecific HLE molecule QVTLKESGPVLVKPTETLTLTCTVSGFSLRNARMAV SWIRQPPGKTLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPVDTATYYCARIVGYGSGWY GYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSVSASVGDRVTITCRASQDIRYDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNFYPLTFGGGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTIS KAKGQ PREP QVYT L P P S RE EMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

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858 . FL_23xCD3scFc Bispecific HLE molecule QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGYDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLIISS LQPEDFATYYCLQHNSFPWTFGQGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNWYVD GVEVHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWDVS Η E D P E VK FNW YVD GVE PP VHN AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 859. FL_23xCD3scFc delGK Bispecific HLE molecule QVTLKESGPALVKPTETLTLTCTVSGFSFRNARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIGYDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLIISS LQPEDFATYYCLQHNSFPWTFGQGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP P VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

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860. FL_36xCD3scFc Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNWYVD GVEVHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE PP VHN AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 861. FL_36xCD3scFc delGK Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGGGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

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862 . FL_39_xCD3scFc Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNWYVD GVEVHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWDVS Η E D P E VK FNW YVD GVE VHN PPV AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 863. FL_39_xCD3scFc delGK Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTLSGFSLNNARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKNRLTIS KDSSKTQWLTMTNVDPVDTATYYCARIVGYGSGWY GFFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASTLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

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864 . FL_42_xCD3scFc Bispecific HLE molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHN PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 865. FL_42_xCD3scFc delGK Bispecific HLE molecule QVTLKESGPALVKPTETLTLTCTLSGFSLNNARMAV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTMTNMDPEDTATYYCARIVGYGTGWY GFFDYWGQGILVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP P VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

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866. EL_46_CCxCD 3-scFc Bispecific HLE molecule QVTLKESGPVLVKPTQTLTLTCTESGESLSNARMGV SWIRQPPGKCLEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQ WLTMTNMD PVDTATYYCARMVGYGS GWY AYEDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRESGSGSGTEETLTISS LQPEDEATYYCLQHNSYPLTEGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGETENKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRETI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKELAPGTPARESGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVEGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVELEPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK ENW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPVLD SDGSEELYSKLTVDKSRWQQGNVESCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVELEPPKPKDT LMIS RT P E VT C VWD VS Η E D P E VK ENW YVD GVE VHN NYKTT PPVLDSDGSEELYSKLTVDKSRWQQGNVESCSVMHE ALHNHYTQKSLSLSPGK 867 . EL_46_CCxCD 3scEc delGK Bispecific HLE molecule QVTLKESGPVLVKPTQTLTLTCTESGESLSNARMGV SWIRQPPGKCLEWLALIYWNDDKRYSPSLKSRLTIT KDT S KNQWLTMTNMD PVDTATYYCARMVGYGS GWY AYEDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPG KAPKRLIYAASSLQSGVPSRESGSGSGTEETLTISS LQPEDEATYYCLQHNSYPLTEGCGTKVEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGETENKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRETI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNEGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKELAPGTPARESGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVEGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVELEPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK ENW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTIS KAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGEYPSDIAVEWESNGQPENNYKTTPPVLD SDGSEELYSKLTVDKSRWQQGNVESCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVELEPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK ENW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGEYPSDIAVEWESNGQPENNY KTTPP VLDSDGSEELYSKLTVDKSRWQQGNVESCSVMHEAL HNHYTQKSLSLSPGK

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868. FL_52_CCxCD 3-scFc Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ kpgqaprgliggtkflapgtparfsgsllggkaa.lt LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT CVWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMIS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHN PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK 869. FL_52_CCxCD 3scFc_delGK Bispecific HLE molecule QVTLKESGPTLVKPTETLTLTCTFSGFSLRYARMGV SWIRQPPGKCLEWLAHIFSNDEKSYSTSLKSRLTIS KDTSKSQWLTLTNMDPVDTATYFCARMPEYSSGWS GAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMT QSPSSLSASVGDRVTITCRASQDIRNDLAWYQQKPG KAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISS LQPEDFATYYCLQHNSYPLTFGCGTKLEIKSGGGGS EVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNW VRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTI SRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSY ISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTW TQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQ KPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALT LSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLGGG GDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR T P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAKT К P CEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVSNKA L PАРIEKTISKAKGQ PREPQVYT L P P S REEMT KNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGGGGSGGGGSGGGGSGGGGSGGGGSG GGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM IS RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHNAK TKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPGK

- 133 -

Claims (19)

870. FL_61xCD3scFc Bispecific HLE molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYEGSNEFYAESVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI S RT P E VT CVWD VS Η E D P E VK FNW YVD GVE VHN AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVT C VWD VS HEDPEVKFNW YVDG VEVHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK 871. FL_61xCD3scFc_delGK Bispecific HLE molecule QVQLVESGGGWQPGRSLRLSCAASGFTFSSYGMHW VRQAPGKGLEWVAVISYEGSNEFYAESVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCARGGEITMVRGV IGYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGS DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY QQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTEFT LTISSLQPEDFATYYCLQHNSYPLTFGGGTKVEIKS GGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNK YAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVK DRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGN FGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGG SQTWTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYP NWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGG KAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLT VLGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDT LMI S RT P E VT C VWD VS Η E D P E VK FNW YVD GVE VHN AKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGGGGSGGGGSGGGGSGGGGSG GGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVT C VWD VS HEDPEVKFNW YVD GVE VHNAKTKPCEEQYGSTYRCVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGK CLAIM 1. A bispecific antibody construct comprising a first binding domain that binds to human FLT3 on the surface of the target cell and a second binding domain that binds to human CD3 on the surface of a T cell, wherein the first binding domain binds to the FLT3 epitope that is contained in the region , shown as SEQ ID NO: 819, and contains a VH region containing CDR-H1, CDR-H2 and CDR-H3, and contains a VL region containing CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of from - 134 040387 SEQ ID NOS:151-156, SEQ ID NOS:181-186, SEQ ID NOS: 211-216, SEQ ID NOS:241-246, SEQ ID NOS: 271-276, SEQ ID N0:301-306, SEQ ID NOS:331-336, SEQ ID NOS:361-366, SEQ ID NOS:391-396, SEQ ID NO:421-426, SEQ ID NOS:451-456, SEQ ID NO:481-486, SEQ ID NOS:511-516, SEQ ID NOS:541-546, SEQ ID NOS:571-576, SEQ ID NOS:601-606, SEQ ID NOS:631-636, SEQ ID NOS:661-666, SEQ ID NOS:721-726, SEQ ID NOS:161-166, SEQ ID NOS:191-196, SEQ ID NOS: 221-226, SEQ ID NOS:251-256, SEQ ID NOS: 281-286, SEQ ID NO:311-316, SEQ ID NOS:341-346, SEQ ID NOS:371-376, SEQ ID N0:401-406, SEQ ID NOS:431-436, SEQ ID NOS: 461-466, SEQ ID NOS: 491-496, SEQ ID NOS:521-526, SEQ ID NOS:551-556, SEQ ID NOS:581-586, SEQ ID NOS:611-616, SEQ ID NOS:641-646, SEQ ID N0:701-706, SEQ ID NO:731-736 SEQ ID NO:171-176, SEQ ID N0:201-206, SEQ ID NO:231-236, SEQ ID NO:261-266, SEQ ID NO:291-296, SEQ ID NO:321-326, SEQ ID NO:351-356, SEQ ID NO:381-386, SEQ ID NO:411-416, SEQ ID NO:441-446, SEQ ID NO:471-476, SEQ ID N0:501-506, SEQ ID NO: 531-536, SEQ ID NO:561-566, SEQ ID NO:591-596, SEQ ID NO:621-626, SEQ ID NO:651-656, SEQ ID NO:711-716, : SEQ ID NO:741 -746. 2. An antibody construct according to claim 1, wherein the first binding domain binds to human and macaque FLT3. 3. An antibody construct according to claim 1 or 2, wherein the antibody construct is in a form selected from the group consisting of (scFv) ₂ , scFv-single domain mAb, diabodies, and oligomers of these forms. 4. An antibody construct according to claim 3, wherein the first binding domain contains a VH region selected from the group consisting of SEQ ID NO:157, SEQ ID NO:197, SEQ ID NO:237, SEQ ID NO:277, SEQ ID NO:317, SEQ ID NO:357, SEQ ID NO:397, SEQ ID NO:437, SEQ ID NO:477, SEQ ID NO:517, SEQ ID NO:557, SEQ ID NO:597, SEQ ID NO:637, SEQ ID NO:707, SEQ ID NO:747. SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO:167, NO:207, NO:247, NO:287, NO:327, NO:367, NO:407, NO:447, NO:487, NO:527, NO:567, NO:607, NO: 647 NO:717 SEQ ID NO:177 SEQ ID NO:217 SEQ ID NO:257 SEQ ID NO:297 SEQ ID NO:337, SEQ ID NO:377, SEQ ID NO:417, SEQ ID NO:457 SEQ ID NO:497 SEQ ID NO:537 SEQ ID NO:577 SEQ ID NO:617, SEQ ID NO:657 SEQ ID NO:727, SEQ ID NO:187 SEQ ID NO:227 SEQ ID NO:267 SEQ ID NO:307 SEQ ID NO:347, SEQ ID NO:387, SEQ ID NO:427, SEQ ID NO:467, SEQ ID NO:507 SEQ ID NO:547, SEQ ID NO:587 SEQ ID NO:627, SEQ ID NO:667, SEQ ID NO:737 and 5. An antibody construct according to claim 3 or 4, wherein the first binding domain contains a VL region selected from the group consisting of - 135 040387 SEQ ID NO:158, SEQ ID NO:198, SEQ ID NO:238, SEQ ID NO:278, SEQ ID NO:318, SEQ ID NO:358, SEQ ID NO:398, SEQ ID NO:438, SEQ ID NO:478, SEQ ID NO:518, SEQ ID NO:558, SEQ ID NO:598, SEQ ID NO:638, SEQ ID NO:708, SEQ ID NO:748. SEQ ID NO:168, SEQ ID NO:208 SEQ ID NO:248, SEQ ID NO:288, SEQ ID NO:328, SEQ ID NO:368, SEQ ID NO:408, SEQ ID NO:448, SEQ ID NO:488, SEQ ID NO:528, SEQ ID NO:568, SEQ ID NO:608, SEQ ID NO:648, SEQ ID NO:718, SEQ ID NO:178, SEQ ID NO:218 SEQ ID NO:258, SEQ ID NO:298, SEQ ID NO:338, SEQ ID NO:378, SEQ ID NO:418, SEQ ID NO:458, SEQ ID NO:498, SEQ ID NO:538, SEQ ID NO:578, SEQ ID NO:618, SEQ ID NO:658, SEQ ID NO:728, SEQ ID NO:188, SEQ ID NO:228, SEQ ID NO:268, SEQ ID NO:308, SEQ ID NO:348, SEQ ID NO:388, SEQ ID NO:428, SEQ ID NO:468, SEQ ID NO:508, SEQ ID NO:548, SEQ ID NO:588, SEQ ID NO:628, SEQ ID NO:668, SEQ ID NO:738 and 6. The design of the antibody according to any one of claims 3-5, where VH and VL region selected from the group consisting of pairs of VH and VL regions the first binding domain contains the region SEQ ID NO:157+158, SEQ ID NO:187+188, SEQ ID NO:217+218, SEQ ID NO:247+248, SEQ ID NO:277+278, SEQ ID N0:307+308, SEQ ID NO:337+338, SEQ ID NO:367+368, SEQ ID NO:397+398, SEQ ID NO:427+428, SEQ ID NO:457+458, SEQ ID NO:487+488, SEQ ID NO:517+518, SEQ ID NO:547+548, SEQ ID NO:577+578, SEQ ID NO:607+608, SEQ ID NO:637+638, SEQ ID NO:667+668, SEQ ID NO:167+168, SEQ ID NO:197+198, SEQ ID NO:227+228, SEQ ID NO:257+258, SEQ ID NO:287+288, SEQ ID NO:317+318, SEQ ID NO:347+348, SEQ ID NO:377+378, SEQ ID N0:407+408, SEQ ID NO:437+438, SEQ ID NO:467+468, SEQ ID NO:497+498, SEQ ID NO:527+528, SEQ ID NO:557+558, SEQ ID NO:587+588, SEQ ID NO:617+618, SEQ ID NO:647+648, SEQ ID N0:707+708, SEQ ID NO:177+178, SEQ ID N0:207+208, SEQ ID NO:237+238, SEQ ID NO:267+268, SEQ ID NO:297+298, SEQ ID NO:327+328, SEQ ID NO:357+358, SEQ ID NO:387+388, SEQ ID NO:417+418, SEQ ID NO:447+448, SEQ ID NO:477+478, SEQ ID N0:507+508, SEQ ID NO: 537+538, SEQ ID NO:567+568, SEQ ID NO:597+598, SEQ ID NO:627+628, SEQ ID NO:657+658, SEQ ID NO:717+718, SEQ ID NO:727+728, SEQ ID NO:737+738 and SEQ ID NO:747+748. 7. An antibody construct according to any one of claims 3-6, wherein the first binding domain comprises a polypeptide selected from the group consisting of - 136 040387 SEQ ID NO:159 SEQ ID NO:199 SEQ ID NO:239 SEQ ID NO:269 SEQ ID NO:299 SEQ ID NO:339, SEQ ID NO:369 SEQ ID NO:399 SEQ ID NO:439, SEQ ID NO:469 SEQ ID NO:499, SEQ ID NO:539, SEQ ID NO:569, SEQ ID NO:599 SEQ ID NO:639, SEQ ID NO:669, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749. 8. An antibody construct according to any one of claims 1 to 7, wherein the second binding domain binds to human CD3 epsilon and to Callithrix jacchus, Saguinus Oedipus or Saimiri sciureus CD3 epsilon. 9. An antibody construct according to any one of claims 1 to 8, comprising: (a) a polypeptide containing in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9; and a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NQ:100, and SEQ ID NO:103; And b) a polypeptide containing in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of - 137 040387 SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NQ:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NQ:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NO: 1-9; a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NQ:100 and SEQ ID NO:103; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NO:1-9; a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 104-134; and (c) a polypeptide containing in the following order, starting from the N-terminus, a polypeptide with the amino acid sequence QRFVTGHFGGLX ₁ PANG (SEQ ID NO: 135), where X ₁ represents Y or H; and a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9; a polypeptide with an amino acid sequence selected from the group consisting of - 138 040387 SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:100 and SEQ ID NO:103; a polypeptide with the amino acid sequence QRFVTGHFGGLHPANG (SEQ ID NO: 137) or QRFCTGHFGGLHPCNG (SEQ ID NO: 139) and (d) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98 and SEQ ID NO:101; a peptide linker with the amino acid sequence of SEQ ID NO: 8; a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:198, SEQ ID NO:238, SEQ ID NO:278, SEQ ID NO:318, SEQ ID NO:358, SEQ ID NO:398, SEQ ID NO:438, SEQ ID NO:478, SEQ ID NO:518, SEQ ID NO:558, SEQ ID NO:598, SEQ ID NO:638, SEQ ID NO:708, SEQ ID NO:168, SEQ ID NO:208, SEQ ID NO:248, SEQ ID NO:288, SEQ ID NO:328, SEQ ID NO:368, SEQ ID NO:408, SEQ ID NO:448, SEQ ID NO:488, SEQ ID NO:528, SEQ ID NO:568, SEQ ID NO:608, SEQ ID NO:648, SEQ ID NO:718, SEQ ID NO:178, SEQ ID NO:218, SEQ ID NO:258, SEQ ID NO:298, SEQ ID NO:338, SEQ ID NO:378, SEQ ID NO:418, SEQ ID NO:458, SEQ ID NO:498, SEQ ID NO:538, SEQ ID NO:578, SEQ ID NO:618, SEQ ID NO:658, SEQ ID NO:728, SEQ ID NO:188, SEQ ID NO:228, SEQ ID NO:268, SEQ ID NO:308, SEQ ID NO:348, SEQ ID NO:388, SEQ ID NO:428, SEQ ID NO:468, SEQ ID NO:508, SEQ ID NO:548, SEQ ID NO:588, SEQ ID NO:628, SEQ ID NO:668, SEQ ID NO:738 and SEQ ID NO:748, and a serine residue at the C-terminus; a polypeptide with the amino acid sequence of SEQ ID NO: 140 and (e) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:157 SEQ ID NO:167 SEQ ID NO:177 SEQ ID NO:187 SEQ ID NO:197 SEQ ID NO:207 SEQ ID NO:217 SEQ ID NO:227 SEQ ID NO:237 SEQ ID NO:247 SEQ ID NO:257 SEQ ID NO:267 SEQ ID NO:277 SEQ ID NO:2 8 7 SEQ ID NO:297 SEQ ID NO:307 SEQ ID NO:317 SEQ ID NO:327 SEQ ID NO:337 SEQ ID NO:347 SEQ ID NO:357 SEQ ID NO:367 SEQ ID NO:377 SEQ ID NO:387 SEQ ID NO:397 SEQ ID NO:407 SEQ ID NO:417 SEQ ID NO:427 SEQ ID NO:437 SEQ ID NO:447 SEQ ID NO:457 SEQ ID NO:467 SEQ ID NO:477 SEQ ID NO:4 8 7 SEQ ID NO:497 SEQ ID NO:507 SEQ ID NO:517 SEQ ID NO:527 SEQ ID NO:537 SEQ ID NO:547 SEQ ID NO:557 SEQ ID NO:567 SEQ ID NO:577 SEQ ID NO:587 SEQ ID NO:597 SEQ ID NO:607 SEQ ID NO:617 SEQ ID NO:627 SEQ ID NO:637 SEQ ID NO:647 SEQ ID NO:657 SEQ ID NO:667 SEQ ID NO:707 SEQ ID NO:717 SEQ ID NO:727 SEQ ID NO:737 and SEQ ID NO:747; a peptide linker with the amino acid sequence of SEQ ID NO: 8; a polypeptide with an amino acid sequence selected from the group consisting of - 139 040387 SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:99 and SEQ ID NO:102 and a serine residue at the C-terminus; a polypeptide with the amino acid sequence of SEQ ID NO: 141; (f) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:62, SEQ ID NO:71, SEQ ID NO:80, SEQ ID NO:89, SEQ ID NO:98 and SEQ ID NO:101; a peptide linker with the amino acid sequence of SEQ ID NO: 8; a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:158, SEQ ID NO:198, SEQ ID NO:238, SEQ ID NO:278, SEQ ID NO:318, SEQ ID NO:358, SEQ ID NO:398, SEQ ID NO:438, SEQ ID NO:478, SEQ ID NO:518, SEQ ID NO:558, SEQ ID NO:598, SEQ ID NO:638, SEQ ID NO:708, SEQ ID NO:168, SEQ ID NO:178, SEQ ID NO:188, SEQ ID NO:208, SEQ ID NO:218, SEQ ID NO:248, SEQ ID NO:258, SEQ ID NO:288, SEQ ID NO:298, SEQ ID NO:328, SEQ ID NO:338, SEQ ID NO:368, SEQ ID NO:378, SEQ ID NO:408, SEQ ID NO:418, SEQ ID NO:448, SEQ ID NO:458, SEQ ID NO:488, SEQ ID NO:498, SEQ ID NO:528, SEQ ID NO:538, SEQ ID NO:568, SEQ ID NO:578, SEQ ID NO:608, SEQ ID NO:618, SEQ ID NO:648, SEQ ID NO:658, SEQ ID NO:718, SEQ ID NO:728, SEQ ID NO:228, SEQ ID NO:268, SEQ ID NO:308, SEQ ID NO:348, SEQ ID NO:388, SEQ ID NO:428, SEQ ID NO:468, SEQ ID NO:508, SEQ ID NO:548, SEQ ID NO:588, SEQ ID NO:628, SEQ ID NO:668, SEQ ID NO:738 and SEQ ID NO:748; a polypeptide having the amino acid sequence of SEQ ID NO: 142 and (g) a polypeptide comprising, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:157 SEQ ID NO:197 SEQ ID NO:237 SEQ ID NO:277 SEQ ID NO:317 SEQ ID NO:357 SEQ ID NO:397 SEQ ID NO:437 SEQ ID NO:477, SEQ ID NO:517, SEQ ID NO:557, SEQ ID NO:597, SEQ ID NO:637, SEQ ID NO:707 SEQ ID NO:167 SEQ ID NO:207 SEQ ID NO:247, SEQ ID NO:287, SEQ ID NO:327 SEQ ID NO:367 SEQ ID NO:407 SEQ ID NO:447, SEQ ID NO:487, SEQ ID NO:527 SEQ ID NO:567, SEQ ID NO:607 SEQ ID NO:647, SEQ ID NO:717, SEQ ID NO:177 SEQ ID NO:217 SEQ ID NO:257 SEQ ID NO:297 SEQ ID NO:337, SEQ ID NO:377, SEQ ID NO:417, SEQ ID NO:457 SEQ ID NO:497 SEQ ID NO:537 SEQ ID NO:577 SEQ ID NO:617, SEQ ID NO:657 SEQ ID NO:727, SEQ ID NO:187 SEQ ID NO:227 SEQ ID NO:267 SEQ ID NO:307 SEQ ID NO:347, SEQ ID NO:387, SEQ ID NO:427, SEQ ID NO:467, SEQ ID NO:507 SEQ ID NO:547, SEQ ID NO:587 SEQ ID NO:627, SEQ ID NO:667, SEQ ID NO:737 and SEQ ID NO:747; a peptide linker with the amino acid sequence of SEQ ID NO: 8; a polypeptide with an amino acid sequence selected from the group consisting of - 140 040387 SEQ ID NO:18, SEQ ID NO:27, SEQ ID NO:36, SEQ ID NO:45, SEQ ID NO:54, SEQ ID NO:63, SEQ ID NO:72, SEQ ID NO:81, SEQ ID NO:90, SEQ ID NO:99 and SEQ ID NO:102 and a serine residue at the C-terminus; a polypeptide with the amino acid sequence of SEQ ID NO: 143; (h) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9; and a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NO:100 and SEQ ID NO:103; and a polypeptide with the amino acid sequence of SEQ ID NO: 144 and a polypeptide with the amino acid sequence of SEQ ID NO: 145; (i) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NQ:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NQ:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; and a polypeptide with the amino acid sequence of SEQ ID NO: 146 and - 141 040387 (j) a polypeptide containing in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19 SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46 SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82 SEQ ID NO:91 SEQ ID NO:100 and SEQ ID NO:103; and a polypeptide with the amino acid sequence of SEQ ID NO: 147; (k) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159 SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189 SEQ ID NO:199 SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229 SEQ ID NO :2 3 9 SEQ ID NO:249 and SEQ ID NO:259 SEQ ID NO:269 SEQ ID NO:279 and SEQ ID NO:289 SEQ ID NO:299 SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329 SEQ ID NO:339 SEQ ID NO:349 and SEQ ID NO:359 SEQ ID NO:369 SEQ ID NO:379 and SEQ ID NO:389 SEQ ID NO:399 SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429 SEQ ID NO :4 3 9 SEQ ID NO:449 and SEQ ID NO:459 SEQ ID NO:469 SEQ ID NO:479 and SEQ ID NO:489 SEQ ID NO:499 SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529 SEQ ID NO:539 SEQ ID NO:549 and SEQ ID NO:559 SEQ ID NO:569 SEQ ID NO:579 and SEQ ID NO:589 SEQ ID NO:599 SEQ ID NO:609 SEQ ID NO:619 and SEQ ID NO:629 SEQ ID NO:639 SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669 SEQ ID NO:709 SEQ ID NO:719 and SEQ ID NO:729 SEQ ID NO:739 and SEQ ID NO:749; and a polypeptide with the amino acid sequence of SEQ ID NO: 148 and (l) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19 SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46 SEQ ID NO: 55 SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82 SEQ ID NO:91 SEQ ID NO:100 and SEQ ID NO:103; and a polypeptide with the amino acid sequence of SEQ ID NO: 149 or (m) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:74 9; a peptide linker with an amino acid sequence selected from the group consisting of - 142 040387 SEQ ID NO: 1-9; and a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:19, SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:46, SEQ ID NO:55, SEQ ID NO:64, SEQ ID NO:73, SEQ ID NO:82, SEQ ID NO:91, SEQ ID NQ:100 and SEQ ID NO:103; and a polypeptide with the amino acid sequence of SEQ ID NO: 150; (n) a polypeptide containing, in the following order, starting from the N-terminus: a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO:159, SEQ ID NO:169, SEQ ID NO:179, SEQ ID NO:189, SEQ ID NO:199, SEQ ID NO:209, SEQ ID NO:219 and SEQ ID NO:229, SEQ ID NO:239, SEQ ID NO:249 and SEQ ID NO:259, SEQ ID NO:269, SEQ ID NO:279 and SEQ ID NO:289, SEQ ID NO:299, SEQ ID NO:309, SEQ ID NO:319 and SEQ ID NO:329, SEQ ID NO:339, SEQ ID NO:349 and SEQ ID NO:359, SEQ ID NO:369, SEQ ID NO:379 and SEQ ID NO:389, SEQ ID NO:399, SEQ ID NO:409, SEQ ID NO:419 and SEQ ID NO:429, SEQ ID NO:439, SEQ ID NO:449 and SEQ ID NO:459, SEQ ID NO:469, SEQ ID NO:479 and SEQ ID NO:489, SEQ ID NO:499, SEQ ID NO:509, SEQ ID NO:519 and SEQ ID NO:529, SEQ ID NO:539, SEQ ID NO:549 and SEQ ID NO:559, SEQ ID NO:569, SEQ ID NO:579 and SEQ ID NO:589, SEQ ID NO:599, SEQ ID NO:609, SEQ ID NO:619 and SEQ ID NO:629, SEQ ID NO:639, SEQ ID NO:649 and SEQ ID NO:659, SEQ ID NO:669, SEQ ID NO:709, SEQ ID NO:719 and SEQ ID NO:729, SEQ ID NO:739 and SEQ ID NO:749; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9; and a polypeptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 64, SEQ ID NO: 73, SEQ ID NO: 82, SEQ ID NO: 91, SEQ ID NO: 100 and SEQ ID NO: 103; a peptide linker with an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9; and a third domain with an amino acid sequence selected from the group consisting of SEQ ID NO: 843-850. 10. An antibody construct according to claim 9, wherein the polypeptide has a His-tag with the sequence of SEQ ID NO: 10. 11. An antibody construct according to any one of claims 1-10, containing or consisting of a polypeptide with any sequence from SEQ ID NO: 856-869. 12. An antibody construct according to any one of claims 1 to 11, wherein the binding of the first binding domain to human FLT3 is reduced by <25%, preferably <20%, more preferably <15%, even more preferably <10% by the FLT3 ligand. %, even more preferably <8%, more preferably <6% and most preferably <2%. 13. A polynucleotide encoding an antibody construct according to any one of the preceding paragraphs. 14. An expression vector containing a polynucleotide according to claim 13. 15. A host cell for the production of an antibody construct according to any one of claims 1 to 12, wherein the cell is transformed or transfected with the polynucleotide of claim 13 or the vector of claim 14. 16. A method for producing an antibody construct according to any one of claims 1-12, comprising culturing a host cell according to claim 15 under conditions allowing expression of the antibody construct according to claims 1-12, and recovering the antibody construct from the culture. 17. Pharmaceutical composition, characterized in that it contains the antibody construct according to any one of claims 1 to 12 or obtained by the method according to claim 16. 18. The use of an antibody construct according to any one of claims 1 to 12 for the prevention, treatment or amelioration of blood cancers or malignancies. 19. The use of an antibody construct obtained by the method of claim 16 for the prevention, treatment, or amelioration of blood cancers or malignancies. - 143 -