Antibody constructs against CLDN18.2 and CD3

Abstract

The present invention relates to an antibody construct comprising a domain that binds to tight junction protein 18.2 (CLDN18.2) and another domain that binds to CD3. In addition, the present invention provides a polynucleotide encoding the antibody construct, a vector comprising the polynucleotide, and a host cell transformed or transfected with the polynucleotide or vector. In addition, the present invention provides a method for producing the antibody construct of the present invention, the medical use of the antibody construct, and a kit comprising the antibody construct.

Description

[0001] The present invention relates to an antibody construct comprising a domain that binds to claudin 18.2 (CLDN18.2) and another domain that binds to CD3. Furthermore, the present invention provides polynucleotides encoding the antibody constructs, vectors comprising the polynucleotides, and host cells transformed or transfected with the polynucleotides or vectors. Furthermore, the present invention provides methods for producing the antibody constructs of the present invention, medical uses of the antibody constructs, and kits comprising the antibody constructs. Background Art

[0002] Claudin is a key structural and functional component of epithelial tight junctions, regulating cell-cell permeability, maintaining ion homeostasis, and supporting cell adhesion and polarity. Claudin is a 22-27 kDa tetraspan transmembrane protein that polymerizes within or across the cell membrane to form a protective barrier. The 24 reported claudin proteins differ in their tissue localization specificity and in their interactions with other proteins.

[0003] Claudin 18 (CLDN18) was initially identified as a target gene of the transcription factor T / EBP / NKX2.1. Consistent with its homology to other members of the claudin family, CLDN18 has been shown to localize to tight junctions in both mice and humans. CLDN18 has been shown to encode two isoforms generated by alternative splicing: CLDN18.1, which is specifically expressed in normal lung, and CLDN18.2, which is expressed in differentiated cells of the gastric mucosa.

[0004] CLDN18.2 is a 261-amino acid protein with two extracellular loops and shares 92% sequence identity with CLDN18.1. Unlike the second extracellular loop, the first extracellular loop of CLDN18.2 differs from CLDN18.1 by eight amino acids. CLDN18.2 shares more limited homology with other family members, with an overall identity of 29%-34% with CLDN1, CLDN6, and CLDN7.

[0005] CLDN18.2 is expressed in several tumor types, including gastric, pancreatic, esophageal, mucinous ovarian, and non-small cell lung cancer. CLDN18.2 expression in gastric cancer includes the invasive front and metastatic sites, but absolute levels of CLDN18 are reported to be reduced in these settings. The expression of CLDN18.2 in multiple tumor types, with normal tissue expression primarily restricted to differentiated cells in the stomach, has led to the consideration of CLDN18.2 as a therapeutic target for gastric cancer and other indications.

[0006] Gastric and gastroesophageal cancers remain indications with high unmet medical need, with at least 1.4 million new cases and 1.1 million deaths reported worldwide annually (Lordick and Janjigian, Nat Rev Cancer 2016). Typical first-line treatments include surgery and combination chemotherapy (including platinum and fluoropyrimidine compounds). While this regimen can potentially improve quality of life and extend median survival by 8-10 months, the 5-year survival rate remains low.

[0007] Targeted therapy provides an alternative treatment strategy. Anti-Her2 monoclonal antibody trastuzumab (trastuzumab) is approved for the first-line treatment of Her2-positive gastric cancer and gastroesophageal cancer in combination with chemotherapy (Bang et al., The Lancet [Lancet] 2010). Anti-VEGFR2 antibody ramucirumab (ramucirumab) is approved for the treatment of gastric cancer and gastroesophageal cancer that have progressed after chemotherapy (Fuchs et al., The Lancet [Lancet] 2014). Although these targeted agents further increase survival compared to chemotherapy alone, their efficacy is limited by the heterogeneity of target expression and resistance mechanisms.

[0008] Recently, immune checkpoint therapy has shown activity in selected settings: pembrolizumab was approved in the United States for the treatment of microsatellite instability-high (MSI-H) tumors, including gastric cancer (Le et al., Science 2017), and for the treatment of unresectable advanced or recurrent gastric cancer that has progressed after two or more lines of chemotherapy (Fuchs et al., J Clin Oncol 2017). Nivolumab was approved in Japan for the treatment of unresectable advanced or recurrent gastric cancer that has progressed after chemotherapy (Kang et al., The Lancet 2017). In these studies, only 1%-2% of patients in the unselected population and 60% of the MSI-H (microsatellite instability-high) population (accounting for 9% of the overall gastric and gastroesophageal cases) showed complete remission. Therefore, there is still a need for new therapies with the potential to provide durable responses for larger patient populations.

[0009] Compared with gastric cancer or gastroesophageal cancer, pancreatic cancer has been shown to have an even lower response to available therapies. Globally, at least 338,000 cases and 331,000 deaths from pancreatic cancer are reported each year, with a median survival of 6 months (Ilic and Ilic, World J Gastroenterol [World Journal of Gastroenterology] 2016). Only 20%-30% of patients are candidates for surgical resection. Until recently, when the combination chemotherapy regimen FOLFIRINOX (5-fluorouracil, leucovorin, oxaliplatin, irinotecan) and gemcitabine combined with albumin-bound paclitaxel (nab-paclitaxel) showed that overall survival increased by about 2-5 months (Uccello et al., Curr Oncol [Modern Oncology] 2018) compared to gemcitabine treatment alone, gemcitabine (gemcitabine) has been considered as first-line therapy. Other chemotherapy combinations are typically used for second-line therapy. Several targeted therapies and immunotherapeutics have been evaluated in advanced pancreatic cancer, but with limited success. Pancreatic tumors are characterized by desmoplasia and an immunosuppressive immune infiltrate, which contributes to a lack of response. New therapies with the potential to overcome this immunosuppressive environment and prolong survival are needed.

[0010] A bispecific antibody construct comprising a domain that binds to CD3 on T cells and a domain that binds to a protein expressed on target cells directly connects T cells to target cells to induce T cell redirected lysis. This mechanism of action is different from chemotherapy, targeted therapy, and other immunotherapies because it can work with any CD3-positive T cell without relying on co-stimulatory activation signals (Klinger et al., Immunol Reviews 2016). The expression of CLDN18.2 on the cell surface of gastric cancer, gastroesophageal cancer, and pancreatic cancer provides a basis for targeting these tumor types with CLDN18.2 x CD3 antibody constructs. In addition, the CLDN18.2 x CD3 antibody construct has the potential to target other tumor types expressing CLDN18.2 (including mucinous ovarian cancer, colorectal cancer, and non-small cell lung cancer). DETAILED DESCRIPTION

[0011] Thus, in one aspect, the present invention provides an antibody construct comprising a first domain that binds to CLDN18.2 on the surface of a target cell and a second domain that binds to CD3 on the surface of a T cell.

[0012] It is also envisaged that:

[0013] (1) The first domain of the antibody construct of the present invention binds to the first extracellular loop (loop 1) of CLDN18.2;

[0014] (2) The first domain of the antibody construct of the present invention binds to the same CLDN18.2 epitope as the following antibody or antibody construct, which comprises the following domain, which binds to CLDN18.2 on the surface of the target cell and comprises:

[0015] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0016] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0017] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0018] d) a VH region as described in SEQ ID NO: 139, and a VL region as described in SEQ ID NO: 140;

[0019] (3) The antibody construct of the present invention competes for binding with an antibody or antibody construct comprising a domain that binds to CLDN18.2 on the surface of a target cell and comprises:

[0020] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0021] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0022] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0023] d) a VH region as described in SEQ ID NO: 139, and a VL region as described in SEQ ID NO: 140;

[0024] (4) The first domain of the antibody construct of the present invention binds to the CLDN18.2 mutant having the amino acid sequence as described in SEQ ID NO: 22 on the surface of the target cell, and optionally also binds to the CLDN18.2 mutant having the amino acid sequence as described in SEQ ID NO: 24 on the surface of the target cell, but does not bind to the CLDN18.2 mutant having the amino acid sequence as described in SEQ ID NO: 23 on the surface of the target cell;

[0025] (5) The first domain of the antibody construct of the present invention binds to a CLDN18.2 mutant having an amino acid sequence as described in SEQ ID NO: 14 on the surface of a target cell and / or a CLDN18.2 mutant having an amino acid sequence as described in SEQ ID NO: 15 on the surface of a target cell, and optionally also binds to one or more CLDN18.2 mutants having an amino acid sequence selected from the group consisting of those described in SEQ ID NO: 11, 12, 13, 16, 17, 19, 20 and 21 on the surface of a target cell, but does not bind to a CLDN18.2 mutant having an amino acid sequence as described in SEQ ID NO: 18 on the surface of a target cell;

[0026] (6) The first domain of the antibody construct of the present invention binds to human CLDN18.2 on the surface of a target cell, wherein Glu (E) at position 56 of human CLDN18.2 is essential for the binding of the first domain, and Ala (A) at position 42 and / or Asn (N) at position 45 of human CLDN18.2 are not essential for the binding of the first domain; and / or

[0027] (7) The first domain of the antibody construct of the present invention binds to an epitope of CLDN18.2, which comprises the amino acid sequence as described in SEQ ID NO: 266, but does not comprise the amino acid sequence as described in SEQ ID NO: 265, and optionally does not further comprise the amino acid sequence as described in SEQ ID NO: 267.

[0028] Advantageously, targeting the epitope of CLDN18.2 recognized by the antibody constructs of the present invention (see also Example 2) provides the following benefits:

[0029] (1) The selectivity of the CLDN18.2 x CD3 antibody construct is superior to that of CLDN18.1 (see Example 6), and

[0030] (2) Unexpectedly high cytotoxic potency towards the CLDN18.2 x CD3 antibody construct (see Example 7.4).

[0031] The term "antibody construct" refers to a molecule wherein structure and / or function is based on antibody, such as the structure and / or function of full-length immunoglobulin molecules. Therefore, the antibody construct immunospecifically binds to its target or antigen, and / or comprises the heavy chain variable region (VH) and / or the light chain variable region (VL) of the antibody, or comprises a domain derived therefrom. Antibody constructs according to the present invention comprise the antibody minimum structural requirement for allowing immunospecific target binding. This minimum requirement can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 in the VH region), preferably all six CDRs. Therefore, the feature of the antibody construct may be that there are three or six CDRs in one or two binding domains, and the technician knows where those CDRs are located (in what order) in the binding domains.

[0032] The definition of "antibody" according to the present invention includes full-length antibodies, and also includes camelid antibodies and other immunoglobulins produced by biotechnology or protein engineering methods or processes. These full-length antibodies can be, for example, monoclonal antibodies, recombinant antibodies, chimeric antibodies, deimmunized antibodies, humanized antibodies and human antibodies, as well as antibodies from other species (such as mouse, hamster, rabbit, rat, goat or non-human primate).

[0033] The "antibody constructs" of the present invention may have naturally occurring full-length immunoglobulin structures. For example, they may comprise (at least) two full-length antibody heavy chains and two full-length antibody light chains. However, given that the antibody constructs according to the present invention comprise one domain that binds to CLDN18.2 and another domain that binds to CD3, they do not occur in nature, and their functions differ significantly from those of naturally occurring products. Therefore, the antibody constructs of the present invention are artificial "hybrid" molecules comprising at least two different binding domains with different specificities.

[0034] The "antibody constructs" of the present invention may also comprise fragments of full-length antibodies, such as VH, VHH, VL, (s) dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "rIgG" (a "half antibody" consisting of a heavy chain and a light chain). The antibody constructs according to the present invention may also comprise modified antibody fragments, also referred to as antibody variants or antibody derivatives. Examples include, but are not limited to, scFv, di-scFv or linked (s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, minibodies (exemplified by the following structures: (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multibodies (such as triabodies or tetrabodies), and single domain antibodies (such as nanobodies or single variable domain antibodies) (comprising only one variable region, which can be VHH, VH or VL, which specifically binds to an antigen or target independently of other variable regions or domains). Further possible formats of the antibody constructs according to the invention are cross bodies, maxi bodies, hetero-Fc constructs, single Fc constructs and scFc constructs. Examples of those formats will be described herein below.

[0035] In addition, the definition of the term "antibody construct" includes bivalent and multivalent constructs and bispecific and multispecific constructs that specifically bind to two, three or more antigenic structures through different binding domains. Antibody constructs can have more binding valencies than specificities, for example, where two binding domains are directed against a first target (Cldn18.2) and one binding domain is directed against a second target (CD3) or vice versa, in which case the construct is trivalent and bispecific. Generally, the term "bispecific" includes the meaning that an antibody construct binds to (at least) two different antigens (such as Cldn18.2 and CD3).

[0036] Furthermore, the definition of the term "antibody construct" includes molecules consisting of only one polypeptide chain as well as molecules consisting of two, three, four or more polypeptide chains, which chains may be identical (homodimers, homotrimers or homooligomers) or different (heterodimers, heterotrimers or heterooligomers). Examples of the above-identified antibodies and their fragments, variants, derivatives and antibody constructs derived therefrom are described, inter alia, in Harlow and Lane, Antibodies: A laboratory manual, CSHL Press (1988); Kontermann and Dübel, Antibody Engineering, Springer, 2nd edition 2010; and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009.

[0037] The term "binding domain" or "domain that binds to..." refers to a domain of an antibody construct that immunospecifically binds to / interacts with / recognizes an epitope on a target or antigen (here: CLDN18.2 in the case of the first domain and CD3 in the case of the second domain). The structure and function of the first domain (binding to CLDN18.2), and preferably also the structure and / or function of the second domain (binding to CD3), are based on the structure and / or function of an antibody (e.g., a full-length immunoglobulin molecule). Thus, a "binding domain" or "domain that binds to..." may comprise the minimum structural requirements of an antibody that allow immunospecific target binding. Such minimum structural requirements of the first domain can be defined, for example, by the presence of at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. It is envisioned that the second domain also comprises such minimum structural requirements of an antibody that allow immunospecific target binding. More preferably, the second domain further comprises at least three light chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VL region) and / or three heavy chain CDRs (i.e., CDR1, CDR2, and CDR3 of the VH region), preferably all six CDRs. A "domain that binds to..." (or "binding domain") typically can comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not necessarily comprise both, but can comprise only one of VH or VL. For example, an Fd fragment typically retains some antigen-binding function of an intact antigen-binding domain.

[0038] Examples of the form of a "domain that binds to..." (or "binding domain") include, but are not limited to, a full-length antibody, a fragment of a full-length antibody (e.g., VH, VHH, VL), (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2, or "r IgG" ("half antibodies"), antibody variants or derivatives (such as scFv, di-scFv or linked (s)-scFv, scFv-Fc, scFv-zipper, scFab, Fab2, Fab3, diabodies, single-chain diabodies, tandem diabodies (Tandab's), tandem di-scFv, tandem tri-scFv, "minibodies" (selected from formats such as (VH-VL-CH3)2, (scFv-CH3)2, ((scFv)2-CH3+CH3)), ((scFv)2-CH3) or (scFv-CH3-scFv)2), multibodies (such as triabodies or tetrabodies), and single domain antibodies (such as nanobodies or single variable domain antibodies) (comprising only one variable region, which can be VHH, VH or VL). Further examples of the form of a "domain that binds to..." (or "binding domain") include (1) an antibody fragment or variant comprising VL, VH, CL and CH1 (such as Fab); (2) an antibody fragment or variant comprising two linked Fab fragments (such as F(ab')2); (3) an antibody fragment or variant comprising VH and CH1 (such as Fd); (4) an antibody fragment or variant comprising VL and CL (such as a light chain); (5) an antibody fragment or variant comprising VL and VH (such as Fv); (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) an antibody variant comprising at least three isolated CDRs of a heavy chain and / or a light chain; and (7) a single-chain Fv (scFv). Examples of embodiments of antibody constructs or binding domains according to the invention are e.g. described 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.

[0039] For the antibody constructs of the present invention, it is envisaged that

[0040] a) the antibody construct is a single-chain polypeptide or single-chain antibody construct,

[0041] b) the first domain is in the form of a scFv,

[0042] c) the second domain is in the form of a scFv,

[0043] d) the first domain and the second domain are connected via a linker, preferably a peptide linker, more preferably a glycine / serine linker, and / or

[0044] e) The antibody construct comprises a domain that provides for extended serum half-life, such as an Fc-based domain.

[0045] The antibody constructs of the present invention are preferably "in vitro generated antibody constructs" and / or "recombinant antibody constructs". In the context of the present invention, the term "in vitro generated" refers to an antibody construct according to the above definition, wherein all or part of a binding domain or variable region (e.g., at least one CDR) is produced in a non-immune cell selection (e.g., in an in vitro phage display, on a protein chip, or in any other method in which the ability of a candidate amino acid sequence to bind to an antigen can be tested). Thus, this term preferably excludes sequences produced solely by genomic rearrangements in immune cells in an animal. It is envisaged that the first and / or second domain of the antibody construct is produced or obtainable by phage display or library screening methods, rather than by transplanting CDR sequences from a pre-existing (monoclonal) antibody into a scaffold. A "recombinant antibody construct" is an antibody construct produced or produced using (in particular) recombinant DNA technology or genetic engineering.

[0046] It is envisioned that antibody construct of the present invention is monoclonal.As used herein, the antibody or the antibody construct of named " monoclonal " (mAb) are obtained from the colony of the antibody / antibody construct of homogeneity basically, i.e. except the possible natural generation sudden change and / or post-translational modification (for example, isomerization, amidation) that can exist in a small amount, each antibody / antibody construct comprised in colony is identical (particularly, about their amino acid sequence).Compared with the polyclonal antibody preparation that typically comprises the different antibodies for different determinants (or epi-position), monoclonal antibody / antibody construct has high specificity for the single epi-position in antigen.Except their specificity, monoclonal antibody is also synthesized by hybridoma culture at them, and is therefore not polluted by other immunoglobulins and is advantageous.The modifier " monoclonal " indicates the feature of the antibody / antibody construct obtained from the antibody colony of homogeneity basically, and should not be construed as requiring to produce antibody by any ad hoc method.

[0047] For the preparation of monoclonal antibodies, any technology providing antibodies produced by continuous cell line cultures can be used. For example, monoclonal antibodies to be used can be by the hybridoma method first described by Koehler et al., Nature [nature], 256:495 (1975), or can be prepared by recombinant DNA methods (see, for example, U.S. Patent number 4,816,567). The example of other technology for producing human monoclonal antibodies includes tri-source hybridoma technology, human B cell hybridoma technology (Kozbor, Immunology Today [today's immunology] 4 (1983), 72) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy [monoclonal antibodies and cancer therapy], Alan R.Liss company (1985), 77-96).

[0048] Standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE) can then be used to determine the TM ) analysis and screening of hybridomas to identify one or more hybridomas that produce antibodies that immunospecifically bind to a given antigen. Any form of the relevant antigen can be used as an immunogen, such as a recombinant antigen, a naturally occurring form, any variant or fragment thereof, and an antigenic peptide thereof. TM Surface plasmon resonance employed in the system can be used to increase the efficiency of phage antibody / antibody construct binding to epitopes of target antigens (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0049] Another exemplary method for preparing antibody constructs or binding domains includes screening protein expression libraries, such as phage display or ribosome display libraries. Phage display is described, for example, in Ladner et al., U.S. Patent No. 5,223,409; 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).

[0050] In addition to using display libraries, related antigens can also be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits or rats). In one embodiment, the non-human animal includes at least a portion of a human immunoglobulin gene. For example, it is possible to engineer a mouse strain with a defective mouse antibody production using a large fragment of a human Ig (immunoglobulin) locus. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes with desired specificity can be produced and selected. See, for example, Xenomouse TM ; Green et al. (1994) Nature Genetics 7: 13-21; US ​​2003-0070185; WO 96 / 34096 and WO 96 / 33735.

[0051] Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, e.g., humanized, deimmunized, rendered chimeric, etc. Examples of modified antibody constructs or binding domains include humanized variants of non-human antibodies / antibody constructs, "affinity matured" antibody constructs or binding domains (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al. Biochemistry 30, 10832-10837 (1991)), and antibody variants or mutants with altered effector function (see, e.g., U.S. Pat. No. 5,648,260; Kontermann and Dübel (2010), loc. cit.; and Little (2009), loc. cit.).

[0052] In immunology, affinity maturation is such a process: through this process, during the immune response, B cells produce antibodies with increased affinity for antigens. Due to repeated exposure to the same antigen, the host will produce antibodies with greater affinity in turn. Like natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize antibodies, antibody fragments, antibody variants, antibody constructs or binding domains. Random mutations are introduced into CDRs using radiation, chemical mutagens or error-prone PCR. In addition, genetic diversity can be increased by chain shuffling. Two or three rounds of mutations and selection are performed using display methods (such as phage display) to generally produce antibodies, antibody fragments, antibody variants, antibody constructs or binding domains with affinity in the low nanomolar range.

[0053] The preferred type of amino acid substitution change of the antibody construct of the present invention or binding domain relates to one or more residues in the hypervariable region of replacement parent antibody structure (such as humanized or human antibody structure). Generally speaking, one or more obtained variants selected for further development will have improved biological properties relative to the parent antibody structure that produces them. A convenient way for producing such substitution variants relates to affinity maturation using phage display. In short, several sites (such as 6-7 sites) of the hypervariable region are mutated to produce all possible amino acid substitutions at each site. The variant thus produced is displayed as a fusion of the gene III product of the M13 packaged in each particle from filamentous phage particles in a monovalent manner. The biological activity (such as binding affinity) of the variant displayed by phage is then screened as disclosed herein. In order to identify candidate hypervariable region sites (modification candidates) that have a significant contribution to antigen binding, alanine scanning mutagenesis can also be performed. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the complex between the antigen and the antibody construct or binding domain to identify the contact points between the binding domain and its specific antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques described herein. Once such variants are generated, the panel of variants is screened as described herein, and antibodies, antigen-binding fragments thereof, antibody constructs, or binding domains that have superior properties in one or more relevant assays can be selected for further development.

[0054] The antibody constructs and binding domains of the present invention specifically include "chimeric" forms (wherein a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass), as well as fragments or variants of such antibodies, as long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 81: 6851-6855 (1984)). Chimeric antibody constructs or binding domains of interest herein include "primatized" antibody constructs comprising variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and human constant region sequences. A variety of methods for preparing chimeric antibodies or antibody constructs have been described. See, e.g., Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0055] Antibodies, antibody constructs, antibody fragments, antibody variants or binding domains can also be modified by specifically deleting human T cell epitopes using methods such as those disclosed in WO 98 / 52976 or WO 00 / 34317 (a method known as "deimmunization"). Briefly, the heavy and light chain variable regions of antibodies, antibody constructs or binding domains can be analyzed for peptides that bind to MHC class II; these peptides represent potential T cell epitopes (as defined, for example, in WO 98 / 52976 and WO 00 / 34317). In order to detect potential T cell epitopes, a computer modeling method known as "peptide threading" can be applied, and in addition, databases 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 therefore constitute potential T cell epitopes. The detected potential T cell epitopes can be eliminated by replacing a small number of amino acid residues in the variable domain or variable region, or preferably by single amino acid substitution. Typically, conservative substitutions are performed. Generally, but not exclusively, amino acids common to the positions in the human germline antibody sequence can be used. Human germline sequences are disclosed, for example, in: Tomlinson et al. (1992) J. Mol. Biol. [Journal of Molecular Biology] 227: 776-798; Cook, GP et al. (1995) Immunol. Today [Today's Immunology] Vol. 16 (5): 237-242; and Tomlinson et al. (1995) EMBO J. [Journal of the European Molecular Biology Association] 14: 14: 4628-4638. The V BASE directory (www2.mrc-lmb.cam.ac.uk / vbase / list2.php) provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. et al., MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequences, for example, for framework regions and CDRs. Consensus human framework regions can also be used, for example, as described in U.S. Patent No. 6,300,064.

[0056] " Humanized " antibody, its variant or fragment, antibody construct and binding domain are immunoglobulins based on major human sequences, and these major human sequences contain one or more minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibody, its variant or fragment, antibody construct and binding domain are based on human immunoglobulin (receptor antibody), wherein the residue from hypervariable region or CDR is replaced by the residue from non-human species (such as rodent (such as mouse, hamster, rat or rabbit)) (donor antibody) hypervariable region or CDR with desired specificity, affinity, ability and / or bioactivity. In some cases, the Fv framework region (FR) residues of human immunoglobulin are replaced by corresponding non-human residues. In addition, " humanized " antibody, its variant or fragment, antibody construct and binding domain as used herein can also be included in the residue not found in receptor antibody and donor antibody. Carry out these modifications to further improve and optimize antibody performance. Humanized antibodies, variants or fragments thereof, antibody constructs and binding domains may also comprise at least a portion of an immunoglobulin constant region (e.g., Fc), typically at least a portion of a human immunoglobulin. For further details, see 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).

[0057] Humanized antibodies, variants or fragments thereof, antibody constructs and binding domains can be produced by replacing sequences of (Fv) variable regions that are not directly involved in antigen binding with equivalent sequences of human (Fv) variable regions. Exemplary methods for producing such molecules are provided by: Morrison (1985) Science [Science] 229: 1202-1207; Oi et al. (1986) BioTechniques [Biotechnology] 4: 214; and US 5,585,089; US 5,693,761; US ​​5,693,762; US 5,859,205; and US 6,407,213. These methods include isolating, manipulating and expressing nucleic acid sequences encoding all or part of an immunoglobulin (Fv) variable region from at least one of a heavy chain or a light chain. Such nucleic acids can be obtained from hybridomas that produce antibodies against a predetermined target as described above, as well as other sources. The recombinant DNA encoding the humanized antibody, variant or fragment thereof, antibody construct or binding domain can then be cloned into an appropriate expression vector.

[0058] Humanized antibodies, its variant or fragment, antibody construct and binding domain can also use transgenic animals (as expressing people's heavy chain and light chain genes but can not express the mouse of endogenous mouse immunoglobulin heavy chain and light chain genes) to produce.Winter has described the exemplary CDR transplantation method (U.S. Patent number 5,225,539) that can be used for preparing humanized molecule as herein described.All CDRs of specific people sequence can be replaced with at least a portion of non-human CDR, or only some CDRs can be replaced with non-human CDR.Only need to substitute for humanized molecule and predetermined antigen combination required CDR quantity.

[0059] Humanized antibodies, variants or fragments thereof, antibody constructs or binding domains can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations. Such altered immunoglobulin molecules can be prepared by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States], 80:7308-7312, 1983; Kozbor et al., Immunology Today [Today's Immunology], 4:7279, 1983; Olsson et al., Meth. Enzymol. [Enzymology Methods], 92:3-16, 1982, and EP 239 400).

[0060] Human anti-mouse antibody (HAMA) responses have led the industry to produce chimeric or other humanized antibodies / antibody constructs. However, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly with long-term or multi-dose use of antibodies or antibody constructs. Therefore, it is desirable to provide antibody constructs that contain human binding domains to CLDN18.2 and / or human binding domains to CD3 in order to eliminate the problems and / or effects of HAMA or HACA responses.

[0061] Therefore, according to one embodiment, antibody construct, the first binding domain and / or the second binding domain are "people". The terms "human antibody", "human antibody construct" and "human binding domain" include antibodies, antibody constructs and binding domains with antibody-derived regions, respectively, which are variable and constant regions or domains substantially corresponding to human germline immunoglobulin sequences as known in the art, including those described by Kabat et al. (1991) (cited above). Human antibody constructs or binding domains of the present invention can include, for example, amino acid residues (such as those introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) in CDR and particularly CDR3 that are not encoded by human germline immunoglobulin sequences. Human antibody constructs or binding domains can have at least 1, 2, 3, 4, 5 or more positions that are replaced by amino acid residues that are not encoded by human germline immunoglobulin sequences. However, the definition of human antibodies, antibody constructs, and binding domains as used herein also encompasses fully human antibodies, antibody constructs, and binding domains that comprise only non-artificially and / or genetically altered human antibody sequences, such as those that can be derived through the use of techniques or systems such as Xenomouse.

[0062] Antibodies and antibody constructs comprising at least one human binding domain avoid some of the problems associated with antibody constructs having non-human, such as rodent (e.g., murine, rat, hamster, or rabbit) variable and / or constant regions. The presence of such rodent-derived proteins can result in rapid clearance of the antibody or antibody construct or can result in an immune response against the antibody or antibody construct in the patient. In order to avoid the use of rodent-derived antibody constructs, humanized or fully human antibody constructs can be produced by introducing human antibody functions into rodents so that the rodents produce fully human antibodies.

[0063] The ability to clone and reconstruct megabase-sized human loci in YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional components of very large or poorly mapped loci and for generating useful human disease models. Furthermore, using this technology to replace mouse loci with their human equivalents can provide unique insights into the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

[0064] The important practical application of this strategy is " humanization " of mouse humoral immune system. Human immunoglobulin (Ig) locus is introduced into the mouse in which endogenous Ig gene has been inactivated to provide the programmed expression of research antibody and the fundamental mechanism of assembly and the opportunity of its effect in B cell development. In addition, this strategy can provide ideal source for the generation of fully human monoclonal antibodies (mAb)-this is an important milestone that helps to realize the prospect of antibody therapy in human disease. Expected fully human antibodies or antibody constructs derived therefrom will minimize the immunogenicity and allergic response inherent to mouse or mouse-derived mAb, and increase the effect and safety of the antibody / antibody construct used thus. It can be expected that using fully human antibodies or antibody constructs can provide significant advantages in the chronic and recurrent human diseases (such as inflammation, autoimmunity and cancer) that need repeated administration of compound for treatment.

[0065] One method to achieve this goal is to use a large fragment of the human Ig locus to engineer mouse antibodies to produce defective mouse strains. It is expected that such mice will produce a large human antibody repertoire in the absence of mouse antibodies. Large human Ig fragments will maintain large variable gene diversity and appropriate regulation of antibody production and expression. By utilizing mouse mechanisms to achieve antibody diversification and selection and lack of immune tolerance to human proteins, the human antibody repertoire regenerated in these mouse strains should produce high-affinity antibodies for any antigen of interest (including human antigens). Using hybridoma technology, antigen-specific human mAbs with desired specificity can be easily produced and selected. This general strategy was demonstrated in conjunction with the generation of the first XenoMouse mouse strain (see Green et al. Nature Genetics [Nature Genetics] 7: 13-21 (1994)). The XenoMouse strain was engineered using yeast artificial chromosomes (YACs) containing 245kb and 190kb size germline configuration fragments of the human heavy chain locus and κ light chain locus, which contain core variable and constant region sequences. Prove that YAC containing human Ig is compatible with mouse system to rearrange and express antibody, and these YAC can replace the mouse Ig gene of inactivation.This is proved by its ability to induce B cell development, produce the adult sample human repertoire of fully human antibody and produce antigen-specific human mAb.These results also show that the human Ig locus that introduces the larger part containing more V gene, additional regulatory elements and human Ig constant region can basically reproduce the complete repertoire as the feature of human humoral response to infection and immunity.The work of Green et al. is expanded to introduce the human antibody repertoire greater than about 80% by introducing the germline configuration YAC fragment of human heavy chain locus and kappa light chain locus of megabase size respectively.See Mendez et al. Nature Genetics [natural genetics] 15:146-156 (1997) and U.S. patent application serial number 08 / 759,620.

[0066] The generation of the XenoMouse model is further discussed and described in the following: U.S. Patent Applications Serial Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 466,649. 4,582, Serial No. 08 / 463,191, Serial No. 08 / 462,837, Serial No. 08 / 486,853, Serial No. 08 / 486,857, Serial No. 08 / 486,859, Serial No. 08 / 462,513, Serial No. 08 / 724,752 and Serial No. 08 / 759,620; and U.S. Patent Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181 and 5,939,598 and Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2 and 3 068 507 B2. See also Mendez et al. Nature Genetics 15: 146-156 (1997) and Green and Jakobovits J. Exp. Med. 188: 483-495 (1998), EP 0 463 151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310 and WO 03 / 47336.

[0067] In an alternative approach, other companies, including GenPharm International, Inc., have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a μ constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal. This method is described in U.S. Pat. No. 5,545,807 to Surani et al.; and U.S. Pat. Nos. 5,545,806; 5,625,825; 5,625,126; 5,633,425; 5,661,016; 5,770,429; 5,789,650; 5,814,318; 5,877,397; 5,874,299; and 6,255,458, each to Lonberg and Kay; U.S. Pat. Nos. 5,591,669 and 6,023,010 to Krimpenfort and Berns; U.S. Pat. Nos. 5,612,205; 5,721,367; and 5,789,215 to Berns et al.; and U.S. Pat. No. 5,643,763 to Choi and Dunn; and GenPharm International's U.S. patent applications serial numbers 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741. See also EP 0 546 073 Bl, 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 U.S. Pat. No. 5,981,175. See further 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).

[0068] Kirin has also demonstrated the production of human antibodies from mice into which large segments of chromosomes or entire chromosomes have been introduced by microcell fusion. See European Patent Applications Nos. 773 288 and 843 961. Xenerex Biosciences is developing a technology for the potential production of human antibodies. In this technology, SCID mice are reconstituted with human lymphocytes (e.g., B and / or T cells). The mice are then immunized with an antigen and can generate an immune response against the antigen. See U.S. Patents Nos. 5,476,996; 5,698,767; and 5,958,765.

[0069] In some embodiments, the antibody constructs of the present invention are "isolated" or "substantially pure" antibody constructs. When used to describe the antibody constructs disclosed herein, "isolated" or "substantially pure" means that the antibody construct has been identified, separated and / or recovered from the components of its production environment. Preferably, the antibody construct is not associated with or substantially not associated with all other components from its production environment. The contaminating components of its production environment, such as those produced by recombinant transfected cells, are substances that may interfere with the diagnosis or therapeutic use of the antibody construct and may include enzymes, hormones and other proteins or non-protein compounds. It should be understood that, depending on the circumstances, an isolated or substantially pure antibody construct can constitute from 5% to 99.9% by weight of the total protein / polypeptide content in a given sample. By using an inducible promoter or a high expression promoter, the desired antibody construct can be produced at significantly higher concentrations. This definition includes producing antibody constructs in a variety of organisms and / or host cells known in the art. In certain embodiments, the antibody construct is (1) purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence using a spinning cup sequenator, or (2) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. However, the isolated antibody construct is typically prepared by at least one purification step.

[0070] According to one embodiment, the entire antibody construct and / or binding domain is in the form of one or more polypeptides or in the form of a protein. In addition to the protein portion, such polypeptides or proteins may include non-protein portions (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde).

[0071] Peptides are short chains of amino acid monomers connected by covalent peptide (amide) bonds. Therefore, peptides are classified as a wide chemical category of bio-oligomers and polymers. The amino acids that are part of a peptide or polypeptide chain are referred to as "residues" and can be numbered consecutively. All peptides except cyclic peptides have an N-terminal residue at one end of the peptide and a C-terminal residue at the other end. Oligopeptides are composed of only a few amino acids (usually between two and twenty). Polypeptides are longer, continuous and unbranched peptide chains. Peptides differ from proteins in size and can be understood as containing about 50 or fewer amino acids as an arbitrary benchmark. Proteins are composed of one or more polypeptides that are usually arranged in a biological function manner. Although the various aspects of the laboratory techniques applied to peptides, polypeptides, and proteins are different (e.g., electrophoretic properties, chromatography, etc.), the size boundaries that distinguish peptides from polypeptides and proteins are not absolute. Therefore, in the context of the present invention, the terms "peptide," "polypeptide," and "protein" can be used interchangeably, and the term "polypeptide" is generally preferred.

[0072] Polypeptide can further form multimers such as dimers, trimers and higher oligomers, and these multimers are composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. can be identical or different. Therefore, the corresponding higher-order structures of such multimers are referred to as homo- or hetero-dimers, homo- or hetero-trimers, etc. An example of a hetero-multimer is an antibody or immunoglobulin molecule, whose 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, wherein the modification is achieved, for example, by post-translational modification (such as glycosylation, acetylation, phosphorylation, etc.). When mentioned in this article, "peptide", "polypeptide" or "protein" can also be chemically modified, such as pegylated. Such modifications are well known in the art and are described below.

[0073] The terms "(specifically or immunospecifically) bind", "(specifically or immunospecifically) recognize", or "(specifically or immunospecifically) react with" according to the present invention mean that an antibody construct or binding domain interacts or (immuno)specifically interacts with a given epitope on a target molecule (antigen) (here: CLDN18.2 and CD3, respectively). This interaction or association occurs more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above, for the epitope on a specific target than for an alternative substance (non-target molecule). However, due to sequence similarity between homologous proteins in different species, an antibody construct or binding domain that immunospecifically binds to its target (e.g., a human target) may cross-react with homologous target molecules from different species (e.g., from non-human primates). Therefore, the term "specifically / immunospecifically binds" can include binding of an antibody construct or binding domain to epitopes or structurally related epitopes in more than one species.

[0074] In the context of the present invention, the term "epitope" refers to a part or region of an antigen that is identified / immunospecifically by a binding domain. An "epitope" is antigenic, and therefore the term epitope is sometimes referred to as an "antigenic structure" or "antigenic determinant." The binding domain portion that is combined with the epitope is referred to as a paratope. It is believed that specific binding is achieved by the specific motif in the amino acid sequence of the binding domain and the antigen. Therefore, as a result of the potential secondary modification of their one-level, secondary and / or tertiary structure and the structure, combination is achieved. The specific interaction of the paratope and its antigenic determinant can cause the simple combination of the site and the antigen. In some cases, specific interaction can alternatively or additionally cause the initiation of a signal, for example, due to the change of inducing antigen conformation, the oligomerization of the antigen, etc.

[0075] Based on the structure of protein antigen and the interaction with paratope, the epitope of protein antigen is divided into two major categories: conformational epitope and linear epitope. Conformational epitope is made up of the discontinuous part of the amino acid sequence of antigen. These epitopes interact with paratope based on the three-dimensional surface characteristics and shape or tertiary structure (folding) of antigen. The method for determining epitope conformation includes but is not limited to x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy and fixed-point spin labeling and electron paramagnetic resonance (EPR) spectroscopy. On the contrary, linear epitope interacts with paratope based on its primary structure. Linear epitope is formed by the continuous amino acid sequence from antigen, and typically includes at least 3 or at least 4 and more generally at least 5 or at least 6 or at least 7, for example, about 8 to about 10 amino acids in unique sequence.

[0076] The following describes a method for CLDN18.2 epitope mapping: A predefined region (contiguous stretch of amino acids) within the extracellular loop of the human CLDN18.2 protein is exchanged / replaced with the corresponding region of a CLDN18.2 paralog (such as human CLDN6 or human CLDN9, but other paralogs are also conceivable as long as the binding domain does not cross-react with the paralog used). These human CLDN18.2 / paralog chimeras are expressed on the surface of host cells (such as CHO cells). The binding of the antibody or antibody construct can be tested via FACS analysis. When binding of the antibody or antibody construct to the chimeric molecule is completely abolished, or when a significant reduction in binding is observed, it can be concluded that the region of human CLDN18.2 removed from this chimeric molecule is relevant for immunospecific epitope-paratope recognition. The reduction in binding compared to human (wild-type) CLDN18.2 is preferably at least 10%, 20%, 30%, 40%, or 50%; more preferably at least 60%, 70%, or 80%, and most preferably 90%, 95%, or even 100%, whereby binding to human CLDN18.2 is set to 100%. Alternatively, the epitope mapping analysis described above can be improved by introducing one or more point mutations into the sequence of CLDN18.2 (particularly in the sequence of extracellular loop 1 or loop 2). These point mutations can, for example, reflect differences between CLDN18.2 and its closely related paralog CLDN18.1. For example, the mutation can be selected from the group consisting of: Q29M, N37D, A42S, N45Q, Q47E, E56Q, G65P, and L69I. See Examples 1 and 2.

[0077] Another method for determining the contribution of specific residues of the target antigen to recognition by an antibody construct or binding domain is alanine scanning (see, e.g., Morrison KL and Weiss GA. Curr Opin Chem Biol. 2001 Jun;5(3):302-7), in which each residue to be analyzed is replaced with alanine, for example, via site-directed mutagenesis. Alanine is used because it has a non-bulky, chemically inert methyl functional group, but still mimics the secondary structure references possessed by many other amino acids. In cases where the size of the mutated residue is desired to be conserved, bulky amino acids (such as valine or leucine) can sometimes be used.

[0078] The interaction between the binding domain and the epitope of the target antigen means that the binding domain exhibits considerable or significant affinity for the epitope / target antigen (here: CLDN18.2 and CD3, respectively), and generally does not exhibit significant affinity for proteins or antigens other than the target antigen (here: CLDN18.2 / CD3) - although cross-reactivity with, for example, homologous targets from other species is discussed above. "Significant affinity" includes binding with an affinity of ≤ 10 -6 Preferably, when the binding affinity is ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, or even ≤10 -11 M, or ≤10 -12 M, the binding is considered specific. Whether a binding domain reacts or binds (immunospecifically) to the target can be easily tested, for example, by comparing the affinity of the binding domain for its desired target protein or antigen with the affinity of the binding domain for a non-target protein or antigen (here: a protein other than CLDN18.2 or CD3, respectively). Preferably, the antibody constructs of the present invention do not significantly bind to proteins or antigens other than CLDN18.2 or CD3, respectively (i.e., the first domain does not bind to proteins other than CLDN18.2, and the second domain does not bind to proteins other than CD3) - unless any one or more additional binding domains directed against additional targets are deliberately introduced into the antibody constructs of the present invention, in which case the present invention also provides for the binding of this binding domain to its specific target.

[0079] It is envisioned that the first domain has an affinity for CLDN18.2 (e.g., human CLDN18.2) of ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, or ≤20 nM. These values ​​are preferably measured in a cell-based assay (e.g., a Scatchard assay). See Example 4. Other methods for determining affinity are also well known. It is further envisioned that the second domain has an affinity for CD3 (e.g., human CD3) of ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM, or ≤10 nM. These values ​​are preferably measured in a surface plasmon resonance assay (e.g., a Biacore assay). See Example 3.

[0080] The term "does not significantly bind" means that the antibody construct or binding domain of the present invention does not bind to proteins or antigens other than CLDN18.2 or CD3 when the proteins or antigens are expressed on the cell surface. Thus, the antibody construct exhibits a reactivity of ≤30%, preferably ≤20%, more preferably ≤10%, particularly preferably ≤9%, ≤8%, ≤7%, ≤6%, ≤5%, ≤4%, ≤3%, ≤2% or ≤1% with proteins or antigens other than CLDN18.2 or CD3 (when the proteins or antigens are expressed on the cell surface), whereby binding to CLDN18.2 or CD3, respectively, is set to 100%. "Reactivity" can be expressed, for example, as an affinity value (see above).

[0081] It is envisioned that the antibody constructs of the present invention (and more specifically, the first domain thereof) do not bind or do not significantly bind to CLDN18.2 paralogs, more specifically human CLDN18.2 paralogs and / or macaque / cynomolgus monkey CLDN18.2 paralogs. It is also envisioned that the antibody constructs do not bind or do not significantly bind to (human or macaque / cynomolgus monkey) CLDN18.2 paralogs on the surface of target cells. CLDN18.2 paralogs include, but are not limited to, CLDN18.1, CLDN1, CLDN2, CLDN3, CLDN4, CLDN6, and CLDN9. According to one embodiment, the human paralogs of CLDN18.2 have sequences as described in SEQ ID NOs: 2-10. See Examples 6 and 7. Figure 6 Therefore, it is envisaged that the first domain of the antibody construct of the present invention does not bind or does not significantly bind to CLDN18.1, CLDN1, CLDN2, CLDN3, CLDN4, CLDN6 and / or CLDN9 (on the surface of a target cell).

[0082] The first domain of the antibody construct of the present invention binds to CLDN18.2 on the surface of a target cell. A "target cell" can be any prokaryotic or eukaryotic cell that expresses CLDN18.2 on its surface; preferably, the target cell is a cell that is part of the human or animal body, such as a cancer or tumor cell that expresses a specific CLDN18.2, or a cell that is a CLDN18.2-positive neoplasm. It should be understood that in the context of the present invention, the term "on the surface" means that the first domain of the antibody construct specifically binds to an epitope contained within the first CLDN18.2 extracellular loop (CLDN18.2 ECL1), within the second CLDN18.2 extracellular loop (CLDN18.2 ECL2), or within a combination of both loops. Thus, it is contemplated that the first domain of the antibody construct of the present invention binds to an extracellular loop of CLDN18.2 (preferably human CLDN18.2). The extracellular loop can be the first loop or the second loop. It is also contemplated that both loops contribute to binding. In this case, it is possible that one loop (e.g., the first loop) represents the primary binding partner of the antibody construct, while another loop (e.g., the second loop) contributes to binding, for example as a stabilizing partner, but is not absolutely necessary for binding. Thus, when CLDN18.2 is expressed by naturally expressing cells or cell lines (e.g., human gastric cancer cell lines SNU-601, SNU-620, or also SNU-16, NUGC, NUG-C4, GSU, or IM95) and / or by cells or cell lines transformed or (stably / transiently) transfected with CLDN18.2, the first domain according to the present invention can bind to CLDN18.2. In one embodiment, in a cell-based binding assay such as a Scatchard assay, when CLDN18.2 is used as a target molecule, the first domain binds to CLDN18.2 (see, e.g., Example 4). Furthermore, it is envisioned that the antibody construct / its first domain binds to human CLDN18.2 on the surface of a target cell. A preferred amino acid sequence of human CLDN18.2 is described in SEQ ID NO: 1.

[0083] It is envisioned that the antibody constructs according to the present invention (and more specifically, the first domain of said antibody constructs) bind to the first extracellular loop (ECL1, loop 1) of CLDN18.2. This does not necessarily exclude that the second extracellular loop also contributes to the paratope-epitope interaction site, albeit to a lesser extent. The term "CLDN18.2 ECL" (ECL = extracellular loop) refers to those portions of CLDN18.2 that are substantially free of the transmembrane and cytoplasmic domains of CLDN18.2. It will be understood that the transmembrane domains identified for the CLDN18.2 polypeptides of the present invention are identified according to the criteria routinely used in the art for identifying such hydrophobic domain types. The exact boundaries of the transmembrane domains may vary, but most likely will vary by no more than about 5 amino acids at either end of the domains specifically mentioned herein. A preferred human CLDN18.2 ECL1 is shown in SEQ ID NO: 2, and a preferred human CLDN18.2 ECL2 is shown in SEQ ID NO: 3.

[0084] Furthermore, the present invention provides:

[0085] The first domain of the antibody construct of the present invention binds to the same CLDN18.2 epitope as an antibody or antibody construct comprising a domain that binds to CLDN18.2 on the surface of a target cell and comprises:

[0086] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0087] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0088] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0089] d) a VH region as described in SEQ ID NO: 139, and a VL region as described in SEQ ID NO: 140;

[0090] The antibody construct of the present invention competes for binding with an antibody or antibody construct comprising a domain that binds to CLDN18.2 on the surface of a target cell and comprises:

[0091] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0092] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0093] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0094] d) a VH region as described in SEQ ID NO: 139, and a VL region as described in SEQ ID NO: 140;

[0095] The first domain of the antibody construct of the present invention binds to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 22 on the surface of the target cell, and optionally also binds to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 24 on the surface of the target cell, but does not bind to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 23 on the surface of the target cell;

[0096] The first domain of the antibody construct of the present invention binds to a CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 14 on the surface of a target cell and / or a CLDN18.2 mutant having the amino acid sequence of SEQ ID NO: 15 on the surface of a target cell, and optionally also binds to one or more CLDN18.2 mutants having an amino acid sequence selected from the group consisting of those depicted in SEQ ID NO: 11, 12, 13, 16, 17, 19, 20 and 21 on the surface of a target cell, but does not bind to a CLDN18.2 mutant having the amino acid sequence depicted in SEQ ID NO: 18 on the surface of a target cell;

[0097] The first domain of the antibody construct of the invention binds to human CLDN18.2 on the surface of a target cell, wherein Glu (E) at position 56 of human CLDN18.2 is essential for the binding of the first domain, and Ala (A) at position 42 and / or Asn (N) at position 45 of human CLDN18.2 are not essential for the binding of the first domain; and / or

[0098] The first domain of the antibody construct of the present invention binds to an epitope of CLDN18.2 that comprises the amino acid sequence as depicted in SEQ ID NO: 266, but does not comprise the amino acid sequence as depicted in SEQ ID NO: 265, and optionally does not further comprise the amino acid sequence as depicted in SEQ ID NO: 267.

[0099] The CLDN18.2 binding specificity of other anti-CLDN18.2 binders (CL-3, CL-4) was also analyzed in epitope mapping (see Example 2). It was found that these CLDN18.2 x CD3 antibody constructs had different epitope specificities and at the same time showed significantly inferior cytotoxic potential compared to the antibody constructs of the present invention. In Example 7.4, it was demonstrated that despite having similar affinity for CLDN18.2, the antibody constructs of the present invention showed EC values ​​in the double picomolar range. 50 values, while the comparative constructs showed EC values ​​in the 3- to 5-digit picomolar range. 50 Value. Antibody constructs showing cytotoxic activity in the latter range may not be effective enough for therapeutic use in directing the patient's immune system (more specifically the cytotoxic activity of T cells against cancer cells). On the other hand, the antibody constructs according to the present invention are present with a very favorable epitope-activity relationship, thus supporting effective antibody construct-mediated cytotoxic activity.

[0100] Whether an antibody, antibody construct or binding domain binds to the same CLDN18.2 epitope on the target cell surface as another given antibody, antibody construct or binding domain can be measured by various assays as described herein (e.g., by epitope mapping with chimeric or mutant CLDN18.2 molecules), as described herein above or in Examples 1 and 2. Other methods for determining epitopes, such as alanine scanning, are described herein.

[0101] Whether an antibody or antibody construct competes with another given antibody or antibody construct for binding to an antigen (such as CLDN18.2) on the surface of a target cell can be measured in a competitive assay such as a competitive ELISA. Avidin-coupled microparticles (beads) can also be used. Similar to avidin-coated ELISA plates, each of these beads can be used as a substrate on which an assay can be performed when reacting with a biotinylated protein. The antigen is coated on the beads and then pre-coated with the first antibody. The second antibody is added and any additional binding is determined. Reading occurs via flow cytometry. Preferably, a cell-based competition assay is used, using cells naturally expressing CLDN18.2 or cells stably or transiently transformed with CLDN18.2. In the context of the present invention, the term "competitive binding" means that competition occurs between two test antibodies by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90%, as determined by any of the assays disclosed above (preferably cell-based assays). Of course, the same analysis can be applied to other targets, such as CD3.

[0102] Competitive antibody binding assays include assays that determine the competitive binding of two antibodies / antibody constructs to cell surface-bound antigens. A common method designed to detect the binding of two antibodies / antibody constructs A and B to the same antigen on the cell surface may include the following steps:

[0103] a) blocking cell surface antigen by pre-incubating cells with antibody / antibody construct A, followed by submaximal addition of labeled antibody / antibody construct B, and detecting binding of B compared to binding in the absence of A;

[0104] b) titrating (i.e. adding varying amounts of) antibody / antibody construct A in the presence of a submaximal amount of labelled antibody / antibody construct B and measuring the effect on binding of B; or

[0105] c) Co-titration of A and B, where both antibodies / antibody constructs are incubated together at maximal concentration and the total binding is determined to equal or exceed that of either A or B alone, i.e. a method that is not affected by the order of addition or the relative amounts of the antibodies / antibody constructs.

[0106] When two antibodies / antibody constructs A and B compete for the antigen bound to the cell surface, the antibodies will often compete with each other in a blocking assay that is independent of the order in which the antibodies are added. In other words, if assayed in either direction, competition is detected. However, this is not always the case, and in some cases, the order in which the antibodies are added or the direction in which they are assayed may have an impact on the signal produced. This may be due to differences in the affinity or avidity of the potential competing antibodies / antibody constructs. If the order in which they are added has a significant impact on the signal produced, it can be inferred that if competition is detected in at least one order, the two antibodies / antibody constructs do compete.

[0107] According to one embodiment, the first domain of the antibody construct of the present invention binds to human CLDN18.2 on the surface of the target cell, wherein Glu (E) at position 56 of human CLDN18.2 is essential for the binding of the first domain, and Ala (A) at position 42 and / or Asn (N) at position 45 of human CLDN18.2 are not essential for the binding of the first domain. In this context, the term "essential for binding" means that a specific amino acid (Glu at position 56) is required for the binding of the antibody construct to CLDN18.2 to occur. If the amino acid Glu at position 56 is exchanged for example to Gln (→E56Q), the binding of the antibody construct is abolished or significantly reduced (see Figure 4 , column designated "P6"). The term "not essential for binding" means that the specific amino acid (Ala at position 42 and / or Asn at position 45) is not essential for the binding of the antibody construct to CLDN18.2 to occur. If the amino acid Ala at position 42 and / or Asn at position 45 is exchanged for, for example, Ser at position 42 (→A42S) and / or Gln at position 45 (→N45Q), the binding of the antibody construct is not reduced or is not significantly reduced (see Figure 4 , columns named "P3" and "P4").

[0108] According to one embodiment, the first domain of the antibody construct of the present invention comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of:

[0109] a) CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126; and

[0110] b) CDR-H1 as described in SEQ ID NO: 133, CDR-H2 as described in SEQ ID NO: 134, and CDR-H3 as described in SEQ ID NO: 135, and CDR-L1 as described in SEQ ID NO: 136, CDR-L2 as described in SEQ ID NO: 137, and CDR-L3 as described in SEQ ID NO: 138.

[0111] In the context of the present invention, the term "variable" refers to those portions of an antibody or immunoglobulin domain that exhibit sequence variability and are involved in determining the specificity and binding affinity of a particular antibody (i.e., "variable region(s)"). Typically, a pairing of a heavy chain variable region (VH) and a light chain variable region (VL) together forms a single antigen binding site.

[0112] Variability is not evenly distributed throughout the variable region of an antibody; it is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conservative (i.e., non-hypervariable) portions of the variable region are called "framework" (FR) regions, and provide a scaffold for the six CDRs in three-dimensional space to form an antigen-binding surface. The variable regions of naturally occurring antibody heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4) that primarily adopt a β-sheet configuration. Together with the CDRs, they form the following sequence within the variable heavy or light chain: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable regions in each chain are closely brought together by the framework regions and typically contribute to the formation of the antigen-binding site together with the hypervariable regions from the other chain (see Kabat et al., cited above).

[0113] The term "CDR" and its plural "CDR" refer to the complementarity determining regions, three of which constitute the binding characteristics of the light chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three of which constitute the binding characteristics of the heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). The CDRs contain most of the residues that are responsible for the specific interaction of the antibody (or antibody construct or binding domain) with the antigen, and therefore contribute to the functional activity of the antibody molecule: they are the main determinants of antigen specificity.

[0114] The accurate definition of CDR boundaries and length is subject to different classifications and numbering systems. Therefore, CDR can be quoted by Kabat, Chothia, contact or any other boundary definition (including numbering systems described herein). Although there are different boundaries, each of these systems has a certain degree of overlap in terms of constituting the so-called "hypervariable region" in the variable sequence. Therefore, the CDR definitions according to these systems can be different in length and boundary regions relative to adjacent framework regions. See, for example, Kabat (based on a method for cross-species sequence variability), Chothia (based on a method for crystallographic studies of antigen-antibody complexes), and / or MacCallum (Kabat et al., above-mentioned citations; Chothia et al., J.MoI.Biol [Journal of Molecular Biology], 1987, 196: 901-917; and MacCallum et al., J.MoI.Biol [Journal of Molecular Biology], 1996, 262: 732). Another standard for characterizing antigen binding sites is the AbM definition used by the AbM antibody modeling software of Oxford University Molecular Company (OxfbrdMolecular). See, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains In: Antibody Engineering Lab Manual (Editors: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). To the extent that the two residue identification techniques define overlapping rather than identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

[0115] Typically, CDRs form a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the main chain conformation adopted by the antigen binding (CDR) loop. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Therefore, the corresponding loops between antibodies can have very similar three-dimensional structures, although most of the loops have high amino acid sequence variability (Chothia and Lesk, J. Mol. Biol. [J. MoI. Biol.], 1987, 196: 901; Chothia et al., Nature [Nature], 1989, 342: 877; Martin and Thornton, J. Mol. Biol. [J. MoI. Biol.], 1996, 263: 800). In addition, there is a relationship between the adopted loop structure and the amino acid sequence around it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues located at key positions within the loop and within the conserved framework (i.e., outside the loop). Therefore, specific canonical classes can be assigned based on the presence of these key amino acid residues.

[0116] The term "canonical structure" can also include considerations about the linear sequence of an antibody, for example, as catalogued by Kabat (Kabat et al., cited above). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues in the variable region of an antibody in a consistent manner, and is a preferred embodiment of the present invention, as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences that the Kabat numbering is not fully reflected can be described by the numbering system of Chothia et al., and / or revealed by other techniques (such as crystallography and two-dimensional or three-dimensional computational modeling). Therefore, a given antibody sequence can be placed in a canonical category that especially allows identification of an appropriate category sequence (for example, based on the expectation of including a variety of canonical structures in a library). The Kabat numbering of antibody amino acid sequences and the structural considerations as described by Chothia et al., cited above, and their participation in explaining the canonical aspects of antibody structure are described in the literature. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known in the art. For a review of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow et al., eds., 1988.

[0117] The CDR3 of light chain and particularly heavy chain can constitute the most important determinant in antigen binding in light chain variable region and heavy chain variable region.In some antibodies or antibody constructs / binding domains, heavy chain CDR3 seems to constitute the main contact area between antigen and antibody.Wherein only the in vitro selection scheme that changes CDR3 can be used to change the binding characteristics of antibody or antibody constructs / binding domains or determine which residues help the combination of antigen.Therefore, CDR3 is typically the largest source of molecular diversity in antibody binding site.For example, CDR-H3 can be as short as two amino acid residues or greater than 26 amino acid.

[0118] In a classical full-length antibody or immunoglobulin, each light (L) chain is linked to a heavy (H) chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. The heavy chain constant (CH) domain closest to VH is usually named CH1. The constant ("C") domain does not directly participate in antigen binding, but exhibits various effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation (complement-dependent cytotoxicity, CDC). The Fc region of an antibody is the "tail" region of a classical antibody that interacts with cell surface receptors known as Fc receptors and some proteins of the complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains (CH2 and CH3) of the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH2, CH3, and CH4) in each polypeptide chain. The Fc region also contains part of the so-called "hinge" region, which is held together by one or more disulfide bonds and non-covalent interactions. The Fc region of naturally occurring IgG has highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is required for Fc receptor-mediated activity.

[0119] ADCC is a cell-mediated immune defense mechanism in which the effector cells of the immune system actively lyse target cells whose membrane surface antigens have been bound by specific antibodies. ADCC requires immune effector cells, which are generally known to be natural killer (NK) cells that typically interact with IgG antibodies. However, ADCC can also be mediated by macrophages, neutrophils, and eosinophils. ADCC involves activating effector cells expressing Fc receptors by antibodies expressing the Fc portion. For example, the most common Fc receptor on the surface of NK cells is known as CD16 or FcγRIII. Once the Fc receptor binds to the Fc region of IgG, NK cells release cytotoxic factors that cause target cell death. Similarly, the Fc receptor (FceRI) of eosinophils will recognize IgE. In contrast, in CDC, due to classical complement activation, the molecule "C1q" of the complement system binds to the antibody Fc region, and this binding triggers the complement cascade, which results in the formation of a membrane attack complex (MAC) on the surface of the target cell. In therapeutic antibodies or antibody constructs, both ADCC and CDC can be modulated by Fc isotype engineering, Fc gene mutations, or modification of the Fc glycosylation profile.

[0120] The sequences of antibody genes after assembly and somatic mutation are highly altered, and it is estimated that these altered genes encode 10 10 The invention relates to a method for producing a repertoire of different antibody molecules (Immunoglobulin Genes [immunoglobulin genes], 2nd edition, Jonio et al., ed., Academic Press [Academic Press], San Diego, CA [San Diego, California], 1995). Therefore, the immune system provides an immunoglobulin repertoire. 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. One or more sequences can be produced by rearrangement in vivo of the V, D and J segments of the heavy chain and the V and J segments of the light chain. Alternatively, one or more sequences can be produced from cells in response to rearrangement (e.g., in vitro stimulation). Alternatively, a portion or all of one or more sequences can be obtained by DNA splicing, nucleotide synthesis, mutagenesis and other methods, see, for example, U.S. Patent No. 5,565,332. The repertoire can include only one sequence or can include multiple sequences, including sequences in a genetic diversity set.

[0121] It is envisioned that the antibody construct has a cysteine ​​clamp within the first domain. Such a cysteine ​​clamp can be introduced to improve the stability of the construct. See, for example, US 2016 / 0193295.

[0122] In one embodiment of the present invention, the first domain of the antibody construct of the present invention comprises a VH region having an amino acid sequence as described in SEQ ID NO: 127 or SEQ ID NO: 139. In another embodiment, the first domain of the antibody construct of the present invention comprises a VL region having an amino acid sequence as described in SEQ ID NO: 128 or SEQ ID NO: 140. In another embodiment, the first domain of the antibody construct of the present invention comprises a VH region and a VL region, and the VH region and the VL region have an amino acid sequence as described in SEQ ID NO: 127+128 (VH+VL) or SEQ ID NO: 139+140 (VH+VL). In yet another embodiment, the first domain of the antibody construct of the present invention comprises a polypeptide having an amino acid sequence as described in SEQ ID NO: 129 or SEQ ID NO: 141.

[0123] As described above herein, the present invention provides the following embodiments, wherein the antibody construct is in the form of the following group, which is composed of the following (scFv) 2, scFv-single domain mAb, diabodies and oligomers of any of the above forms. The term "in ... form" does not exclude constructs that can be further modified (e.g., by attachment or fusion to other parts), as described herein. According to one embodiment of the antibody construct of the present invention, the first domain and / or the second domain are in the form of scFv. In scFv, the VH region and the VL region are arranged in the order of VH-VL or VL-VH (from N-terminus to C-terminus). It is envisioned that the VH and VL regions of the first and / or second binding domains are connected via a joint, preferably a peptide joint. According to one embodiment of the first and / or second domain, the VH region is located at the N-terminus of the joint, and the VL region is located at the C-terminus of the joint. In other words, in one embodiment of the first and / or second domain, scFv comprises from N-terminus to C-terminus: VH-joint-VL. It is further envisioned that the first domain and the second domain of the antibody construct are connected via a joint, preferably a peptide joint. The antibody construct can, for example, comprise domains in the order of the first domain-joint-second domain (from N-terminal to C-terminal). The reverse order (second domain-joint-first domain) is also possible.

[0124] The joint is preferably a peptide joint, more preferably a short peptide joint. According to the present invention, " peptide joint " comprises the amino acid sequence of a domain of antibody construct and another (variable and / or combined) domain (such as variable domain or binding domain) connected amino acid sequence. The basic technical characteristics of this peptide joint are that it does not comprise any polymerization activity. Suitable peptide joints are those described in U.S. Patents 4,751,180 and 4,935,233 or WO 88 / 09344. Peptide joints can also be used for other domains or modules or districts (such as half-life extension domain) being connected to the antibody construct of the present invention. The example of a useful peptide joint is shown in SEQ ID NO:155-168. In the context of the present invention, " short " joint has between 2 and 50 amino acids, preferably between 3 and 35, between 4 and 30, between 5 and 25, between 6 and 20 or between 6 and 17 amino acids. The linker between the two variable regions of a binding domain can have a different length than the linker between the two binding domains (e.g., it can be longer). For example, the linker between the two variable regions of a binding domain can have a length of between 7 and 15 (preferably between 9 and 13) amino acids, and the linker between the two binding domains can have a length of between 3 and 10 (preferably between 4 and 8) amino acids. It is further envisioned that the peptide linker is a glycine / serine linker, such as those described in SEQ ID NOs: 156 and 158-168. Most of the amino acids in the glycine / serine linker are selected from glycine and serine.

[0125] In the case of using a joint, the joint preferably has a length and sequence sufficient to ensure that each of the first domain and the second domain can retain its difference binding specificity independently of each other. For the peptide joint of at least two binding domains (or two variable regions forming a binding domain) in the connection antibody construct, it is envisioned that those peptide joints comprising only a few amino acid residues (such as 12 amino acid residues or less) are included. Therefore, the peptide joints of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. It is envisioned that the peptide joint with less than 5 amino acid comprises 4, 3, 2 or 1 amino acid, wherein the joint rich in Gly is preferred. "Single amino acid" joint in the context of the "peptide joint" is Gly. Another embodiment of a peptide joint is characterized in that the amino acid sequence Gly-Gly-Gly-Gly-Ser (i.e., Gly4Ser (SEQ ID NO: 156)) or its polymer (i.e., (Gly4Ser) x, wherein x is an integer of 1 or larger (e.g., 2 or 3)). Useful linkers are described in SEQ ID NOs: 155-163. The characteristics of such peptide linkers 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). Peptide linkers that do not promote any secondary structure are preferred. The connection of the domains to each other can be provided, for example, by genetic engineering. Methods for preparing fused and operably linked bispecific single chain constructs and expressing them in mammalian cells or bacteria are well known in the art (e.g., WO 99 / 54440 or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001).

[0126] According to one embodiment of the present invention, the antibody construct of the present invention is a "single-chain antibody construct". It is also envisioned that the first binding domain or the second binding domain or both binding domains can be in the form of a "single-chain Fv" (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by independent genes, these two domains can be joined by an artificial linker using recombinant methods. As described above, this artificial linker enables them to be made into a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule; see, for example, Huston et al. (1988) Proc. Natl. Acad. Sci USA [Proceedings of the National Academy of Sciences of the United States of America] 85: 5879-5883. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the function of the fragments is evaluated in the same manner as for full-length antibodies or IgG. Therefore, a single-chain variable fragment (scFv) is a fusion protein of the heavy chain (VH) and light chain (VL) variable regions of an immunoglobulin, typically connected by a short linker peptide. The linker is typically glycine-rich for flexibility and serine- or threonine-rich for solubility and can connect the N-terminus of VH to the C-terminus of VL, or vice versa. Despite the removal of the constant regions and the introduction of the linker, the protein retains the specificity of the original immunoglobulin.

[0127] 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; Blood, (2000), 95, 6, 2098-2103; Brühl, Immunol., (2001), 166, 2420-2426; Kipriyanov, J. Mol. Biol., (1999), 293, 41-56. Techniques described for the production of single-chain antibody constructs (see, inter alia, U.S. Patent No. 4,946,778; Kontermann and Dübel (2010), loc. cit., and Little (2009), loc. cit.) can be adapted to produce single-chain antibody constructs that specifically recognize one or more selected targets.

[0128] Bivalent (also known as bivalent) or bispecific single chain variable fragments (linked scFv or bi-scFv of the form (scFv) 2) can be engineered by linking two scFv molecules (e.g., using a linker as described above). Linking can be performed by creating a single polypeptide chain with two VH regions and two VL regions, thereby creating a tandem scFv (see, e.g., Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244). Another possibility is to create scFv molecules with linker peptides that are too short for the two variable regions to fold together (e.g., about five amino acids), thereby forcing the scFv to dimerize. In this case, the VH and VL binding domains (binding to the target antigen CLDN18.2 or CD3) are not directly linked via a peptide linker. Thus, the VH of the CD3 binding domain can be fused to the VL of the CLDN18.2 binding domain, for example, via a peptide linker, and the VH of the CLDN18.2 binding domain can be fused to the VL of the CD3 binding domain via this peptide linker. This type of antibody is called a diabody (see, e.g., Hollinger, Philipp et al., (July 1993) Proceedings of the National Academy of Sciences of the United States of America 90(14):6444-8).

[0129] Antibody constructs designated as "single domain antibodies" contain one (monomeric) antibody variable region that is capable of selectively binding to a specific antigen independently of other variable regions. The first single domain antibodies were engineered from heavy chain antibodies found in camels, and these are called V H H fragment. Cartilaginous fish also have heavy chain antibodies (IgNARs), from which V NAR Single domain antibodies of fragments. An alternative approach is to split the dimer variable regions from common immunoglobulins into monomers, thus obtaining VH or VL as single domain Abs. Although most studies on single domain antibodies are currently based on heavy chain variable regions, nanobodies derived from light chains have also been shown to specifically bind to target epitopes. Examples of single domain antibodies are so-called sdAbs, nanobodies or single variable domain antibodies. Therefore, (single domain mAb)2 is a monoclonal antibody construct consisting of (at least) two single domain monoclonal antibody constructs individually selected from the group consisting of: VH, VL, V H H and V NARThe linker is preferably in the form of a peptide linker. Similarly, a "scFv-single domain mAb" is a monoclonal antibody construct composed of at least one single domain antibody as described above and one scFv molecule as described above. Again, the linker is preferably in the form of a peptide linker.

[0130] It is also envisioned that the antibody constructs of the present invention have additional functions in addition to the function of binding to the target molecules CLDN18.2 and CD3. In this form, the antibody constructs can be trifunctional or multifunctional antibody constructs by binding to target cells through CLDN18.2, mediating cytotoxic T cell activity through CD3 binding, and providing additional functions (such as means or domains for enhancing or extending serum half-life, fully functional or modified Fc constant domains that mediate ADCC by recruiting effector cells, labels (fluorescence, etc.), therapeutic agents such as toxins or radionuclides, etc.).

[0131] The example of the means or domain of extending the serum half-life of the antibody construct of the present invention includes the peptide, protein or protein domain fused or otherwise attached to the antibody construct. The group of peptide, protein or protein domain is included in the human body with preferred pharmacokinetic characteristics and other protein-bound peptides, such as serum albumin (see WO2009 / 127691). The alternative concept of the peptide of this type of half-life extension includes the peptide combined with neonatal Fc receptor (FcRn, see WO2007 / 098420), which can also be used in the antibody construct of the present invention. The concept of attaching a larger protein domain or a complete protein includes 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 fusions of its domains, and fusions of immunoglobulin constant regions (Fc domains) and variants thereof. Such variants of the Fc domain are referred to as Fc-based domains and can, for example, be optimized / modified to allow desired dimer or multimer pairing, to eliminate Fc receptor binding (e.g., to avoid ADCC or CDC) or for other reasons. Another concept known in the art for extending the half-life of a substance or molecule in the human body is the pegylation of those molecules (e.g., the antibody constructs of the present invention).

[0132] In one embodiment, the antibody construct according to the present invention is linked (e.g., via a peptide bond) to a fusion partner (e.g., a protein, polypeptide, or peptide), e.g., for the purpose of extending the serum half-life of the construct. These fusion partners can be selected from human serum albumin ("HSA" or "HALB") and sequence variants thereof, peptides that bind to HSA, peptides that bind to FcRn ("FcRn BP"), or constructs comprising an (antibody-derived) Fc region. Exemplary sequences of these fusion partners are described in SEQ ID NOs: 170-232. In general, the fusion partners can be linked directly (e.g., via a peptide bond) or through a peptide linker such as (GGGGS) n (wherein "n" is an integer of 2 or greater, such as 2 or 3 or 4) is linked to the N-terminus or C-terminus of the antibody construct according to the present invention. Suitable peptide linkers are described in SEQ ID NOs: 155-163.

[0133] Thus, it is envisaged that the antibody construct according to the present invention comprises:

[0134] (a) A polypeptide composed from N-terminus to C-terminus in the following order:

[0135] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0136] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163; and

[0137] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0138] Optionally a His-tag, for example selected from SEQ ID NO: 169, 269, 270 or 271;

[0139] (b) A polypeptide composed from N-terminus to C-terminus in the following order:

[0140] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0141] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163;

[0142] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0143] Optionally, a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163;

[0144] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 170 and 176-205; and

[0145] Optionally a His-tag, for example selected from SEQ ID NO: 169, 269, 270 or 271;

[0146] (c) A polypeptide composed from N-terminus to C-terminus in the following order:

[0147] A polypeptide having the amino acid sequence QRFVTGHFGGLX1PANG (SEQ ID NO: 171), wherein X1 is Y or H; and

[0148] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0149] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163;

[0150] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0151] A polypeptide having an amino acid sequence as set forth in SEQ ID NO: 173 or SEQ ID NO: 175; and

[0152] Optionally a His-tag, for example selected from SEQ ID NO: 169, 269, 270 or 271;

[0153] (d) A polypeptide composed from N-terminus to C-terminus in the following order:

[0154] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, and SEQ ID NO:118;

[0155] A peptide linker having the amino acid sequence described in SEQ ID NO: 162;

[0156] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 140, followed by a serine residue at the C-terminus;

[0157] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 206; and

[0158] A polypeptide composed from N-terminus to C-terminus in the following order:

[0159] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and SEQ ID NO: 139;

[0160] A peptide linker having the amino acid sequence described in SEQ ID NO: 162;

[0161] a polypeptide having an amino acid sequence selected from the group consisting of 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, SEQ ID NO:107, SEQ ID NO:116, and SEQ ID NO:119; followed by a serine residue at the C-terminus; and

[0162] A polypeptide having the amino acid sequence described in SEQ ID NO: 207;

[0163] (e) A polypeptide composed from N-terminus to C-terminus in the following order:

[0164] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:34, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:61, SEQ ID NO:70, SEQ ID NO:79, SEQ ID NO:88, SEQ ID NO:97, SEQ ID NO:106, SEQ ID NO:115, and SEQ ID NO:118;

[0165] A peptide linker having the amino acid sequence described in SEQ ID NO: 162;

[0166] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 128 and SEQ ID NO: 140; and

[0167] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 208; and

[0168] A polypeptide composed from N-terminus to C-terminus in the following order:

[0169] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 127 and SEQ ID NO: 139;

[0170] A peptide linker having the amino acid sequence described in SEQ ID NO: 162;

[0171] a polypeptide having an amino acid sequence selected from the group consisting of 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, SEQ ID NO:107, SEQ ID NO:116, and SEQ ID NO:119, followed by a serine residue at the C-terminus; and

[0172] A polypeptide having the amino acid sequence described in SEQ ID NO: 209;

[0173] (f) A polypeptide composed from N-terminus to C-terminus in the following order:

[0174] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0175] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163;

[0176] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0177] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 210; and

[0178] A polypeptide having the amino acid sequence described in SEQ ID NO: 211;

[0179] (g) A polypeptide composed from N-terminus to C-terminus in the following order:

[0180] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0181] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 212; and

[0182] A peptide consisting of the following sequence starting from the N-terminus:

[0183] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0184] A polypeptide having the amino acid sequence described in SEQ ID NO: 213;

[0185] (h) A polypeptide composed from N-terminus to C-terminus in the following order:

[0186] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0187] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 214; and

[0188] A peptide consisting of the following sequence starting from the N-terminus:

[0189] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0190] A polypeptide having the amino acid sequence set forth in SEQ ID NO: 215; or

[0191] (i) A polypeptide composed from N-terminus to C-terminus in the following order:

[0192] A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0193] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155-163;

[0194] a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168; and

[0195] A polypeptide having the amino acid sequence described in SEQ ID NO: 216.

[0196] According to another embodiment, the antibody construct of the present invention comprises (in addition to the first and second domains) a third domain, which comprises two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker. It is envisioned that the third domain comprises, in the order from N-terminus to C-terminus: hinge-CH2-CH3-linker-hinge-CH2-CH3. The amino acid sequence that can be used for the third domain is described in SEQ ID NO: 225-232. Each of the polypeptide monomers can have an amino acid sequence selected from the group consisting of SEQ ID NO: 217-224, or at least 90% identical to those sequences. In another embodiment, the first and second domains of the antibody construct of the present invention are fused to the third domain via a peptide linker, and the peptide linker is, for example, selected from the group consisting of SEQ ID NO: 155, 156, 157, 158, 159, 160, 161, 162 or 163.

[0197] According to the present invention, a "hinge" is an IgG hinge region. This region can be identified by analogy using Kabat numbering, see, for example, Kabat positions 223-243. Consistent with the above, the minimum requirement for a "hinge" is the amino acid residues corresponding to the IgG1 sequence extension from D231 to P243 according to Kabat numbering. The terms "CH2" and "CH3" refer to immunoglobulin heavy chain constant regions 2 and 3. These regions can also be identified by analogy using Kabat numbering, see, for example, Kabat positions 244-360 (for CH2) and Kabat positions 361-478 (for CH3). It will be appreciated that there is some variation among immunoglobulins in their IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region (see, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). The term Fc region refers to the last two heavy chain constant regions of IgA, IgD, and IgG, and the last three heavy chain constant regions of IgE and IgM. The Fc region may also include a flexible hinge at the N-termini of these domains. For IgA and IgM, the Fc region may include a J chain. For IgG, the Fc region comprises the immunoglobulin domains CH2 and CH3, and a hinge between the first two domains and CH2. Although the boundaries of the Fc region of an immunoglobulin may vary, an example of a human IgG heavy chain Fc portion comprising the functional hinge, CH2, and CH3 domains can be defined, e.g., to comprise residues D231 (residues of the hinge domain) to P476 (residues at the C-terminus of the CH3 domain), or D231 to L476 for IgG4, respectively, where the numbering is according to Kabat.

[0198] Therefore, the antibody construct of the present invention may comprise, in order from N-terminus to C-terminus:

[0199] (a) the first domain;

[0200] (b) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 162, and 163;

[0201] (c) the second domain;

[0202] (d) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155, 156, 158, 159, 160, 162, and 163;

[0203] (e) a first polypeptide monomer of the third domain (comprising hinge, CH2, and CH3 domains);

[0204] (f) a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 165, 166, 167, and 168; and

[0205] (g) a second polypeptide monomer of the third domain (comprising the hinge, CH2, and CH3 domains).

[0206] It is also envisaged that the antibody constructs of the present invention comprise, in order from N-terminus to C-terminus:

[0207] a first domain having an amino acid sequence selected from the group consisting of SEQ ID NO: 129 and SEQ ID NO: 141; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0208] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 162, and 163;

[0209] a second domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254, and 255; the peptide linker contained in those sequences and having SEQ ID NO: 163 may be replaced by any one of SEQ ID NOs: 155-162 and 164-168;

[0210] a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs: 155, 156, 158, 159, 160, 162, and 163; and

[0211] • a third domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 225-232.

[0212] Thus, in one embodiment, the antibody construct of the present invention comprises or consists of a polypeptide having an amino acid sequence selected from the group of those amino acid sequences depicted in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 143 or SEQ ID NO: 144.

[0213] The covalent modification of antibody constructs is also included in the scope of the present invention, and is usually but not always carried out after translation. For example, by making the specific amino acid residues of the antibody construct react with an organic derivatizing agent that can react with the side chain of selection or with the N-terminal or C-terminal residue, several types of covalent modification of the antibody construct are introduced into the molecule. Derivatization with a bifunctional agent can be used to cross-link the antibody construct of the present invention to a water-insoluble support matrix or surface for use in a variety of methods. Glutaminyl residues and asparaginyl residues are usually deamidated to corresponding glutamyl residues and aspartyl residues respectively. Alternatively, these residues are deamidated under weakly acidic conditions. Any form of these residues all belongs to 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 α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0214] Another type of covalent modification of antibody constructs included within the scope of the present invention includes altering the glycosylation pattern of the protein. As is known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues discussed below) or the host cell or organism in which the protein is produced. Specific expression systems are discussed below. Glycosylation of polypeptides is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (wherein X is any amino acid other than proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0215] Addition of glycosylation sites to the antibody construct is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding one or more serine or threonine residues to the starting sequence or replacing them with one or more serine or threonine residues (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antibody construct can be altered by changes at the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases to produce codons that will translate into the desired amino acids.

[0216] Another means of increasing the number of carbohydrate moieties on the antibody construct is by chemical or enzymatic coupling of glycosides to proteins. The advantage of these programs is that they do not need to produce proteins in host cells with glycosylation capabilities for N- and O-linked glycosylation. Depending on the coupling mode used, one or more sugars may be connected to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as those of cysteine, (d) free hydroxyl groups, such as those of serine, threonine or hydroxyproline, (e) aromatic residues, such as those of phenylalanine, tyrosine or tryptophan, or (f) amide groups of glutamine. These methods are described in WO 87 / 05330 and Aplin and Wriston, 1981, CRC Crit. Rev. Biochem. [CRC Biochemistry Key Review], pp. 259-306.

[0217] Removal of carbohydrate moieties present on the starting antibody construct can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described by 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 using a variety of endoglycosidases and exoglycosidases, as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by using the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

[0218] Other modifications of the antibody construct are also contemplated herein. For example, another type of covalent modification of the antibody construct includes connecting the antibody construct to various non-protein polymers (including polyols) in a manner described in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 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 the antibody construct, for example to facilitate the addition of polymers such as polyethylene glycol (PEG).

[0219] In some embodiments, the covalent modification of the antibody constructs of the present invention comprises the addition of one or more labels. The labeling group can be coupled to the antibody construct via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to perform the present invention. The term "label" or "labeling group" refers to any detectable label. Generally speaking, labels fall into a variety of categories, depending on the assay in which they are to be detected - the following examples include, but are not limited to:

[0220] a) Isotopic labels, which may be radioactive or heavy isotopes, such as radioisotopes or radionuclides (e.g. 3 H. 14 C. 15 N. 35 S. 89 Zr, 90 Y. 99 Tc, 111 In, 125 I. 131 I)

[0221] b) Magnetic labels (e.g., magnetic particles)

[0222] c) Redox-active moiety

[0223] d) Optical dyes (including but not limited to chromophores, phosphors, and fluorophores), such as fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), chemiluminescent groups, and fluorophores, which can be "small molecule" fluorescent agents or protein fluorescent agents

[0224] e) Enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase)

[0225] f) Biotinylation group

[0226] g) a predetermined polypeptide epitope recognized by a second reporter gene (e.g., a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag, etc.)

[0227] "Fluorescent label" means any molecule that can be detected via its intrinsic fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, fluorescein yellow, Cascade Blue J, 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 R-phycoerythrin (PE) (Molecular Probes, Eugene, Oregon). 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 by Richard P. Haugland.

[0228] Suitable protein fluorescent labels also include, but are not limited to, green fluorescent proteins, including GFP from Renilla, Ptilosarcus, or Aequorea species (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Accession No. U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9) 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), beta-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607), and Renilla (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Patent Nos. 5,292,658; 5,418,155; 5,683,888; 5,741,668; 5,777,079; 5,804,387; 5,874,304; 5,876,995; 5,925,558).

[0229] Leucine zipper domains are peptides that promote the oligomerization of proteins in which they are found. Leucine zippers were initially identified in several DNA binding proteins (Landschulz et al., 1988, Science [Science] 240: 1759), and have since been found in a variety of different proteins. Known leucine zippers include naturally occurring peptides and their dimerized or trimerized derivatives. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and leucine zippers derived from surfactant protein D (SPD) are described in Hoppe et al., 1994, FEBS Letters [European Federation of Biochemical Societies Communications] 344: 191. The use of modified leucine zippers that allow stable trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. [Immunology Research Collection] 6: 267-78.

[0230] The antibody construct of the present invention can also include other domains, which for example contribute to separating molecules or relate to the adaptive pharmacokinetic curve of molecules. The domain that contributes to separating antibody constructs can be selected from peptide motifs or the part introduced auxiliaryly, and these parts can be captured in separation methods (such as separation columns). The non-limiting examples of this type of other domains include peptide motifs referred to as Myc-tags, HAT-tags, HA-tags, TAP-tags, GST-tags, chitin binding domains (CBD-tags), maltose binding protein (MBP-tags), Flag-tags, Strep-tags and variants thereof (such as StrepII-tags) and His tags. All antibody constructs disclosed herein (featured by the CDR identified) can include His-tag domains, which are generally referred to as the repetitive sequences of the continuous His residues in the amino acid sequence of molecules, such as the repetitive sequences (SEQ ID NO:269) of five His residues or the repetitive sequences (six histidines, SEQ ID NO:169) of six His residues. The His-tag can be located, for example, at the N-terminus or the C-terminus of the antibody construct. In one embodiment, the hexahistidine tag (HHHHHH) is linked to the C-terminus of the antibody construct according to the invention via a peptide bond.

[0231] It is also envisioned that the antibody construct of the present invention comprises or consists of a polypeptide having an amino acid sequence selected from the group consisting of those described in SEQ ID NOs: 130 and 142 and linked to a protein purification tag at its N-terminus or its C-terminus (preferably via a peptide bond (amide bond)). Attachment of the protein purification tag to the C-terminus of the polypeptide is preferred. It is envisioned that the protein purification tag is a short peptide. For example, the length of the short peptide can be 2-30 amino acids, 4-25 amino acids, 5-20 amino acids, or 6-19 amino acids. Examples of protein purification tags include, but are not limited to, an AU1 epitope (e.g., as described in SEQ ID NO: 272), an AU5 epitope (e.g., as described in SEQ ID NO: 273), a T7-tag (e.g., as described in SEQ ID NO: 274), a V5-tag (e.g., as described in SEQ ID NO: 275), a B-tag (e.g., as described in SEQ ID NO: 276), an E2 epitope (e.g., as described in SEQ ID NO: 277), a FLAG epitope / FLAG tag (e.g., as described in SEQ ID NO: 278), a Glu-Glu tag (e.g., as described in SEQ ID NO: 279 or 280), an HA tag, a histidine affinity tag (e.g., as described in SEQ ID NO: 281), an HSV epitope (e.g., as described in SEQ ID NO: 282), a KT3 epitope (e.g., as described in SEQ ID NO: 283), a Myc epitope (e.g., as described in SEQ ID NO: 284), a NO: 284), a polyarginine tag (5-6 Arg residues), a polyaspartic acid tag (5-16 Asp residues), a polyhistidine tag (2-10 His residues, typically 6 His residues, see, e.g., SEQ ID NOs: 169 and 269-271), a polyphenylalanine tag (typically 11 Phe residues), an S1 tag (e.g., as described in SEQ ID NO: 285), an S-tag (e.g., as described in SEQ ID NO: 286), a Strep-tag (e.g., as described in SEQ ID NO: 287 or 288), a universal tag (e.g., as described in SEQ ID NO: 289), VSV-G (e.g., as described in SEQ ID NO: 290), protein C (e.g., as described in SEQ ID NO: 291), and protein A. Histidine tags are preferred, especially the 6x His tag (SEQ ID NO: 169).Therefore, it is further envisioned that the antibody construct of the present invention consists of a polypeptide having an amino acid sequence selected from the group consisting of those described in SEQ ID NOs: 130 and 142 and linked to a 6x His tag at its C-terminus via a peptide bond. An embodiment of the antibody construct of the present invention has an amino acid sequence as described in SEQ ID NO: 131 or SEQ ID NO: 143.

[0232] T cells or T lymphocytes are a type of lymphocyte (itself a type of white blood cell) that plays a central role in cell-mediated immunity. There are several T cell subgroups, each with different functions. T cells can be distinguished from other lymphocytes (such as B cells and NK cells) by the presence of T cell receptors (TCRs) on the cell surface. TCRs are responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules and are composed of two different protein chains. In 95% of T cells, TCRs are composed of alpha (α) and beta (β) chains. When TCRs engage with antigenic peptides and MHC (peptide / MHC complexes), T lymphocytes are activated through a series of biochemical events mediated by related enzymes, co-receptors, specialized adapter molecules, and activated or released transcription factors.

[0233] The antibody construct of the present invention comprises a domain that binds to CD3 on the T cell surface. "CD3" (cluster of differentiation 3) is a T cell co-receptor consisting of four chains. In mammals, the CD3 protein complex contains CD3γ (gamma) chain, CD3δ (delta) chain and two CD3ε (ipsilon) chains. These four chains associate with the T cell receptor (TCR) and the so-called ζ (truncation tower) chain to form a "T cell receptor complex" and produce activation signals in T lymphocytes. CD3γ (gamma), CD3δ (delta) and CD3ε (ipsilon) chains are highly related cell surface proteins of the immunoglobulin superfamily, and each contains a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif necessary for the signal transduction ability of the TCR, referred to as an immunoreceptor tyrosine-based activation motif (ITAM). The CD3ε molecule is a polypeptide that is encoded by the CD3E gene on chromosome 11 in humans.

[0234] Redirected target cell lysis is typically achieved by recruiting T cells with antibody constructs that bind to CD3 on the T cells and to a target protein on the target cell, and typically involves cytolytic synapse formation and the delivery of perforins and granzymes. Engaged T cells are capable of continuous target cell lysis and are unaffected by immune escape mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation; see, e.g., WO 2007 / 042261.

[0235] Cytotoxicity mediated by the CLDN18.2 x CD3 antibody construct can be measured in various ways. See Example 7. "Half maximal effective concentration" (EC 50 ) is often used as a measure of the potency of a biologically active molecule, such as an antibody construct of the present invention. It can be expressed in molar units. In the present case of measuring cytotoxicity, EC 50 The value refers to the concentration of the antibody construct that induces a cytotoxic response (target cell lysis) midway between the baseline and the maximum value. The effector cells in the cytotoxicity assay can be, for example, stimulated enriched (human) CD8 positive T cells or unstimulated (human) peripheral blood mononuclear cells (PBMCs). When stimulated / enriched CD8+ T cells are used as effector cells, EC is generally expected to be higher than that of unstimulated PBMCs. 50 The value is lower. If the target cells are of macaque origin or express or transfected with macaque CLDN18.2, the effector cells should also be of macaque origin, such as a macaque T cell line, for example 4119LnPx. The target cells should express CLDN18.2 on the cell surface, such as human or macaque CLDN18.2. Preferably, the target cells should express at least one or more extracellular loops of CLDN18.2, such as CLDN18.2 loop 1 and / or loop 2, on the cell surface. The target cells can be cell lines (such as CHO) stably or transiently transfected with CLDN18.2 (e.g., human or macaque CLDN18.2). Alternatively, the target cells can be CLDN18.2-positive naturally expressing cell lines, such as the human gastric cancer cell lines SNU-601 or SNU-620, or SNU-16, NUGC, NUG-C4, GSU ​​or IM95. In general, ECs are expected to be more resilient when target cells expressing higher levels of CLDN18.2 on the cell surface are used compared to target cells with lower target expression rates. 50 The value is lower.

[0236] The effector cell to target cell (E:T) ratio in cytotoxicity assays is typically about 10:1, but can vary. The cytotoxic activity of the CLDN18.2 x CD3 antibody construct can be measured in a 51-chromium release assay (e.g., with an incubation time of about 18 hours) or in a FACS-based cytotoxicity assay (e.g., with an incubation time of about 48 hours). Modification of the incubation time (cytotoxic response) is also contemplated. Other methods for measuring cytotoxicity are well known and include MTT or MTS assays, ATP-based assays (including bioluminescence assays), sulforhodamine B (SRB) assays, WST assays, clonogenic assays, and ECIS techniques.

[0237] According to one embodiment, the cytotoxic activity mediated by the CLDN18.2 x CD3 antibody construct of the present invention is measured in a cell-based cytotoxicity assay. It can also be measured in a 51-chromium release assay. It is envisaged that the EC of the antibody construct of the present invention 50 The value is ≤300pM, ≤280pM, ≤260pM, ≤250pM, ≤240pM, ≤220pM, ≤200pM, ≤180pM, ≤160pM, ≤150pM, ≤140pM, ≤120pM, ≤100pM, ≤90pM, ≤80pM, ≤70pM, ≤60pM, ≤50pM, ≤40pM, ≤30pM, ≤20pM, ≤15pM, ≤10pM or ≤5pM.

[0238] The EC given above can be measured in different assays and under different conditions. 50 For example, when human PBMCs are used as effector cells and CLDN18.2 transfected cells such as CHO cells are used as target cells, it is envisaged that the EC of the CLDN18.2 x CD3 antibody construct 50 The EC values ​​of the CLDN18.2 x CD3 antibody constructs are ≤500 pM, ≤400 pM, ≤300 pM, ≤280 pM, ≤260 pM, ≤250 pM, ≤240 pM, ≤220 pM, ≤200 pM, ≤180 pM, ≤160 pM, ≤150 pM, ≤140 pM, ≤120 pM, ≤100 pM, ≤90 pM, ≤80 pM, ≤70 pM, ≤60 pM, ≤50 pM, ≤40 pM, ≤30 pM, ≤20 pM, ≤15 pM, ≤10 pM or ≤5 pM. When human PBMCs are used as effector cells and when the target cells are CLDN18.2-positive cell lines such as SNU-601 or SNU-620, it is envisioned that the EC values ​​of the CLDN18.2 x CD3 antibody constructs are ≤500 pM, ≤400 pM, ≤300 pM, ≤20 pM, ≤15 pM, ≤10 pM or ≤5 pM. 50 The value is ≤300pM, ≤280pM, ≤260pM, ≤250pM, ≤240pM, ≤220pM, ≤200pM, ≤180pM, ≤160pM, ≤150pM, ≤140pM, ≤120pM, ≤100pM, ≤90pM, ≤80pM, ≤70pM, ≤60pM, ≤50pM, ≤40pM, ≤30pM, ≤20pM, ≤15pM, ≤10pM or ≤5pM.

[0239] According to one embodiment, the CLDN18.2 x CD3 antibody construct of the present invention does not induce / mediate lysis of cells that do not express CLDN18.2 on their surface (CLDN18.2-negative cells), such as CHO cells, or does not substantially induce / mediate lysis. The terms "do not induce lysis," "do not substantially induce lysis," "do not mediate lysis," or "do not substantially mediate lysis" mean that the antibody construct of the present invention does not induce or mediate lysis of more than 30%, preferably no more than 20%, more preferably no more than 10%, and particularly preferably no more than 9%, 8%, 7%, 6%, or 5% of CLDN18.2-negative cells, whereby lysis of target cells expressing CLDN18.2 (e.g., cells transformed or transfected with CLDN18.2 or naturally expressing cell lines such as the human gastric cancer cell lines SNU-601 or SNU-620) is set to 100%. This generally applies to antibody constructs at concentrations up to 500 nM. Cell lysis measurements are conventional techniques. In addition, this specification provides specific instructions for measuring cell lysis.

[0240] The difference in cytotoxic activity between monomeric and dimeric isoforms of a single CLDN18.2 x CD3 antibody construct is referred to as the "potency gap." This potency gap can be calculated, for example, as the EC of the monomeric versus dimeric forms of the molecule. 50 In one method for determining this gap, an 18-hour 51-chromium release assay or a 48-hour FACS-based cytotoxicity assay was performed using purified antibody construct monomers and dimers as described below (Examples 7.1 and 7.2). Effector cells were stimulated enriched human CD8+ T cells or unstimulated human PBMCs. Target cells were huCLDN18.2-transfected CHO cells. The effector cell to target cell (E:T) ratio was 10:1. The potency gap of the CLDN18.2 x CD3 antibody constructs of the present invention is preferably ≤5, more preferably ≤4, even more preferably ≤3, even more preferably ≤2, and most preferably ≤1.

[0241] The first and / or second domains of the antibody constructs of the present invention preferably have cross-species specificity for members of the order Primates, such as macaques. Cross-species specific CD3 binding domains are described, for example, in WO 2008 / 119567. According to one embodiment, in addition to binding to human CD3, the second domain will also bind to CD3 from primates, including, but not limited to, New World primates (such as common marmosets (Callithrix jacchus), tamarins (Saguinus Oedipus), or squirrel monkeys (Saimiri sciureus), Old World primates (such as baboons and macaques), gibbons, orangutans, and non-human homininae. It is contemplated that the second domain that binds to human CD3 on the surface of T cells also binds to at least macaque CD3. A preferred macaque is the cynomolgus macaque (Macaca fascicularis). Macaca mulatta (rhesus monkey) is also contemplated. An antibody construct of the present invention comprises a first domain that binds to human CLDN18.2 on the surface of a target cell and a second domain that binds to human CD3 and at least macaque CD3 on the surface of a T cell.

[0242] In one embodiment, the antibody construct according to the invention has an affinity difference for binding to macaque CD3 and human CD3 [KD ma CD3:KD hu CD3] (as determined by, e.g., BiaCore or Scatchard analysis) of between 0.01 and 100, preferably between 0.1 and 10, more preferably between 0.2 and 5, more preferably between 0.3 and 4, even more preferably between 0.5 and 3 or between 0.5 and 2.5, and most preferably between 0.5 and 1. See Example 3.

[0243] The second domain of the antibody construct of the present invention binds to CD3. More preferably, it binds to CD3 on the surface of T cells. In addition, it is envisioned that the second domain binds to human CD3, preferably to human CD3 on the surface of T cells. It is also envisioned that the second domain binds to CD3ε. More preferably, it binds to human CD3ε, for example, to human CD3ε on the surface of T cells. A preferred amino acid sequence of the extracellular domain of human CD3ε is set forth in SEQ ID NO: 256.

[0244] In one embodiment of the present invention, the second domain of the antibody construct binds to human CD3ε (or human CD3ε on the surface of T cells) and to common marmoset (Callithrix jacchus) or squirrel monkey (Saimiri sciureus) CD3ε. It is also envisioned that the second domain binds to an extracellular epitope of CD3ε, preferably to an extracellular epitope of human CD3ε. It is also envisioned that the second domain binds to an extracellular epitope of the human and macaque CD3ε chain. A preferred epitope of CD3ε is contained within amino acid residues 1-27 of the extracellular domain of human CD3ε (see SEQ ID NO: 257). Even more specifically, the epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu. The common marmoset is a New World primate belonging to the family Callitrichidae, while the squirrel monkey is a New World primate belonging to the family Cebidae. Conjugates with such characteristics are described in detail in WO 2008 / 119567.

[0245] Antibodies or bispecific antibody constructs directed against (human) CD3 or specifically against CD3ε are known in the art, and their CDR, VH, and VL sequences can serve as the basis for the second binding domain of the antibody constructs of the present invention. For example, in 1979, Kung et al. reported the development of OKT3 (Ortho Kung T3), the first mAb to recognize CD3 on human T cells (particularly the epsilon chain of CD3). OKT3 (muromonab) is the first murine monoclonal antibody that can be used for human therapy. Newer anti-CD3 monoclonal antibodies include otelixizumab (TRX4), teplizumab (MGA031), foralumab, and visilizumab, all of which target the epsilon chain of CD3. Bispecific antibody constructs for (cancer) targets and CD3 are also being developed and (pre) clinically tested, and their CD3 binding domains (CDR, VH, VL) can be used as the basis of the second binding domain of the antibody construct of the present invention. Examples include but are not limited to Blinatumomab, Solitomab (MT110, AMG 110), Catumaxomab, Duvortuxizumab, Ertumaxomab, Mosunetuzumab, FBTA05 (Bi20, TPBs05), CEA-TCB (RG7802, RO6958688), AFM11, and MGD006 (S80880). Other examples of CD3 binding domains are disclosed in, for example, US 7,994,289 B2, US 7,728,114 B2, US 7,381,803 Bl, US 6,706,265 Bl.

[0246] For the antibody constructs of the present invention, it is envisaged that the second domain that binds to CD3 on the surface of T cells comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of:

[0247] (a) CDR-L1 as described in SEQ ID NO: 37, CDR-L2 as described in SEQ ID NO: 38, and CDR-L3 as described in SEQ ID NO: 39;

[0248] (b) CDR-L1 as described in SEQ ID NO:82, CDR-L2 as described in SEQ ID NO:83, and CDR-L3 as described in SEQ ID NO:84; and

[0249] (c) CDR-L1 as depicted in SEQ ID NO: 100, CDR-L2 as depicted in SEQ ID NO: 101, and CDR-L3 as depicted in SEQ ID NO: 102.

[0250] For the antibody constructs of the present invention, it is also envisioned that the second domain that binds to CD3 on the surface of T cells comprises a VH region comprising a CDR-H1, CDR-H2, and CDR-H3 selected from the group consisting of:

[0251] (a) CDR-H1 as described in SEQ ID NO:31, CDR-H2 as described in SEQ ID NO:32, and CDR-H3 as described in SEQ ID NO:33;

[0252] (b) CDR-H1 as described in SEQ ID NO:40, CDR-H2 as described in SEQ ID NO:41, and CDR-H3 as described in SEQ ID NO:42;

[0253] (c) CDR-H1 as described in SEQ ID NO:49, CDR-H2 as described in SEQ ID NO:50, and CDR-H3 as described in SEQ ID NO:51;

[0254] (d) CDR-H1 as described in SEQ ID NO:58, CDR-H2 as described in SEQ ID NO:59, and CDR-H3 as described in SEQ ID NO:60;

[0255] (e) CDR-H1 as described in SEQ ID NO:67, CDR-H2 as described in SEQ ID NO:68, and CDR-H3 as described in SEQ ID NO:69;

[0256] (f) CDR-H1 as described in SEQ ID NO:76, CDR-H2 as described in SEQ ID NO:77, and CDR-H3 as described in SEQ ID NO:78;

[0257] (g) CDR-H1 as described in SEQ ID NO:85, CDR-H2 as described in SEQ ID NO:86, and CDR-H3 as described in SEQ ID NO:87;

[0258] (h) CDR-H1 as described in SEQ ID NO:94, CDR-H2 as described in SEQ ID NO:95, and CDR-H3 as described in SEQ ID NO:96;

[0259] (i) CDR-H1 as described in SEQ ID NO: 103, CDR-H2 as described in SEQ ID NO: 104, and CDR-H3 as described in SEQ ID NO: 105; and

[0260] (j) CDR-H1 as depicted in SEQ ID NO:112, CDR-H2 as depicted in SEQ ID NO:113, and CDR-H3 as depicted in SEQ ID NO:114.

[0261] Furthermore, for the antibody constructs of the present invention, it is envisaged that the second domain that binds to CD3 comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group consisting of: and a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of:

[0262] (a) CDR-L1 as described in SEQ ID NO:28, CDR-L2 as described in SEQ ID NO:29, CDR-L3 as described in SEQ ID NO:30, CDR-H1 as described in SEQ ID NO:31, CDR-H2 as described in SEQ ID NO:32, and CDR-H3 as described in SEQ ID NO:33;

[0263] (b) CDR-L1 as described in SEQ ID NO:37, CDR-L2 as described in SEQ ID NO:38, CDR-L3 as described in SEQ ID NO:39, CDR-H1 as described in SEQ ID NO:40, CDR-H2 as described in SEQ ID NO:41, and CDR-H3 as described in SEQ ID NO:42;

[0264] (c) CDR-L1 as described in SEQ ID NO:46, CDR-L2 as described in SEQ ID NO:47, CDR-L3 as described in SEQ ID NO:48, CDR-H1 as described in SEQ ID NO:49, CDR-H2 as described in SEQ ID NO:50, and CDR-H3 as described in SEQ ID NO:51;

[0265] (d) CDR-L1 as described in SEQ ID NO:55, CDR-L2 as described in SEQ ID NO:56, CDR-L3 as described in SEQ ID NO:57, CDR-H1 as described in SEQ ID NO:58, CDR-H2 as described in SEQ ID NO:59, and CDR-H3 as described in SEQ ID NO:60;

[0266] (e) CDR-L1 as described in SEQ ID NO:64, CDR-L2 as described in SEQ ID NO:65, CDR-L3 as described in SEQ ID NO:66, CDR-H1 as described in SEQ ID NO:67, CDR-H2 as described in SEQ ID NO:68, and CDR-H3 as described in SEQ ID NO:69;

[0267] (f) CDR-L1 as described in SEQ ID NO:73, CDR-L2 as described in SEQ ID NO:74, CDR-L3 as described in SEQ ID NO:75, CDR-H1 as described in SEQ ID NO:76, CDR-H2 as described in SEQ ID NO:77, and CDR-H3 as described in SEQ ID NO:78;

[0268] (g) CDR-L1 as described in SEQ ID NO:82, CDR-L2 as described in SEQ ID NO:83, CDR-L3 as described in SEQ ID NO:84, CDR-H1 as described in SEQ ID NO:85, CDR-H2 as described in SEQ ID NO:86, and CDR-H3 as described in SEQ ID NO:87;

[0269] (h) CDR-L1 as described in SEQ ID NO:91, CDR-L2 as described in SEQ ID NO:92, CDR-L3 as described in SEQ ID NO:93, CDR-H1 as described in SEQ ID NO:94, CDR-H2 as described in SEQ ID NO:95, and CDR-H3 as described in SEQ ID NO:96;

[0270] (i) CDR-L1 as described in SEQ ID NO: 100, CDR-L2 as described in SEQ ID NO: 101, CDR-L3 as described in SEQ ID NO: 102, CDR-H1 as described in SEQ ID NO: 103, CDR-H2 as described in SEQ ID NO: 104, and CDR-H3 as described in SEQ ID NO: 105; and

[0271] (j) CDR-L1 as described in SEQ ID NO: 109, CDR-L2 as described in SEQ ID NO: 110, CDR-L3 as described in SEQ ID NO: 111, CDR-H1 as described in SEQ ID NO: 112, CDR-H2 as described in SEQ ID NO: 113, and CDR-H3 as described in SEQ ID NO: 114.

[0272] For the antibody constructs of the present invention, it is envisioned that the second domain that binds to CD3 on the surface of T cells comprises a VL region selected from the group consisting of a VL region as described in any one of SEQ ID NO: 44, SEQ ID NO: 89, SEQ ID NO: 107, SEQ ID NO: 233, SEQ ID NO: 234 and SEQ ID NO: 235.

[0273] It is also envisioned that the second domain that binds to CD3 on the surface of T cells comprises a VH region selected from the group consisting of a VH region as described in any one of SEQ ID NO: 34, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 61, SEQ ID NO: 70, SEQ ID NO: 79, SEQ ID NO: 88, SEQ ID NO: 97, SEQ ID NO: 106, SEQ ID NO: 115, SEQ ID NO: 118, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, and SEQ ID NO: 245.

[0274] More preferably, the antibody construct of the invention is characterized in that the second domain that binds to CD3 on the surface of T cells comprises a VL region and a VH region selected from the group consisting of:

[0275] (a) a VL region as described in SEQ ID NO: 35 or 233 and a VH region as described in SEQ ID NO: 34 or 236;

[0276] (b) a VL region as described in SEQ ID NO: 44 or 233 and a VH region as described in SEQ ID NO: 43 or 237;

[0277] (c) a VL region as described in SEQ ID NO: 53 or 233 and a VH region as described in SEQ ID NO: 52 or 238;

[0278] (d) a VL region as described in SEQ ID NO: 62 or 233 and a VH region as described in SEQ ID NO: 61 or 239;

[0279] (e) a VL region as described in SEQ ID NO: 71 or 234 and a VH region as described in SEQ ID NO: 70 or 240;

[0280] (f) a VL region as described in SEQ ID NO: 80 or 233 and a VH region as described in SEQ ID NO: 79 or 241;

[0281] (g) a VL region as described in SEQ ID NO: 89 or 234 and a VH region as described in SEQ ID NO: 88 or 242;

[0282] (h) a VL region as described in SEQ ID NO: 98 or 233 and a VH region as described in SEQ ID NO: 97 or 243;

[0283] (i) a VL region as described in SEQ ID NO: 107 or 235 and a VH region as described in SEQ ID NO: 106 or 244;

[0284] (j) a VL region as described in SEQ ID NO: 116 or 235 and a VH region as described in SEQ ID NO: 115 or 245; and

[0285] (k) the VL region as depicted in SEQ ID NO: 119 and the VH region as depicted in SEQ ID NO: 118.

[0286] Preferred embodiments of the antibody constructs of the invention described above are characterized in that the second domain that binds to CD3 on the surface of T cells comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 36, 45, 54, 63, 72, 81, 90, 99, 108, 117, 120, 246, 247, 248, 249, 250, 251, 252, 253, 254 and 255.

[0287] Amino acid sequence modifications of the antibody constructs described herein are also contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody construct. Amino acid sequence variants of the antibody construct are prepared by peptide synthesis or by introducing appropriate nucleotide changes into the nucleic acid molecule encoding the antibody construct. All of the amino acid sequence modifications described below should result in antibody constructs that retain the desired biological activity of the unmodified parent molecule (e.g., binding to CLDN18.2 and CD3, inducing cytotoxicity against CLDN18.2-positive target cells).

[0288] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art-recognized definition, such as 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 (Ile 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 as desired. There are essentially four different classes of amino acids, defined by their side chains:

[0289] (1) Non-polar and neutral (uncharged): Ala, Gly, Ile, Leu, Met, Phe, Pro, Val

[0290] (2) Polar and neutral (uncharged): Asn, Cys (only slightly polar), Gln, Ser, Thr, Trp (only slightly polar), Tyr

[0291] (3) Acidic and polar (negatively charged): Asp and Glu

[0292] (4) Basicity and polarity (positive charge): Arg, His, Lys

[0293] Hydrophobic amino acids can be divided according to whether they have aliphatic or aromatic side chains. Phe and Trp (very hydrophobic), Tyr and His (less hydrophobic) are classified as aromatic amino acids. Strictly speaking, aliphatic means that the side chain contains only hydrogen and carbon atoms. By this strict definition, the amino acids with aliphatic side chains are alanine, isoleucine, leucine (also norleucine), proline, and valine. Alanine's side chain is very short, meaning it is not particularly hydrophobic, and proline has an unusual geometry that gives it a special role in proteins. It is often convenient to consider methionine to be in the same category as isoleucine, leucine, and valine, even though it also contains a sulfur atom. The unifying theme is that these amino acids contain primarily non-reactive and flexible side chains. The amino acids alanine, cysteine, glycine, proline, serine, and threonine are usually grouped together because they are all very small in size. Gly and Pro may affect chain orientation.

[0294] Amino acid modifications include, for example, deletions, insertions, and / or substitutions of residues within the amino acid sequence of the antibody construct. Any combination of deletions, insertions, and / or substitutions is performed to obtain the final antibody construct, provided that the final construct possesses the desired characteristics, for example, the biological activity of the unmodified parent molecule (e.g., binding to CLDN18.2 and CD3, inducing cytotoxicity against CLDN18.2-positive target cells). Amino acid changes can also alter post-translational processes of the antibody construct, such as changing the number or position of glycosylation sites.

[0295] For example, 1, 2, 3, 4, 5 or 6 amino acids may be inserted, deleted and / or substituted in each CDR (depending, of course, on their respective lengths), while 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 inserted, deleted and / or substituted in each FR. Amino acid sequence insertions also include N-terminal and / or C-terminal amino acid additions ranging in length from, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to polypeptides containing more than 10, for example, one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertion variants of the antibody constructs of the present invention include fusion of a polypeptide that increases or prolongs the serum half-life of the antibody construct to the N-terminus or C-terminus of the antibody construct. It is also conceivable that such insertion occurs within the antibody construct, for example between the first and second domains.

[0296] The most interesting sites for amino acid modification (particularly for amino acid substitution) include hypervariable regions, particularly each CDR of heavy chain and / or light chain, but FR changes in heavy chain and / or light chain are also contemplated. Substitutions can be conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids can be substituted in CDR, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be substituted in framework region (FR), depending on the length of CDR or FR, respectively. For example, if the CDR sequence comprises 6 amino acids, it is envisioned that 1, 2 or 3 of these amino acids are substituted. Similarly, if a CDR sequence comprises 15 amino acids, it is envisaged that 1, 2, 3, 4, 5 or 6 of these amino acids are substituted.

[0297] A useful method for identifying certain residues or regions within an antibody construct as preferred locations for mutagenesis is called "alanine scanning mutagenesis" and is described, for example, in Cunningham BC and Wells JA (Science [Science]. 1989 Jun 2; 244(4908): 1081-5). Here, residues or groups of residues within an antibody construct (e.g., charged residues such as Arg, His, Lys, Asp, and Glu) are identified and replaced with neutral or non-polar amino acids (most preferably alanine or polyalanine) to affect the interaction of each amino acid with the target protein epitope. Alanine scanning is a technique for determining the contribution of a particular residue to the stability or function of a given protein. Alanine is used because it has a non-bulky, chemically inert methyl functional group, but still mimics the secondary structure preferences of many other amino acids. In cases where the size of the mutated residue is conservative, bulky amino acids (such as valine or leucine) can sometimes be used. This technique can also be used to determine whether the side chain of a particular residue plays an important role in biological activity. Alanine scanning is typically performed by site-directed mutagenesis or randomly by creating PCR libraries. Furthermore, computational methods have been developed to estimate thermodynamic parameters based on theoretical alanine substitutions. The data can be validated using IR and NMR spectroscopy, mathematical methods, bioassays, and more.

[0298] Then, by introducing additional or other variants at or for the substitution site, those amino acid positions that exhibit functional sensitivity to the substitution (as determined, for example, by alanine scanning) are precisely targeted. Thus, while the site or region for introducing amino acid sequence changes is predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze or optimize the performance of mutations at a given site, alanine scanning or random mutagenesis can be performed at the target codon or region, and the expressed antibody construct variants are screened for the optimal combination of desired activity. Techniques for performing substitution mutations at predetermined sites in DNA with a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. For example, mutant screening is performed using assays for antigen (e.g., CLDN18.2 or CD3) binding activity and / or cytotoxic activity.

[0299] In general, if an amino acid is substituted in one or more or all CDRs of the heavy and / or light chain, it is envisaged that the then obtained "substituted" sequence is at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, and particularly preferably 90% or 95% identical / homologous to the "original" or "parent" CDR sequence. This means that the degree of identity / homology between the original sequence and the substituted sequence depends on the length of the CDR. For example, a CDR having a total of 5 amino acids and comprising one amino acid substitution is 80% identical to the "original" or "parent" CDR sequence, while a CDR having a total of 10 amino acids and comprising one amino acid substitution is 90% identical to the "original" or "parent" CDR sequence. Thus, the substituted CDRs of the antibody constructs of the present invention may have different degrees of identity with their original sequences, for example, CDRL1 may have 80% homology, while CDRL3 may have 90% homology. Similar considerations apply to the framework regions and the entire VH and VL regions.

[0300] A "variant CDR" is a CDR that has a certain sequence homology, similarity or identity with a parent CDR of the present invention, and shares a biological function with the parent CDR, including but not limited to 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% of the specificity and / or activity of the parent CDR. Generally, the amino acid homology, similarity or identity between each variant CDR is at least 60% of the parent sequence described herein, and more typically has at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, and most preferably 95%, 96%, 97%, 98%, 99%, and almost 100% increasing homology, similarity or identity. The same applies to "variant VH" and "variant VL". According to one embodiment, the sequence changes within "variant VH" and / or "variant VL" do not extend to the CDRs. Therefore, the present invention relates to antibody constructs as defined herein, which antibody constructs comprise VH and VL sequences that have certain sequence homology (see above) with specific sequences defined herein ("parent" VH and VL), wherein these CDR sequences are 100% identical to the specific CDR sequences defined herein ("parent" CDRs).

[0301] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "Exemplary Substitutions" listed in Table 1 below) is contemplated as long as the antibody construct retains its ability to bind to CLDN18.2 via the first domain and to CD3 or CD3ε via the second domain, and / or as long as its CDR, FR, VH and / or VL sequence has a degree of identity of at least 60% or 65%, more preferably at least 70% or 75%, even more preferably at least 80% or 85%, and particularly preferably at least 90% or 95% to the original or parent sequence.

[0302] Conservative substitutions (also referred to as conservative mutations or conservative substitutions) are amino acid substitutions that change a given amino acid into a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, size). Conservative substitutions in proteins generally have less impact on protein function than non-conservative substitutions. Conservative substitutions are shown in Table 1. Exemplary conservative substitutions are shown as "Exemplary Substitutions." If such substitutions result in changes in biological activity, more substantial changes can be introduced as further described herein with reference to the amino acid classes, and the desired characteristics of the product can be screened.

[0303] Table 1: Amino acid substitutions (aa = amino acid)

[0304]

[0305]

[0306] Substantial modification of the biological properties of the antibody constructs of the present invention is accomplished by selecting substitutions that differ significantly in maintaining the following effects: (a) the structure of the polypeptide backbone in the region of the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) a substantial portion of the side chain. Non-conservative substitutions generally entail exchanging a member of one of the above-defined amino acid classes (e.g., polar, neutral, acidic, basic, aliphatic, aromatic, small, ...) for another class. Any cysteine ​​residue that is not involved in maintaining the proper conformation of the antibody construct can generally be substituted with a serine to improve the oxidative stability of the antibody construct.

[0307] Sequence identity, homology and / or similarity of amino acid sequences are determined using standard techniques known in the art, preferably using default settings or by inspection, including but not limited to the local sequence identity algorithm of Smith and Waterman (1981, Adv. Appl. Math. 2:482), the sequence identity alignment algorithm of Needleman and Wunsch (J Mol Biol. 1970 Mar;48(3):443-53), the similarity search method of Pearson and Lipman (Proc Natl Acad Sci USA. 1988 Apr;85(8):2444-8), computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Avenue, Madison, WI). Group, 575 Science Drive, Madison, Wis.), a best fit sequence program described by Devereux et al. (Nucleic Acids Res. 1984 Jan 11; 12(1 Pt 1): 387-95). It is contemplated that percent identity is calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and ligation penalty of 30. See also "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss Company.

[0308] An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also draw a dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J Mol Evol. 1987; 25(4): 351-60); this method is similar to the method described by Higgins and Sharp (Comput Appl Biosci. 1989 Apr; 5(2): 151-3). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0309] Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al. (J Mol Biol. 1990 Oct 5;215(3):403-10); Altschul et al. (Nucleic Acids Res. 1997 Sep 1;25(17):3389-402); and Karlin and Altschul (Proc Natl Acad Sci USA. 1993 Jun 15;90(12):5873-7). A particularly useful BLAST program is the WU-Blast-2 program available from Altschul et al. (Methods Enzymol. 1996;266:460-80). WU-Blast-2 uses a number of search parameters, most of which are set to default values. Adjustable parameters are set to the following values: overlap span = 1, overlap fraction = 0.125, word threshold (T) = II. The HSP S and HSP S2 parameters are dynamic values ​​and are established by the program itself based on the composition of the specific sequence and the composition of the specific database against which the sequence of interest is being searched; however, these values ​​can be adjusted to increase sensitivity.

[0310] Another useful algorithm is Gapped BLAST, as reported by Altschul et al. (Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Gapped BLAST uses BLOSUM-62 instead of scoring; the threshold T parameter is set to 9; a double-click method is used to trigger non-gap extensions, with a cost of 10+k for a gap length of k; Xu is set to 16, and Xg is set to 40 (for the database search phase) and 67 (for the output phase of the algorithm). Gapped alignments are triggered by a score corresponding to approximately 22 bits.

[0311] In accordance with this, the term "percentage (%) of nucleic acid sequence identity / homology / similarity" with respect to the nucleic acid sequences encoding the antibody constructs identified herein is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody construct. One method of aligning two sequences and thereby determining their homology uses the BLASTN module of WU-Blast2 (set to default parameters), with overlap span and overlap fraction set to 1 and 0.125, respectively. Generally speaking, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding each variant CDR and the nucleotide sequence described herein is at least 60%, and more typically has 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 100% increasing homology, similarity or identity. Again, this also applies to the nucleic acid sequences encoding "variant VH" and / or "variant VL".

[0312] In one embodiment, the percent identity to the human germline of the antibody constructs according to the invention or the human germline of the first and second domains (binding domains) of these antibody constructs is ≥70% or ≥75%, more preferably ≥80% or ≥85%, even more preferably ≥90%, and most preferably ≥91%, ≥92%, ≥93%, ≥94%, ≥95% or even ≥96%. Identity to human antibody germline gene products is considered an important feature to reduce the risk of therapeutic proteins eliciting an immune response against the drug in patients during treatment. Hwang WY and Foote J. (Methods. 2005 May; 36(1): 3-10) demonstrated that the reduction of the non-human portion of a drug-antibody construct resulted in a reduced risk of inducing anti-drug antibodies in patients during treatment. By comparing numerous clinically evaluated antibody drugs and corresponding immunogenicity data, the following trend is shown: humanization of the variable regions of antibodies / antibody constructs results in lower protein immunogenicity (average 5.1% of patients) than antibodies / antibody constructs carrying unchanged non-human variable regions (average 23.59% of patients). Therefore, protein therapeutics based on variable regions and in the form of antibody constructs need to have a high degree of identity with human sequences. For the purpose of determining germline identity, the V region of the VL can be aligned with the amino acid sequences of human germline V segments and J segments (http: / / www2.mrc-lmb.cam.ac.uk / vbase / ) using Vector NTI software and the amino acid sequence of the VL can be calculated by dividing the identical amino acid residues by the total number of amino acid residues in the VL (in percentage). The same can be done for the VH segment (http: / / www2.mrc-lmb.cam.ac.uk / vbase / ), except that the VH CDR3 can be excluded due to the high diversity of the VH CDR3 and the lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase sequence identity to the human antibody germline genes.

[0313] In another embodiment, the antibody constructs of the present invention exhibit high monomer yields under standard research scale conditions, such as in a standard two-step purification process. It is envisioned that the monomer yield of the antibody constructs according to the present invention is ≥0.25 mg / L supernatant (SN), preferably ≥0.5 mg / L SN, more preferably ≥1 mg / L SN, even more preferably ≥2 mg / L SN, and most preferably ≥3 mg / L SN. The yield of the antibody construct designated "CL-1 x I2C-6His" was shown to be 4.1 mg / L supernatant, and the yield of the antibody construct designated "CL-1 x I2C-scFc" was shown to be 36.5 mg / L supernatant.

[0314] Similarly, the yield of the dimeric antibody construct isotype of the antibody construct can be determined, and from this the monomer percentage (i.e., monomer: (monomer + dimer)). The productivity of monomeric and dimeric antibody constructs and the calculated monomer percentage can be obtained, for example, in an SEC purification step of culture supernatants from standardized research-scale production in roller bottles. According to one embodiment, the monomer percentage of the antibody construct of the present invention is ≥80%, more preferably ≥85%, even more preferably ≥90%, and most preferably ≥95%.

[0315] According to one embodiment, the plasma stability of the antibody construct of the present invention (with an EC of plasma) 50 EC without plasma 50 The plasma stability of the antibody constructs can be tested by incubating the purified constructs in human plasma at a concentration of 2-20 μg / ml for 24 to 96 hours at 37° C., followed by EC in an 18 h 51-chromium release or 48 h FACS cytotoxicity assay (e.g., as described in Examples 7.1 and 7.2). 50 Determine. The effector cells in the cytotoxicity assay can be stimulated enriched human CD8 positive T cells (preferred) or unstimulated human PBMCs. The target cells can be, for example, CHO cells transfected with human CLDN18.2. The ratio of effector cells to target cells (E:T) can be 10:1. The starting concentration of the antibody construct in the cytotoxicity assay can be 0.01-0.1 μg / ml. The human plasma pool for this purpose is derived from the blood of healthy donors collected by EDTA-coated syringes. The cellular components are removed by centrifugation, and the upper plasma phase is collected and subsequently pooled. As a control, the unincubated antibody construct is diluted immediately in an appropriate culture medium such as RPMI-1640 before the cytotoxicity assay. The plasma stability is calculated as EC 50 (after plasma incubation) with EC 50 Ratio of (control / no incubation).

[0316] In addition, it is envisioned that the monomer and dimer conversion of the antibody construct of the present invention is lower. Conversion can be measured under different conditions and analyzed by high performance size exclusion chromatography. Referring to Example 8. For example, the incubation of the monomer isotype of the antibody construct can be carried out in an incubator in a universal formulation buffer and with a concentration of, for example, 100 μg / ml or 250 μg / ml at 37 ° C for 7 days, followed by high performance SEC to determine the percentage of the initial monomer antibody construct converted into a dimeric antibody construct. Under these conditions, it is envisioned that the antibody construct of the present invention shows a dimer percentage of ≤8%, preferably ≤6%, more preferably ≤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%.

[0317] Likewise, it is envisioned that the antibody constructs of the present invention present very low dimer conversion after several freeze / thaw cycles. For example, it is 250 μg / ml for the antibody construct monomer to be adjusted to a concentration of, for example, 250 μg / ml in a general formulation buffer, and three freeze / thaw cycles are performed (freezing 30 min at -80°C, thawing 30 min at room temperature thereafter), followed by high performance SEC to determine the percentage of the initial monomer antibody construct that has been converted into a dimeric antibody construct. It is envisioned that, for example, after three freeze / thaw cycles, the dimer percentage of the antibody construct is ≤ 8%, preferably ≤ 6%, more preferably ≤ 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%.

[0318] According to one embodiment, the antibody constructs of the present invention show a favorable thermal stability with an aggregation temperature of ≥45°C or ≥46°C, more preferably ≥47°C or ≥48°C, even more preferably ≥49°C or ≥50°C, and most preferably ≥51°C. The thermal stability parameter can be determined as follows based on the antibody aggregation temperature: an antibody solution with a concentration of 250 μg / ml is transferred to a disposable cuvette and placed in a dynamic light scattering (DLS) device. The sample is heated from 40°C to 70°C at a heating rate of 0.5°C / min, and the measured radius is constantly acquired. The aggregation temperature of the antibody is calculated using the increase in radius indicating the melting of the protein and aggregates. See Example 9.

[0319] Alternatively, the temperature melting curve can be determined by differential scanning calorimetry (DSC) to determine the intrinsic biophysical protein stability of the antibody construct. These experiments can be performed using a MicroCal LLC VP-DSC device. The energy uptake of a sample containing the antibody construct compared to a sample containing only formulation buffer is recorded from 20°C to 90°C. For example, the antibody construct is adjusted to a final concentration of 250 μg / ml in SEC running buffer. In order to record the corresponding melting curve, the entire sample temperature is gradually increased. The energy absorption of the sample and formulation buffer reference is recorded at each temperature. The energy absorption Cp (kcal / mole / °C) of the sample minus the difference in the reference is plotted against the corresponding temperature. The melting temperature is defined as the temperature at the first maximum energy absorption.

[0320] It is also envisaged that the antibody constructs of the present invention have a turbidity of ≤0.2 or ≤0.15, preferably ≤0.10 or ≤0.08, more preferably ≤0.06 or ≤0.05, and most preferably ≤0.04 or ≤0.03. Turbidity can be measured by OD340 at an antibody construct concentration of 2.5 mg / ml and incubated at 5°C for 16 h. See Example 10.

[0321] The change in the efficacy of the target x CD3 antibody construct can be measured as a function of pre-incubation on target cells in the absence of T cells. If the antibody construct is internalized, it is expected that it will undergo lysosomal degradation. Therefore, the effective concentration is expected to decrease over time, and therefore the apparent efficacy should also decrease. This effect has been observed for some targets, for which this is a known phenomenon. It is envisaged that the antibody construct of the present invention is not internalized by the target cells or does not undergo significant target cell internalization. The internalization rate can be determined, for example, as follows: T cells are counted and diluted to 1x10 in assay medium. 5 The target positive target cells are counted and plated, for example, at 2500 cells / well (cpw). The antibody construct is serially diluted, for example, at a starting concentration of 100 nM at 1:2. The antibody construct is added to the culture assay plate to allow incubation for 0 hours, 1 hour, or 2 hours before adding the T cells. The T cells are then plated at 25000 cpw (E:T=10:1) and the assay is incubated at 37°C for 48 hours. For example, using Preferably, after 2 hours of (pre)incubation of the antibody construct with the target cells, the internalization rate (e.g., measured as a reduction in cytotoxicity) is ≤ 20%, more preferably ≤ 15%, even more preferably ≤ 10%, and most preferably ≤ 5%.

[0322] In addition, for the antibody constructs of the present invention, it is envisioned that the soluble target that comes off or does not significantly impair its efficacy or biological activity. This can be measured, for example, in a cytotoxicity assay, where the soluble target is added to the assay at increasing concentrations (e.g., 0nM-0.3nM-0.7nM-1nM-3nM-7nM-12nM). An exemplary E:T value is 10: 1. In the presence of a soluble target, the EC50 value of the antibody construct tested should not significantly increase.

[0323] In another embodiment, antibody construct according to the present invention is stable under acidic pH. The more tolerant the performance of antibody construct under non-physiological pH (such as pH 5.5) (pH required for operation such as cation exchange chromatography), the higher the recovery of the antibody construct eluted from the ion exchange column relative to the total amount of loaded protein. The recovery of antibody construct from the ion (such as cation) exchange column of pH 5.5 is preferably ≥30%, more preferably ≥40%, more preferably ≥50%, even more preferably ≥60%, even more preferably ≥70%, even more preferably ≥80% and most preferably ≥95%. Percentage represents the area under the curve (=AUC) of the main peak. Referring to Example 11.

[0324] In addition, it is envisioned that the antibody constructs of the present invention exhibit therapeutic efficacy, which is manifested as anti-tumor activity or tumor growth inhibition. This can be evaluated, for example, in the studies disclosed in Example 13 or 14. In one embodiment, the tumor growth inhibition T / C [%] of the antibody constructs of the present invention is ≤70, ≤60, ≤50, ≤40, ≤30, ≤20, ≤10, ≤5, ≤4, ≤3 or ≤2. It is also envisioned that certain parameters of these studies (such as the number of injected tumor cells, injection site, number of transplanted human T cells, amount of antibody construct to be administered, and timeline) can be modified or adjusted while still obtaining meaningful and reproducible results.

[0325] The present invention further provides a polynucleotide / nucleic acid molecule that encodes an antibody construct of the present invention. Nucleic acid molecules are biopolymers made up of nucleotides. Polynucleotides are biopolymers made up of 13 or more nucleotide monomers covalently bonded in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotide / nucleic acid molecules with different biological functions. Nucleotides are organic molecules that act as monomers or subunits of nucleic acid molecules such as DNA or RNA. The nucleic acid molecules or polynucleotides of the present invention can be double-stranded or single-stranded, linear or circular. It is envisioned that the nucleic acid molecules or polynucleotides are contained in a vector. In addition, it is envisioned that such a vector is contained in a host cell. The host cell, for example, can express the antibody construct after transformation or transfection with the vector or polynucleotide / nucleic acid molecule of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0326] The genetic code is a set of rules for translating the information encoded within genetic material (nucleic acids) into proteins. Biological decoding in living cells is accomplished by ribosomes, which link amino acids in the order specified by mRNA, using tRNA molecules to carry the amino acids and read the mRNA three nucleotides at a time. This code defines how the sequence of these nucleotide triplets (called codons) specifies which amino acid will be added next during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies a single amino acid. Because the vast majority of genes are encoded using the exact same code, this particular code is often referred to as the canonical or standard genetic code.

[0327] Codon degeneracy is the redundancy of the genetic code, manifested as a variety of three-base-pair codon combinations that specify amino acids. Degeneracy occurs because there are more codons than there are coded amino acids. The codons encoding an amino acid can differ in any of their three positions; however, this difference is usually in the second or third position. For example, the codons GAA and GAG both specify glutamate and exhibit redundancy; however, neither specifies any other amino acid and therefore exhibits no ambiguity. The genetic codes of different organisms may favor the use of one of several codons encoding the same amino acid over others—that is, one codon will be found more frequently than would be expected by chance. For example, leucine is specified by six different codons, some of which are rarely used. Codon usage tables detailing the frequency of codon usage in the genomes of most organisms are available. Recombinant gene technology generally utilizes this effect by implementing a technique known as codon optimization, in which those codons are used to design polynucleotides that are preferred by respective host cells (e.g., cells of human hamster origin, Escherichia coli cells, or Saccharomyces cerevisiae cells), for example to increase protein expression. Therefore, it is envisioned that the polynucleotides / nucleic acid molecules of the present disclosure are codon optimized. However, any codon encoding a desired amino acid can be used to design the polynucleotides / nucleic acid molecules encoding the antibody constructs of the present invention.

[0328] According to one embodiment, the polynucleotide / nucleic acid molecule of the present invention encoding the antibody construct of the present invention is in the form of a single molecule or in the form of two or more separate molecules. If the antibody construct of the present invention is a single-chain antibody construct, the polynucleotide / nucleic acid molecule encoding such a construct will most likely also be in the form of a single molecule. However, it is also envisioned that the different components of the antibody construct (such as different domains, such as domains that bind to CLDN18.2, domains that bind to CD3, and / or additional domains such as antibody constant domains) are located on separate polypeptide chains, in which case the polynucleotide / nucleic acid molecule is most likely in the form of two or more separate molecules.

[0329] This also applies to the vector comprising the polynucleotide / nucleic acid molecule of the present invention.If antibody construct of the present invention is a single-chain antibody construct, then a vector can comprise the polynucleotide of encoding antibody construct in a single position (as a single open reading frame, ORF).A vector can also comprise two or more polynucleotide / nucleic acid molecules in a separated position (with independent ORF), the different components of each of which encodes antibody construct of the present invention.It is envisioned that the vector comprising the polynucleotide / nucleic acid molecule of the present invention is in the form of a single vector or in the form of two or more separated vectors. In one embodiment, and for the purpose of expressing antibody construct in host cell, host cell of the present invention should comprise the polynucleotide / nucleic acid molecule of encoding antibody construct or the vector comprising this polynucleotide / nucleic acid molecule is whole, this means that all components of antibody construct-no matter being encoded as a single molecule or encoding at independent molecule / position-will be assembled after translation and form together biologically active antibody construct of the present invention.

[0330] The present invention also provides vectors comprising the polynucleotide / nucleic acid molecules of the present invention. Vectors are nucleic acid molecules used as the carrier for transferring (external) genetic material into cells, usually for the purpose of replication and / or expression. The term "vector" encompasses but is not limited to plasmids, viruses, cosmids and artificial chromosomes. Some vectors are specifically designed for cloning (cloning vectors), and other vectors are designed for protein expression (expression vectors). So-called transcription vectors are mainly used to amplify their inserts. DNA manipulation is usually carried out on an E. coli vector containing the elements required for maintaining it in E. coli. However, vectors can also have elements that allow them to be maintained in another organism such as yeast, plant or mammalian cells, and these vectors are referred to as shuttle vectors. Inserting a vector into a target or host cell is usually referred to as transformation (for bacterial cells) and transfection (for eukaryotic cells), and the insertion of a viral vector is usually referred to as transduction.

[0331] Generally speaking, engineered vectors contain an origin of replication, a multiple cloning site, and a selective marker. The vector itself is typically a nucleotide sequence (usually a DNA sequence) comprising an insert (transgene) and a larger sequence that serves as a vector "backbone." While the genetic code determines the polypeptide sequence of a given coding region, other genomic regions may affect the time and place of production of these polypeptides. Therefore, modern vectors can include additional features in addition to the transgenic insert and backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors known as expression vectors (expression constructs) are particularly useful for expressing transgenes in target cells and typically have control sequences.

[0332] The term "control sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, Kozak sequences, and enhancers.

[0333] When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the presequence or secretory leader is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned so as to facilitate translation, the ribosome binding side is operably linked to the coding sequence. Generally speaking, "operably linked" means that the nucleotide sequences being linked are continuous, and in the case of a secretory leader, are continuous and in the reading phase. However, enhancers do not have to be continuous. Connection is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or joints are used according to conventional practice.

[0334] "Transfection" is the process of intentionally introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is generally used to describe the transfer of virally mediated nucleic acid molecules or polynucleotides. Transfecting animal cells typically involves opening transient pores or "holes" in the cell membrane to allow the uptake of substances. Transfection can be performed using bioparticles (such as viral transfection, also referred to as viral transduction), chemical-based methods (such as using calcium phosphate, lipofection, Fugene, cationic polymers, nanoparticles), or physical treatments (such as electroporation, microinjection, gene guns, cell squeezing, magnetic transfection, hydrostatic pressure, impalefection, ultrasound, optical transfection, heat shock).

[0335] 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 therefore a genetic alteration of a bacterial or non-animal eukaryotic cell resulting from the direct uptake of foreign genetic material (nucleic acid molecules) from its surroundings through one or more cell membranes and subsequent incorporation. Transformation can be achieved by artificial means. For transformation to occur, the cell or bacterium must be in a competent state, which may occur as a time-limited response to environmental conditions such as starvation and cell density, and can also be artificially induced.

[0336] Furthermore, the present invention provides a host cell transformed or transfected with the polynucleotide / nucleic acid molecule of the present invention or with the vector of the present invention.

[0337] As used herein, the term "host cell" or "recipient cell" is intended to include any individual cell or cell culture that may be or has been a receptor for a polynucleotide encoding an antibody construct of the present invention and / or the antibody construct itself. Corresponding substances are introduced into the cell (see above) by modes such as transformation and transfection. The term "host cell" is also intended to include the offspring or potential offspring of a unicellular cell. Because some changes may occur in subsequent generations due to natural, unexpected or intentional mutations or due to environmental influences, such offspring may not in fact be identical (in morphology or genome or entire DNA complement) with the parental cell, but is still included in the scope of the term used herein. Suitable host cells include prokaryotic or eukaryotic cells, and also include but are not limited to bacteria (such as Escherichia coli), yeast cells, fungal cells, plant cells and animal cells, such as insect cells and mammalian cells, for example hamsters, rodents, rats, macaques or people.

[0338] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for the antibody constructs of the invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyce 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 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium; and Aspergillus hosts, such as A. nidulans and A. niger.

[0339] Suitable host cells for expressing glycosylated antibody constructs are derived from multicellular organisms. Examples of invertebrate cells include plants and insect cells. Many baculovirus strains and variants from hosts such as Spodopterafrugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (fruit flies) and Bombyx mori (Bombyx mori) and corresponding permissive insect host cells have been identified. Multiple virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and according to the present invention, such viruses can be used as viruses herein, particularly for transfecting Spodoptera frugiperda cells.

[0340] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis thaliana, and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell cultures are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen et al. (1992) Bio / Technology 10:790-794; Artsaenko et al. (1995) The Plant J 8:745-750; and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0341] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (cell culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed with SV40 (e.g., COS-7, ATCC CRL1651); human embryonic kidney lines (e.g., 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (e.g., CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (e.g., TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (e.g., CVI ATCC CCL 70); African green monkey kidney cells (e.g., VERO-76, ATCC CRL1587); human cervical cancer cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); Buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL75); human hepatocytes (e.g., Hep G2, 1413 8065); mouse mammary tumor (e.g., MMT 060562, ATCC CCL-51); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and human hepatoma cell lines (e.g., Hep G2).

[0342] In another 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 allowing expression of the antibody construct of the present invention and recovering the produced antibody construct from the culture.

[0343] As used herein, the term "culture" refers to the maintenance, differentiation, growth, proliferation and / or propagation of cells in vitro in a culture medium under suitable conditions. Cells are grown and maintained in a cell growth medium under a suitable temperature and gas mixture. For each cell type, culture conditions vary greatly. Typical growth conditions are a temperature of approximately 37°C, a CO concentration of approximately 5% and a humidity of approximately 95%. The formula of the growth medium can, for example, vary in the presence of pH, carbon source (such as glucose) concentration, growth factor properties and concentration, and other nutrients (such as amino acids or vitamins). The growth factor used to supplement the culture medium is typically derived from the serum of animal blood, such as fetal bovine serum (FBS), bovine calf serum (FCS), horse serum and porcine serum. Cells can be grown in suspension or as adherent cultures. There are also cell lines that have been modified to survive in suspension cultures, so they can be grown at a higher density than adherent conditions would allow.

[0344] The term "expression" includes any step involved in producing the antibody construct of the present invention, including but not limited to transcription, post-transcriptional modification, translation, folding, post-translational modification, targeting to a specific subcellular or extracellular location, and secretion. The term "recovery" refers to a series of processes intended to separate the antibody construct from cell culture. A "recovery" or "purification" process can separate the protein and non-protein fractions of the cell culture and ultimately separate the desired antibody construct from all other polypeptides and proteins. Separation steps typically utilize differences in protein size, physicochemical properties, binding affinity, and biological activity. Preparative purification is intended to produce relatively large amounts of purified protein for subsequent use, while analytical purification produces relatively small amounts of protein for various research or analytical purposes.

[0345] When using recombinant technology, the antibody construct can be produced intracellularly in the periplasmic space or directly secreted into the culture medium. If the antibody construct is produced intracellularly, then as a first step, the host cells or particulate fragments of the dissolved fragments are removed, for example, by centrifugation or ultrafiltration. The antibody construct of the present invention can be produced, for example, in bacteria such as Escherichia coli. After expression, the construct is separated from the bacterial cell paste in the soluble fraction and can be purified, for example, via affinity chromatography and / or size exclusion. Final purification can be performed in a manner similar to the method for purifying antibody constructs expressed in mammalian cells and secreted into the culture medium. Carter et al. (Biotechnology (NY) [Biotechnology (NY)] February 1992; 10 (2): 163-7) describe a procedure for separating antibodies secreted into the periplasmic space of Escherichia coli.

[0346] Where the antibody is secreted into the culture medium, supernatants from such expression systems are typically first concentrated using commercially available protein concentration filters (e.g., ultrafiltration units).

[0347] Antibody constructs of the invention prepared from host cells can be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Depending on the antibody construct to be recovered, other techniques for protein purification are also available, such as fractionation on ion exchange columns, mixed-mode ion exchange, HIC, ethanol precipitation, size exclusion chromatography, reversed-phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), immunoaffinity (such as protein A / G / L) chromatography, chromatofocusing, SDS-PAGE, ultracentrifugation, and ammonium sulfate precipitation.

[0348] Protease inhibitors may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of contaminants.

[0349] Furthermore, the present invention provides a pharmaceutical composition or formulation comprising the antibody construct of the present invention or the antibody construct produced according to the method of the present invention.

[0350] As used herein, the term "pharmaceutical composition" relates to a composition suitable for administration to a patient, preferably a human patient. Particularly preferred pharmaceutical compositions of the present invention comprise preferably a therapeutically effective amount of one or more antibody constructs of the present invention. Preferably, the pharmaceutical composition further comprises a suitable formulation of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives and / or adjuvants. The acceptable ingredients of the composition are preferably non-toxic to the recipient at the dosage and concentration employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0351] These compositions can include a pharmaceutically acceptable carrier. Generally speaking, as used herein, "pharmaceutically acceptable carrier" means any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers (e.g., phosphate buffered saline (PBS) solutions), water, suspensions, emulsions (such as oil / water emulsions), various types of wetting agents, liposomes, dispersion media, and coatings that are compatible with drug administration, particularly parenteral administration. The use of such media and medicaments in pharmaceutical compositions is well known in the art, and compositions comprising such carriers can be formulated by well-known conventional methods.

[0352] Certain embodiments provide pharmaceutical compositions comprising the antibody constructs of the present invention and one or more excipients, such as those described illustratively in this section and elsewhere herein. Excipients may be used for a variety of purposes in the present invention, such as adjusting the physical, chemical, or biological properties of the formulation, such as adjusting viscosity and / or the methods of the present invention to improve effectiveness and / or stabilize such formulations and methods to prevent, for example, degradation and spoilage due to pressure during manufacture, transportation, storage, preparation before use, administration, and subsequent processes. Excipients should generally be used at their lowest effective concentration.

[0353] In certain embodiments, pharmaceutical compositions may contain formulation substances for the purpose of altering, maintaining, or preserving certain characteristics of the composition, such as pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeability (see Remington's Pharmaceutical Sciences, 18th ed., 1990, Mack Publishing Company). In such embodiments, suitable formulation substances may include, but are not limited to:

[0354] Amino acids

[0355] Antimicrobial agents, such as antibacterial and antifungal agents

[0356] Antioxidants

[0357] Buffers, buffer systems, and buffering agents for maintaining compositions at physiological pH or slightly lower pH (typically within the pH range of about 5 to about 8 or 9)

[0358] Non-aqueous solvents, vegetable oils and injectable organic esters

[0359] Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media

[0360] Biodegradable polymers such as polyester

[0361] ·Building agent

[0362] Chelating agents

[0363] Isotonicity agents and absorption delaying agents

[0364] Complexing agent

[0365] Fillers

[0366] ·carbohydrate

[0367] (Low molecular weight) proteins, polypeptides or protein carriers, preferably of human origin

[0368] Coloring and flavoring agents

[0369] Sulfur-containing reducing agent

[0370] Diluent

[0371] Emulsifier

[0372] Hydrophilic polymers

[0373] Salt-forming counterions

[0374] ·preservative

[0375] Metal complexes

[0376] Solvents and cosolvents

[0377] Sugars and sugar alcohols

[0378] Suspension concentrate

[0379] Surfactants or wetting agents

[0380] Stability enhancer

[0381] Tension enhancers

[0382] Parenteral delivery vehicles

[0383] Intravenous delivery vehicles

[0384] It is well known that different components of a pharmaceutical composition can have different effects, and that, for example, amino acids can act as buffers, stabilizers, and / or antioxidants; mannitol can act as a bulking agent and / or tonicity enhancing agent; sodium chloride can act as a delivery vehicle and / or tonicity enhancing agent; and so on.

[0385] In the context of the present invention, a pharmaceutical composition may comprise:

[0386] (a) an antibody construct as described herein,

[0387] (b) at least one buffer,

[0388] (c) at least one sugar, and

[0389] (d) at least one surfactant;

[0390] The pH of the pharmaceutical composition is in the range of 3.5 to 6.

[0391] In the composition described above, the first domain preferably has an isoelectric point (pI) in the range of 4 to 9.5; the second domain has a pI in the range of 8 to 10 (preferably 8.5 to 9.0); and the antibody construct optionally comprises a third domain comprising two polypeptide monomers, each polypeptide monomer comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker;

[0392] In the above-described compositions, it is further contemplated that the at least one buffering agent is present in a concentration range of 5 mM to 200 mM, more preferably in a concentration range of 10 mM to 50 mM. It is also contemplated that the at least one sugar is selected from the group consisting of a monosaccharide, a disaccharide, a cyclic polysaccharide, a sugar alcohol, a linear branched glucan, or a linear unbranched glucan. It is also contemplated that the disaccharide is selected from the group consisting of sucrose, trehalose, and mannitol, sorbitol, and combinations thereof. It is further contemplated that the sugar alcohol is sorbitol. It is also contemplated that the at least one sugar is present in a concentration range of 1% to 15% (m / v), preferably in a concentration range of 9% to 12% (m / v). It is further contemplated that the antibody construct is present in a concentration range of 0.1 mg / ml to 8 mg / ml, preferably 0.2-2.5 mg / ml, and more preferably 0.25-1.0 mg / ml.

[0393] According to one embodiment of the composition described above, the at least one surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, poloxamer 188, Pluronic F68, Triton X-100, polyoxyethylen, PEG 3350, PEG 4000, and combinations thereof. It is further contemplated that the at least one surfactant is present at a concentration in the range of 0.004% to 0.5% (m / V), preferably in the range of 0.001% to 0.01% (m / V). It is contemplated that the pH of the composition is in the range of 4.0 to 5.0, preferably 4.2. It is also contemplated that the pharmaceutical composition has an osmotic pressure of 150 mOsm to 500 mOsm. It is further contemplated that the pharmaceutical composition further comprises an excipient selected from the group consisting of one or more polyols and one or more amino acids. In the context of the present invention, it is envisaged that the one or more excipients are present in a concentration range of 0.1% to 15% (w / V).

[0394] The present invention also provides a pharmaceutical composition comprising

[0395] (a) an antibody construct as described herein, preferably in a concentration range of 0.1 mg / ml to 8 mg / ml, preferably 0.2 mg / ml-2.5 mg / ml, more preferably 0.25 mg / ml-1.0 mg / ml;

[0396] (b) 10 mM glutamate or acetate;

[0397] (c) 9% (m / V) sucrose or 6% (m / V) sucrose and 6% (m / V) hydroxypropyl-β-cyclodextrin;

[0398] (d) 0.01% (m / V) polysorbate 80;

[0399] The pH of the liquid pharmaceutical composition is 4.2.

[0400] It is envisioned that in addition to the antibody constructs of the present invention defined herein, the compositions of the present invention may comprise additional bioactive agents, depending on the intended use of the composition. Such agents may be drugs that act on the gastrointestinal system, drugs that act as cytostatics, drugs that prevent hyperuricemia, drugs that suppress immune responses, drugs that regulate inflammatory responses, drugs that act on the circulatory system, and / or agents known in the art such as cytokines. It is also envisioned that the antibody constructs of the present invention will be used in co-therapy, i.e., in combination with another anticancer drug.

[0401] In this context, it is envisioned that the pharmaceutical composition of the present invention (which comprises an antibody construct comprising a first domain that binds to CLDN18.2 on the surface of a target cell and a second domain that binds to CD3 on the surface of a T cell, as described in more detail herein above) further comprises an agent (preferably an antibody or antibody construct) that binds to a protein of an immune checkpoint pathway (such as PD-1 or CTLA-4) or to a co-stimulatory immune checkpoint receptor (such as 4-1BB). The present invention also relates to an antibody construct according to the present invention (which comprises an antibody construct comprising a first domain that binds to CLDN18.2 on the surface of a target cell and a second domain that binds to CD3 on the surface of a T cell, as described in more detail herein above) and a combination of an agent (preferably an antibody or antibody construct) that binds to a protein of an immune checkpoint pathway (such as PD-1 or CTLA-4) or to a co-stimulatory immune checkpoint receptor (such as 4-1BB). Due to the properties of at least two components of the combination, i.e., their pharmaceutical activity, the combination may also be referred to as a therapeutic combination. In some embodiments, the combination may be in the form of a pharmaceutical composition or a kit. According to one embodiment, the pharmaceutical composition or the combination comprises an antibody construct of the present invention and an antibody or antibody construct that binds to PD-1. For example, anti-PD-1 binding proteins that can be used for this purpose are described in detail in PCT / US2019 / 013205.

[0402] Therefore, in a further aspect, the present invention relates to a pharmaceutical composition or combination comprising:

[0403] an antibody construct comprising a first domain that binds to CLDN18.2 on the surface of a target cell and a second domain that binds to CD3 on the surface of a T cell, as described in more detail herein above, and

[0404] An antibody or antibody construct that binds to PD-1 and comprises:

[0405] a) a VH region and / or a VL region, the VH region comprising CDR-H1 as described in SEQ ID NO: 292, CDR-H2 as described in SEQ ID NO: 293, and CDR-H3 as described in SEQ ID NO: 294, the VL region comprising CDR-L1 as described in SEQ ID NO: 295, CDR-L2 as described in SEQ ID NO: 296, and CDR-L3 as described in SEQ ID NO: 297;

[0406] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 302, a CDR-H2 as described in SEQ ID NO: 303, and a CDR-H3 as described in SEQ ID NO: 304, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 305, a CDR-L2 as described in SEQ ID NO: 306, and a CDR-L3 as described in SEQ ID NO: 307;

[0407] c) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 312, a CDR-H2 as described in SEQ ID NO: 313, and a CDR-H3 as described in SEQ ID NO: 314, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 315, a CDR-L2 as described in SEQ ID NO: 316, and a CDR-L3 as described in SEQ ID NO: 317;

[0408] d) a VH region and / or a VL region, the VH region comprising CDR-H1 as described in SEQ ID NO: 322, CDR-H2 as described in SEQ ID NO: 323, and CDR-H3 as described in SEQ ID NO: 324, and the VL region comprising CDR-L1 as described in SEQ ID NO: 325, CDR-L2 as described in SEQ ID NO: 326, and CDR-L3 as described in SEQ ID NO: 327;

[0409] e) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 332, a CDR-H2 as described in SEQ ID NO: 333, and a CDR-H3 as described in SEQ ID NO: 334, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 335, a CDR-L2 as described in SEQ ID NO: 336, and a CDR-L3 as described in SEQ ID NO: 337;

[0410] f) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 342, a CDR-H2 as described in SEQ ID NO: 343, and a CDR-H3 as described in SEQ ID NO: 344, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 345, a CDR-L2 as described in SEQ ID NO: 346, and a CDR-L3 as described in SEQ ID NO: 347;

[0411] g) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 352, a CDR-H2 as described in SEQ ID NO: 353, and a CDR-H3 as described in SEQ ID NO: 354, the VL region comprising a CDR-L1 as described in SEQ ID NO: 355, a CDR-L2 as described in SEQ ID NO: 356, and a CDR-L3 as described in SEQ ID NO: 357; or

[0412] h) a VH region and / or a VL region, the VH region comprising the CDR-H1 as described in SEQ ID NO: 362, the CDR-H2 as described in SEQ ID NO: 363, and the CDR-H3 as described in SEQ ID NO: 364, and the VL region comprising the CDR-L1 as described in SEQ ID NO: 365, the CDR-L2 as described in SEQ ID NO: 366, and the CDR-L3 as described in SEQ ID NO: 367.

[0413] In one embodiment, the antibody or antibody construct described above that binds to PD-1 comprises

[0414] a) a VH region as depicted in SEQ ID NO: 298, and a VL region as depicted in SEQ ID NO: 299;

[0415] b) a VH region as depicted in SEQ ID NO: 308, and a VL region as depicted in SEQ ID NO: 309;

[0416] c) a VH region as depicted in SEQ ID NO: 318, and a VL region as depicted in SEQ ID NO: 319;

[0417] d) a VH region as described in SEQ ID NO: 328, and a VL region as described in SEQ ID NO: 329;

[0418] e) a VH region as depicted in SEQ ID NO: 338, and a VL region as depicted in SEQ ID NO: 339;

[0419] f) a VH region as described in SEQ ID NO: 348, and a VL region as described in SEQ ID NO: 349;

[0420] g) a VH region as depicted in SEQ ID NO: 358, and a VL region as depicted in SEQ ID NO: 359; or

[0421] h) a VH region as depicted in SEQ ID NO: 368, and a VL region as depicted in SEQ ID NO: 369.

[0422] In one embodiment, the above-mentioned antibody or antibody construct that binds to PD-1 comprises:

[0423] a) a heavy chain as described in SEQ ID NO: 300, and a light chain as described in SEQ ID NO: 301;

[0424] b) a heavy chain as described in SEQ ID NO: 310, and a light chain as described in SEQ ID NO: 311;

[0425] c) a heavy chain as described in SEQ ID NO: 320, and a light chain as described in SEQ ID NO: 321;

[0426] d) a heavy chain as described in SEQ ID NO: 330, and a light chain as described in SEQ ID NO: 331;

[0427] e) a heavy chain as described in SEQ ID NO: 340, and a light chain as described in SEQ ID NO: 341;

[0428] f) a heavy chain as described in SEQ ID NO: 350, and a light chain as described in SEQ ID NO: 351;

[0429] g) a heavy chain as described in SEQ ID NO: 360, and a light chain as described in SEQ ID NO: 361; or

[0430] h) a heavy chain as depicted in SEQ ID NO: 370, and a light chain as depicted in SEQ ID NO: 371.

[0431] In certain embodiments, the optimal pharmaceutical composition will be determined based on, for example, the intended route of administration, delivery form, and desired dosage. See, for example, Remington's Pharmaceutical Sciences, supra. In certain embodiments, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the antibody constructs of the present invention. In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition can be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier can be water for injection or a physiological saline solution, which may be supplemented with other substances commonly found in compositions for parenteral administration. In certain embodiments, compositions comprising the antibody constructs of the present invention can be prepared for storage by mixing the selected components with the desired purity with an optional formulation (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. In addition, in certain embodiments, the antibody constructs of the present invention can be formulated into a lyophilized product using a suitable excipient.

[0432] When considering parenteral administration, the form of the acceptable aqueous solution of parenteral without pyrogen can be provided for therapeutic composition of the present invention, and this aqueous solution is included in the desired antibody construct of the present invention in the pharmaceutically acceptable vehicle.The especially suitable vehicle for parenteral injection is sterile distilled water, wherein the antibody construct of the present invention is mixed with the sterile isotonic solution of suitable preservation.In certain embodiments, preparation can comprise and prepare desired molecule with the reagent preparation of controlled release or sustained release of product, and this product can be sent via accumulating injection, or can promote lasting persistent period in circulation.In certain embodiments, implantable drug delivery device can be used for introducing desired antibody construct.

[0433] Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving the antibody constructs of the present invention in sustained or controlled delivery formulations. Techniques for formulating various sustained or controlled delivery means are known to those skilled in the art. The antibody constructs may also be embedded in microcapsules, for example, prepared by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems, or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

[0434] The pharmaceutical composition for in vivo administration is typically provided as a sterile preparation. Sterilization can be completed by filtration with a sterile filter membrane. When the composition is freeze-dried, the sterilization using the method can be carried out before or after freeze-drying and reconstruction. The composition for parenteral administration can be stored in a lyophilized form or in a solution. Usually the parenteral composition is placed in a container with a sterile access port (e.g., an intravenous solution bag or a bottle with a stopper that can be pierced by a hypodermic needle).

[0435] Another aspect of the present invention comprises self-buffering formulations comprising the antibody constructs of the present invention, which can be used as pharmaceutical compositions, as described in International Patent Application WO 2006 / 138181. There are a variety of publications available on protein stabilization and formulation materials and methods that can be used in this regard, such as Arawaka T. et al., Pharm Res. 1991 Mar;8(3):285-91; Kendrick et al., "Physical stabilization of proteins inaqueous solution" In: Rational Design of Stable Protein Formulations: Theory and Practice, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13:61-84 (2002), and Randolph and Jones, Pharm Biotechnol. 2002;13:159-75, seeing in particular the sections relating to excipients and methods for self-buffering protein formulations, particularly with respect to protein pharmaceutical products and methods for veterinary and / or human medical use.

[0436] According to some embodiments of the present invention, salt can be used for example to adjust the ionic strength and / or isotonicity of compositions or preparation and / or improve the solubility and / or the physical stability of the antibody construct or other compositions of compositions according to the present invention.Ion can be by being combined with the charged residue on the protein surface and by shielding the charged group and polar group in the protein and reducing its electrostatic interaction, attracting and repelling the interacting intensity to come the native state of stabilizing protein.Ion can also be by being combined with the denatured peptide bond (--CONH) of protein especially to come the denatured state of stabilizing protein.In addition, can also reduce intermolecular electrostatic interaction with the ionic interaction of the charged group and polar group in the protein, and thereby prevent or reduce protein aggregation and insoluble.

[0437] Ionic species have significantly different effects on proteins. A variety of ion species that can be used to prepare pharmaceutical compositions according to the present invention and their classification ratings for their effects on proteins have been developed. An example is the Hofmeister series, which rates ions and polar non-ionic solutes by their effects on the conformational stability of proteins in solution. Stable solutes are called "lyophiles." Unstable solutes are called "chaotropics." High concentrations of lyophiles are commonly used to precipitate proteins from solution ("salting out"). Chaotropic agents are commonly used to denature and / or dissolve proteins ("salting out"). The relative effectiveness of ion pairs "salting out" and "salting out" defines their position in the Hofmeister series.

[0438] According to various embodiments of the present invention, free amino acids can be used in formulations or compositions comprising the antibody constructs of the present invention as bulking agents, stabilizers, and antioxidants, as well as for other standard uses. Certain amino acids can be used to stabilize the protein in the formulation, while other amino acids can be used during the lyophilization process to ensure the correct cake structure and properties of the active ingredient. Some amino acids can be used to inhibit protein aggregation in liquid and lyophilized formulations, while other amino acids can be used as antioxidants.

[0439] Polyols are lyophilic and can be used as stabilizers in liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols can also be used to adjust the tension of formulations and to prevent freeze-thaw stress during transportation or to prevent lumps during manufacturing. In the context of the present invention, polyols can also be used as cryoprotectants.

[0440] Certain embodiments of the preparation or composition comprising the antibody construct of the present invention may include a surfactant. Proteins may be easily adsorbed on the surface and are prone to denaturation and aggregation at air-liquid, solid-liquid and liquid-liquid interfaces. These harmful interactions are usually inversely proportional to protein concentration and are typically exacerbated by physical oscillations (such as those produced during product transportation and handling). Conventional surfactants are used to prevent, minimize or reduce surface adsorption. Surfactants are also commonly used in controlling protein conformational stability. Using a surfactant in this respect is protein-specific because a specific surfactant typically stabilizes some proteins and makes other proteins unstable.

[0441] The preparation comprising the antibody construct of the present invention or certain embodiments of the composition can include one or more antioxidants. By maintaining the ambient oxygen and temperature of the appropriate level and avoiding exposure to light, the harmful oxidation of proteins in the pharmaceutical formulation can be prevented to a certain extent. Antioxidant excipients can also be used to prevent the oxidative degradation of proteins. It is envisioned that the antioxidant for the therapeutic protein formulation according to the present invention can be water-soluble and keep its activity throughout the shelf life of the product (composition comprising the antibody construct). Antioxidants may also destroy proteins, and therefore should be selected in a way, among other things, to eliminate or fully reduce the possibility that the antioxidant destroys the antibody construct or other proteins in the preparation.

[0442] Certain embodiments of the formulation or composition comprising the antibody construct of the present invention may include one or more preservatives. For example, when development involves extracting more than one multi-dose parenteral formulation from the same container, a preservative is necessary. Its primary function is to inhibit microbial growth and ensure product sterility throughout the entire shelf life or service life of the drug product. Although preservatives have a long history of use with small molecule parenteral drugs, developing protein formulations including preservatives may be challenging. Preservatives typically have an unstable effect (aggregation) on proteins, and this has become a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs are only formulated for single-use. However, when multi-dose formulations are possible, they have the added advantage of making patients convenient and the marketability of the increase. A good example is human growth hormone (hGH), where the development of preservative formulations has led to the commercialization of more convenient, reusable injection pens. Several aspects need to be considered during the formulation and development of preservative formulations. The effective preservative concentration in the drug product must be optimized. This requires testing a given preservative in a dosage form at a concentration range that confers antimicrobial effectiveness without compromising protein stability.

[0443] As can be expected, the development of liquid formulations containing preservatives is more challenging than freeze-dried formulations. Freeze-dried products can be freeze-dried in the absence of preservatives and reconstituted with a diluent containing a preservative when in use. This shortens the time when the preservative contacts the antibody construct, thereby significantly minimizing the relevant stability risk. In the case of liquid formulations, preservative effectiveness and stability should be maintained throughout the product shelf life. The key point to be pointed out is that preservative effectiveness should be confirmed in the final formulation containing active drug and all excipient components. Once a pharmaceutical composition is prepared, it can be stored in a sterile bottle as a solution, suspension, gel, emulsion, solid, crystal or as a dehydrated or freeze-dried powder. Such formulations can be stored in a ready-to-use form or in a form (such as freeze-dried form) reconstructed before administration.

[0444] The biological activity of the pharmaceutical compositions defined herein can be determined, for example, by cytotoxicity assays, as described in the following examples, WO 99 / 54440, or by Schlereth et al. (Cancer Immunol. Immunother. [Cancer Immunology Immunotherapy] 20 (2005), 1-12). As used herein, "efficacy" or "in vivo efficacy" refers to the response to treatment with the pharmaceutical composition or formulation of the present invention using, for example, standardized NCI response criteria. The success or in vivo efficacy of a therapy using the pharmaceutical composition of the present invention refers to the effectiveness of the composition for its intended use, i.e., the ability of the composition to cause its desired effect, i.e., to deplete pathological cells (e.g., tumor cells). In vivo efficacy can be monitored by established standard methods for the corresponding disease entity, including but not limited to white blood cell counts, differentials, fluorescence activated cell sorting, bone marrow aspiration. In addition, various disease-specific clinical chemistry parameters and other established standard methods can be used. In addition, computer-assisted tomography, X-rays, magnetic resonance tomography, positron emission tomography scans, lymph node biopsy / histology, and other established standard methods can be used.

[0445] Another major challenge in developing drugs, such as the pharmaceutical compositions of the present invention, is the predictable regulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of a candidate drug can be established, i.e., a profile of pharmacokinetic parameters that influence the ability of a particular drug to treat a given condition. Pharmacokinetic parameters of drugs that influence the ability of a drug to treat a certain disease entity include, but are not limited to, half-life, volume of distribution, first-pass metabolism in the liver, and serum binding. The efficacy of a given pharmaceutical agent can be affected by each of the parameters mentioned above.

[0446] "Half-life" is the time required for an amount to be reduced to half of its initial value. In medical science, it refers to the half-life of a substance or drug in the human body. In a medical context, half-life can refer to the time it takes for a substance / drug to lose half of its activity (e.g., pharmacological, physiological, or radiological activity). Half-life can also describe the time it takes for the concentration of a drug or substance (e.g., an antibody construct of the present invention) in plasma / serum to reach half of its steady-state value ("serum half-life"). Typically, clearance or removal of an administered substance / drug refers to cleansing of the body by biological processes such as metabolism, excretion, etc. (also involving the functions of the kidneys and liver). "First-pass metabolism" is the phenomenon by which a drug is metabolized, thereby reducing its concentration before it reaches the circulation. It is the portion of the drug lost during the absorption process. Thus, "hepatic first-pass metabolism" means the tendency of a drug to be metabolized when it first comes into contact with the liver (i.e., during its first passage through the liver). "Volume of distribution" (V D ) refers to the extent to which the drug is distributed in body tissues rather than in plasma. A higher V DIndicates a large amount of tissue distribution. Drug retention can occur throughout the body's various compartments, such as intracellular and extracellular spaces, tissues, and organs. "Serum binding" refers to the tendency of a drug to interact and bind to serum proteins (such as albumin), resulting in a reduction or loss of the drug's biological activity.

[0447] Pharmacokinetic parameters also include bioavailability, lag time (T lag), Tmax, absorption rate, and / or Cmax for a given amount of the drug administered. "Bioavailability" refers to the fraction of the administered drug / substance dose that reaches the systemic circulation (blood compartment). When a drug is administered intravenously, its bioavailability is considered to be 100%. However, when a drug is administered via other routes (such as orally), its bioavailability is typically reduced. "Lag time" means the time delay between drug administration and its detection and measurability in blood or plasma. Cmax is the maximum plasma concentration reached by the drug after its administration (and before administering the second dose). Tmax is the time to reach Cmax. The time to reach the blood or tissue concentration of the drug required for its biological effect is affected by all parameters. As outlined above and as shown, for example, in Schlereth et al. (ibid.), the pharmacokinetic parameters of antibody constructs exhibiting cross-species specificity can be determined in preclinical animal testing of non-chimpanzee primates.

[0448] One embodiment provides an antibody construct of the present invention (or an antibody construct produced according to the method of the present invention) for use in preventing, treating or alleviating a disease (preferably a vegetation). Another embodiment provides an antibody construct of the present invention (or an antibody construct produced according to the method of the present invention) for use in the manufacture of a medicine for preventing, treating or alleviating a disease (preferably a vegetation). It is also envisioned to provide a method for preventing, treating or alleviating a disease (preferably a vegetation), the method comprising the step of administering an antibody construct of the present invention (or an antibody construct produced according to the method of the present invention) to a subject in need thereof. Those of the terms "subject in need", "patient" or "needing treatment" include those already suffering from the disease, and those to be prevented from the disease. These terms also include people and other mammalian subjects receiving preventive or therapeutic treatments.

[0449] Antibody constructs of the present invention and formulations / pharmaceutical compositions described herein can be used for treating, alleviating and / or preventing medical conditions as described herein in patients in need thereof.Term " treatment " refers to both therapeutic treatment and prophylactic (prophylactic or preventative) measures.Treatment includes that antibody construct / pharmaceutical composition is used or is applied to and suffers from disease / disorder as described herein, has the symptom of this disease / disorder or has the patient of the tendency of suffering from this disease / disorder or has experimenter in need thereof, separates tissue or cell, and purpose is to cure, recover, alleviate, alleviate, change, remedy, alleviate, improve or affect this disease, this disease symptom or the tendency of suffering from this disease.As used herein, term " alleviating " refers to by being applied to such patient or experimenter in need thereof according to antibody construct of the present invention any improvement to the patient's morbid state.Such improvement can be regarded as the prevention of the progress of patient's disease slowing down or stopping and / or the severity of disease symptoms reduces, the frequency of no disease symptom phase or duration increases or the damage or disability caused due to disease. As used herein, the term "prevention" means avoiding the occurrence or recurrence of a disease as specified herein by administering an antibody construct according to the present invention to a subject in need thereof.

[0450] The term "disease" refers to any condition that would benefit from treatment with the antibody constructs or pharmaceutical compositions described herein. This includes chronic and acute conditions or diseases, including those pathological conditions that predispose a mammal to the disease in question. The disease is preferably a neoplasm, cancer, or tumor. The disease, neoplasm, cancer, or tumor is preferably CLDN18.2 positive, i.e., characterized by expression or overexpression of CLDN18.2.

[0451] " Vegetation " is the abnormal growth of tissue, usually but not always forms a mass. When also forming a mass, it is usually referred to as "tumor". In brain tumors, uncontrolled cell division means that the size of the vegetation mass increases, and in confined spaces such as the intracranial cavity, this soon becomes problematic because the mass invades the brain space and pushes it aside, causing brain tissue compression and increased intracranial pressure and the destruction of brain substance. Vegetation or tumor can be benign, potentially malignant (precancerous) or malignant (cancerous). Malignant vegetation / tumor is usually referred to as cancer. They usually invade and destroy surrounding tissues, and may form metastasis, that is, they spread to other parts of the body, tissues or organs. "Primary tumor" is a tumor that grows at the anatomical site where tumor progression begins and continues to produce a cancerous mass. For example, when abnormal cells are formed in the brain, a brain tumor will occur. Most cancers develop in their primary site, but then continue to metastasize or spread to other parts of the body (such as tissues and organs). These other tumors are "secondary tumors". Most cancers continue to be called after their primary site, even after they have spread to other parts of the body.

[0452] Lymphomas and leukemias are lymphoid neoplasms. For the purposes of the present invention, they are also encompassed by the terms "tumor" or "cancer." For the purposes of the present invention, the terms "neoplasm," "tumor," and "cancer" are used interchangeably and encompass both primary tumors / cancers and secondary tumors / cancers (or "metastases"), as well as mass-forming neoplasms (tumors) and lymphoid neoplasms (such as lymphomas and leukemias), and MRD.

[0453] The term "minimal residual disease" (MRD) refers to evidence of the presence of small amounts of residual cancer cells that remain in a patient's body after treatment for cancer, such as when the patient is in remission (no signs or symptoms of the disease). Very small amounts of remaining cancer cells are not usually detectable by conventional means because standard tests used to assess or detect cancer are not sensitive enough to detect MRD. Today, very sensitive molecular biology tests for MRD are available, such as flow cytometry, PCR, and next-generation sequencing. These tests can measure the lowest levels of cancer cells in a tissue sample, sometimes as low as one cancer cell in a million normal cells. In the context of the present invention, it is contemplated that the terms "prevention," "treatment," or "relief" of cancer also encompass "prevention, treatment, or remission of MRD," regardless of whether MRD is detected.

[0454] In one embodiment of the present invention, the neoplasm, cancer, or tumor is selected from the group consisting of, but not limited to, gastrointestinal cancer, ovarian cancer, and lung cancer. According to one embodiment of the present invention, the gastrointestinal cancer is selected from the group consisting of: gastric cancer (stomach cancer), esophageal cancer, gastroesophageal cancer, pancreatic cancer, and colorectal cancer. According to another embodiment of the present invention, the ovarian cancer is mucinous ovarian cancer. According to another embodiment of the present invention, the lung cancer is non-small cell lung cancer.

[0455] Antibody constructs of the present invention are typically designed for specific routes of administration and methods, specific dosages and frequencies, specific treatments for specific diseases, bioavailability, and persistence ranges. The material of the composition is preferably formulated with a concentration acceptable to the site of administration. Therefore, formulations and compositions can be designed according to the present invention to be delivered by any suitable route of administration. In the context of the present invention, routes of administration include, but are not limited to, topical, enteral, and parenteral routes.

[0456] If the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in an appropriate liquid before administration. The lyophilized material can be reconstituted in, for example, bacteriostatic water for injection (BWFI), normal saline, phosphate buffered saline (PBS), or the same formulation in which the protein was before lyophilization. The pharmaceutical compositions and antibody constructs of the present invention are particularly useful for parenteral administration, such as intravenous delivery, for example, by injection or infusion. The pharmaceutical composition can be administered using a medical device. Examples of medical devices for administering pharmaceutical compositions are described in U.S. Patent Nos. 4,475,196; 4,439,196; 4,447,224; 4,447,233; 4,486,194; 4,487,603; 4,596,556; 4,790,824; 4,941,880; 5,064,413; 5,312,335; 5,312,335; 5,383,851; and 5,399,163.

[0457] The compositions of the present invention can be administered to a subject at an appropriate dose, which can be determined, for example, in a dose escalation study. As described above, the antibody constructs of the present invention that exhibit cross-species specificity as described herein can also be advantageously used in preclinical testing in non-chimpanzee primates. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, time and route of administration, general health, and other drugs administered concurrently.

[0458] An "effective dose" is an amount of a therapeutic agent sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective dose" is an amount sufficient to cure or at least partially prevent the disease and its complications, signs and symptoms in a patient suffering from the disease. The amount or dose effective for this purpose will depend on the disease to be treated (indication), the antibody construct being delivered, the treatment context and goal, the severity of the disease, previous therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the patient's size (weight, body surface) and / or condition (age and general health) and the general state of the patient's autoimmune system. The appropriate dosage can be adjusted at the discretion of the attending physician to obtain the best therapeutic effect.

[0459] A therapeutically effective amount of the antibody constructs of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency or duration of disease-free periods, or prevention of impairment or disability due to the disease. In the treatment of CLDN18.2-expressing tumors, a therapeutically effective amount of the antibody constructs of the invention preferably inhibits tumor cell growth by at least about 20%, at least about 40%, 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 tumor growth can be evaluated in animal models predictive of efficacy in human tumors.

[0460] In another embodiment, the invention provides a test 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 "test kit" means that two or more components (one of which corresponds to an antibody construct of the present invention, a pharmaceutical composition, a polynucleotide, a vector or a host cell) are packaged together in a container, a receptacle or other. Therefore, a test kit can be described as a group of products and / or appliances sufficient to achieve a certain goal, which can be sold as a single unit.

[0461] It is envisioned that additional components of the kit of the present invention are agents (preferably antibodies or antibody constructs) that bind to proteins of the immune checkpoint pathway (such as PD-1 or CTLA-4) or to costimulatory immune checkpoint receptors (such as 4-1BB). These agents are described in more detail above herein. According to one embodiment, the kit comprises an antibody construct of the present invention and an antibody or antibody construct that binds to PD-1. For example, anti-PD-1 binding proteins that can be used for this purpose are described in detail in PCT / US2019 / 013205. In certain embodiments, the kit allows for simultaneous and / or sequential administration of the components.

[0462] The kit may include one or more receptacles (such as vials, ampoules, containers, syringes, bottles, bags) of any suitable shape, size, and material (preferably waterproof, e.g., plastic or glass) containing an antibody construct or pharmaceutical composition of the invention (see above) in a dose suitable for administration. The kit may further contain instructions for use (e.g., in the form of a brochure or instruction manual), a device for administering the antibody construct or pharmaceutical composition of the invention (e.g., a syringe, pump, infusion set, etc.), a device for reconstitution of the antibody construct of the invention, and / or a device for diluting the antibody construct of the invention.

[0463] The present invention also provides a kit for a single-dose administration unit. The kit of the present invention may also contain a first receptacle containing a dried / lyophilized antibody construct or pharmaceutical composition and a second receptacle containing an aqueous formulation. In certain embodiments of the present invention, a kit containing a single-chamber and multi-chamber prefilled syringe is provided.

[0464] The present invention relates to the following projects:

[0465] Item 1. An antibody construct comprising a first domain that binds to CLDN18.2 (preferably having SEQ ID NO: 1) on the surface of a target cell and a second domain that binds to CD3 on the surface of a T cell.

[0466] Item 2. The antibody construct according to Item 1, wherein the first domain binds to the first extracellular loop (loop 1) of CLDN18.2, which loop preferably has SEQ ID NO: 2.

[0467] Item 3. The antibody construct according to Item 1 or 2, comprising

[0468] a first domain that binds to CLDN18.2 on the surface of target cells, and

[0469] A second domain that binds to human CD3 on the surface of T cells, wherein

[0470] This first domain binds to the same CLDN18.2 epitope as an antibody or antibody construct comprising a domain that binds to CLDN18.2 on the surface of a target cell and comprises:

[0471] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0472] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0473] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0474] d) a VH region as depicted in SEQ ID NO: 139, and a VL region as depicted in SEQ ID NO: 140.

[0475] Item 4. The antibody construct according to any one of the preceding items, wherein the antibody construct competes for binding with an antibody or antibody construct comprising the following domain, which binds to CLDN18.2 on the surface of a target cell and comprises:

[0476] a) a VH region comprising CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, and a VL region comprising CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126;

[0477] b) a VH region and / or a VL region, the VH region comprising a CDR-H1 as described in SEQ ID NO: 133, a CDR-H2 as described in SEQ ID NO: 134, and a CDR-H3 as described in SEQ ID NO: 135, and the VL region comprising a CDR-L1 as described in SEQ ID NO: 136, a CDR-L2 as described in SEQ ID NO: 137, and a CDR-L3 as described in SEQ ID NO: 138;

[0478] c) the VH region as depicted in SEQ ID NO: 127, and the VL region as depicted in SEQ ID NO: 128; or

[0479] d) a VH region as depicted in SEQ ID NO: 139, and a VL region as depicted in SEQ ID NO: 140.

[0480] Item 5. The antibody construct according to any one of the preceding items, wherein the first domain of the antibody construct binds to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 22 on the surface of the target cell, and optionally also binds to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 24 on the surface of the target cell, but does not bind to the CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 23 on the surface of the target cell;

[0481] Item 6. The antibody construct according to any one of the preceding items, wherein the first domain of the antibody construct binds to a CLDN18.2 mutant having the amino acid sequence as depicted in SEQ ID NO: 14 on the surface of a target cell and / or a CLDN18.2 mutant having the amino acid sequence of SEQ ID NO: 15 on the surface of a target cell, and optionally also binds to one or more CLDN18.2 mutants having an amino acid sequence selected from the group consisting of those described in SEQ ID NO: 11, 12, 13, 16, 17, 19, 20 and 21 on the surface of a target cell, but does not bind to a CLDN18.2 mutant having the amino acid sequence depicted in SEQ ID NO: 18 on the surface of a target cell;

[0482] Item 7. An antibody construct according to any one of the preceding items, wherein the first domain of the antibody construct binds to human CLDN18.2 on the surface of a target cell, wherein Glu (E) at position 56 of human CLDN18.2 is essential for the binding of the first domain, and Ala (A) at position 42 and / or Asn (N) at position 45 of human CLDN18.2 are not essential for the binding of the first domain; and / or

[0483] Item 8. An antibody construct according to any one of the preceding items, wherein the first domain of the antibody construct binds to an epitope of CLDN18.2, which epitope comprises the amino acid sequence as described in SEQ ID NO: 266, but does not comprise the amino acid sequence as described in SEQ ID NO: 265, and optionally does not further comprise the amino acid sequence as described in SEQ ID NO: 267.

[0484] Item 9. The antibody construct according to any one of the preceding items, wherein the second domain binds to human CD3ε and common marmoset or squirrel monkey CD3ε.

[0485] Item 10. The antibody construct according to any one of the preceding items, wherein

[0486] a) the antibody construct, the first domain and / or the second domain are human or humanized,

[0487] b) the antibody construct is a single-chain antibody construct,

[0488] c) the first domain is in the form of a scFv,

[0489] d) the second domain is in the form of a scFv,

[0490] e) the first domain and the second domain are connected via a linker, preferably a peptide linker, more preferably a glycine / serine linker, and / or

[0491] f) The antibody construct comprises a domain that provides for extended serum half-life, such as an Fc-based domain.

[0492] Item 11. The antibody construct according to any one of the preceding items, wherein the first domain does not bind or does not significantly bind to CLDN18.1, CLDN1, CLDN2, CLDN3, CLDN4, CLDN6 and / or CLDN9.

[0493] Item 12 An antibody construct according to any one of the preceding items, wherein

[0494] a) the first domain has an affinity for CLDN18.2 (e.g., hu CLDN18.2) of ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM or ≤20 nM, as preferably measured in a cell-based assay such as a Scatchard assay;

[0495] b) the second domain has an affinity for CD3 (e.g., hu CD3, e.g., hu CD3ε) of ≤100 nM, ≤90 nM, ≤80 nM, ≤70 nM, ≤60 nM, ≤50 nM, ≤40 nM, ≤30 nM, ≤20 nM or ≤10 nM, as preferably measured in a surface plasmon resonance assay such as a Biacore assay; and / or

[0496] c) EC of the antibody construct 50 The values ​​are ≤500pM, ≤400pM, ≤300pM, ≤280pM, ≤260pM, ≤250pM, ≤240pM, ≤220pM, ≤200pM, ≤180pM, ≤160pM, ≤150pM, ≤140pM, ≤120pM, ≤100pM, ≤90pM, ≤80pM, ≤70pM, ≤60pM, ≤50pM, ≤4 0 pM, ≤30 pM, ≤20 pM, ≤15 pM, ≤10 pM or ≤5 pM, as preferably measured in a cytotoxicity assay performed with CLDN18.2-positive target cells (such as CHO cells transfected with SNU-601, SNU-620 or CLDN18.2) and stimulated enriched (human) CD8-positive T cells or unstimulated (human) peripheral blood mononuclear cells as effector cells.

[0497] Item 13. The antibody construct according to any one of the preceding items, wherein the first domain comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 selected from the group consisting of:

[0498] a) CDR-H1 as described in SEQ ID NO: 121, CDR-H2 as described in SEQ ID NO: 122, and CDR-H3 as described in SEQ ID NO: 123, CDR-L1 as described in SEQ ID NO: 124, CDR-L2 as described in SEQ ID NO: 125, and CDR-L3 as described in SEQ ID NO: 126; and

[0499] b) CDR-H1 as described in SEQ ID NO: 133, CDR-H2 as described in SEQ ID NO: 134, and CDR-H3 as described in SEQ ID NO: 135, and CDR-L1 as described in SEQ ID NO: 136, CDR-L2 as described in SEQ ID NO: 137, and CDR-L3 as described in SEQ ID NO: 138.

[0500] Item 14. The antibody construct according to any one of the preceding items, wherein the first domain comprises a VH region having an amino acid sequence as described in SEQ ID NO: 127 or SEQ ID NO: 139.

[0501] Item 15. The antibody construct according to one of the preceding items, wherein the first domain comprises a VL region having an amino acid sequence as described in SEQ ID NO: 128 or SEQ ID NO: 140.

[0502] Item 16. The antibody construct according to one of the preceding items, wherein the first domain comprises a VH region and a VL region, and the VH region and the VL region have the amino acid sequence as described in SEQ ID NO: 127+128 or SEQ ID NO: 139+140.

[0503] Item 17. The antibody construct according to one of the preceding items, wherein the first domain comprises a polypeptide having an amino acid sequence as depicted in SEQ ID NO: 129 or SEQ ID NO: 141.

[0504] Item 18. The antibody construct according to one of the preceding items, comprising or consisting of a polypeptide having an amino acid sequence selected from the group consisting of those described in SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 143 and SEQ ID NO: 144.

[0505] Item 19. A polynucleotide encoding an antibody construct as defined in any of the preceding items.

[0506] Item 20. A vector comprising the polynucleotide as defined in Item 19.

[0507] Item 21. A host cell transformed or transfected with the polynucleotide as defined in Item 19 or the vector as defined in Item 20.

[0508] Item 22. A method for producing an antibody construct as defined in any one of Items 1 to 18, the method comprising culturing a host cell as defined in Item 21 under conditions allowing expression of the antibody construct and recovering the produced antibody construct from the culture.

[0509] Item 23. A pharmaceutical composition comprising an antibody construct as defined in any one of Items 1 to 18 or produced according to the method of Item 22.

[0510] Item 24. An antibody construct according to any one of Items 1 to 18 or produced according to the method of Item 22, for use in preventing, treating or ameliorating a disease, preferably a neoplasm, more preferably a CLDN18.2-positive neoplasm.

[0511] Item 25. The antibody construct according to Item 24, wherein the disease or neoplasm is selected from the group consisting of gastrointestinal cancer, ovarian cancer and lung cancer.

[0512] Item 26. The antibody construct according to Item 25, wherein the gastrointestinal cancer is selected from the group consisting of gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer and colorectal cancer.

[0513] Item 27. A kit comprising an antibody construct as defined in any one of items 1 to 18, an antibody construct produced according to the method of item 22, a polynucleotide as defined in item 19, a vector as defined in item 20, and / or a host cell as defined in item 21.

[0514] Item 28. A pharmaceutical composition as described in Item 23 or a kit as described in Item 27, which further comprises an agent (preferably an antibody or antibody construct) that binds to a protein of the immune checkpoint pathway (such as PD-1 or CTLA-4) or to a co-stimulatory immune checkpoint receptor (such as 4-1BB).

[0515] Item 29. The pharmaceutical composition according to Item 23 or 28 or the kit according to Item 27 or 28, for therapeutic use, preferably for use in preventing, treating or alleviating a disease, preferably a neoplasm, more preferably a CLDN18.2-positive neoplasm.

[0516] ***** Sequence Listing <110> Amgen Research & Development (Munich) GmbH Amgen <120> Antibody constructs against CLDN18.2 and CD3 <130> AMG16502PCT <150> US 62 / 714,366 <151> August 3, 2018 <160> 371 <170> PatentIn Version 3.5 <210> 1 <211> 261 <212> PRT <213> Homo sapiens <400> 1 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 甘氨酸-亮氨酸-半胱氨酸-丙氨酸-异亮氨酸-丙氨酸-甘氨酸-缬氨酸-丝氨酸-缬氨酸-苯丙氨酸-丙氨酸-天冬酰胺-甲硫氨酸-亮氨酸-缬氨酸 130 135 140 苏氨酸-天冬酰胺-苯丙氨酸-色氨酸-甲硫氨酸-丝氨酸-苏氨酸-丙氨酸-天冬酰胺-甲硫氨酸-酪氨酸-苏氨酸-甘氨酸-甲硫氨酸-甘氨酸-甘氨酸 145 150 155 160 甲硫氨酸-缬氨酸-谷氨酰胺-苏氨酸-缬氨酸-谷氨酰胺-苏氨酸-精氨酸-酪氨酸-苏氨酸-苯丙氨酸-甘氨酸-丙氨酸-丙氨酸-亮氨酸-苯丙氨酸 165 170 175 缬氨酸-甘氨酸-色氨酸-缬氨酸-丙氨酸-甘氨酸-甘氨酸-亮氨酸-苏氨酸-亮氨酸-异亮氨酸-甘氨酸-甘氨酸-缬氨酸-甲硫氨酸-甲硫氨酸 180 185 190 半胱氨酸-异亮氨酸-丙氨酸-半胱氨酸-精氨酸-甘氨酸-亮氨酸-丙氨酸-脯氨酸-谷氨酸-谷氨酸-苏氨酸-天冬酰胺-酪氨酸-赖氨酸-丙氨酸 195 200 205 缬氨酸-丝氨酸-酪氨酸-组氨酸-丙氨酸-丝氨酸-甘氨酸-组氨酸-丝氨酸-缬氨酸-丙氨酸-酪氨酸-赖氨酸-脯氨酸-甘氨酸-甘氨酸 210 215 220 苯丙氨酸-赖氨酸-丙氨酸-丝氨酸-苏氨酸-甘氨酸-苯丙氨酸-甘氨酸-丝氨酸-天冬酰胺-苏氨酸-赖氨酸-天冬酰胺-赖氨酸-赖氨酸-异亮氨酸 225 230 235 240 酪氨酸-天冬氨酸-甘氨酸-甘氨酸-丙氨酸-精氨酸-苏氨酸-谷氨酸-天冬氨酸-天冬氨酸-缬氨酸-谷氨酰胺-丝氨酸-酪氨酸-脯氨酸-丝氨酸 245 250 255 赖氨酸-组氨酸-天冬氨酸-酪氨酸-缬氨酸 260 <210> 2 <211> 53 <212> PRT <213> Homo sapiens <400> 2 Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val 1 5 10 15 Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly 20 25 30 Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met 35 40 45 Leu Gln Ala Val Arg 50 <210> 3[[ID=十七]] <211> 31 <212> PRT <213> Homo sapiens <400> 3 Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly 1 5 10 15 Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala 20 25 30 <210> 4 <211> 261 <212> PRT <213> Homo sapiens <400> 4 Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly It should be noted that there seems to be an incorrect "十七" in the translation of ID=17 which should be " ". Please check and correct if necessary. 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260<000123s><210> 5 <211> 211<| <212> PRT <213> Homo sapiens <400> 5 Met Ala Asn Ala Gly Leu Gln Leu Leu Gly Phe Ile Leu Ala Phe Leu 1 5 10 15 Gly Trp Ile Gly Ala Ile Val Ser Thr Ala Leu Pro Gln Trp Arg Ile 20 25 30 Tyr Ser Tyr Ala Gly Asp Asn Ile Val Thr Ala Gln Ala Met Tyr Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Ser Gln Ser Thr Gly Gln Ile Gln Cys 50 55 60 Lys Val Phe Asp Ser Leu Leu Asn Leu Ser Ser Thr Leu Gln Ala Thr 65 70 75 80 It should be noted that there seems to be a misspelling in the tag "<000123s>" in the original text. It is assumed to be " " for the purpose of translation. If this is not the case, the translation may need to be adjusted accordingly after the correct tag is provided.Arg Ala Leu Met Val Val Gly Ile Leu Leu Gly Val Ile Ala Ile Phe 85 90 95 Val Ala Thr Val Gly Met Lys Cys Met Lys Cys Leu Glu Asp Asp Glu 100 105 110 Val Gln Lys Met Arg Met Ala Val Ile Gly Gly Ala Ile Phe Leu Leu 115 120 125 Ala Gly Leu Ala Ile Leu Val Ala Thr Ala Trp Tyr Gly Asn Arg Ile 130 135 140 Val Gln Glu Phe Tyr Asp Pro Met Thr Pro Val Asn Ala Arg Tyr Glu 145 150 155 160 Phe Gly Gln Ala Leu Phe Thr Gly Trp Ala Ala Ala Ser Leu Cys Leu 165 170 175 Leu Gly Gly Ala Leu Leu Cys Cys Ser Cys Pro Arg Lys Thr Thr Ser 180 185 190 Tyr Pro Thr Pro Arg Pro Tyr Pro Lys Pro Ala Pro Ser Ser Gly Lys 195 200 205 Asp Tyr Val 210 <210> 6 <211> 230 <212> PRT <213> Homo sapiens <400> 6 Met Ala Ser Leu Gly Leu Gln Leu Val Gly Tyr Ile Leu Gly Leu Leu 1 5 10 15 Gly Leu Leu Gly Thr Leu Val Ala Met Leu Leu Pro Ser Trp Lys Thr 20 25 30 Ser Ser Tyr Val Gly Ala Ser Ile Val Thr Ala Val Gly Phe Ser Lys 35 40 45 Gly Leu Trp Met Glu Cys Ala Thr His Ser Thr Gly Ile Thr Gln Cys 50 55 60 Asp Ile Tyr Ser Thr Leu Leu Gly Leu Pro Ala Asp Ile Gln Ala Ala 65 70 75 80 Gln Ala Met Met Val Thr Ser Ser Ala Ile Ser Ser Leu Ala Cys Ile 85 90 95 Ile Ser Val Val Gly Met Arg Cys Thr Val Phe Cys Gln Glu Ser Arg 100 105 110 Ala Lys Asp Arg Val Ala Val Ala Gly Gly Val Phe Phe Ile Leu Gly 115 120 125 Gly Leu Leu Gly Phe Ile Pro Val Ala Trp Asn Leu His Gly Ile Leu 130 135 140 Arg Asp Phe Tyr Ser Pro Leu Val Pro Asp Ser Met Lys Phe Glu Ile 145 150 155 160 Gly Glu Ala Leu Tyr Leu Gly Ile Ile Ser Ser Leu Phe Ser Leu Ile 165 170 175 Ala Gly Ile Ile Leu Cys Phe Ser Cys Ser Ser Gln Arg Asn Arg Ser 180 185 190 Asn Tyr Tyr Asp Ala Tyr Gln Ala Gln Pro Leu Ala Thr Arg Ser Ser 195 200 205 Pro Arg Pro Gly Gln Pro Pro Lys Val Lys Ser Glu Phe Asn Ser Tyr ’ 210 215 220 Ser Leu Thr Gly Tyr Val 225 230 <210> 7 <211> 220 <212> PRT <213> Homo sapiens <400> 7 Met Ser Met Gly Leu Glu Ile Thr Gly Thr Ala Leu Ala Val Leu Gly 1 5 10 15 Trp Leu Gly Thr Ile Val Cys Cys Ala Leu Pro Met Trp Arg Val Ser 20 25 30 Ala Phe Ile Gly Ser Asn Ile Ile Thr Ser Gln Asn Ile Trp Glu Gly 35 40 45 Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys 50 55 60 It should be noted that there seems to be a small error in the original text where "205" is likely a typo and should be "205". The above translation is based on the corrected understanding.Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg 65 70 75 80 Ala Leu Ile Val Val Ala Ile Leu Leu Ala Ala Phe Gly Leu Leu Val 85 90 95 Ala Leu Val Gly Ala Gln Cys Thr Asn Cys Val Gln Asp Asp Thr Ala 100 105 110 Lys Ala Lys Ile Thr Ile Val Ala Gly Val Leu Phe Leu Leu Ala Ala 115 120 125 Leu Leu Thr Leu Val Pro Val Ser Trp Ser Ala Asn Thr Ile Ile Arg 130 135 140 Asp Phe Tyr Asn Pro Val Val Pro Glu Ala Gln Lys Arg Glu Met Gly 145 150 155 160 Ala Gly Leu Tyr Val Gly Trp Ala Ala Ala Ala Leu Gln Leu Leu Gly 165 170 175 Gly Ala Leu Leu Cys Cys Ser Cys Pro Pro Arg Glu Lys Lys Tyr Thr 180 185 190 Ala Thr Lys Val Val Tyr Ser Ala Pro Arg Ser Thr Gly Pro Gly Ala 195 200 205 Ser Leu Gly Thr Gly Tyr Asp Arg Lys Asp Tyr Val 210 215 220 <210> 8 <211> 209 <212> PRT <213> Homo sapiens <400> 8 Met Ala Ser Met Gly Leu Gln Val Met Gly Ile Ala Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Ala Val Met Leu Cys Cys Ala Leu Pro Met Trp Arg Val 20 25 30 Thr Ala Phe Ile Gly Ser Asn Ile Val Thr Ser Gln Thr Ile Trp Glu 35 40 45 Gly Leu Trp Met Asn Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Val Ile Ile Ser Ile Ile Val Ala Ala Leu Gly Val Leu 85 90 95 Leu Ser Val Val Gly Gly Lys Cys Thr Asn Cys Leu Glu Asp Glu Ser 100 105 110 Ala Lys Ala Lys Thr Met Ile Val Ala Gly Val Val Phe Leu Leu Ala 115 120 125 Gly Leu Met Val Ile Val Pro Val Ser Trp Thr Ala His Asn Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Ser Gly Gln Lys Arg Glu Met 145 150 155 160 Gly Ala Ser Leu Tyr Val Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Asn Cys Pro Pro Arg Thr Asp Lys Pro 180 185 190 Tyr Ser Ala Lys Tyr Ser Ala Ala Arg Ser Ala Ala Ala Ser Asn Tyr 195 200 205 Val <210> 9 <211> 220 <212> PRT <213> Homo sapiens <400> 9 Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu 1 5 10 15 Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu 85 90 95 Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp 100 105 110 Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser 115 120 125 Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly 180 185 190 Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser 195 200 205 Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val 210 215 220 <210> 10 <211> 217 <212> PRT <213> Homo sapiens <400> 10 Met Ala Ser Thr Gly Leu Glu Leu Leu Gly Met Thr Leu Ala Val Leu 1 5 10 15 Gly Trp Leu Gly Thr Leu Val Ser Cys Ala Leu Pro Leu Trp Lys Val 20 25 30 Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu 35 40 45 Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys 50 55 60 Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala 65 70 75 80 Arg Ala Leu Cys Val Ile Ala Leu Leu Leu Ala Leu Leu Gly Leu Leu 85 90 95 Val Ala Ile Thr Gly Ala Gln Cys Thr Thr Cys Val Glu Asp Glu Gly 100 105 110 Ala Lys Ala Arg Ile Val Leu Thr Ala Gly Val Ile Leu Leu Leu Ala 115 120 125 Gly Ile Leu Val Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ala Ala Leu Leu Met Leu 165 170 175 Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Pro Pro Gln Val Glu Arg 180 185 190 Pro Arg Gly Pro Arg Leu Gly Tyr Ser Ile Pro Ser Arg Ser Gly Ala 195 200 205 Ser Gly Leu Asp Lys Arg Asp Tyr Val 210 215 <210> 11 <211> 261 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 11 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 12 <211> 261 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 12 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 13 <211> 261 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 13 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 14 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 14 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ser Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val<00​​​​​​​​​​​​​​​​​​​​​​​​​​​ Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 16 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 16 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15<\ Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg \65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 17 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 17 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 18 <211> 261 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 18 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 20 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 20 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 21 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 21 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 22 <211> 261 <212> PRT <213> Synthetic sequence <220> <223> Synthetic polypeptide <400> 22 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Gln Val Val Trp Glu Gly 35 40 45 Leu Trp Met Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg [[ID=4十一年]]65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 23 <211> 250 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 23 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys Lys 50 55 60 Val Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile 165 170 175 Gly Gly Val Met Met Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu 180 185 190 Thr Asn Tyr Lys Ala Val Ser Tyr His Ala Ser Gly His Ser Val Ala 195 200 205 Tyr Lys Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr 210 215 220 Lys Asn Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val 225 230 235 240 Gln Ser Tyr Pro Ser Lys His Asp Tyr Val 245 250 <210> 24 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 24 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 25 <211> 250 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 25 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Ile 130 135 140 Gln Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Leu Lys Arg Glu Leu 145 150 155 160 Gly Ala Ser Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile 165 170 175 Gly Gly Val Met Met Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu 180 185 190 Thr Asn Tyr Lys Ala Val Ser Tyr His Ala Ser Gly His Ser Val Ala 195 200 205 Tyr Lys Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr 210 215 220 Lys Asn Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val 225 230 235 240 Gln Ser Tyr Pro Ser Lys His Asp Tyr Val 245 250 <210> 26 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 26 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Ile 130 135 140 Arg Asp Phe Tyr Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr 145 150 155 160 Phe Gly Ala Ala Leu Phe Val Gly Trp Val Ala Gly Gly Leu Thr Leu 165 170 175 Ile Gly Gly Val Met Met Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu 180 18� 190 Glu Thr Asn Tyr Lys Ala Val Ser Tyr His Ala Ser Gly His Ser Val 195 200 205 Ala Tyr Lys Pro Gly Gly Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn 210 215 220 [[ID=३1]]Thr Lys Asn Lys Lys Ile Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu 225 230 235 240 Val Gln Ser Tyr Pro Ser Lys His Asp Tyr Val 245 250 <210> 27 <211> 260 <212> PRT <213> Artificial Sequence It should be noted that there may be some unclear or incorrect information in the original text, such as the "18�" which might be a typo. This translation is based on the best understanding of the given content. <220> <223> synthetic peptide <400> 27 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Asn Pro 145 150 155 160 Leu Val Ala Glu Ala Gln Lys Arg Glu Leu Gly Ala Ser Leu Phe Val 165 170 175 Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met Cys 180 185 190 Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala Val 195 200 205 Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly Phe 210 215 220 Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile Tyr 225 230 235 240 Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser Lys 245 250 255 His Asp Tyr Val 260 <210> 28 <211> 14 <212> PRT <213> Synthetic sequence <220> <223> Synthetic polypeptide <400> 28 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Tyr Tyr Pro Asn 1 5 10 <210> 29 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 29 Gly Thr Lys Phe Leu Ala Pro 1 5 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 30 Ala Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 31 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 31 Ile Tyr Ala Met Asn 1 5 <210> 32 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 32 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Ser <210> 33 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 33 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Phe Phe Ala Tyr 1 5 10 <210> 34 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ile Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Phe Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 35 <211> 109 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 35 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Tyr Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 36 <211> 249 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Ile Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Ser Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Phe Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Tyr Tyr Pro Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Leu Ile Gly Gly 180 185 190 Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe Ser Gly Ser Leu 195 200 205 Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val Gln Pro Glu Asp 210 215 220 Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn Arg Trp Val Phe 225 230 235 240 Gly Gly Gly Thr Lys Leu Thr Val Leu 245 <210> 37 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 37 Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Tyr Tyr Pro Asn 1 5 10 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 38 Gly Thr Lys Phe Leu Ala Pro 1 5 <210> 39 <211> 9 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 39 Ala Leu Trp Tyr Ser Asn Arg Trp Val 1 5 <210> 40 <211> 5 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 40 Lys Tyr Ala Met Asn 1 5 <210> 41 <211> 19 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 41 Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser 1 5 10 15 Val Lys Asp <210> 42 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic peptides <400> 42 His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp Ala Tyr 1 5 10 <210> 43 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 44 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 44 Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly 20 25 30 Tyr Tyr Pro Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Lys Phe Leu Ala Pro Gly Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Val 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Arg Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu <210> 45 <211> 249 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr It should be noted that in the original text, there is an incorrect sequence number in the tag <210>. It is <210> 45 in the original, but in the translation, it is assumed that the correct number should be <210> 45 as well. Also, the sequence in the original text for the part starting from line 26 seems a bit unclear as it repeats the sequence from line 24. The translation is done based on the best understanding of the overall text and following the given rules. If there are specific clarifications needed for this text, it would be beneficial for a more accurate translation.20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Ile Ser Tyr Trp 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly 115 120 125 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Thr Val Val 130 135 140 Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly Thr Val Thr Leu 145 150 155 160 Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Ser Gly Tyr Tyr Pro Asn 165 170 175 Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly Le...

Claims

1. An antibody construct comprising a first domain that binds to CLDN18.2 on the surface of a target cell, and A second domain that binds to human CD3 on the surface of T cells, wherein The first domain comprises a VH region and a VL region, the VH region comprises CDR-H1 as shown in SEQ ID NO:121, CDR-H2 as shown in SEQ ID NO:122 and CDR-H3 as shown in SEQ ID NO:123, and the VL region comprises CDR-L1 as shown in SEQ ID NO:124, CDR-L2 as shown in SEQ ID NO:125 and CDR-L3 as shown in SEQ ID NO:

126.

2. The antibody construct of claim 1, wherein the second domain binds to human CD3ε and binds to common marmoset or squirrel monkey CD3ε.

3. The antibody construct according to claim 1 or 2, wherein a) the antibody construct is a single-chain antibody construct, b) the first domain is in the form of a scFv, c) the second domain is in the form of a scFv, d) the first domain and the second domain are connected via a linker, and / or e) The antibody construct comprises a domain that provides for prolonged serum half-life.

4. The antibody construct according to claim 1 or 2, wherein the first domain does not bind or does not significantly bind to CLDN18.1, CLDN1, CLDN2, CLDN3, CLDN4, CLDN6 and / or CLDN9.

5. The antibody construct according to claim 1 or 2, wherein the first domain comprises a VH region having an amino acid sequence as shown in SEQ ID NO: 127 or SEQ ID NO:

139.

6. The antibody construct according to claim 1 or 2, wherein the first domain comprises a VL region having an amino acid sequence as shown in SEQ ID NO: 128 or SEQ ID NO:

140.

7. The antibody construct according to claim 1 or 2, wherein the first domain comprises: (1) a VH region as shown in SEQ ID NO: 127 and a VL region as shown in SEQ ID NO: 128, or (2) a VH region as shown in SEQ ID NO: 139 and a VL region as shown in SEQ ID NO:

140.

8. The antibody construct according to claim 1 or 2, wherein the first domain comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 129 or SEQ ID NO:

141.

9. The antibody construct according to claim 1 or 2, comprising or consisting of a polypeptide having an amino acid sequence selected from SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 143 or SEQ ID NO:

144.

10. A polynucleotide encoding an antibody construct as defined in any one of the preceding claims.

11. A vector comprising a polynucleotide as defined in claim 10.

12. A host cell transformed or transfected with a polynucleotide as defined in claim 10 or with a vector as defined in claim 11.

13. A method for producing an antibody construct as defined in any one of claims 1 to 9, the method comprising: The host cell as defined in claim 12 is cultured under conditions allowing expression of the antibody construct and the produced antibody construct is recovered from the culture.

14. A pharmaceutical composition comprising an antibody construct as defined in any one of claims 1 to 9 or produced according to the method of claim 13.

15. Use of the antibody construct according to claim 1 or 2 or the antibody construct produced according to the method of claim 13 in the preparation of a medicament for treating a disease, wherein the disease is selected from CLDN18.2-positive gastrointestinal cancer, ovarian cancer and lung cancer.

16. The use according to claim 15, wherein the gastrointestinal cancer is selected from the group consisting of: Gastric cancer, esophageal cancer, gastroesophageal cancer, pancreatic cancer and colorectal cancer.

17. A kit comprising an antibody construct as defined in any one of claims 1 to 9, an antibody construct produced according to the method of claim 13, a polynucleotide as defined in claim 10, a vector as defined in claim 11, and / or a host cell as defined in claim 12.

Images