CN116685606A

CN116685606A - Polypeptide constructs that selectively bind CLDN6 and CD3

Info

Publication number: CN116685606A
Application number: CN202180079883.8A
Authority: CN
Inventors: C·达尔霍夫; T·劳姆; J·安拉尔; C·布鲁梅尔; L·加埃德科; S·夸利亚; J·霍纳; J·拜里斯; E·D·范; C·M·穆劳斯基; B·M·阿尔巴
Original assignee: Amgen Research Munich GmbH; Amgen Inc
Current assignee: Amgen Research Munich GmbH; Amgen Inc
Priority date: 2020-11-06
Filing date: 2021-11-08
Publication date: 2023-09-01

Abstract

The present invention relates to a polypeptide or polypeptide construct comprising a domain that binds to claudin 6 (CLDN 6) and another domain that binds to CD 3. In addition, polynucleotides encoding the constructs, vectors comprising the polynucleotides, and host cells transformed or transfected with the polynucleotides or vectors are provided. Furthermore, the invention provides a process for producing the construct of the invention, the medical use of the construct, and a kit comprising the construct.

Description

Polypeptide constructs that selectively bind CLDN6 and CD3

The present invention relates to polypeptide/polypeptide constructs comprising a domain comprising a paratope that binds to claudin 6 (CLDN 6) and another domain comprising a paratope that binds to CD 3. In addition, polynucleotides encoding these polypeptides/polypeptide constructs, vectors comprising the polynucleotides, and host cells transformed or transfected with the polynucleotides or vectors are provided. Furthermore, the invention provides a process for producing the polypeptide/polypeptide construct of the invention, the medical use of said polypeptide/polypeptide construct, and a kit comprising said construct.

Background

Tight junction proteins are key structural and functional components of epithelial tight junctions between two adjacent cells that regulate cell-cell permeability, maintain ion homeostasis, and support cell adhesion and polarity. The tight junction proteins are 22-27kDa four-molecule cross-linked (tetraspan) transmembrane proteins that multimerize within or across the cell membrane to form a protective barrier. The 24 tight junction proteins that have been reported differ depending on their tissue localization and expression, as a result of their interactions with other proteins.

Claudin 6 (CLDN 6) was originally identified in similarity studies on other genes and proteins belonging to the claudin family of genes and proteins (Morita et al, proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci., USA ], vol.96, pages 511-516, 1999). The expression of the claudin 6mRNA was not detected in adult tissues, but only in embryonic tissues. mRNA and protein expression was then detected in a variety of tumors and tumor cell lines. Consistent with this finding, claudin 6 is considered carcinoembryonic transmembrane protein, which is absent in normal adult tissues. CLDN6 expression is abnormally activated in various cancer types such as ovarian, lung, gastric, breast, germ cell and pediatric cancers (Stadler et al, oncoimmunology [ tumor immunology ]2016, volume 5, stage 3, e1091555 and references cited therein, e.g., mick et al, int.j.cancer [ international journal of cancer ]2014:2206-14; rend n-Huerta et al, j.gastoist.cancer [ gastrointestinal tract journal of cancer ]2010;41:52-59; ushiku et al, histology [ tissue ]2012,61:1043-56; ben-David et al, nature communication ]2013;4:1992; birks et al, pin hol. BRAIN pathology ]2010;20:140-50; sun et al, am.j.j.j.Surgu.36:80, U.S. Path.80).

CLDN6 is a 220 amino acid protein with two extracellular loops (ECLs) that has a high degree of sequence identity to CLDN9, with only three amino acid residues differing in these two ECLs.

Expression of CLDN6 in a variety of tumor types (normal tissue expression is limited to fetal development) has led to the consideration of CLDN6 as a therapeutic target for various cancer types, such as ovarian cancer and non-small cell lung cancer (NSCLC) and other indications.

Ovarian cancer and NSCLC cancer remain highly unmet medical indications.

Ovarian cancer is the seventh most common cancer worldwide. In 2018, there were 295,414 new cases worldwide and 184,799 deaths, the mortality rate in northern hemisphere countries was higher than in asia or africa (Bray et al, CA Cancer J Clin [ journal of clinical tumors ] 2018). Typical first line treatments include surgery and combination chemotherapy (including platinum and paclitaxel or docetaxel). Recently, anti-VEGF antibodies bevacizumab and PARP inhibitors have been approved as maintenance treatments following first line chemotherapy. However, despite the initial response, up to 70% of patients experience disease recurrence due to development of chemotherapy resistance and/or tumor immune evasion. Ovarian tumors are characterized by a highly immunosuppressive tumor microenvironment; although there is evidence that ovarian tumors are immunogenic, immune checkpoint therapies that alter the standard of treatment for other solid tumor types have limited persistence in ovarian cancer (Rodriguez et al, cancer 2018). Despite the progress in clinical trials of ovarian cancer in a variety of new therapies and combination therapies, survival rates for 5 years remain low and therapies that produce a sustained response are urgently needed.

Lung cancer is one of the most common cancers worldwide, with over 200 thousands of new cases and 170 thousands of deaths reported in 2018 (Bray et al, CA: ACancer Journal for Clinicians [ journal of clinician cancers ] 2018). Non-small cell lung cancer (NSCLC) accounts for the majority (85%) of lung cancer cases and is commonly associated with smoking and environmental exposure such as asbestos (Zappa and Mousa, transl Lung Cancer Res [ lung cancer transformation study ] 2016). For patients with tumor expressing PD-L1, the recommended first line treatment of NSCLC is immune checkpoint blockade and platinum dual drug chemotherapy, although targeted therapy may be preferred for initial treatment of tumors with driving mutations (Ettinger et al, JNCCN, 2019). While these advances are promising, and some patients achieve long-lasting responses with immune checkpoint blockade (Santini and Hellman, cancer J2018), further evaluation of immunotherapeutic combinations is required, and treatment of most patients requires the progress of additional new therapies.

Thus, treatment of ovarian cancer and/or NSCLC, particularly treatment of any type of cancer that expresses CLDN6, more particularly treatment of cancer patients in second-line therapy or higher (such as patients who have previously received chemotherapy or immunotherapy and patients with recurrent disease), still requires new therapies that potentially provide a persistent response to a larger patient population.

Bispecific (and multispecific) constructs comprising an antigen-binding (more precisely, epitope-binding) domain that binds to CD3 on a T cell and an antigen-binding (more precisely, epitope-binding) domain that binds to a protein expressed on a target cell directly connect T cells to target cells to induce T cell directed lysis. This mechanism of action differs from chemotherapy, targeted therapy and other immunotherapies in that it works with any CD3 positive T cell, independent of the co-stimulatory activation signal (Klinger et al, immunoreviews [ immunoreviews ] 2016).

Expression of CLDN6 on the cell surface of germ cell tumors, ovarian cancer, and non-small cell lung cancer provides a basis for targeting these tumor types with CLDN6 x CD3 polypeptide/polypeptide constructs. Furthermore, CLDN6 x CD3 polypeptide/polypeptide constructs have the potential to target additional tumor types expressing CLDN6, particularly any type of cancer expressing CLDN6, more particularly for the treatment of cancer patients in secondary or higher therapies (such as patients previously receiving chemotherapy or immunotherapy and patients suffering from recurrent disease).

Detailed Description

The present invention provides novel polypeptides/polypeptide constructs as compounds that selectively and preferably specifically bind to CLDN6 (SEQ ID NO: 1) or any subtype thereof, compositions comprising such compounds, methods of treating and preventing neoplastic disease using the products disclosed herein, kits comprising the products disclosed herein, products for use as medicaments, particularly products for treating and preventing neoplastic disease. The amino acid sequence of human CLDN6 and related information can be found in the UniProt database under accession number P56747.

Compounds of the invention

In one aspect, the invention provides a polypeptide/polypeptide construct comprising or consisting of a domain that binds to CLDN6 (SEQ ID NO:1 or a fragment or amino acid sequence variant thereof) on the surface of a target cell and a domain that binds to CD3 on the surface of a T cell, binding to both CLDN6 and CD3 allowing activation of the T cell. Binding of the polypeptide construct according to the invention engages T cells, i.e. with CD3, and brings T cells into close contact with target cells, allowing the activated T cells to induce a cytotoxic/cytolytic mechanism leading to destruction of the target cells (T cell dependent cytotoxicity).

Further, the present invention provides a polypeptide/polypeptide construct comprising or consisting of a domain comprising a paratope (i.e. an antigen binding domain, more particularly an epitope binding structure) that binds to CLDN6, wherein optionally the domain comprising a paratope (i.e. an antigen binding (epitope binding) structure) of the polypeptide/polypeptide construct of the present invention is capable of binding to CLDN6 on the surface of a cell expressing CLDN6, the polypeptide/polypeptide construct binding to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1). Accordingly, the present invention provides a polypeptide/polypeptide construct comprising or consisting of a domain that binds to CLDN6, wherein optionally the domain is capable of binding to CLDN6 on the surface of cells expressing CLDN6, the polypeptide/polypeptide construct binding to E1A and/or E2B regions, the sequence of which corresponds to the E1A and/or E2B regions of the loops depicted in SEQ ID NOs 9 and 10.

In an embodiment of the invention, a polypeptide/polypeptide construct comprising or consisting of a domain comprising a paratope (i.e. an antigen binding domain, more particularly an epitope binding structure) that binds to CLDN6 binds to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1) and does not bind to amino acids 138-150 of CLDN6 depicted in SEQ ID NO:1, wherein optionally a domain comprising a paratope (i.e. an antigen binding (epitope binding) structure) of a polypeptide/polypeptide construct of the invention is capable of binding to CLDN6 on the surface of cells expressing CLDN 6. In further embodiments of the invention, a polypeptide/polypeptide construct comprising or consisting of a domain that binds to CLDN6 binds to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1) and does not bind to amino acids 138-150 of CLDN6 depicted in SEQ ID NO: 1.

Accordingly, the present invention provides a polypeptide/polypeptide construct comprising or consisting of a domain comprising a paratope (i.e. an antigen binding domain, more particularly an epitope binding structure) that binds to an epitope region of an amino acid comprising extracellular loop 1 (ECL 1) of CLDN6, preferably comprising amino acids 29-39 of SEQ ID NO:1, and/or an epitope region of an amino acid comprising extracellular loop 2 (ECL 2) of CLDN6 corresponding to amino acids 151-160 of SEQ ID NO:1 on the surface of a target cell. Accordingly, the present invention provides a polypeptide/polypeptide construct comprising or consisting of a domain that binds to an epitope region comprising amino acids of extracellular loop 1 (ECL 1) of CLDN6, preferably comprising amino acids 29-39 of SEQ ID NO:1, and/or an epitope region comprising amino acids 151-160 of extracellular loop 2 (ECL 2) of CLDN6, corresponding to amino acids 151-160 of SEQ ID NO:1, on the surface of a target cell.

Accordingly, the present invention provides polypeptide/polypeptide constructs as defined in any of the preceding paragraphs, comprising a further domain recognizing an extracellular epitope of the CD3 epsilon chain (preferably, human and cynomolgus CD3 epsilon chains) and/or the paratope bound thereto, i.e. the antigen binding structure (epitope binding structure), and a domain extending the polypeptide half-life (HLE domain) after administration to an individual, the domain extending the polypeptide half-life optionally comprising two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain. Accordingly, the present invention provides polypeptide/polypeptide constructs as defined in any of the preceding paragraphs, comprising a further domain that recognizes and/or binds to an extracellular epitope of the CD3 epsilon chain (preferably the human and cynomolgus CD3 epsilon chain) and a domain that extends the half-life of the polypeptide (HLE domain) after administration to an individual, optionally comprising two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain.

According to the present invention there is provided a polypeptide/polypeptide construct wherein the domain of the construct immunoselectively binds to an epitope of CLDN6 recognized and/or bound by a paratope (antigen binding or epitope binding structure) comprised in any one of the sequences mentioned in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are very preferred:

a) A VH region comprising CDR-H1 depicted in SEQ ID NO. 13, CDR-H2 depicted in SEQ ID NO. 14 and CDR-H3 depicted in SEQ ID NO. 15, a VL region comprising CDR-L1 depicted in SEQ ID NO. 16, CDR-L2 depicted in SEQ ID NO. 17 and CDR-L3 depicted in SEQ ID NO. 18;

b) A VH region comprising CDR-H1 depicted in SEQ ID NO. 27, CDR-H2 depicted in SEQ ID NO. 28 and CDR-H3 depicted in SEQ ID NO. 29, a VL region comprising CDR-L1 depicted in SEQ ID NO. 30, CDR-L2 depicted in SEQ ID NO. 31 and CDR-L3 depicted in SEQ ID NO. 32;

c) A VH region comprising CDR-H1 depicted in SEQ ID NO. 41, CDR-H2 depicted in SEQ ID NO. 42 and CDR-H3 depicted in SEQ ID NO. 43, a VL region comprising CDR-L1 depicted in SEQ ID NO. 44, CDR-L2 depicted in SEQ ID NO. 45 and CDR-L3 depicted in SEQ ID NO. 46;

d) A VH region comprising CDR-H1 depicted in SEQ ID NO. 55, CDR-H2 depicted in SEQ ID NO. 56 and CDR-H3 depicted in SEQ ID NO. 57, a VL region comprising CDR-L1 depicted in SEQ ID NO. 58, CDR-L2 depicted in SEQ ID NO. 59 and CDR-L3 depicted in SEQ ID NO. 60;

e) A VH region comprising CDR-H1 depicted in SEQ ID NO. 69, CDR-H2 depicted in SEQ ID NO. 70 and CDR-H3 depicted in SEQ ID NO. 71, a VL region comprising CDR-L1 depicted in SEQ ID NO. 72, CDR-L2 depicted in SEQ ID NO. 73 and CDR-L3 depicted in SEQ ID NO. 74;

f) A VH region comprising CDR-H1 depicted in SEQ ID NO. 83, CDR-H2 depicted in SEQ ID NO. 84 and CDR-H3 depicted in SEQ ID NO. 85, a VL region comprising CDR-L1 depicted in SEQ ID NO. 86, CDR-L2 depicted in SEQ ID NO. 87 and CDR-L3 depicted in SEQ ID NO. 88;

g) A VH region comprising CDR-H1 depicted in SEQ ID NO. 97, CDR-H2 depicted in SEQ ID NO. 98 and CDR-H3 depicted in SEQ ID NO. 99, a VL region comprising CDR-L1 depicted in SEQ ID NO. 100, CDR-L2 depicted in SEQ ID NO. 101 and CDR-L3 depicted in SEQ ID NO. 102;

h) A VH region comprising CDR-H1 depicted in SEQ ID NO. 111, CDR-H2 depicted in SEQ ID NO. 112 and CDR-H3 depicted in SEQ ID NO. 113, a VL region comprising CDR-L1 depicted in SEQ ID NO. 114, CDR-L2 depicted in SEQ ID NO. 115 and CDR-L3 depicted in SEQ ID NO. 116;

i) A VH region comprising CDR-H1 depicted in SEQ ID NO. 125, CDR-H2 depicted in SEQ ID NO. 126 and CDR-H3 depicted in SEQ ID NO. 127, a VL region comprising CDR-L1 depicted in SEQ ID NO. 128, CDR-L2 depicted in SEQ ID NO. 129 and CDR-L3 depicted in SEQ ID NO. 130;

j) A VH region comprising CDR-H1 depicted in SEQ ID NO. 139, CDR-H2 depicted in SEQ ID NO. 140 and CDR-H3 depicted in SEQ ID NO. 141, a VL region comprising CDR-L1 depicted in SEQ ID NO. 142, CDR-L2 depicted in SEQ ID NO. 143 and CDR-L3 depicted in SEQ ID NO. 144;

k) A VH region comprising CDR-H1 depicted in SEQ ID NO. 153, CDR-H2 depicted in SEQ ID NO. 154 and CDR-H3 depicted in SEQ ID NO. 155, a VL region comprising CDR-L1 depicted in SEQ ID NO. 156, CDR-L2 depicted in SEQ ID NO. 157 and CDR-L3 depicted in SEQ ID NO. 158;

l) a VH region comprising CDR-H1 depicted in SEQ ID NO. 167, CDR-H2 depicted in SEQ ID NO. 168 and CDR-H3 depicted in SEQ ID NO. 169, and a VL region comprising CDR-L1 depicted in SEQ ID NO. 170, CDR-L2 depicted in SEQ ID NO. 171 and CDR-L3 depicted in SEQ ID NO. 172;

m) a VH region comprising CDR-H1 as depicted in SEQ ID NO:181, CDR-H2 as depicted in SEQ ID NO:182 and CDR-H3 as depicted in SEQ ID NO:183, and a VL region comprising CDR-L1 as depicted in SEQ ID NO:184, CDR-L2 as depicted in SEQ ID NO:185 and CDR-L3 as depicted in SEQ ID NO: 186;

n) a VH region comprising CDR-H1 as depicted in SEQ ID NO. 195, CDR-H2 as depicted in SEQ ID NO. 196 and CDR-H3 as depicted in SEQ ID NO. 197, and a VL region comprising CDR-L1 as depicted in SEQ ID NO. 198, CDR-L2 as depicted in SEQ ID NO. 199 and CDR-L3 as depicted in SEQ ID NO. 200;

o) a VH region comprising CDR-H1 as depicted in SEQ ID NO. 209, CDR-H2 as depicted in SEQ ID NO. 210 and CDR-H3 as depicted in SEQ ID NO. 211, and a VL region comprising CDR-L1 as depicted in SEQ ID NO. 212, CDR-L2 as depicted in SEQ ID NO. 213 and CDR-L3 as depicted in SEQ ID NO. 214;

p) a VH region comprising CDR-H1 as depicted in SEQ ID NO. 223, CDR-H2 as depicted in SEQ ID NO. 224 and CDR-H3 as depicted in SEQ ID NO. 225, and a VL region comprising CDR-L1 as depicted in SEQ ID NO. 226, CDR-L2 as depicted in SEQ ID NO. 227 and CDR-L3 as depicted in SEQ ID NO. 228;

q) a VH region comprising CDR-H1 as depicted in SEQ ID NO. 237, CDR-H2 as depicted in SEQ ID NO. 238 and CDR-H3 as depicted in SEQ ID NO. 239, and a VL region comprising CDR-L1 as depicted in SEQ ID NO. 240, CDR-L2 as depicted in SEQ ID NO. 241 and CDR-L3 as depicted in SEQ ID NO. 242;

r) a VH region comprising CDR-H1 as depicted in SEQ ID NO. 251, CDR-H2 as depicted in SEQ ID NO. 252 and CDR-H3 as depicted in SEQ ID NO. 253, and a VL region comprising CDR-L1 as depicted in SEQ ID NO. 254, CDR-L2 as depicted in SEQ ID NO. 255 and CDR-L3 as depicted in SEQ ID NO. 256,

s) a VH region comprising CDR-H1 depicted in SEQ ID NO. 265, CDR-H2 depicted in SEQ ID NO. 266 and CDR-H3 depicted in SEQ ID NO. 267, and a VL region comprising CDR-L1 depicted in SEQ ID NO. 268, CDR-L2 depicted in SEQ ID NO. 269 and CDR-L3 depicted in SEQ ID NO. 270, and

t) a VH region comprising CDR-H1 as depicted in SEQ ID NO:680, CDR-H2 as depicted in any of SEQ ID NO:681, 682 or 683 and CDR-H3 as depicted in any of SEQ ID NO:684, 685, 686 or 687, and a VL region comprising CDR-L1 as depicted in any of SEQ ID NO:688 or 689, CDR-L2 as depicted in SEQ ID NO:690 and CDR-L3 as depicted in any of SEQ ID NO:691, 692, 693 or 694, and any possible combination with the CDRs of the heavy and light chains described herein.

The construct of the preceding paragraph accordingly preferably comprises at least one domain comprising a paratope that binds CLDN6 as defined in parts (a) to(s), optionally further comprising a domain that binds CD3, e.g. the construct of the preceding paragraph accordingly preferably binds CLDN6 and has VL and/or VH regions comprising CDRs as defined in parts (a) to(s), optionally further comprising a domain that binds CD 3.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:13, CDR-H2 as depicted in SEQ ID NO:14 and CDR-H3 as depicted in SEQ ID NO:15 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:16, CDR-L2 as depicted in SEQ ID NO:17 and CDR-L3 as depicted in SEQ ID NO: 18. Thus, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:13, CDR-H2 as depicted in SEQ ID NO:14 and CDR-H3 as depicted in SEQ ID NO:15 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:16, CDR-L2 as depicted in SEQ ID NO:17 and CDR-L3 as depicted in SEQ ID NO: 18.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:27, CDR-H2 as depicted in SEQ ID NO:28 and CDR-H3 as depicted in SEQ ID NO:29 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:30, CDR-L2 as depicted in SEQ ID NO:31 and CDR-L3 as depicted in SEQ ID NO: 32. In other words, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:27, CDR-H2 as depicted in SEQ ID NO:28 and CDR-H3 as depicted in SEQ ID NO:29 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:30, CDR-L2 as depicted in SEQ ID NO:31 and CDR-L3 as depicted in SEQ ID NO: 32.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:41, CDR-H2 as depicted in SEQ ID NO:42 and CDR-H3 as depicted in SEQ ID NO:43 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:44, CDR-L2 as depicted in SEQ ID NO:45 and CDR-L3 as depicted in SEQ ID NO: 46. Thus, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:41, CDR-H2 as depicted in SEQ ID NO:42 and CDR-H3 as depicted in SEQ ID NO:43 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:44, CDR-L2 as depicted in SEQ ID NO:45 and CDR-L3 as depicted in SEQ ID NO: 46.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:69, CDR-H2 as depicted in SEQ ID NO:70 and CDR-H3 as depicted in SEQ ID NO:71 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:72, CDR-L2 as depicted in SEQ ID NO:73 and CDR-L3 as depicted in SEQ ID NO: 74. Thus, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:69, CDR-H2 as depicted in SEQ ID NO:70 and CDR-H3 as depicted in SEQ ID NO:71 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:72, CDR-L2 as depicted in SEQ ID NO:73 and CDR-L3 as depicted in SEQ ID NO: 74.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:195, CDR-H2 as depicted in SEQ ID NO:196 and CDR-H3 as depicted in SEQ ID NO:197 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:198, CDR-L2 as depicted in SEQ ID NO:199 and CDR-L3 as depicted in SEQ ID NO: 200. Thus, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:195, CDR-H2 as depicted in SEQ ID NO:196 and CDR-H3 as depicted in SEQ ID NO:197 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:198, CDR-L2 as depicted in SEQ ID NO:199 and CDR-L3 as depicted in SEQ ID NO: 200.

According to the present invention there are provided polypeptide/polypeptide constructs comprising domains comprising paratope (antigen binding (epitope binding) structure) binding (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:237, CDR-H2 as depicted in SEQ ID NO:238 and CDR-H3 as depicted in SEQ ID NO:239 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:240, CDR-L2 as depicted in SEQ ID NO:241 and CDR-L3 as depicted in SEQ ID NO: 242. Thus, according to the present invention there are provided polypeptide/polypeptide constructs comprising domains that bind (immunoselectively) to an epitope recognized by a domain comprising a VH region comprising CDR-H1 as depicted in SEQ ID NO:237, CDR-H2 as depicted in SEQ ID NO:238 and CDR-H3 as depicted in SEQ ID NO:239 and a VL region comprising CDR-L1 as depicted in SEQ ID NO:240, CDR-L2 as depicted in SEQ ID NO:241 and CDR-L3 as depicted in SEQ ID NO: 242.

According to the present invention there is provided a polypeptide/polypeptide construct wherein

(i) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of the first extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 1 (ECL 1); the epitope region is depicted in SEQ ID NO 9 and this domain optionally comprises any of the sequences mentioned under a) to s) below, and/or

(ii) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of a second extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 2 (ECL 2); the epitope region is depicted in SEQ ID NO. 10 and this domain optionally comprises any of the sequences mentioned under a) to s) below; and/or

(iii) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of ECL1 and ECL2 of CLDN6, preferably those comprising the amino acids of epitope regions comprising SEQ ID NOs 9 and 10, and optionally any one of the structures mentioned in a) to s) below; and/or

(iv) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) the same epitope on CLDN6 as an antibody or polypeptide construct comprising a paratope that binds CLDN6 on the surface of a target cell, and comprises any of the sequences mentioned in a) to s) below:

r) a VH region comprising CDR-H1 as depicted in SEQ ID NO:251, CDR-H2 as depicted in SEQ ID NO:252 and CDR-H3 as depicted in SEQ ID NO:253, and a VL region comprising CDR-L1 as depicted in SEQ ID NO:254, CDR-L2 as depicted in SEQ ID NO:255 and CDR-L3 as depicted in SEQ ID NO:256, and

s) a VH region comprising CDR-H1 depicted in SEQ ID NO. 265, CDR-H2 depicted in SEQ ID NO. 266 and CDR-H3 depicted in SEQ ID NO. 267, and a VL region comprising CDR-L1 depicted in SEQ ID NO. 268, CDR-L2 depicted in SEQ ID NO. 269 and CDR-L3 depicted in SEQ ID NO. 270.

Thus, according to the present invention there is provided a polypeptide/polypeptide construct wherein

(i) The domain binds (immunoselectively) to an epitope region comprising the amino acids of a first extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 1 (ECL 1); the epitope region is depicted in SEQ ID NO 9 and this domain optionally comprises any of the sequences mentioned under a) to s) below, and/or

(ii) The domain binds (immunoselectively) to an epitope region comprising the amino acids of a second extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 2 (ECL 2); the epitope region is depicted in SEQ ID NO. 10 and this domain optionally comprises any of the sequences mentioned under a) to s) below; and/or

(iii) The domain binds (immunoselectively) to epitope regions of amino acids of ECL1 and ECL2 comprising CLDN6, preferably those comprising amino acids comprising epitope regions of SEQ ID NOs 9 and 10, and optionally comprising any of the structures mentioned in a) to s) below; and/or

(iv) The domain binds (immunoselectively) the same epitope on CLDN6 to an antibody or polypeptide construct comprising a paratope that binds to CLDN6 on the surface of a target cell and comprises any of the sequences mentioned in a) to s) below:

According to the present invention there is provided a polypeptide/polypeptide construct wherein:

(i) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of the first extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 1 (ECL 1); the epitope region is depicted in SEQ ID NO 9 and this domain optionally comprises any of the sequences mentioned under a-1) to s-1) below, and/or

(ii) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of a second extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 2 (ECL 2); the epitope region is depicted in SEQ ID NO. 10, and this domain optionally comprises any of the sequences mentioned under a-1) to s-1) below; and/or

(iii) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) to an epitope region comprising the amino acids of ECL1 and ECL2 of CLDN6, preferably those comprising the amino acids of epitope regions comprising SEQ ID NOs 9 and 10, and optionally any one of the structures mentioned in a-1) to s-1) below; and/or

(iv) The domain comprises a paratope (antigen binding (epitope binding) structure) that binds (immunoselectively) the same epitope on CLDN6 as an antibody or polypeptide construct comprising a paratope that binds CLDN6 on the surface of a target cell, and that paratope comprises any of the sequences mentioned in a-1) to s-1) below:

a-1) the VH region depicted in SEQ ID NO. 11, and/or the VL region depicted in SEQ ID NO. 12;

b-1) the VH region depicted in SEQ ID NO. 25, and/or the VL region depicted in SEQ ID NO. 26;

c-1) the VH region depicted in SEQ ID NO. 39, and/or the VL region depicted in SEQ ID NO. 40;

d-1) the VH region depicted in SEQ ID NO. 53, and/or the VL region depicted in SEQ ID NO. 54;

e-1) the VH region depicted in SEQ ID NO. 67, and/or the VL region depicted in SEQ ID NO. 68;

f-1) the VH region depicted in SEQ ID NO. 81, and/or the VL region depicted in SEQ ID NO. 82;

g-1) the VH region depicted in SEQ ID NO. 95, and/or the VL region depicted in SEQ ID NO. 96;

h-1) the VH region depicted in SEQ ID NO. 109, and/or the VL region depicted in SEQ ID NO. 110;

i-1) the VH region depicted in SEQ ID NO. 123, and/or the VL region depicted in SEQ ID NO. 124;

j-1) the VH region depicted in SEQ ID NO. 137, and/or the VL region depicted in SEQ ID NO. 138;

k-1) the VH region depicted in SEQ ID NO. 151, and/or the VL region depicted in SEQ ID NO. 152;

l-1) the VH region depicted in SEQ ID NO. 165, and/or the VL region depicted in SEQ ID NO. 166;

m-1) the VH region depicted in SEQ ID NO. 179, and/or the VL region depicted in SEQ ID NO. 180;

n-1) the VH region depicted in SEQ ID NO. 193, and/or the VL region depicted in SEQ ID NO. 194;

o-1) the VH region depicted in SEQ ID NO. 207, and/or the VL region depicted in SEQ ID NO. 208;

p-1) the VH region depicted in SEQ ID NO. 221, and/or the VL region depicted in SEQ ID NO. 222;

q-1) the VH region depicted in SEQ ID NO. 235, and/or the VL region depicted in SEQ ID NO. 236;

r-1) the VH region depicted in SEQ ID NO. 249, and/or the VL region depicted in SEQ ID NO. 250; and

s-1) the VH region depicted in SEQ ID NO:263, and/or the VL region depicted in SEQ ID NO: 264.

Thus, according to the present invention there is provided a polypeptide/polypeptide construct wherein:

(i) The domain binds (immunoselectively) to an epitope region comprising the amino acids of a first extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 1 (ECL 1); the epitope region is depicted in SEQ ID NO 9 and this domain optionally comprises any of the sequences mentioned under a-1) to s-1) below, and/or

(ii) The domain binds (immunoselectively) to an epitope region comprising the amino acids of a second extracellular loop of CLDN6 (as depicted in SEQ ID NO: 1), also referred to as extracellular loop 2 (ECL 2); the epitope region is depicted in SEQ ID NO. 10, and this domain optionally comprises any of the sequences mentioned under a-1) to s-1) below; and/or

(iii) The domain binds (immunoselectively) to epitope regions of amino acids of ECL1 and ECL2 comprising CLDN6, preferably those comprising amino acids comprising epitope regions of SEQ ID NOs 9 and 10, and optionally comprising any of the structures mentioned in a-1) to s-1) below; and/or

(iv) The domain binds (immunoselectively) the same epitope on CLDN6 to an antibody or polypeptide construct comprising a paratope that binds to CLDN6 on the surface of a target cell, and the paratope comprises any of the sequences mentioned in a-1) to s-1) below:

According to the present invention there are provided polypeptide/polypeptide constructs which compete for binding with a polypeptide construct comprising or consisting of:

Thus, according to the present invention there are provided polypeptide/polypeptide constructs which compete for binding with a polypeptide construct comprising or consisting of:

Further, the polypeptide construct of the invention competes for binding with a construct comprising a domain that selectively binds to CLDN6 on the surface of a target cell and comprises any one of the sequence sets mentioned in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are highly preferred, and the polypeptide construct of the invention competes for binding with a construct comprising a domain comprising a paratope (i.e. an antigen binding (epitope binding) structure that selectively binds CLDN6 on the surface of a target cell, and which paratope comprises any of the sequence groups mentioned in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) being particularly preferred:

Further, the polypeptide construct of the invention binds to or competes for binding with an antibody or polypeptide construct comprising a paratope (i.e., an antigen binding or epitope binding structure) that binds to (immunoselectively) CLDN6 on the surface of a target cell and comprises any of the following sets of sequences, and the polypeptide construct of the invention binds to or competes for binding with an antibody or polypeptide construct that binds to (immunoselectively) CLDN6 on the surface of a target cell and comprises any of the following sets of sequences:

The present invention relates to a polypeptide/polypeptide construct according to any of the preceding paragraphs, wherein the paratope (i.e. antigen binding (epitope binding) structure) that binds CLDN6 consists of a pair of VH and VL regions, and a polypeptide/polypeptide construct according to any of the preceding paragraphs, wherein the domain that binds CLDN6 consists of a pair of VH and VL regions comprising the amino acid sequences depicted in: 11+12, 25+26, 39+40, 53+54, 67+68, 81+82, 95+96, 109+110, 123+124, 137+138, 151+152, 165+166, 179+180, 193+194, 207+208, 221+222, 235+236, 249+250, or 263+264, or the polypeptides or polypeptide constructs compete with the CLDN6 binding polypeptide construct.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequences depicted in: SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 47, SEQ ID NO. 50, SEQ ID NO. 61, SEQ ID NO. 64, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 89, SEQ ID NO. 92, SEQ ID NO. 103, SEQ ID NO. 106, SEQ ID NO. 117, SEQ ID NO. 120, SEQ ID NO. 131, SEQ ID NO. 134, SEQ ID NO. 145, SEQ ID NO. 148, SEQ ID NO. 159, SEQ ID NO. 162, SEQ ID NO. 173, SEQ ID NO. 176, SEQ ID NO. 187, SEQ ID NO. 190, SEQ ID NO. 201, SEQ ID NO. 204, SEQ ID NO. 215, SEQ ID NO. 218, SEQ ID NO. 229, SEQ ID NO. 232, SEQ ID NO. 243, SEQ ID NO. 246, SEQ ID NO. 257, or SEQ ID NO. 260, SEQ ID NO. 271 or 274, or a polypeptide or polypeptide which competes with DN 6.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of amino acid sequences selected from the group of those depicted in:

-SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23 and SEQ ID NO. 24;

-SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37 and SEQ ID NO 38;

-SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51 and SEQ ID NO. 52;

-SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65 and SEQ ID NO. 66;

-SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 78, SEQ ID NO 79 and SEQ ID NO 80;

-SEQ ID NO 89, SEQ ID NO 90, SEQ ID NO 91, SEQ ID NO 92, SEQ ID NO 93 and SEQ ID NO 94;

-SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107 and SEQ ID NO 108;

-SEQ ID NO 117, SEQ ID NO 118, SEQ ID NO 119, SEQ ID NO 120, SEQ ID NO 121 and SEQ ID NO 122;

-SEQ ID NO. 131, SEQ ID NO. 132, SEQ ID NO. 133, SEQ ID NO. 134, SEQ ID NO. 135 and SEQ ID NO. 136;

-SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149 and SEQ ID NO:150;

-SEQ ID NO 159, SEQ ID NO 160, SEQ ID NO 161, SEQ ID NO 162, SEQ ID NO 163 and SEQ ID NO 164;

-SEQ ID NO 173, SEQ ID NO 174, SEQ ID NO 175, SEQ ID NO 176, SEQ ID NO 177 and SEQ ID NO 178;

-SEQ ID NO 187, SEQ ID NO 188, SEQ ID NO 189, SEQ ID NO 190, SEQ ID NO 191 and SEQ ID NO 192;

-SEQ ID NO. 201, SEQ ID NO. 202, SEQ ID NO. 203, SEQ ID NO. 204, SEQ ID NO. 205 and SEQ ID NO. 206;

-SEQ ID NO 215, SEQ ID NO 216, SEQ ID NO 217, SEQ ID NO 218, SEQ ID NO 219 and SEQ ID NO 220;

-SEQ ID NO 229, SEQ ID NO 230, SEQ ID NO 231, SEQ ID NO 232, SEQ ID NO 233 and SEQ ID NO 234;

-SEQ ID NO 243, SEQ ID NO 244, SEQ ID NO 245, SEQ ID NO 246, SEQ ID NO 247 and SEQ ID NO 248;

-SEQ ID NO 257, SEQ ID NO 258, SEQ ID NO 259, SEQ ID NO 260, SEQ ID NO 261 and SEQ ID NO 262; and

-SEQ ID NO:271, 272, 273, 274, 275, 276, or polypeptide constructs competing for binding with CLDN 6.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 21.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 24.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 35.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 38.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 49.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 52.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 63.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 66.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 77.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 80.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO. 234.

The present invention relates to polypeptide/polypeptide constructs according to any of the preceding paragraphs, comprising or consisting of the amino acid sequence depicted in SEQ ID NO 276.

The polypeptide/polypeptide construct according to any one of the preceding embodiments, wherein the polypeptide/polypeptide construct induces at least 100-fold, at least 250-fold, at least 500-fold or at least 1000-fold less cytotoxicity than T-cell-dependent cytotoxicity measured in an in vitro assay using cells expressing CLDN6 depicted in SEQ ID NO:1, as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID NO:1, the mutant comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L.

According to the present invention there is provided a polypeptide/polypeptide construct,

wherein the domain of the polypeptide construct of the invention comprising the paratope, i.e. the antigen binding (epitope binding) structure, is capable of binding and distinguishing between CLDN6 on the surface of a cell expressing CLDN6 depicted in SEQ ID No. 1 and a CLDN6 mutant on the surface of a cell expressing said CLDN6 mutant, wherein said CLDN6 mutant comprises the sequence depicted in SEQ ID No. 1, wherein at least one of residues 31, 38 and 39 is replaced by another amino acid residue, in particular wherein residue 31 is R and/or residue 38 is S and/or residue 39 is N, and/or wherein at least one of residues 31, 38 and 39 is replaced by another amino acid residue, in particular wherein residue 156 is not Q,

wherein optionally the domain of the polypeptide construct of the invention, including the paratope (i.e. antigen binding (epitope binding) structure) binds to CD3, in particular human or non-human primate CD3,

-wherein the polypeptide/polypeptide construct (i.e. antigen binding (epitope binding) structure)) is capable of engaging, activating and inducing T-cell dependent cytotoxicity when the polypeptide/polypeptide construct (paratope) binds to CLDN6 on the surface of a cell expressing CLDN6 and when another antigen binding (epitope binding) domain comprises paratope binding to CD3, and

-wherein the domain that binds CLDN6 (comprising a paratope (i.e., an antigen binding (epitope binding) structure)) comprises a heavy chain CDR3 region comprising the sequence: x1LIVX2APX3 (SEQ ID NO. 667), wherein X1 is A or N; x2 is V or E; and X3 is V or A,

wherein optionally the polypeptide construct does not bind selectively to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9 and/or CLDN18.1,

preferably, the polypeptide/polypeptide construct binds to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1) depicted in SEQ ID NO:9 and 10, and

wherein preferably the polypeptide/polypeptide construct does not bind to an epitope comprising amino acids 138-150 of CLDN6 (SEQ ID NO: 1).

wherein the domain of the polypeptide construct of the invention comprising the paratope, i.e. the antigen binding (epitope binding) structure, is capable of binding and distinguishing between CLDN6 on the surface of a cell expressing CLDN6 depicted in SEQ ID No. 1 and a CLDN6 mutant on the surface of a cell expressing said CLDN6 mutant, wherein said CLDN6 mutant comprises the sequence depicted in SEQ ID No. 1, wherein at least one of residues 31, 38 and 39 is replaced by another amino acid residue, in particular wherein residue 31 is R and/or residue 38 is S and/or residue 39 is N,

Wherein optionally, the domains of the constructs of the invention, including paratope (i.e., antigen binding (epitope binding) structures) bind CD3 (particularly human or non-human primate CD 3),

further, wherein the polypeptide/polypeptide construct is capable of engaging, activating, and inducing T cell-dependent cytotoxicity when the polypeptide/polypeptide construct (e.g., paratope (i.e., antigen binding (epitope binding) structure)) binds to CLDN6 on the surface of a cell expressing CLDN6 and when another antigen binding (epitope binding) domain (comprising paratope) binds to CD3, and

wherein the CLDN6 binding domain (comprising a paratope (i.e., an antigen binding (epitope binding) structure)) comprises a heavy chain CDR3 region comprising the sequence: DX1LIVX2APX3T (SEQ ID NO. 668), wherein X1 is A or N; x2 is V or E; and X3 is V or A,

wherein optionally the domain comprising the paratope (i.e. antigen binding (epitope binding) structure) does not immunospecifically or immunoselectively bind to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9 and/or CLDN18.1,

wherein optionally the domain of the polypeptide construct of the invention comprising a paratope (i.e. an antigen binding (epitope binding) structure) is capable of binding and distinguishing CLDN6 on the surface of a cell expressing CLDN6, binding to the E1A and/or E2B regions of CLDN6 (SEQ ID NO: 1).

wherein the domain of the polypeptide/polypeptide construct comprising the paratope, i.e. the antigen binding (epitope binding) structure, is capable of binding and distinguishing between CLDN6 on the surface of a cell expressing CLDN6 depicted in SEQ ID No. 1 and a CLDN6 mutant on the surface of a cell expressing said CLDN6 mutant, wherein said CLDN6 mutant comprises the sequence depicted in SEQ ID No. 1, wherein at least one of residues 31, 38 and 39 is replaced by another amino acid residue, in particular wherein residue 31 is R and/or residue 38 is S and/or residue 39 is N,

further, wherein the polypeptide/polypeptide construct is capable of engaging, activating, and inducing T cell-dependent cytotoxicity when the polypeptide/polypeptide construct (e.g., paratope (i.e., antigen binding (epitope binding) structure)) binds to CLDN6 on the surface of a cell expressing CLDN6, and when another antigen binding (epitope binding) domain comprises paratope binding to CD3, and

Wherein the CLDN6 binding domain (comprising a paratope (i.e., an antigen binding (epitope binding) structure)) comprises a heavy chain CDR3 region comprising the sequence: DX (Duplex position) ₁ LIVX ₂ APX ₃ TRDYYYYGMDV (SEQ ID NO. 669), wherein X ₁ Is A or N; x is X ₂ Is V or E; and X is ₃ Is V or A,

wherein optionally the domain binding CLDN6, including the paratope (i.e. antigen binding (epitope binding) structure), does not bind (immunospecifically or immunoselectively) to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9, CLDN18.1 and/or CLDN18.2,

wherein optionally the domain of the polypeptide construct of the invention comprising a paratope (i.e. an antigen binding (epitope binding) structure) is capable of binding and distinguishing CLDN6 on the surface of a cell expressing CLDN6, binding to the E1A and/or E2B regions of CLDN6 (SEQ ID NO: 1). In an embodiment, the polypeptide/polypeptide construct is capable of binding to CLDN6 on the surface of a cell expressing CLDN6, which binds to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1), but not to amino acids 138-150 of CLDN6 depicted in SEQ ID NO: 1.

wherein the domain of the polypeptide/polypeptide construct (comprising a paratope, i.e. an antigen binding (epitope binding) structure)) is capable of binding and distinguishing between CLDN6 on the surface of a cell expressing CLDN6 depicted in SEQ ID No. 1 and a CLDN6 mutant on the surface of a cell expressing said CLDN6 mutant, wherein said CLDN6 mutant comprises the sequence depicted in SEQ ID No. 1, wherein at least one of residues 31, 38 and 39 is replaced by another amino acid residue, in particular wherein residue 31 is R and/or residue 38 is S and/or residue 39 is N,

Wherein optionally the domain of the construct of the invention, including the paratope (i.e. antigen binding (epitope binding) structure) binds to CD3, in particular human or non-human primate CD3,

further wherein the polypeptide/polypeptide construct is capable of engaging, activating and inducing T cell dependent cytotoxicity when the polypeptide/polypeptide construct binds (via paratope (i.e., antigen binding (epitope binding) structure)) to CLDN6 on the surface of a cell expressing CLDN6 and when the other antigen binding (epitope binding) domain comprises paratope binding to CD3,

-wherein the domain capable of binding and distinguishing CLDN6 on the surface of a cell expressing CLDN6 depicted in SEQ ID No. 1, comprising a paratope (i.e. an antigen binding (epitope binding) structure), comprises a heavy chain fragment comprising a heavy chain CDR3 region comprising or consisting of a set of sequences selected from any one of SEQ ID nos. 15, 23, 31, 39, 47, 55, 63, 71, 79, 87, 95, 103, 111, 119, 127, 135, 143 and 151, in particular comprising the set of sequences depicted in SEQ ID nos. 15, 23, 31 and 47, very particularly the heavy chain CDR3 region comprises or consists of SEQ ID No. 15;

-wherein optionally the polypeptide construct does not bind selectively to CLDN2 (SEQ ID NO: 5), CLDN3 (SEQ ID NO: 6), CLDN4 (SEQ ID NO: 7), CLDN9 (SEQ ID NO: 8), CLDN18.1 (SEQ ID NO: 2) and/or CLDN18.2 (SEQ ID NO: 3), and/or

-wherein the construct binds to the E1A and/or E2B region of CLDN6 (SEQ ID NO: 1) but not to amino acids 138-150 of CLDN6 depicted in SEQ ID NO: 1.

According to the present invention there is provided a polypeptide/polypeptide construct as defined throughout the specification and claims comprising a domain that binds to human CLDN6 (SEQ ID NO: 1) and a domain that binds to human CD3, and a domain that extends the half-life of the polypeptide. The domain binding to CLDN6 comprises a Variable Light (VL) chain domain comprising the sequence RASQSVX ₁ SX ₂ CDR1 region depicted in YLA (SEQ ID NO: 695), wherein X ₁ Selected from S and R, preferably S, and wherein X ₂ Selected from S and T, preferably S: and/or in the following sequence QQYX ₁ X ₂ SPX ₃ The CDR3 region depicted in T (SEQ ID NO: 696), wherein X ₁ Selected from G, D and Q, preferably G, and wherein X ₂ Selected from S, A and T, preferably S, and X ₃ Selected from L and I, preferably L. At a particular pointIn embodiments, these polypeptides/polypeptide constructs have a VL chain comprising the CDR1 region depicted in SEQ ID No. 16 and the CDR3 region depicted in SEQ ID No. 18, further preferably in combination with the VL CDR2 region depicted in SEQ ID No. 17, further in particular in combination with the CDR1, CDR2, CDR3 regions of the Variable Heavy (VH) chain domains depicted in SEQ ID nos. 13, 14 and/or 15; these polypeptides/polypeptides are identical to the CLDN6 region depicted in SEQ ID NOs 9 and/or 10 as determined in the domain exchange experiments (reference examples section). The polypeptides or polypeptide constructs of the invention are found to be particularly suitable for distinguishing between CLDN6 and CLDN9, and preferably bind to CLDN6 cells (e.g., CHO cells transformed with nucleic acids encoding CLDN6 and/or CLDN 9) and kill them effectively in vitro. Not only is the cytotoxic activity better, but these polypeptides or polypeptide constructs also show surprisingly high protein stability as determined in the DLS ℃ aggregation thermostability test at 1mg/ml when they have the CDRs as indicated in the heading above. These properties are important for polypeptides and/or polypeptides used in immunological oncology (T cell engagement) treatment methods as well as in the preparation and storage of pharmaceutical formulations.

According to the present invention there are provided polypeptide/polypeptide constructs wherein the domain comprising the paratope (i.e. antigen binding (epitope binding) structure) binds to CLDN6 as defined in any of the above parts, these polypeptide/polypeptide constructs further comprising a domain binding to CD3, in particular to the CD3 binding paratope (comprising the paratope (i.e. antigen binding (epitope binding) structure)), as disclosed for example in WO 2019/133961, the polypeptide/polypeptide constructs only show cross-species specificity for the human and cynomolgus, or common marmoset (Callithrix jacchus), tamarius villous (saimius oedaibus) or saimius sciureus) CD3 epsilon chain and do not demonstrate the same degree of T cell non-specific activation as observed for the previous generation of T cell binding antibodies due to the recognition of this specific epitope (instead of the epitope of the CD3 conjugate in the dual specific T cell binding molecule described previously). The sequences of CD3 binding domains/paratopes that can be used in the context of antibodies and constructs of the invention are described in the following respective paragraphs.

Advantageously, an epitope targeting CLDN6 recognized by the constructs of the invention (see also the examples section) provides the following benefits:

(1) Immunospecificity/immunoselectivity of CLDN6xCD3 construct relative to CLDN9 (examples 1 and 5), and

(2) Unexpectedly high cytotoxic potency against CLDN6xCD3 constructs (examples 4, 6 and 7).

According to the invention, the polypeptide/polypeptide construct of the invention comprises an antigen binding (epitope binding) domain comprising a paratope (i.e. an antigen binding (epitope binding) structure), which domain specifically and selectively binds to CD3 normally expressed on T cells.

Examples of CD3 epsilon extracellular domains bound by the current domain/paratope are shown in SEQ ID NOS 442 and 443, respectively. Further, examples of CD3 epsilon binding domains/paratope amino acids, scFv, VH and VL chains comprising them are shown in SEQ ID NOS 444 to 562 and in SEQ ID NOS 670 to 678.

The invention also relates to a polypeptide according to any of the preceding paragraphs, wherein the binding domain that binds to the extracellular domain of the human CD3 epsilon chain comprises or consists of a VH region linked to a VL region, wherein

-i) the VH region comprises:

CDR-H1 sequences of X1YAX N, wherein X1 is K, V, S, G, R, T or I; and X2 is M or I;

CDR-H2 sequences of RIRSKYNNYATYYADX1VK X2, wherein X1 is S or Q; and X2 is D, G, K, S or E; and

CDR-H3 sequence of HX1NFGNSYX2SX3X4AY, wherein X1 is G, R or A; x2 is I, L, V or T; x3 is Y, W or F; and X4 is W, F or Y; and is also provided with

-ii) wherein the VL region comprises:

CDR-L1 sequence of X1SSTGAVTX2X3X4YX5N, wherein X1 is G, R or A; x2 is S or T; x3 is G or S; x4 is N or Y; and X5 is P or a;

CDR-L2 sequence of X1TX2X3X4X5X 6; wherein X1 is G or A; x2 is K, D or N; x3 is F, M or K; x4 is L or R; x5 is A, P or V; and X6 is P or S; and

CDR-L3 sequence of X1LWYSNX2WV, wherein X1 is V, A or T; and X2 is R or L; and is also provided with

-iii) wherein the CDR sequences of i) and/or ii) comprise one or more amino acid substitutions selected from the group consisting of: X24V or X24F in CDR-H1;

d15 (preferably E), X116A in CDR-H2;

h1 (preferably a or N), X12E, F (preferably I) and/or N6 (preferably S or T) in CDR-H3; and

w93 (preferably Y) in CDR-L3.

The present invention relates to compounds that may have linkers, half-life extending peptides, and other structural moieties disclosed in SEQ ID NOS: 563-575 and SEQ ID NOS: 576-666, respectively. Details about the function of these structures are found in the sequence listing after the examples section.

It is contemplated that a polypeptide/polypeptide construct according to the invention, the domain that binds CD3 on the surface of a T cell (comprising paratope) comprises a VL region selected from the group consisting of the VL regions depicted in the corresponding SEQ ID nos 444 to 562 and 677 (in particular in SEQ ID nos 507-512 and 534-541 and 677) exemplified in the sequence listing.

In another embodiment, a polypeptide/polypeptide construct according to the invention, the domain that binds CD3 on the surface of a T cell (comprising paratope) comprises the VL region depicted in SEQ ID NO: 677.

It is envisaged that the polypeptide/polypeptide construct according to the invention, the domain that binds CD3 on the surface of a T cell (comprising paratope) comprises a VH region selected from the group consisting of the VH regions depicted in the corresponding SEQ ID nos 444 to 562 and 676 (in particular in SEQ ID nos 513-533 and 676) as exemplified in the sequence listing.

In another embodiment, a polypeptide/polypeptide construct according to the invention, the domain that binds CD3 on the surface of a T cell (comprising paratope) comprises the VH region depicted in SEQ ID NO. 676.

More preferably, a polypeptide/polypeptide construct according to the invention comprising a domain binding to CD3 on the surface of a T cell (comprising paratope) comprises a VL region and a VH region selected from the group consisting of the VL region and the VH region depicted in the corresponding SEQ ID numbers exemplified in the sequence listing, in particular the following pairs of VL and VH regions, in particular the pairs of VL and VH regions depicted in SEQ ID numbers 507+514, 508+519, 509+521, 510+525, 511+528, 512+532, 534+513, 535+515, 536+516, 537+517, 538+518, 539+520, 540+522 and 541+523, and very particularly the pairs of VL and VH regions, in particular the pairs of VL and VH regions depicted in SEQ ID numbers 676+677.

A preferred embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratope) that binds CD3 on the surface of a T cell, comprising an amino acid sequence selected from the group consisting of SEQ ID NOS 542-562 and SEQ ID NO 678.

One particular embodiment of the above-described polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratope) that binds to CD3 on the surface of a T cell, comprising the amino acid sequence depicted in SEQ ID NO: 678.

One particular embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain that binds to CD3 on the surface of a T cell (comprising the paratope), which domain comprises the amino acid sequence depicted in SEQ ID NO:678, and wherein the domain that binds to CLDN6 (comprising the paratope (i.e. antigen binding (epitope binding) structure)) (or a domain that competes for binding to CLDN 6) consists of a pair of VH and VL domains comprising the amino acid sequences depicted in: SEQ ID NO 11+12, SEQ ID NO 25+26, SEQ ID NO 39+40, SEQ ID NO 53+54, SEQ ID NO 67+68, SEQ ID NO 81+82, SEQ ID NO 95+96, SEQ ID NO 109+110, SEQ ID NO 123+124, SEQ ID NO 137+138, SEQ ID NO 151+152, SEQ ID NO 165+166, SEQ ID NO 179+180, SEQ ID NO 193+194, SEQ ID NO 207+208, SEQ ID NO 221+222, SEQ ID NO 235+236, SEQ ID NO 249+250, or SEQ ID NO 263+264.

A specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratope) that binds to CD3 on the surface of a T cell comprising the amino acid sequence depicted in SEQ ID NO:678, and wherein the domain that binds to CLDN6 (comprising paratope (i.e. antigen binding (epitope binding) structure)) consists of the amino acid sequence depicted in SEQ ID NO, or the domain that competes for binding to CLDN6 comprises the amino acid sequence depicted in SEQ ID NO: SEQ ID NO 19, SEQ ID NO 22, SEQ ID NO 33, SEQ ID NO 36, SEQ ID NO 47, SEQ ID NO 50, SEQ ID NO 61, SEQ ID NO 64, SEQ ID NO 75, SEQ ID NO 78, SEQ ID NO 89, SEQ ID NO 92, SEQ ID NO 103, SEQ ID NO 106, SEQ ID NO 117, SEQ ID NO 120, SEQ ID NO 131, SEQ ID NO 134, SEQ ID NO 145, SEQ ID NO 148, SEQ ID NO 159, SEQ ID NO 162, SEQ ID NO 173, SEQ ID NO 176, SEQ ID NO 187, SEQ ID NO 190, SEQ ID NO 201, SEQ ID NO 204, SEQ ID NO 215, SEQ ID NO 218, SEQ ID NO 229, SEQ ID NO 232, SEQ ID NO 243, SEQ ID NO 246, SEQ ID NO 257, or SEQ ID NO 260, SEQ ID NO 271 or SEQ ID NO 274.

One particular embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratope) that binds to CD3 on the surface of a T cell, comprising the amino acid sequence depicted in SEQ ID No. 678, and wherein the domain that binds to CLDN6, comprising paratope (i.e. antigen binding (epitope binding) structure), is depicted in SEQ ID No. selected from the group consisting of SEQ ID NOs 19, 22, 33, 36, 47, 50, 75, 78, 201 and 204, in particular SEQ ID NOs 19 and 22, very particularly SEQ ID No. 22.

Another specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratope) that binds to CD3 on the surface of a T cell, comprising the amino acid sequence depicted in SEQ ID No. 678, and wherein the domain that binds to CLDN6, comprising paratope (i.e. antigen binding (epitope binding) structure) is depicted in SEQ ID No. 22.

A very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratopes) that binds to CD3 on the surface of a T cell, said domain comprising the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain (comprising paratopes) that binds to CD3 on the surface of a T cell comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in SEQ ID NOs 673, 674 and/or 675, and wherein the domain (comprising paratope (i.e. antigen binding) structure)) that binds to CLDN6 is depicted in any of the sequences depicted in SEQ ID NOs 22, 36, 50, 78 and 204.

Another very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain that binds to CD3 on the surface of a T cell (comprising paratope), wherein the domain comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in SEQ ID NOs 673, 674 and/or 675, and wherein the domain that binds to CLDN6 (comprising paratope (i.e. antigen binding (epitope binding) structure)) comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in SEQ ID NOs 13, 14 and/or 15, and/or wherein the domain (comprising paratope (i.e. antigen binding (epitope binding) structure)) comprises the VL sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences depicted in SEQ ID NOs 16, 17 and/or 18.

Yet another very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratopes) that binds to CD3 on the surface of a T cell, wherein said domain comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences shown in SEQ ID NOs 673, 674 and/or 675, and wherein said domain that binds to CLDN6 (comprising paratopes (i.e. antigen binding) structure)) comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 27, 28 and/or 29, and/or wherein the domain (comprising paratopes (i.e. antigen binding) structure) comprises any of the VH CDR sequences HCDR1, HCDR2 and/or LCDR3 depicted in any of the sequences shown in SEQ ID NOs 30, 31 and/or 29.

Yet another very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratopes) that binds to CD3 on the surface of a T cell, wherein said domain comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences depicted in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences depicted in SEQ ID NOs 673, 674 and/or 675, and wherein said domain that binds to CLDN6 (comprising paratope (i.e. antigen binding) structure)) comprises the VH sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences depicted in SEQ ID NOs 41, 42 and/or 42, and/or wherein the domain (comprising paratope (i.e. antigen binding) structure)) comprises the VL sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences depicted in SEQ ID NOs 44, 45 and/or 46.

Yet another very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratopes) that binds to CD3 on the surface of a T cell, wherein said domain comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences shown in SEQ ID NOs 673, 674 and/or 675, and wherein said domain that binds to CLDN6 (comprising paratopes (i.e. antigen binding) structure)) comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 69, 70 and/or 71, and/or wherein the domain (comprising paratopes (i.e. antigen binding) comprises any of the sequences shown in SEQ ID NOs 72, 73 and/or 74 and/or LCDR2 depicted in any of the sequences HCDR3, LCDR2 and/or LCDR3.

Yet another very specific embodiment of the above polypeptide/polypeptide construct according to the invention is characterized by a domain (comprising paratopes) that binds to CD3 on the surface of a T cell, wherein said domain comprises the VH CDR sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 670, 671 and/or 672, and/or wherein the domain comprises the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences shown in SEQ ID NOs 673, 674 and/or 675, and wherein said domain that binds to CLDN6 (comprising paratope (i.e. an epitope binding) structure) comprises the VH sequences HCDR1, HCDR2 and/or HCDR3 depicted in any of the sequences shown in SEQ ID NOs 195, 196 and/or 197, and/or wherein the domain (comprising paratope (i.e. an epitope binding) structure) comprises any of the VL CDR sequences LCDR1, LCDR2 and/or LCDR3 depicted in any of the sequences shown in SEQ ID NOs 198, 196 and/or 200.

Nucleic acid, host cell and process for producing the compounds of the invention

In a second aspect, it is further envisaged in the context of the present invention to provide a polynucleotide encoding a polypeptide construct of the present invention as depicted in any of the preceding sections.

It is also contemplated in the context of the present invention to provide a vector comprising a polynucleotide of the present invention.

Further, the present invention provides host cells transformed or transfected with the polynucleotides or vectors of the present invention.

It is also contemplated in the context of the present invention to provide a process for producing a polypeptide construct of the present invention, said process comprising culturing a host cell of the present invention under conditions allowing expression of the construct and recovering the produced polypeptide construct from the culture.

Pharmaceutical compositions of the invention

In another aspect, the invention provides a pharmaceutical composition comprising a polypeptide compound of the invention or a polypeptide compound produced according to the process of the invention.

Within this aspect, it is also contemplated in the context of the present invention that the pharmaceutical composition is stable at about-20 ℃ for at least four weeks.

Therapeutic uses/methods of the invention

It is further contemplated in the context of the present invention to provide a polypeptide compound of the present invention and a pharmaceutical composition comprising a polypeptide compound produced according to the process of the present invention for use as a medicament, in particular for the prevention, treatment or alleviation of a disease selected from the group consisting of a proliferative disease, a neoplastic disease, a cancer or an immunological disorder.

It is further contemplated in the context of the present invention to provide a method for treating or alleviating a proliferative disease, a neoplastic disease, a cancer or an immunological disorder, the method comprising the step of administering a polypeptide compound or pharmaceutical composition of the present invention to a subject in need thereof, wherein optionally the compound is produced according to the process of the present invention.

Preferably, the disease is selected from the group comprising various types of CLDN6 expressing cancers selected from the group consisting of: urinary bladder cancer, ovarian cancer (particularly ovarian adenocarcinoma and ovarian teratocarcinoma), lung cancer (including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma), stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer (particularly basal cell carcinoma and squamous cell carcinoma), malignant melanoma, head and neck cancer (particularly malignant polymorphous adenoma), sarcoma (particularly synovial sarcoma and carcinoma sarcoma), cholangiocarcinoma, urinary bladder cancer (particularly transitional cell carcinoma and papillary carcinoma), kidney cancer (particularly renal cell carcinoma, including clear cell renal cell carcinoma and papillary carcinoma), colon cancer, small intestine cancer (including ileocecal carcinoma, in particular small intestine adenocarcinoma and ileal adenocarcinoma), testicular embryo cancer, placental choriocarcinoma, cervical cancer, testicular cancer (in particular testicular seminoma, testicular teratoma and embryonic testicular cancer), uterine cancer, germ cell tumors (such as teratocarcinoma or embryonal carcinoma, in particular testicular germ cell tumors and their metastatic forms, very particularly testicular germ cell cancer), ovarian cancer (in particular ovarian serous cyst adenocarcinoma) and uterine cancer (such as endometrial cancer), lung cancer (including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), such as lung adenocarcinoma), triple negative breast cancer, gastric cancer, cholangiocarcinoma, esophageal cancer, wilms tumor, rhabdoid tumor, in particular ovarian cancer, uterine cancer and/or lung cancer, and more particularly ovarian serous cyst adenocarcinoma, uterine carcinoma sarcoma, endometrial carcinoma, and/or, in particular, squamous cell lung carcinoma and lung adenocarcinoma.

Also provided is the use of a compound as described herein in the manufacture of a medicament for the treatment or prevention or amelioration of a neoplastic disease, particularly ovarian, uterine and/or lung cancer.

It is further contemplated in the context of the present invention to provide a method for treating or alleviating gastrointestinal cancer comprising the step of administering to a subject in need thereof a construct for CLDN6 and CD 3.

It is also contemplated in the context of the present invention to provide polypeptide/polypeptide constructs directed against CLDN6 and CD3 for use as a medicament, in particular for use in the treatment or amelioration of e.g. ovarian cancer, uterine cancer, lung cancer (in particular ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma), lung cancer (including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma).

The kit of the invention

In another aspect, it is also contemplated in the context of the present invention to provide a kit comprising a polypeptide construct of the present invention or an antibody construct produced according to the process 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.

Definition of terms according to the invention

The term "polypeptide construct" (alternatively abbreviated as "compound") refers to an antigen binding (or epitope binding) molecule comprising a domain that itself contains a paratope. In the context of the present invention, a polypeptide construct is understood to be an organic polymer comprising at least one continuous, unbranched amino acid chain which does not occur naturally but is engineered. An example of a polypeptide construct that is a single polypeptide is one that comprises a core structure A molecule comprising at least one functional target binding domain and at least one complete functional CD3 binding domain on a single polypeptide chain, wherein these domains are directly linked via a flexible peptide ("linker") without any further intervening domains, unlike Xmab, which comprises a target conjugate and a CD3 conjugate on different polypeptide chains. In the context of the present invention, such polypeptide constructs comprising more than one amino acid chain are also envisaged. Preferably, surgeryThe term "polypeptide" is used in connection with single-chain forms of the compounds of the invention, whereas "polypeptide construct" may preferably also be more suitable to describe polypeptides comprising more than one polypeptide chain (e.g. two, three or four polypeptide chains). Furthermore, the term "polypeptide construct" is also suitable for describing a compound of the invention comprising one or more non-amino acid based components, such as human serum albumin and the like (HSA). The polypeptide amino acid chain typically comprises at least 50 amino acids, preferably at least 100, 200, 300, 400 or 500 amino acids. It is also envisaged in the context of the present invention that the amino acid chains of the polymer are linked to entities which do not consist of amino acids.

Polypeptides include structural and/or functional features based on the structure and/or function of an antibody (e.g., a full-length immunoglobulin molecule). Thus, the polypeptide construct binds specifically and preferably selectively or immunospecifically to its target or antigen, more precisely to an epitope of said target or target antigen, and/or it comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) naturally occurring in antibodies, or comprises a domain derived therefrom. Thus, the construct may alternatively be considered to comprise paratope structuring (i.e. paratope formation) and epitope binding structures, such as those found in natural antibodies or fragments thereof. The polypeptide constructs according to the invention comprise the minimum antibody structural requirement to allow binding of an immunospecific target, i.e. to immunospecifically or immunoselectively recognize the paratope of an epitope on the target antigen. Such minimum requirements may 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. Thus, a polypeptide construct may be characterized by the presence of three or six CDRs in one or both binding domains, and the skilled artisan knows where (in what order) those CDRs are located within the paratope binding structure. As used herein, the term "antigen binding structure" refers to any polypeptide comprising an antigen binding structure or any molecule having binding activity to an antigen. Peptides and proteins are not limited to those derived from living organisms, for example, they may be polypeptides produced from artificially designed sequences. They may also be any of naturally occurring polypeptides, synthetic polypeptides, recombinant polypeptides, and the like. Since the antigen binding structures according to the invention bind specifically to parts of the antigen, i.e. they bind specifically to epitopes, the antigen (epitope) binding structure may also be defined as a "paratope". Thus, a polypeptide/polypeptide construct according to the invention may also be defined as comprising a domain that preferentially immunospecifically or immunoselectively binds to a target antigen/target epitope and another paratope domain that preferentially immunospecifically or immunoselectively binds to another target antigen/target epitope of a CD3 molecule as defined herein. Thus, whenever the present specification refers to a domain of a construct or molecule of the invention, the construct comprises at least one paratope (or paratope) that binds CLDN6 as defined herein (in particular according to any of the appended claims), and another paratope that binds CD3 as defined herein.

The term "antibody" as used according to the invention comprises full length antibodies, but also 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 (deimmunized) antibodies, humanized antibodies, and human antibodies, as well as antibodies from other species such as mice, hamsters, rabbits, rats, goats, or non-human primates.

The "polypeptide/polypeptide construct" of the invention may also comprise a naturally occurring full-length immunoglobulin structure. For example, they may comprise (at least) two full length antibody heavy chains and two full length antibody light chains. However, whereas the polypeptide/polypeptide construct according to the invention comprises one domain of the paratope binding to CLDN6 and another domain of the paratope binding to CD3, they are not naturally occurring and they differ significantly from naturally occurring products in their function. Thus, a polypeptide or polypeptide construct of the invention is an artificial "hybrid" molecule comprising different binding domains with different specificities and/or selectivities.

As mentioned above, the polypeptides of the invention may comprise more than one polypeptide chain, i.e. polypeptides comprising two or more polypeptide chains are also subject to the present invention, in particular polypeptides forming a three-dimensional proteinaceous structure allowing immunospecific binding to CLDN6 and CD 3. Thus, the definition of the term "polypeptide 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 hetero-oligomers). Examples of Antibodies and fragments, variants, derivatives and constructs derived therefrom identified above are described in particular in Harlow and Lane, antibodies: A laboratory manual [ Antibodies: laboratory Manual ], CSHL Press [ Cold spring harbor laboratory Press ] (1988); kontermann and Dubel, antibody Engineering [ antibody engineering ], springer [ Springer Press ], 2 nd edition 2010; little, recombinant Antibodies for Immunotherapy [ recombinant antibodies for immunotherapy ], cambridge University Press [ Cambridge university Press ]2009

The "polypeptide/polypeptide construct" of the invention may also comprise fragments of full-length antibodies, such as VH, VHH, VL,(s) dabs, fv, light chain (VL-CL), fd (VH-CH 1), heavy chain, fab ', F (ab') 2 or "igg" (a "half antibody" consisting of heavy and light chains). The polypeptide/polypeptide construct according to the invention may also comprise modified fragments of antibodies, also referred to as antibody variants or antibody derivatives. Examples include, but are not limited to, scFv, di-scFv or bi(s) -scFv, scFv-Fc, scFv-zipper (zipper), scFab, fab2, fab3, diabody, single chain diabody, tandem diabody (Tandab), tandem di-scFv, tandem tri-scFv, minibody ", exemplified by the following structures: (VH-VL-CH 3) 2, (scFv-CH 3) 2, ((scFv) 2-ch3+ch3), ((scFv) 2-CH 3) or (scFv-CH 3-scFv) 2, multimeric antibodies such as tri-or tetra-antibodies (tetrabodies), and single domain antibodies (such as nanobodies or single variable domain antibodies comprising only one variable region, which may be VHH, VH or VL, which selectively and preferably specifically bind to an antigen or target independently of other variable regions or domains). Other possible forms of the polypeptide/polypeptide construct according to the invention are a cross-over, a max, an iso-Fc construct, a mono-Fc construct and a scFc construct. Examples of those forms will be described below.

Furthermore, the definition of the term "polypeptide construct" includes bivalent and multivalent (polyvalent/multispecific) polypeptides/polypeptide constructs as well as bispecific/multispecific polypeptides/polypeptide constructs that selectively and preferably specifically bind to two, three or more antigen structures (epitopes) via different binding domains. The polypeptide construct may have a more specific binding valency, for example in the case where two binding domains are directed against one target (CLDN 6) and one binding domain is directed against the other target (CD 3) or vice versa, in which case the polypeptide construct is trivalent and bispecific. In general, the term "bispecific" includes the meaning of a polypeptide construct binding to (at least) two different antigens (e.g., CLDN6 and CD 3).

The terms "paratope", "antigen binding domain", "epitope binding domain", "binding domain" or "domain binding to … …" characterize the domain of the construct in connection with the present invention which selectively and preferably specifically or immunospecifically binds/interacts/recognizes an epitope on the target or antigen (here: CLDN6 in the case of the first domain and CD3 in the case of the second domain). With respect to the "constructs" described herein, the term "binding domain" or "domain that binds to … …" or "domain" characterizes the domain of the construct in connection with the present invention that immunospecifically binds/interacts/recognizes an epitope (i.e., selectively interacts with certain amino acids) on a target or antigen. The structure and function of the first domain (binding to the target antigen) and preferably the structure and/or function of the second domain (binding to CD 3) are based on the structure and/or function of an antibody (e.g. a full length immunoglobulin polypeptide). Thus, a "binding domain" or "domain that binds to … …" may comprise the minimum structural requirements of an antibody that allows for immunospecific target binding. Such minimum structural requirements of the first domain may 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 envisaged that the second domain also comprises such an antibody minimum structural requirement that allows for 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. The "domain that binds … …" (or "binding domain") typically can comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it need not contain both, but may contain only one of VH or VL. For example, fd fragments typically retain some of the antigen binding function of the complete antigen binding domain. As used herein, the terms "paratope", "antigen binding structure" and "epitope binding structure" also refer to a portion of an antibody (or molecule according to the invention) that comprises a region that specifically binds to and is complementary to all or part of an antigen or a portion thereof, i.e., an antibody can only bind to a specific portion of an antigen. This particular moiety is referred to as an "epitope". The antigen binding domain may be provided by one or more antibody variable domains. Preferably, the antigen binding domain comprises an antibody variable region comprising an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). Such preferred antigen binding domains include, for example, "single chain Fv (scFv)", "single chain antibody", "Fv", "single chain Fv2 (scFv 2)", "Fab" and "F (ab') 2". "paratope" is also characterized by a specific amino acid that chemically interacts with a specific amino acid flanking the epitope (antigen/target).

Examples of forms of "domain that binds … …", "domain comprising paratope" (or "binding domain", "antigen binding structure", "epitope binding structure") include, but are not limited to, full length antibodies, fragments of full length antibodies (e.g., VH, VHH, VL),(s) dAb, fv, light chain (VL-CL), fd (VH-CH 1), heavy chain, fab ', F (ab') 2, or "r IgG" ("half antibody")), antibody variants or derivatives (e.g., scFv, di-scFv, or bi(s) -scFv, scFv-Fc, scFv-zipper, scFab, fab2, fab3, diabody, single chain diabody, tandem diabody (Tandab), tandem di-scFv, tandem tri-scFv, "minibody" (selected from forms such as (VH-VL-CH 3) 2, (scFv-CH 3) 2, ((scFv) 2-CH 3) or (scFv-CH 3) 2, multi-antibodies (e.g., tri-or tetrabodibodies), and single domain antibodies (e.g., diabodies or diabodies) (variable region, VH, variable region, or variable region, only). Other examples of forms of "domain that binds to … …" (or "binding domain") include (1) antibody fragments or variants (e.g., fab) comprising VL, VH, CL and CH 1; (2) Antibody fragments or variants (e.g., F (ab') 2) comprising two linked Fab fragments; (3) antibody fragments or variants (e.g., fd) comprising VH and CH 1; (4) Antibody fragments or variants (e.g., light chains) comprising VL and CL; (5) antibody fragments or variants (such as Fv) comprising VL and VH; (5) dAb fragments with VH domains (Ward et al, (1989) Nature [ 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) single chain Fv (scFv). Examples of embodiments of constructs or binding domains according to the invention are described, for example, in the following: WO 00/006605, WO 2005/040220, WO 2008/119567, WO 2010/037838, WO 2013/026837, WO 2013/026833, US 2014/0308285, US 2014/0302037, WO 2014/144722, WO 2014/151910 and WO 2015/048272. In the context of the present invention, paratope is understood as an antigen binding site that is part of a polypeptide as described herein and that recognizes and binds an antigen. Paratopes are typically small regions of about at least 5 amino acids. Paratopes as understood herein typically comprise portions of antibody-derived heavy (VH) and light (VL) chain sequences. Each binding domain of a polypeptide according to the invention provides a paratope comprising a set of 6 complementarity determining regions (CDR loops), each three of which are contained within antibody derived VH and VL sequences, respectively.

For compounds, in particular for the constructs of the invention, it is envisaged that a) the construct is a single chain polypeptide or a single chain construct, b) the first domain is in the form of an scFv, c) the second domain is in the form of an scFv, d) the first and second domains are linked via a linker, preferably a peptide linker, more preferably a glycine/serine linker, and/or e) the construct comprises a domain providing an extended serum half-life, such as an Fc-based domain or Human Serum Albumin (HSA). In the latter case, the following is a preferred embodiment, wherein the term "polypeptide construct" explicitly indicates that it comprises more than one peptide chain.

The constructs of the invention are preferably "in vitro generated constructs" and/or "recombinant constructs". In the context of the present invention, the term "in vitro generated" refers to a construct according to the definition above, wherein all or part of the binding domain or variable region (e.g. at least one CDR) is generated 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 an antigen can be tested). Thus, this term preferably excludes sequences that result solely from genomic rearrangements in animal immune cells. It is envisaged that the first and/or second domains of the construct are produced by or obtainable by phage display or library screening methods, rather than by grafting CDR sequences from pre-existing (monoclonal) antibodies into scaffolds. A "recombinant construct" is a construct that is produced or produced using, inter alia, recombinant DNA technology or genetic engineering.

It is contemplated that the constructs of the invention are monoclonal. As used herein, a polypeptide or construct designated "monoclonal" (mAb) is obtained from a population of substantially homogeneous antibodies/constructs, i.e., the individual antibodies/constructs contained in the population are identical (in particular, with respect to their amino acid sequences) except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies/constructs are highly specific for a single epitope within an antigen, as compared to polyclonal antibody preparations that typically include different antibodies directed against different determinants (or epitopes). In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by hybridoma cultures and are therefore not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody/construct as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures may be used. For example, monoclonal antibodies to be used may be prepared by the hybridoma method described for the first time by Koehler et al, nature [ Nature ],256:495 (1975), or by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Examples of additional techniques for producing human monoclonal antibodies include the triple-source hybridoma technique, the human B-cell hybridoma technique (Kozbor, immunology Today's Immunology ]4 (1983), 72), and the EBV-hybridoma technique (Cole et al Monoclonal Antibodies and Cancer Therapy [ monoclonal antibodies and cancer therapy ], alan R.List company (1985), 77-96).

Standard methods (e.g., enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE) can then be used ^TM ) The hybridomas are screened analytically to identify one or more hybridomas which produce antibodies which selectively and preferably specifically or immunospecifically bind to the indicated antigen. Any form of the relevant antigen may be used as an immunogen, e.g. a recombinant antigen, a naturally occurring form, any variant or fragment thereof and antigenic peptides thereof. Such as in BIAcore ^TM Surface plasmon resonance employed in the system can be used to increase the efficiency of phage antibodies/constructs binding to epitopes of target antigens (Schier, human Antibodies Hybridomas [ human antibody hybridomas]7 (1996), 97-105; malmbrg, J.Immunol.methods [ J.Immunol.methods ]]183(1995),7-13)。

Another exemplary method of making a construct or binding domain includes screening a protein expression library, such as a phage display or ribosome display library. Phage display is described, for example, in the following: ladner et al, U.S. Pat. nos. 5,223,409; smith (1985) Science 228:1315-1317, clackson et al, nature 352:624-628 (1991) and Marks et al, J.mol. Biol. [ J.Mol.molecular biology ],222:581-597 (1991).

In addition to using a display library, a non-human animal, e.g., a rodent (such as a mouse, hamster, rabbit, or rat) can be immunized with the relevant antigen. In one embodiment, the non-human animal comprises at least a portion of a human immunoglobulin gene. For example, it is possible to work with large fragments of the human Ig (immunoglobulin) locusCheng Huaxiao mouse strain deficient in murine antibody production. Using hybridoma technology, antigen-specific monoclonal antibodies derived from genes having the desired specificity can be generated and selected. See, e.g., xenomous ^TM The method comprises the steps of carrying out a first treatment on the surface of the Green et al (1994) Nature Genetics [ Nature Genetics ]]7:13-21; US 2003-007185; WO 96/34096 and WO 96/33735.

Monoclonal antibodies can also be obtained from non-human animals and then modified using recombinant DNA techniques known in the art, such as humanization, deimmunization, presentation of chimerism, and the like. Examples of modified constructs or binding domains include humanized variants of non-human antibodies/constructs, "affinity matured" constructs or binding domains (see, e.g., hawkins et al j. Mol. Biol. [ journal of molecular biology ]254,889-896 (1992) and Lowman et al Biochemistry [ Biochemistry ]30,10832-10837 (1991)), and antibody variants or mutants with altered effector functions (see, e.g., U.S. Pat. No. 5,648,260; kontermann and clubel (2010), the above citations, and Little (2009), the above citations).

In immunology, affinity maturation is the process of: through this process, B cells produce antibodies with increased affinity to the antigen during the course of the immune response. After repeated exposure to the same antigen, the host will produce antibodies with successively greater affinities. Similar to the natural prototype, 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, constructs or binding domains. Random mutations were introduced into the CDRs using radiation, chemical mutagens, or error prone PCR. Furthermore, genetic diversity can be increased by chain shuffling. Two or three rounds of mutation and selection using a display method (e.g., phage display) typically result in antibodies, antibody fragments, antibody variants, constructs, or binding domains with affinities in the low nanomolar range.

The preferred type of amino acid substitution change of the constructs or binding domains of the invention involves substitution of one or more residues within the hypervariable region of the parent antibody structure (e.g., humanized or human antibody structure). Generally, the resulting variant or variants selected for further development will have improved biological properties relative to the parent antibody structure from which they were derived. A convenient way to generate such substitution variants involves affinity maturation using phage display. Briefly, several sites (e.g., 6-7 sites) of the hypervariable region are mutated to produce all possible amino acid substitutions at each site. The variants thus produced are displayed in a monovalent manner from the filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for biological activity (e.g., binding affinity) as disclosed herein. Alanine scanning mutagenesis may also be performed in order to identify candidate hypervariable region sites (modification candidates) that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the complex between the antigen and the construct or binding domain to identify the point of contact between the binding domain and its specific antigen. Such contact residues and adjacent residues are candidates for substitution according to the techniques set forth herein. Once such variants are produced, the set of variants is screened as described herein, and antibodies, antigen binding fragments, constructs, or binding domains thereof having superior properties in one or more relevant assays may be selected for further development.

Constructs and binding domains of the invention specifically include "chimeric" forms (wherein a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass), as well as fragments or variants of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; morrison et al, proc.Natl. Acad. Sci. USA [ national academy of sciences USA ]81:6851-6855 (1984)); chimeric constructs or binding domains of interest herein include "primatized" constructs comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.), and human constant region sequences. Various methods for preparing chimeric antibodies or constructs have been described. See, e.g., morrison et al, proc.Natl. Acad. ScL U.S.A. [ Proc. Natl. Acad. Sci. USA ]81:6851,1985; takeda et al Nature [ Nature ]314:452,1985; cabill et al, U.S. patent nos. 4,816,567; boss et al, U.S. Pat. nos. 4,816,397; tanaguchi et al, EP 0171496; EP 0173494; and GB 2177096.

Antibodies, polypeptide constructs, antibody fragments, antibody variants or binding domains may also be modified by specific deletion of human T cell epitopes (a method known as "deimmunization") using, for example, the methods disclosed in WO 98/52976 or WO 00/34317. Briefly, the heavy and light chain variable regions of an antibody, construct or binding domain 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). To detect potential T cell epitopes, a computer modeling method called "peptide threading" can be applied and furthermore 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 thus constitute potential T cell epitopes. The potential T cell epitope detected may be eliminated by substitution of a small number of amino acid residues in the variable domain or variable region, or preferably by a single amino acid substitution. Typically, conservative substitutions are made. Generally, but not exclusively, amino acids common to positions in the human germline antibody sequence may be used. Human germline sequences are disclosed, for example, in the following: tomlinson et al (1992) J.MoI.biol. [ journal of molecular biology ]227:776-798; cook, G.P. et al (1995) immunol. Today's immunology 16 volume (5): 237-242; and Tomlinson et al (1995) EMBO J. [ J. European molecular biology 14:14:4628-4638). The V BASE catalog (www 2.MRC-lmb.cam.ac.uk/vbase/list2. Php) provides a comprehensive catalog of human immunoglobulin variable region sequences (assembled MRC Centre for Protein Engineering by Tomlinson, LA. et al, cambridge, UK [ Cambridge MRC protein engineering center, UK ]). These sequences can be used as a source of human sequences such as framework regions and CDRs. Common human frame regions may also be used, for example as described in U.S. Pat. No. 6,300,064.

"humanized" antibodies, variants or fragments thereof, constructs and binding domains are immunoglobulins based on primary human sequences which contain one or more minimum sequences derived from non-human immunoglobulins. In most cases, humanized antibodies, variants or fragments thereof, constructs and binding domains are based on human immunoglobulins (recipient antibody) in which residues from a hypervariable region or CDR are replaced by residues from a hypervariable region or CDR of a non-human species (donor antibody), such as a rodent (e.g., mouse, hamster, rat, or rabbit) having the desired specificity, affinity, capacity and/or biological activity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, a "humanized" antibody, variant or fragment thereof, antibody, construct, and binding domain may also comprise residues not found in either the recipient antibody or the donor antibody. These modifications are made to further improve and optimize antibody performance. The humanized antibodies, variants, or fragments thereof, constructs, and binding domains may also comprise at least a portion of an immunoglobulin constant region (e.g., fc), typically that of a human immunoglobulin. For more details, see Jones et al Nature, 321:522-525 (1986); reichmann et al Nature [ Nature ],332:323-329 (1988); and Presta, curr.Op.struct.biol. [ New structural biology ],2:593-596 (1992).

Humanized antibodies, variants or fragments thereof, constructs, and binding domains may be generated by substituting sequences of human (Fv) variable regions for those not directly involved in antigen binding. 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 or light chain. Such nucleic acids may be obtained from hybridomas producing antibodies to the intended target as described above, as well as other sources. Recombinant DNA encoding the humanized antibody, variant or fragment thereof, construct or binding domain may then be cloned into a suitable expression vector.

Humanized antibodies, variants or fragments thereof, constructs, and binding domains may also be produced using transgenic animals (e.g., mice expressing human heavy and light chain genes but not endogenous mouse immunoglobulin heavy and light chain genes). Winter describes an exemplary CDR grafting method that can be used to prepare the humanized molecules described herein (U.S. Pat. No. 5,225,539). All CDRs of a given human sequence may be replaced with at least a portion of the non-human CDRs, or only some CDRs may be replaced with non-human CDRs. Only the number of CDRs required for binding the humanized molecule to the predetermined antigen need be replaced.

Humanized antibodies, variants or fragments thereof, constructs or binding domains may be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and/or back mutations. Such altered immunoglobulin molecules may be prepared by any of several techniques known in the art (e.g., teng et al, proc. Natl. Acad. Sci. U.S.A. [ Proc. Natl. Acad. Sci. U.S. A., U.S. Sci. A., 80:7308-7312,1983; kozbor et al, immunology Today, 4:7279,1983; olsson et al, meth. Enzymol. [ methods of enzymology ],92:3-16,1982, and EP 239 400).

Human anti-mouse antibody (HAMA) responses have led the industry to the preparation of chimeric or other humanized antibodies/constructs. However, it is expected that certain human anti-chimeric antibody (HACA) responses will be observed, particularly in the long-term or multi-dose use of antibodies or constructs. It is therefore desirable to provide constructs comprising a human binding domain for CLDN6 and/or a human binding domain for CD3 to eliminate problems and/or effects of HAMA or HACA reactions.

Thus, according to one embodiment, the polypeptide construct, the first binding domain and/or the further binding domain is "human". The terms "human antibody", "human construct" and "human binding domain" include antibodies, constructs and binding domains, respectively, having antibody derived regions, which are variable and constant regions or domains as substantially correspond to human germline immunoglobulin sequences known in the art, including for example those described by Kabat et al (1991) (citation above). The human constructs or binding domains of the invention may comprise amino acid residues in, for example, CDRs and in particular CDR3 that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). The human construct or binding domain may have at least 1, 2, 3, 4, 5 or more positions replaced by amino acid residues not encoded by the human germline immunoglobulin sequence. However, the definition of human antibodies, constructs and binding domains as used herein also encompasses fully human antibodies, constructs and binding domains that include only non-artificial and/or genetically altered human antibody sequences, such as those that may be derivable by use of techniques or systems such as Xenomouse.

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

The ability to clone and recombine 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 coarsely located loci and for generating useful models of human disease. Furthermore, substitution of the mouse locus with its human equivalent using this technology can provide unique insights about the expression and regulation of human gene products during development, their communication with other systems, and their involvement in disease induction and progression.

An important practical application of this strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which endogenous Ig genes have been inactivated provides an opportunity to study the underlying mechanisms of programmed expression and assembly of antibodies and their role in B cell development. Furthermore, this strategy may provide an ideal source for the production of fully human monoclonal antibodies (mabs) -an important milestone that helps to achieve the prospects of antibody therapies in human disease. The fully human antibodies or constructs derived therefrom are expected to minimize the immunogenic and allergic responses inherent to the mouse or mouse-derived mAb and thereby increase the efficacy and safety of the administered antibodies/constructs. The use of fully human antibodies or constructs can be expected to provide a number of advantages in the treatment of chronic and recurrent human diseases (such as inflammation, autoimmunity and cancer) that require repeated administration of compounds.

One way to achieve this goal is to engineer a mouse strain with defective mouse antibody production with a large fragment of the human Ig locus, which would be expected to produce a large repertoire of human antibodies 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 using a mouse mechanism to achieve antibody diversification and selection and lack of immune tolerance to human proteins, a repertoire of human antibodies regenerated in these mouse strains should produce high affinity antibodies against any antigen of interest, including human antigens. Antigen-specific human mabs with the desired specificity can be readily produced and selected using hybridoma technology. This general strategy was demonstrated in connection with the generation of the first Xenomouse strain (see Green et al Nature Genetics [ Nature Genetics ]7:13-21 (1994)). The XenoMouse strain was engineered with Yeast Artificial Chromosomes (YACs) containing 245kb and 190kb sized germline conformational fragments of the human heavy chain locus and kappa light chain locus, respectively, containing core variable and constant region sequences. YACs containing human Ig proved to be compatible with the mouse system to rearrange and express antibodies and to be able to replace the inactivated mouse Ig genes. This is demonstrated by its ability to induce B cell development, to produce adult-like human repertoires of fully human antibodies, and to produce antigen-specific human mabs. These results also demonstrate that the introduction of a human Ig locus containing a greater number of V genes, additional regulatory elements, and a greater portion of the human Ig constant region can substantially reproduce the complete repertoire as a feature of human fluid responses to infection and immunization. The work of Green et al extends to the introduction of greater than about 80% of human antibody repertoires by the introduction of germline configured YAC fragments of megabase-sized human heavy chain loci and kappa light chain loci, respectively. See Mendez et al Nature Genetics [ Nature Genetics ]15:146-156 (1997) and U.S. patent application Ser. No. 08/759,620.

The generation of the XenoMouse model is further discussed and described in the following: U.S. patent application Ser. No. 07/466,008, ser. No. 07/610,515, ser. No. 07/919,297, ser. No. 07/922,649, ser. No. 08/031,801, ser. No. 08/112,848, ser. No. 08/234,145, ser. No. 08/376,279, ser. No. 08/430,464, ser. No. 08/584, ser. No. 08/464,582, ser. No. 08/463,191, ser. No. 08/462,837, ser. No. 08/486,853, ser. No. 08/486,857, ser. No. 08/486,859, ser. No. 08/462,513, ser. No. 08/724,752, ser. No. 08/759,620; and U.S. patent No. 6,162,963;6,150,584;6,114,598;6,075,181 and 5,939,598, and japanese patent nos. 3 068 180b2, 3 068 506b2, and 3 068507B2. See also Mendez et al Nature Genetics [ Nature Genetics ]15:146-156 (1997), green and Jakobovits J.exp. Med. [ journal of laboratory medicine ]188:483-495 (1998), EP 0 463 B1, WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310 and WO 03/47336.

In an alternative approach, other companies, including the genuine pharmaceutical international company (GenPharm International, inc.), utilize the "microlocus" approach. In the minilocus approach, exogenous Ig loci are mimicked by inclusion of fragments (individual genes) from the Ig loci. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed as constructs for insertion into an animal. This method is described in the following: surani et al, U.S. patent No. 5,545,807 and U.S. patent 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 (Lonberg and Kay, respectively), krimpenfort and Berns, U.S. patent nos. 5,591,669 and 6,023.010, berns et al, U.S. patent nos. 5,612,205;5,721,367; and 5,789,215, and U.S. Pat. No. 5,643,763 to Choi and Dunn, and International patent application Ser. No. 07/574,748 to real medicine (GenPharm), ser. No. 07/575,962, ser. No. 07/810,279, ser. No. 07/853,408, ser. No. 07/904,068, ser. No. 07/990,860, ser. No. 08/053,131, ser. No. 08/096,762, ser. No. 08/155,301, ser. No. 08/161,739, ser. No. 08/165,699, ser. No. 08/209,741. See also EP 0 546 073 B1, WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884 and 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).

Kirin also demonstrates the production of human antibodies from mice that have been introduced into a large chromosome or whole chromosome by minicell fusion. See European patent application Nos. 773 288 and 843 961.Xenerex Biosciences techniques for potential production of human antibodies are being developed. In this technique, SCID mice are reconstituted with human lymphocytes (e.g., B and/or T cells). The mice are then immunized with the antigen and an immune response can be generated against the antigen. See U.S. patent No. 5,476,996;5,698,767; and 5,958,765.

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

According to one embodiment, the entire 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 moiety, such polypeptides or proteins may include non-protein moieties (e.g., chemical linkers or chemical cross-linking agents such as glutaraldehyde).

Peptides are short chains of amino acid monomers linked via covalent peptide (amide) bonds. Thus, peptides belong to a broad chemical class of biological oligomers and polymers. Amino acids that are part of a peptide or polypeptide chain are referred to as "residues" and may 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 consist of only a few amino acids (typically between two and twenty). Polypeptides are longer, continuous and unbranched peptide chains. Peptides differ from proteins based on size; and as an arbitrary basis, a peptide may be understood to contain about 50 amino acids or less. Proteins consist of one or more polypeptides, typically arranged in a biologically functional manner. Although the laboratory techniques applied to peptides and polypeptides and proteins differ in various aspects (e.g., characteristics of electrophoresis, chromatography, etc.), the dimensional boundaries that distinguish peptides from polypeptides and proteins are not absolute. Thus, in the context of the present invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably, and the term "polypeptide" is generally preferred.

As mentioned above, the polypeptide may further form multimers (e.g., dimers, trimers, and higher oligomers) consisting of more than one polypeptide molecule. The polypeptide molecules forming such dimers, trimers, etc. may be identical or different. Accordingly, the corresponding higher order structures of such multimers are referred to as homo-or heterodimers, homo-or heterotrimers, and the like. Examples of heteromultimers are antibodies or immunoglobulin molecules, the naturally occurring form of which 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 effected, for example, by post-translational modification (e.g., glycosylation, acetylation, phosphorylation, etc.). As referred to herein, a "peptide," "polypeptide," or "protein" may also be chemically modified, such as pegylated. Such modifications are well known in the art and are described below.

The terms "selectively" and "preferably selectively", "(specifically or immunospecifically) bind", "(specifically or immunospecifically) recognize", or "react with … … (specifically or immunospecifically)" mean that a construct or binding domain selectively interacts or (immunospecifically) interacts with a given epitope on a target molecule (antigen) (here: CLDN6 and CD3, respectively) according to the invention. This interaction or binding occurs more frequently, more rapidly in an epitope on a particular target (CLDN 6 herein) than in an alternative substance (non-target molecule, e.g., CLDN4, CLDN9, CLDN3 herein, etc.), with a longer duration, with a greater affinity, or with some combination of these parameters. However, due to sequence similarity between homologous proteins in different species, constructs or binding domains that selectively and/or immunospecifically bind their targets (e.g., human targets) may cross-react with homologous target molecules from different species (e.g., from non-human primates). Thus, the terms "specifically bind", "specific/immunospecific binding" and the like may include binding of a construct or binding domain to an epitope or structurally related epitope in more than one species. In the context of the present invention, a polypeptide of the invention binds in a specific manner to its corresponding target structure. Preferably, each binding domain of a polypeptide according to the invention comprises a paratope, which binding domain "specifically or immunospecifically" binds to its corresponding target structure, "(specifically or immunospecifically) recognizes" its corresponding target structure, or reacts "(specifically or immunospecifically) with its corresponding target structure". According to the invention, this means that the polypeptide or binding domain thereof interacts or (immunospecifically) with a given epitope on the target molecule (antigen) and CD3, respectively. This interaction or binding occurs more frequently, more rapidly in epitopes on a particular target than in alternative substances (non-target molecules), with longer duration, with greater affinity, or with some combination of these parameters. However, due to sequence similarity between homologous proteins in different species, antibody constructs or binding domains that immunospecifically bind their targets (e.g., human targets) may cross-react with homologous target molecules from different species (e.g., from non-human primates). Thus, the term "specific/immunospecific binding" may include binding of an antibody construct or binding domain to an epitope and/or a structurally related epitope in more than one species. The term (immunological) selective binding does not include binding to a structurally related epitope.

In the context of the present invention, the term "epitope" refers to a portion or region of an antigen that is selectively recognized/immunospecifically recognized by a binding structure (i.e., paratope). An "epitope" is antigenic, and thus the term epitope is sometimes also referred to as an "antigenic structure" or "antigenic determinant". The portion of the binding domain that binds to an epitope is called the paratope. Specific binding is thought to be achieved by specific motifs in the amino acid sequence of the binding domain and antigen. Thus, binding is achieved due to their primary, secondary and/or tertiary structure and the potential secondary modification of the structure. Specific interactions of paratopes with their antigenic determinants can result in simple binding of the site to the antigen. In some cases, the specific interaction may alternatively or additionally lead to initiation of a signal, e.g. due to induction of a change in antigen conformation, oligomerization of the antigen, etc.

Epitopes of protein antigens are classified into two classes (conformational epitopes and linear epitopes) based on the structure of the protein antigen and the interaction with paratopes. Conformational epitopes consist of an uninterrupted portion of the antigen amino acid sequence. These epitopes interact with complementary bits based on the three-dimensional surface features and shape or tertiary structure (folding) of the antigen. Methods for determining epitope conformation include, but are 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. In contrast, linear epitopes interact with complementary epitopes based on their primary structure. Linear epitopes are formed by sustained amino acid sequences from antigens and typically comprise at least 3 or at least 4, and more usually at least 5 or at least 6 or at least 7, for example from about 8 to about 10 amino acids in a unique sequence.

The following describes the method for CLDN6 epitope mapping: predefined regions (contiguous amino acid extensions) within the extracellular loop of the human CLDN6 protein are exchanged/substituted with corresponding regions of a CLDN6 paralog (e.g., human CLDN4 or human CLDN18.2, although other paralogs are also contemplated, so long as the binding domain does not cross-react with the paralog used). These human CLDN 6/paralog chimeras are expressed on the surface of host cells (e.g., CHO cells). Binding of the antibody or construct can be tested via FACS analysis. When binding of the antibody or construct to the chimeric molecule is completely eliminated, or when a significant decrease in binding is observed, it can be concluded that the human CLDN6 region removed from this chimeric molecule is associated with immunospecific epitope-paratope recognition. The reduction in binding is preferably at least 10%, 20%, 30%, 40% or 50% compared to binding of human (wild-type) CLDN 6; more preferably at least 60%, 70% or 80%, and most preferably 90%, 95% or even 100%, whereby the binding to human CLDN6 is set to 100%. Alternatively, the epitope mapping analysis described above may be modified by introducing one or several point mutations into the sequence of CLDN6, in particular in the sequence of extracellular loop 1 or loop 2, more in particular in the sequences corresponding to the E1A and/or E2B regions of these loops depicted in SEQ ID NOs 9 and 10. These point mutations may, for example, reflect differences between CLDN6 and its closely related paralog CLDN 4.

Another method of determining the contribution of a specific residue of a target antigen to the recognition of a construct or binding domain is alanine scanning (see, e.g., morrison KL and Weiss GA.Cur Opin Chem Biol. [ New Biol. Chemical biology ] 6 month 2001; 5 (3): 302-7), wherein each residue to be analyzed is replaced by alanine, e.g., via site-directed mutagenesis. Alanine is used because it has a non-bulky, chemically inert methyl function, but still mimics the secondary structural references that many other amino acids have. In cases where the size of the conservatively mutated residue is desired, a large amino acid (e.g., valine or leucine) may sometimes be used.

The interaction between the binding domain and an epitope of the target antigen means that the binding domain exhibits considerable or significant affinity for the epitope/target antigen (here: CLDN6 and CD3, respectively) and typically does not exhibit significant affinity for proteins or antigens other than the target antigen (here: CLDN6/CD 3) -although cross-reactions with homologous targets, e.g., from other species, or CLDN9 of the same species (particularly human CLDN6 and CLDN 9) are discussed above. "significant affinity" includes binding with an affinity of 10-6M (dissociation constant, KD). Preferably, binding is considered specific when the binding affinity is 10-7M, 10-8M, 10-9M, 10-10M, or even 10-11M, or 10-12M. Whether a binding domain specifically reacts with or binds to a target (immune) can be readily tested, for example, by comparing the affinity of the binding domain for its desired target protein or antigen to the affinity of the binding domain for non-target proteins or antigens (here: proteins other than CLDN6 or CD3, respectively). Preferably, the construct of the invention does not bind significantly to proteins or antigens other than CLDN6 or CD3, respectively (i.e. the first domain does not bind to proteins other than CLDN6 and the second domain does not bind to proteins other than CD 3) -unless any one or more additional binding domains for additional targets are deliberately introduced into the construct of the invention, in which case the invention also provides for binding of the binding domain to its specific target.

It is contemplated that the first domain has an affinity for CLDN6 (e.g., human CLDN 6) of 100nM, 90nM, 80nM, 70nM, 60nM, 50nM, 40nM, 30nM or 20nM. These values are preferably measured in a cell-based assay (e.g., a Scatchard assay). Other methods of determining affinity are also well known. It is further contemplated that the second domain has an affinity for CD3 (e.g., human CD 3) of 100nM or less, 90nM or less, 80nM or less, 70nM or less, 60nM or less, 50nM or less, 40nM or less, 30nM or less, 20nM or less, or 10nM or less. These values are preferably measured in a surface plasmon resonance assay (e.g., biacore assay).

The terms "do not significantly bind" and "do not selectively bind" mean that the construct or binding domain of the invention does not bind to proteins or antigens other than CLDN6 or CD3 when the proteins or antigens are expressed on the cell surface. Thus, the construct exhibits a reactivity of 30%, preferably 20%, more preferably 10%, particularly preferably 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% with a protein or antigen other than CLDN6 or CD3 (when expressed on the cell surface), whereby binding to CLDN6 or CD3, respectively, is set to 100%. "reactivity" may be expressed, for example, in terms of affinity values (see above).

It is contemplated that the constructs of the invention (and more specifically, the domains comprising paratopes that bind to CLDN 6) do not bind or do not significantly bind to CLDN6 paralogs, more specifically human CLDN6 paralogs and/or cynomolgus monkey CLDN6 paralogs. It is also contemplated that the construct does not bind or does not significantly bind to (human or cynomolgus monkey/cynomolgus monkey) CLDN6 paralogs on the surface of target cells. CLDN6 paralogs include-but are not limited to-CLDN 1, CLDN2, CLDN3, CLDN4, CLDN18.1, CLDN18.2 and, in particular, CLDN9. According to one embodiment, the human paralog of CLDN6 has the sequence as depicted in SEQ ID NOS.2-8. Thus, it is contemplated that the first domain of the construct of the invention does not bind or does not bind significantly selectively to CLDN1, CLDN2, CLDN3, CLDN4, CLDN18.1, CLDN18.2 and/or CLDN9 (on the surface of a cell). It is contemplated that the constructs of the invention do not substantially bind CLDN9 expressed on various organs. Selectively binds to CLDN6 and does not substantially bind to CLDN9, avoiding potential adverse events that may result from off-target binding.

One domain comprising the paratope (antigen binding (epitope binding) structure) of the polypeptide construct of the invention specifically and/or selectively binds CLDN6 on the surface of target cells. The "target cell" may be any prokaryotic or eukaryotic cell expressing CLDN6 on its surface; preferably, the target cell is a cell that is part of the human or animal body, such as a specific CLDN6 expressing cancer cell or tumor cell or CLDN6 positive neoplasm cell or an artificially generated CLDN6 expressing cell (the latter may be used, for example, in an ex vivo assay). It is to be understood that in the context of the present invention, the term "on the surface" means that the first antigen binding domain of the construct binds selectively and preferably specifically to an epitope comprised within the first CLDN6 extracellular loop (CLDN 6 ECL 1), within the second CLDN6 extracellular loop (CLDN 6 ECL 2), in particular it binds to an epitope formed within the combination of the two loops. It is therefore envisaged that the domain of the construct of the invention comprising a paratope (antigen binding (epitope binding) structure) binds to an epitope formed by one or both extracellular loops of CLDN6, preferably human CLDN6. The extracellular loop may be the first loop and/or the second loop. It is also contemplated that both rings may facilitate bonding. In this case, it is possible that one loop (e.g. the first loop) represents the primary binding partner of the construct, while the other loop (e.g. the second loop) e.g. serves as a stabilizing partner to facilitate binding, but is not necessary for binding. Thus, when CLDN6 is expressed by naturally expressed cells or cell lines (e.g., human cancer cell lines OVCAR-3, OAW28, LCLC97TM1, and NCI-H1435) and/or by cells or cell lines transformed or (stably/transiently) transfected with CLDN6, domains comprising paratope (antigen binding (epitope binding) structures) according to the invention can bind to CLDN6. In one embodiment, when CLDN6 is used as a target molecule in a cell-based binding assay such as Scatchard, the domain comprises a paratope (antigen binding (epitope binding) structure) that binds to CLDN6. Furthermore, it is envisaged that the construct/its first domain binds to human CLDN6 on the surface of target cells. The preferred amino acid sequence of human CLDN6 is depicted in SEQ ID NO. 1.

It is envisaged that a polypeptide construct according to the invention (and more specifically, that this domain comprises a paratope (i.e. antigen binding (epitope binding) structure) of CLDN6 binding to said construct) binds to a first extracellular loop (ECL 1, loop 1) of CLDN 6. This does not necessarily exclude that the second extracellular loop also contributes to the paratope-epitope interaction site, although to a different, e.g. lesser extent. The term "CLDN6 ECL" (ecl=extracellular loop) refers to those portions of CLDN6 that are substantially free of transmembrane and cytoplasmic domains of CLDN 6. It will be appreciated that the transmembrane domain identified for the CLDN6 binding polypeptides of the invention is identified according to criteria conventionally used in the art for identifying the type of hydrophobic domain. The exact boundaries of the transmembrane domains may vary, but most likely do not vary by more than about 5 amino acids at either end of the domains explicitly mentioned herein. Preferred human CLDN6ECL1 is shown in SEQ ID NO. 9 and preferred human CLDN6ECL 2 is shown in SEQ ID NO. 10. In a very specific embodiment, the construct according to the invention (and more particularly the first domain of the construct) binds to the E1A domain of the first extracellular loop (ECL 1, loop 1) and the E2B domain of the second extracellular loop (ECL 2, loop 2) of CLDN6 (preferably human CLDN 6), which is advantageously expressed on the surface of cancer cells or cells that have been induced (by transformation or transfection) to express CLDN6 (e.g. human CLDN 6), and does not bind to amino acids 138-150 of CLDN6 depicted in SEQ ID No. 1.

The invention further provides that the domain comprising a paratope (antigen binding (epitope binding) structure) of the construct of the invention preferably selectively binds to the same epitope on CLDN6 as an antibody or construct comprising a domain that binds to CLDN6 on the surface of a target cell, and that domain of the construct of the invention comprises:

r) a VH region comprising CDR-H1 as depicted in SEQ ID NO:251, CDR-H2 as depicted in SEQ ID NO:252 and CDR-H3 as depicted in SEQ ID NO:253, and a VL region comprising CDR-L1 as depicted in SEQ ID NO:254, CDR-L2 as depicted in SEQ ID NO:255 and CDR-L3 as depicted in SEQ ID NO:256, or

s) a VH region comprising CDR-H1 depicted in SEQ ID NO. 265, CDR-H2 depicted in SEQ ID NO. 266 and CDR-H3 depicted in SEQ ID NO. 267, and a VL region comprising CDR-L1 depicted in SEQ ID NO. 268, CDR-L2 depicted in SEQ ID NO. 269 and CDR-L3 depicted in SEQ ID NO. 270,

Or (b)

a-1) the VH region depicted in SEQ ID NO. 11, and the VL region depicted in SEQ ID NO. 12;

b-1) the VH region depicted in SEQ ID NO. 19, and the VL region depicted in SEQ ID NO. 20;

c-1) the VH region depicted in SEQ ID NO. 27, and the VL region depicted in SEQ ID NO. 28;

d-1) the VH region depicted in SEQ ID NO. 35, and the VL region depicted in SEQ ID NO. 36;

e-1) the VH region depicted in SEQ ID NO. 43, and the VL region depicted in SEQ ID NO. 44;

f-1) the VH region depicted in SEQ ID NO. 51, and the VL region depicted in SEQ ID NO. 52;

g-1) the VH region depicted in SEQ ID NO. 59, and the VL region depicted in SEQ ID NO. 60;

h-1) the VH region depicted in SEQ ID NO. 67, and the VL region depicted in SEQ ID NO. 68;

i-1) the VH region depicted in SEQ ID NO. 75, and the VL region depicted in SEQ ID NO. 76;

j-1) the VH region depicted in SEQ ID NO. 83, and the VL region depicted in SEQ ID NO. 84;

k-1) the VH region depicted in SEQ ID NO. 91, and the VL region depicted in SEQ ID NO. 92;

l-1) the VH region depicted in SEQ ID NO. 99, and the VL region depicted in SEQ ID NO. 100;

m-1) the VH region depicted in SEQ ID NO. 107, and the VL region depicted in SEQ ID NO. 108;

n-1) the VH region depicted in SEQ ID NO. 115, and the VL region depicted in SEQ ID NO. 116;

o-1) the VH region depicted in SEQ ID NO. 123, and the VL region depicted in SEQ ID NO. 124;

p-1) the VH region depicted in SEQ ID NO. 131, and the VL region depicted in SEQ ID NO. 132;

q-1) the VH region depicted in SEQ ID NO. 139, and the VL region depicted in SEQ ID NO. 140; or (b)

r-1) the VH region depicted in SEQ ID NO:147, and the VL region depicted in SEQ ID NO:148, or

In further embodiments, polypeptide constructs of the invention comprise a domain that binds CLDN6 comprising any CDR region depicted in SEQ ID NO:680 to 694, such as heavy chain CDR1 depicted in SEQ ID NO:680, or CDR2 depicted in heavy chain SEQ ID NO:681, or heavy chain CDR2 depicted in SEQ ID NO:682, or heavy chain CDR2 depicted in SEQ ID NO:683, or heavy chain CDR3 depicted in SEQ ID NO:684, or heavy chain CDR3 depicted in SEQ ID NO:685, or heavy chain CDR3 depicted in SEQ ID NO:686, or heavy chain CDR3 depicted in SEQ ID NO:687, and/or light chain CDR1 depicted in SEQ ID NO:688, or light chain CDR1 depicted in SEQ ID NO:689, light chain CDR2 depicted in SEQ ID NO:690, light chain CDR3 depicted in SEQ ID NO:691, light chain CDR3 depicted in SEQ ID NO:692, or heavy chain CDR3 depicted in SEQ ID NO: 696, or light chain CDR3 depicted in SEQ ID NO:688, and/or a combination of any of the sequences depicted in SEQ ID NO:684, comprising any of the sequences depicted in SEQ ID NO:684 to 684. Those skilled in the art will appreciate that the binding domain that binds CLDN6 may comprise only one of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which may be present in a combination according to the invention.

CLDN6 binding specificities of other anti-CLDN 6 binders were also analyzed in epitope mapping (see example 2). These CLDN6xCD3 constructs were found to have different epitope specificities and at the same time showed significantly poorer cytotoxic potential compared to the constructs of the invention. In example 4, it was demonstrated that the constructs of the invention showed EC50 values in the 2-digit picomolar range while the comparative constructs showed EC50 values in the 3-digit to 5-digit picomolar range, despite similar affinities for CLDN 6. Constructs exhibiting the latter range of cytotoxic activity may not be sufficiently effective for therapeutic use in guiding the patient's immune system, more specifically the cytotoxic activity of T cells on cancer cells. On the other hand, the constructs according to the invention are present in a very favourable epitope-activity relationship and thus support potent construct-mediated cytotoxic activity.

Whether an antibody, polypeptide construct, or domain comprising a paratope (antigen binding (epitope binding) structure) binds the same CLDN6 epitope on the surface of a target cell as another given antibody, construct, or binding domain can be measured by different assays as described herein (e.g., by epitope mapping with chimeric or mutated CLDN6 molecules), as described herein above or in examples 1 and 2. Other methods of determining epitopes, such as alanine scanning, are described herein.

Whether an antibody or polypeptide construct or domain comprising a paratope (antigen binding (epitope binding) structure) competes with another given antibody or construct for binding to an antigen (e.g., CLDN 6) on the surface of a target cell can be measured in a competition assay, such as a competition ELISA. Avidin coupled microparticles (beads) may also be used. Similar to avidin coated ELISA plates, each of these beads can be used as a substrate upon which an assay can be performed when reacting with biotinylated proteins. The antigen is coated on the beads and then pre-coated with the first antibody. The secondary antibody is added and any additional binding is determined. Readout was performed via flow cytometry. Preferably, a cell-based competition assay is used, using cells naturally expressing CLDN6 or cells stably or transiently transformed with CLDN 6. In the context of the present invention, the term "competitive binding" means that competition occurs between 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% of the two test antibodies, 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.

Competitive antibody/polypeptide construct binding assays include assays that determine the competitive binding of two antibodies/constructs to a cell surface binding antigen. A common method aimed at detecting the binding of two antibodies/constructs a and B to the same antigen on the cell surface may comprise the following steps:

a) Blocking cell surface antigen by pre-incubating the cells with antibody/polypeptide construct a followed by a second maximum addition of labeled antibody/polypeptide construct B and detecting binding of B compared to binding in the absence of a;

b) Titrating (i.e., adding different amounts of) antibody/polypeptide construct a in the presence of a sub-maximum amount of labeled antibody/polypeptide construct B, and detecting the effect on B binding; or (b)

c) Co-titrating a and B, wherein the two antibody/polypeptide constructs are incubated together at maximum concentration, and detecting whether the total binding equals or exceeds that of a or B alone, i.e., independent of the order of addition or the relative amounts of the antibodies/constructs.

When two antibody/polypeptide constructs a and B compete for cell surface bound antigen, the antibodies will often compete in a blocking assay that is independent of the order in which the antibodies are added. In other words, if the assay is performed in either direction, competition is detected. However, this is not always the case and in some cases the order of addition of antibodies or the direction of the assay may affect the signal generated. This may be due to differences in affinity or avidity of the potentially competing antibodies/constructs. If the order of addition has a significant effect on the signal generated, it is inferred that the two antibodies/constructs do compete if competition is detected in at least one order.

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

Variability is not evenly distributed throughout the variable regions of the antibody; it concentrates in the subdomain of each of the heavy chain variable region and the light chain variable region. These subdomains are referred to as "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable region are referred to as the "framework" (FR) regions, and provide scaffolds for the six CDRs in three-dimensional space to form the antigen-binding surface. The variable regions of naturally occurring antibody heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR 4) that adopt predominantly a β -sheet configuration. Together with CDRs, they form the following sequences within the variable heavy or light chain: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The hypervariable regions in each strand are brought together by a framework region and often together with the hypervariable regions from the other strand contribute to the formation of an antigen binding site (see Kabat et al, above-referenced). As used herein, the polypeptide/polypeptide construct of the invention may have modifications in the framework regions. These modifications may be substitutions of one or more amino acid residues of the sequences disclosed herein with other amino acid residues. Possible further modifications are deletions, inversions, additions of amino acid residues if these modifications do not prevent the selective binding of the polypeptide/polypeptide construct to CLDN6 and/or as long as these modifications do not prevent the selective binding of the polypeptide/polypeptide construct to target cells expressing CLDN6 and the ability of the construct to engage and activate T cells and induce T cell mediated cytotoxicity. Thus, when compared to a non-framework modified antibody, the modification of the framework regions of the constructs disclosed herein still allows for at least 100%, at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85%, at least 84%, at least 83%, at least 82%, at least 81%, at least 80%, at least 79%, at least 78%, at least 77%, at least 76%, at least 75%, at least 74%, at least 73%, at least 72%, at least 71%, at least 70%, at least 65%, at least 60%, at least 55%, at least 50%, at least 45%, at least 40%, at least 35%, at least 30%, at least 25%, at least 20%, at least 15%, at least 10%, at least 5% of the ability to bind T cells and induce T cell mediated cytotoxicity. Modifications in the framework regions may also be associated with higher activity than unmodified constructs. It is also contemplated that the framework regions of the constructs of the invention are modified for purposes such as increasing the solubility of the construct in a given medium, increasing the stability of the construct, and the like.

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

The exact definition of CDR boundaries and lengths is subject to different classification and numbering systems. Thus, CDRs may be referenced by Kabat, chothia, contact or any other boundary definition (including the numbering system described herein). Each of these systems, although having different boundaries, has a degree of overlap in terms of what constitutes a so-called "hypervariable region" within the variable sequence. Thus, CDR definitions according to these systems may differ in length and boundary region relative to adjacent framework regions. See, e.g., kabat (a method based on cross-species sequence variability), chothia (a method based on crystallographic studies of antigen-antibody complexes) and/or MacCallum (Kabat et al, supra; 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). Yet another criterion for characterizing antigen binding sites is the AbM definition used by AbM antibody modeling software of Oxfbrd Molecular corporation (Oxfbrd Molecular). See, e.g., protein Sequence and Structure Analysis of Antibody Variable Domains [ protein sequence and structural analysis of antibody variable domains ] in: antibody Engineering Lab Manual [ handbook of antibody engineering laboratories ] (editions: duebel, S. And Kontermann, R., springer-Verlag, sea Derburg). To the extent that two residue identification techniques define overlapping regions rather than identical regions, they can be combined to define hybrid CDRs. However, numbering according to the so-called Kabat system is preferred.

Typically, CDRs form a loop structure that can be classified as a canonical structure. The term "canonical structure" refers to the backbone conformation used by the antigen binding (CDR) loop. From comparative structural studies, five of the six antigen binding loops have been found to have only a limited pool of available conformations. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Thus, the corresponding loops between antibodies can have very similar three-dimensional structures, despite high amino acid sequence variability in most of the loops (Chothia and Lesk, J. Mol. Biol. [ journal of molecular biology ],1987,196:901; chothia et al, nature [ Nature ],1989,342:877; martin and Thorton, J. Mol. Biol [ journal of molecular biology ],1996, 263:800). Furthermore, there is a relationship between the loop structure used and the amino acid sequence surrounding it. The conformation of a particular canonical class is determined by the length of the loop and amino acid residues that are located at key positions within the loop and within a conserved framework (i.e., outside the loop). Thus, assignment to specific canonical categories can be made based on the presence of these critical amino acid residues.

The term "canonical structure" may also include considerations regarding the linear sequence of an antibody, e.g., as programmed by Kabat (Kabat et al, above-referenced). The Kabat numbering scheme (system) is a widely used standard for numbering amino acid residues of antibody variable regions in a consistent manner and is a preferred scheme for the use of the invention, as also referred to elsewhere herein. Additional structural considerations may also be used to determine the canonical structure of an antibody. For example, those differences that are not fully reflected by Kabat numbering may be described by the numbering system of Chothia et al, and/or revealed by other techniques (e.g., crystallography and two-dimensional or three-dimensional computational modeling). Thus, a given antibody sequence may be placed in a canonical class that allows, among other things, identification of the appropriate class sequence (e.g., based on the desire to include multiple canonical structures in the library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia et al, the foregoing citations, and their involvement in interpreting canonical aspects of antibody structure are described in the literature. Subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the structure of Antibodies, see Antibodies ALaboratory Manual [ Antibodies: laboratory Manual ], cold Spring Harbor Laboratory [ Cold spring harbor laboratory ], harlow et al, editions, 1988.

CDR3 of the light chain, and particularly CDR3 of the heavy chain, may constitute the most important determinant in antigen binding within the light chain variable region and the heavy chain variable region. In some antibodies or construct/binding domains, the heavy chain CDR3 appears to constitute the primary contact region between antigen and antibody. In vitro selection schemes in which only CDR3 is altered may be used to alter the binding characteristics of an antibody or construct/binding domain or to determine which residues contribute to antigen binding. Thus, CDR3 is typically the greatest source of molecular diversity in the binding site of an antibody. For example, CDR-H3 may be as short as two amino acid residues or greater than 26 amino acids.

In classical full length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one 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 typically designated CH1. The constant ("C") domain is not directly involved 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, which interacts with cell surface receptors called Fc receptors and some proteins of the complement system. In IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH 2 and CH 3) of the two heavy chains of the antibody. IgM and IgE Fc regions contain three heavy chain constant domains (CH 2, CH3 and CH 4) in each polypeptide chain. The Fc region also contains portions of the so-called "hinge" region 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 necessary for Fc receptor mediated activity.

ADCC is a cell-mediated immune defense mechanism whereby 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 as Natural Killer (NK) cells that typically interact with IgG antibodies. ADCC, however, may also be mediated by macrophages, neutrophils and eosinophils. Naturally occurring ADCC involves activation of 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 fcyriii. Once the Fc receptor binds to the Fc region of IgG, NK cells release the cytotoxic factors that lead to target cell death. Likewise, the Fc receptor of eosinophils (FcerI) will recognize IgE. In contrast, in CDC, the molecule "C1q" of the complement system binds to the antibody Fc region due to classical pathway complement activation, and this binding triggers the complement cascade, which results in the formation of a Membrane Attack Complex (MAC) on the target cell surface. In therapeutic antibodies, both ADCC and CDC may be modulated by Fc isotype engineering, fc gene mutation, or Fc glycosylation profile modification. As used herein, the polypeptide/polypeptide construct of the invention does not induce ADCC as is commonly understood. In contrast, polypeptides can bind T cells and induce T cell-mediated cytotoxicity, e.g., by secreting perforin and/or inducing apoptosis.

The sequences of the assembled and somatic mutated antibody genes were highly altered and these altered genes were estimated to encode 1010 different antibody molecules (Immunoglobulin Genes [ immunoglobulin genes ], 2 nd edition, edited by Jonio et al, academic Press [ Academic Press ], san Diego, calif., san [ San Diego, calif., 1995). Thus, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived, in whole or in part, from at least one sequence encoding at least one immunoglobulin. One or more sequences may be generated by in vivo rearrangement 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 may be produced from the cell in response to a rearrangement (e.g., in vitro stimulation) occurring. Alternatively, part or all of one or more sequences may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, for example, U.S. Pat. No. 5,565,332. The repertoire may include only one sequence or may include a variety of sequences, including sequences in a collection of genetic diversity.

It is contemplated that the polypeptide constructs of the invention have a cysteine clamp (cysteine clamp) within the first domain. Such cysteine fixtures may be introduced to improve the stability of the construct. See, for example, US 2016/0193295.

In one embodiment of the invention, CLDN6 binding paratope (antigen binding (epitope binding) structure) of one domain of the constructs of the invention comprises a VH region with the amino acid sequence depicted in: SEQ ID NO. 11, SEQ ID NO. 25, SEQ ID NO. 39, SEQ ID NO. 67 or SEQ ID NO. 193.

In another embodiment, the CLDN 6-specific paratope (i.e., antigen binding (epitope binding) domain) of a construct of the invention comprises a VL region having an amino acid sequence depicted in seq id no: SEQ ID NO. 12, SEQ ID NO. 26, SEQ ID NO. 40, SEQ ID NO. 68 or SEQ ID NO. 194.

In another embodiment, the CLDN 6-specific paratope (i.e., antigen-binding) domain of a construct of the invention comprises a VH region and a VL region having the amino acid sequences depicted in seq id no:11+12 (VH+VL), 25+26, 39+40, 67+68 or 193+194 (VH+VL).

In yet another embodiment, the CLDN 6-specific paratope (i.e., antigen binding (epitope binding) domain) of a construct of the invention comprises a polypeptide having the amino acid sequence depicted in seq id no: SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 47, SEQ ID NO. 50, SEQ ID NO. 76, SEQ ID NO. 78, SEQ ID NO. 201 or SEQ ID NO. 204, in particular SEQ ID NO. 19 and 22.

As described herein above, the present invention provides embodiments wherein the polypeptide construct is in a form selected from the group consisting of: (scFv) 2, scFv-single domain mabs, diabodies and oligomers of any of the above forms. The term "in … … form" as described herein does not exclude constructs that may be further modified (e.g. by attachment or fusion to other parts). According to one embodiment of the polypeptide construct of the invention, the domain comprising paratope as described herein is in the form of an scFv. In scFv, the VH and VL domains are arranged in the order VH-VL or VL-VH (from N-terminal to C-terminal). It is envisaged that the VH and VL regions comprising paratope domains as described herein are connected via a linker, preferably a peptide linker. According to one embodiment of the domain comprising the paratope as described herein, the VH region is located at the N-terminus of the linker and the VL region is located at the C-terminus of the linker. In other words, in one embodiment of a domain comprising a paratope as described herein, the scFv comprises from N-terminus to C-terminus: VH-linker-VL. It is further contemplated that the paratope-containing domains of the constructs as described herein are connected via a linker, preferably a peptide linker. A construct may for example comprise domains in the order of one domain-linker-second other domain (from N-terminal to C-terminal). Reverse order (other domain-linker-first domain) is also possible.

The linker is preferably a peptide linker, more preferably a short peptide linker. According to the invention, a "peptide linker" comprises an amino acid sequence that links the amino acid sequence of one domain of a construct to another (variable and/or binding) domain (e.g., a variable domain or binding domain). The basic technical feature of this peptide linker is that it does not comprise any polymerization activity. Suitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180 and 4,935,233 or WO 88/09344. Peptide linkers can also be used to attach other domains or modules or regions (e.g., half-life extending domains) to the constructs of the invention. Examples of useful peptide linkers are shown in SEQ ID NO:563-575 and SEQ ID NO: 679. In the context of the present invention, a "short" linker 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 one binding domain may have a different length (e.g., may be longer) than the linker between the two binding domains. For example, the linker between the two variable regions of one binding domain may have a length of between 7 and 15 (preferably between 9 and 13) amino acids, and the linker between the two binding domains may have a length of between 3 and 10 (preferably between 4 and 8) amino acids. It is further contemplated that the peptide linker is a glycine/serine linker such as those depicted in SEQ ID NO:563-575 and SEQ ID NO: 679. Most of the amino acids in the glycine/serine linker are selected from glycine and serine.

If a linker is used, the linker preferably has a length and sequence that ensures that each of the first domain and the second domain can retain their differential binding specificity independently of each other. For peptide linkers that connect at least two binding domains (or two variable regions that form one binding domain) in a construct, those peptide linkers are contemplated that comprise only a few amino acid residues (e.g., 12 amino acid residues or less). Thus, peptide linkers of 12, 11, 10, 9, 8, 7, 6 or 5 amino acid residues are preferred. Peptide linkers with less than 5 amino acids are contemplated to comprise 4, 3, 2 or 1 amino acid, with Gly-rich linkers being preferred. The "single amino acid" linker in the context of the "peptide linker" is Gly. Another example of a peptide linker is characterized by the amino acid sequence Gly-Gly-Gly-Gly-Ser (i.e.Gly4Ser (SEQ ID NO: 563)) or a polymer thereof (i.e.(Gly4Ser) x, where x is an integer of 1 or more (e.g.2 or 3). Useful linkers are depicted in SEQ ID NO:563-575 and SEQ ID NO: 679. The characteristics of the peptide linkers are known in the art and are described, for example, in Dall' Acqua et al (Biochem. [ biochemistry ] (1998) 37, 9266-9273), cheadle et al (Mol Immunol ] (1992) 29,21-30), and Raag and Whitlow (FASEB [ Proc. Natl. Acad. Sci. USA ] (1995) 9 (1), 73-80). Peptide linkers that do not promote any secondary structure are preferred. The linking of the domains to each other may 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 [ molecular cloning: A laboratory Manual ], cold Spring Harbor Laboratory Press [ Cold spring harbor laboratory Press ], cold Spring Harbor, new York [ New York Cold spring harbor ], 2001).

According to one embodiment of the invention, the polypeptide construct of the invention is a "single-stranded construct". It is also contemplated that the first binding domain or the second binding domain or both binding domains may be in the form of a "single chain Fv" (scFv). Although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by an artificial linker that enables them to be made as a single protein chain, as described above, in which the VL and VH regions pair to form monovalent molecules; see, e.g., huston et al (1988) Proc. Natl. Acad. Sci USA [ Proc. Natl. Acad. Sci. USA, U.S. national academy of sciences ]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 full length antibodies or IgG. Thus, a single chain variable fragment (scFv) is a fusion protein of the heavy (VH) and light (VL) variable regions of an immunoglobulin, typically linked by a short linker peptide. For flexibility, the linker is usually rich in glycine, and for solubility is usually rich in serine or also threonine, and can link the N-terminus of VH with the C-terminus of VL, or vice versa. The protein retains the original immunoglobulin specificity despite removal of the constant region and introduction of the linker.

Bispecific single chain molecules are known in the art and are described in the following: WO 99/54440; mack, J.Immunol. [ J.Immunol.](1997) 158,3965-3970; mack, PNAS [ Proc of national academy of sciences USA ]](1995), 92,7021-7025; kufer, cancer immunol. Immunother [ Cancer immunology immunotherapy ]],(1997),45,193-197；Blood [ Blood ]](2000), 95,6,2098-2103; bruhl, immunol [ immunology ]](2001), 166,2420-2426; kipriyanov, J.mol.biol. [ journal of molecular biology ]], (1999),293,41-56. The techniques described for generating single-stranded constructs (see, inter alia, U.S. Pat. No. 4,946,778; kontermann and Dubel (2010), the above-mentioned citations, and Little (2009), the above-mentioned citations) may be adapted to generate single-stranded constructs that selectively and preferably specifically recognize one or more selected targets.

Divalent (also known as bivalent) or bispecific single chain variable fragments (bi-scFv or bi-scFv with (scFv) 2 format) can be engineered by ligating two scFv molecules (e.g. using linkers as described above). Ligation may be performed by generating a single polypeptide chain having two VH and two VL regions, thereby generating a tandem scFv (see, e.g., kufer P. Et al, (2004) Trends in Biotechnology [ Biotechnology trend ]22 (5): 238-244). Another possibility is to generate scFv molecules with linker peptides that are too short for the two variable regions to fold together (e.g. about five amino acids), forcing scFv dimerization. In this case, VH and VL of the binding domain (binding to target antigen CLDN6 or CD 3) are not directly linked via a peptide linker. Thus, the VH of the CD3 binding domain may be fused to the VL of the CLDN6 binding domain, e.g., via a peptide linker, and the VH of the CLDN6 binding domain fused to the VL of the CD3 binding domain via such a peptide linker. This type is known as diabodies (see, e.g., hollinger, philipp et al (7. 1993) Proceedings of the National Academy of Sciences of the United States of America [ Proc. Natl. Acad. Sci. USA ]90 (14): 6444-8).

Constructs designated "single domain constructs" (or occasionally "antibody constructs") comprise one (monomeric) antibody variable region that can selectively bind a particular antigen independently of other variable regions. The first single domain antibodies were engineered from heavy chain antibodies found in camels and these are referred to as VHH fragments. Cartilaginous fish also have heavy chain antibodies (IgNAR) from which single domain antibodies, known as VNAR fragments, can be obtained. An alternative approach is to split the dimeric variable region from the common immunoglobulin into monomers, thus obtaining VH or VL as single domain abs. While most studies on single domain antibodies are currently based on heavy chain variable regions, nanobodies derived from light chains have also been shown to bind specifically to target epitopes. Examples of single domain antibodies are so-called sdabs, nanobodies or single variable domain antibodies. Thus, (single domain mAb) 2 is a monoclonal construct consisting of (at least) two single domain monoclonal constructs, which are individually selected from the group comprising VH, VL, VHH and VNAR. The linker is preferably in the form of a peptide linker. Similarly, a "scFv-single domain mAb" is a monoclonal construct consisting of at least one single domain antibody as described above and one scFv molecule as described above. Likewise, the linker is preferably in the form of a peptide linker.

It is also contemplated that the polypeptide constructs of the invention have additional functions in addition to the function of binding to the target molecules CLDN6 and CD 3. In this form, targeting the target cells by CLDN6 binding, mediating cytotoxic T cell activity by CD3 binding, and providing additional functions (e.g., means or domains to enhance or prolong serum half-life, full-function or modified Fc constant domains to mediate cytotoxicity by recruiting effector cells, labels (fluorescent, etc.), therapeutic agents such as toxins or radionuclides, etc.), the construct may be a tri-or multi-functional construct.

Examples of means or domains that extend the serum half-life of the polypeptide/polypeptide constructs of the invention include peptides, proteins, or domains of proteins fused or otherwise attached to the polypeptide/polypeptide construct. The group of peptides, proteins or domains of proteins comprises peptides that bind to other proteins in humans with preferred pharmacokinetic characteristics, such as serum albumin (see WO 2009/127691). Alternative concepts of such half-life extended peptides include peptides that bind to neonatal Fc receptors (FcRn, see WO 2007/098420), which may also be used in the constructs of the invention. Concepts of attaching larger protein domains or intact proteins include 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 fusion of domains thereof, and fusion of immunoglobulin constant regions (Fc domains) and variants thereof. Such variants of Fc domains are referred to as Fc-based domains, and may, for example, be optimized/modified to allow for desired dimer or multimer pairing, to eliminate Fc receptor binding (e.g., avoid ADCC or CDC), or for other reasons. An additional concept known in the art to extend the half-life of a substance or molecule in humans is the pegylation of those molecules (e.g., constructs of the invention).

In one embodiment, for example, to extend the serum half-life of the construct, a polypeptide/polypeptide construct according to the invention is linked (e.g., via a peptide bond) to a fusion partner (e.g., a protein, polypeptide, or peptide). These fusion partners may 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 for these fusion partners are described in SEQ ID NOS 576-637. Typically, the fusion partner may be linked to the N-terminus or C-terminus of the construct according to the invention either directly (e.g. via a peptide bond) or by a peptide linker such as (GGGGS) N (where "N" is an integer of 2 or more, e.g. 2 or 3 or 4). Suitable peptide linkers are shown and discussed in SEQ ID NO. 563-575.

Thus, a polypeptide comprising, from N-terminus to C-terminus, in the following order, is envisaged for the polypeptide construct according to the invention:

a) VL (comprising part of CLDN6 binding paratope) - (G4S) 3-VH (comprising part of CLDN6 binding paratope) -peptide linker (SG 4S) -VH (comprising part of CD3 binding paratope) - (G4S) 3-VL (comprising part of CD3 binding paratope) -peptide linker (G4) -Fc monomer (part of HLE domain) - (G4S) 6-Fc monomer (part of HLE domain); or (b)

b) VH (comprising part of CLDN6 binding paratope) - (G4S) 3-VL (comprising part of CLDN6 binding paratope) -peptide linker (SG 4S) -VH (comprising part of CD3 binding paratope) - (G4S) 3-VL (comprising part of CD3 binding paratope) -peptide linker (G4) -Fc monomer (part of HLE domain) - (G4S) 6-Fc monomer (part of HLE domain).

Thus, it is envisaged that the polypeptide construct according to the invention comprises:

(a) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 12, SEQ ID NO. 26, SEQ ID NO. 40, SEQ ID NO. 68 and SEQ ID NO. 194;

a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID No. 563, which may be substituted by any one of: 564-575 of SEQ ID NO; or 679; and

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 11, SEQ ID NO. 25, SEQ ID NO. 39, SEQ ID NO. 67 and SEQ ID NO. 193;

(b) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 11, SEQ ID NO. 25, SEQ ID NO. 39, SEQ ID NO. 67 and SEQ ID NO. 193

(c) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 12 and SEQ ID NO. 194, in particular SEQ ID NO. 12;

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 11 and SEQ ID NO. 193, in particular SEQ ID NO. 11;

(d) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 25, SEQ ID NO. 39 and SEQ ID NO. 67,

a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID No. 563, which may be substituted by any one of: 564-575 of SEQ ID NO; and 679; and

A polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO. 26, SEQ ID NO. 40 and SEQ ID NO. 68;

(e) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

(f) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO 565, which may be substituted by any one of: SEQ ID NOS 563, 564, 566-575; or 679; and

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 19, 22, 33, 36, 47, 50, 75, 78, 201 and 204, in particular 19 and 22;

(g) A polypeptide comprising, in the following order from the N-terminus to the C-terminus:

a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs 19, 22, 33, 36, 47, 50, 75, 78, 201 and 204, in particular 19 and 22.

According to another embodiment, the construct of the invention comprises (in addition to the domain comprising the paratope described herein that binds CLDN6 and CD 3) an additional domain comprising two polypeptide monomers, each comprising a hinge, CH2 and CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker. It is envisaged that the third domain comprises, in order from N-terminus to C-terminus: hinge-CH 2-CH 3-linker-hinge-CH 2-CH3. The amino acid sequence that can be used for the third domain is depicted in SEQ ID NO 581-637. Each of the polypeptide monomers may have an amino acid sequence selected from the group consisting of SEQ ID NOS: 630-637, or at least 90% identical to those sequences.

One preferred polypeptide monomer depicted in SEQ ID NO. 622, and a preferred third domain depicted in SEQ ID NO. 630.

In another embodiment, the first and second domains of the constructs of the invention are fused to the third domain via a peptide linker, e.g., selected from the group consisting of SEQ ID NO:563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, and 575 and SEQ ID NO: 679.

According to the invention, a "hinge" is an IgG hinge region. This region can be identified by analogy using Kabat numbering, see, e.g., 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 of 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 CH 2) and Kabat positions 361-478 (for CH 3). It is understood that there are some variations between immunoglobulins in their IgG1 Fc region, igG2 Fc region, igG3 Fc region, igG4Fc region, igM Fc region, igAFc region, igD Fc region and IgE Fc region (see, e.g., padlan, molecular Immunology [ 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-terminus of these domains. For IgA and IgM, the Fc region may include the J chain. For IgG, the Fc region comprises immunoglobulin domains CH2 and CH3, and a hinge between the first two domains and CH 2. Although the boundaries of the Fc region of an immunoglobulin may vary, examples of human IgG heavy chain Fc portions comprising functional hinge, CH2 and CH3 domains may be defined as comprising residues D231 (residues of the hinge domain) to P476 (residues of the C-terminal end of the CH3 domain), or D231 to L476, respectively, for IgG4, wherein numbering is according to Kabat.

Thus, the polypeptide construct of the invention may comprise, in order from N-terminus to C-terminus:

(a) A domain comprising a paratope (antigen binding (epitope binding) structure) that binds to a CLDN6 epitope;

(b) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID nos. 563-575, particularly 563, 568, 570 to 575, more particularly SEQ ID No. 570;

(c) Another domain comprising a paratope (antigen binding (epitope binding) structure) that binds a CD3 epitope;

(d) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO. 563-575 and SEQ ID NO. 679, in particular SEQ ID NO. 679;

(e) A polypeptide monomer of a half-life extending domain (including hinge, CH2 and CH3 domains) having an amino acid sequence selected from the group consisting of SEQ ID NOs 630-637, in particular SEQ ID NO 630;

(f) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO. 563-575, in particular SEQ ID NO. 573; and

(g) Another polypeptide monomer of the half-life extending domain (including hinge, CH2 and CH3 domains) has an amino acid sequence selected from the group consisting of SEQ ID NOS: 630-637, in particular SEQ ID NO: 630.

It is also contemplated that the polypeptide construct of the invention comprises, in order from N-terminus to C-terminus:

a domain comprising a paratope (antigen binding (epitope binding) structure) that binds to an epitope of CLDN6, having an amino acid sequence selected from the group consisting of: SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 47, SEQ ID NO. 50, SEQ ID NO. 75, SEQ ID NO. 78, SEQ ID NO. 201, SEQ ID NO. 204, in particular SEQ ID NO. 19, SEQ ID NO. 22, SEQ ID NO. 33, SEQ ID NO. 36, SEQ ID NO. 47, more in particular SEQ ID NO. 19, SEQ ID NO. 22; wherein the peptide linker contained in those sequences and having SEQ ID NO. 570 may be replaced by any of SEQ ID NO. 563-575;

a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NO. 563, 565, 566, 569, 570, in particular SEQ ID NO. 565;

another domain comprising the paratope (antigen binding (epitope binding) structure) of an epitope that binds CD3, having an amino acid sequence selected from the group consisting of: 542-562 and 678; wherein the peptide linkers contained in those sequences are selected from the group consisting of amino acids having SEQ ID NOs 563-575 and 679;

A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs 563-575; and

another domain comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOS: 630-637, in particular SEQ ID NO:630, and a peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOS: 563-575, in particular SEQ ID NO: 573.

Thus, in one embodiment, the polypeptide construct of the invention comprises or consists of a polypeptide having an amino acid sequence selected from the group consisting of: 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, 105, 108, 119, 122, 133, 136, 147, 150, 161, 164, 175, 178, 189, 192, 203, 206, 217, 220, 231, 234, 245, 148, 259, 262, 273, 276, 287, 290, 301, 304, 315, 318, 329, 332, 343, 346, 357, 360, 371, 374, 385, 388, 399, 402, 413, 416, 427 and 430, in particular 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, more in particular 21, 24, 35, 38, 49, 52, 77 and 80, in particular 21, 35, 49 and 77.

Covalent modification of polypeptides/polypeptide constructs is also included within the scope of the invention and is usually, but not always, performed post-translationally. For example, several types of covalent modifications of the construct are introduced into the molecule by reacting specific amino acid residues of the construct with an organic derivatizing agent that can react with selected side chains or with N-terminal or C-terminal residues. Derivatization with bifunctional agents can be used to crosslink the constructs of the invention to a water-insoluble carrier matrix or surface for use in a variety of methods. Glutaminyl and asparaginyl residues are typically deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under weakly acidic conditions. Any of these forms of residues is within the scope of the invention. Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains (T.E. CreutIn, proteins: structure and Molecular Properties [ protein: structure and molecular characteristics ], W.H. Freeman & Co. [ W.H. Frieman Co., san Francisco, 1983, pages 79-86), acetylation of N-terminal amines and amidation of any C-terminal carboxyl groups.

Another species of covalent modification of constructs included within the scope of the invention includes altering the glycosylation pattern of the protein. As known in the art, the glycosylation pattern can depend on the sequence of the protein (e.g., the presence or absence of a particular glycosylated amino acid residue 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. Tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences that enzymatically attach a carbohydrate moiety to an asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites (for N-ligation) to the construct is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above. Alterations may also be made by adding or substituting one or more serine or threonine residues to the starting sequence (for the O-linked glycosylation site). For convenience, the amino acid sequence of the construct may be altered by variations in the DNA level, particularly by mutating the DNA encoding the polypeptide at preselected bases, so as to produce codons that will translate into the desired amino acid.

Another means of increasing the number of carbohydrate moieties on a construct is by chemically or enzymatically coupling a glycoside to a protein. These procedures are advantageous in that they do not require the production of proteins in host cells with glycosylation capabilities for N-and O-linked glycosylation. Depending on the coupling mode used, one or more saccharides may be attached 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, aplin and Wriston,1981,CRC Crit.Rev.Biochem [ CRC biochemistry key comment ], pages 259-306.

Removal of the carbohydrate moiety present on the starting construct may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposing the protein to the compound trifluoromethanesulfonic acid, or an equivalent compound. This treatment results in cleavage of most or all of the sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimudin et al, 1987, arch. Biochem. Biophys. [ Biochem and biophysical Proc. ]259:52, edge et al, 1981, anal. Biochem. [ analytical biochemistry ] 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be accomplished using a variety of endo-and exo-glycosidases, as described by Thoakura et al, 1987, meth. Enzymol. [ methods of enzymology ] 138:350. The compound tunicamycin can be used to prevent glycosylation at potential glycosylation sites, as described by Duskin et al, 1982, J.biol.chem. [ J.Biochem ] 257:3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

Other modifications of the construct are also contemplated herein. For example, another type of covalent modification of the construct includes that described in U.S. Pat. nos. 4,640,835, 4,496,689;4,301,144;4,670,417;4,791,192 or 4,179,337 to various non-protein polymers (including polyols). Furthermore, amino acid substitutions may be made at various positions within the construct, for example to facilitate the addition of polymers such as polyethylene glycol (PEG), as known in the art.

In some embodiments, covalent modification of the constructs of the invention includes the addition of one or more labels. The labeling group may be coupled to the construct via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and may be used to carry out the present invention. The term "label" or "labeling group" refers to any detectable label. Typically, labels fall into a variety of categories, depending on the assay in which they are to be detected—examples below include, but are not limited to:

a) Isotopic labeling, which may be a radioisotope or heavy isotope, such as a radioisotope or radionuclide (e.g. ³ H、 ¹⁴ C、 ¹⁵ N、 ³⁵ S、 ⁸⁹ Zr、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I)

b) Magnetic labels (e.g. magnetic particles)

c) Redox active moiety

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

e) Enzymatic groups (e.g. horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase)

f) Biotinylation group

g) A predetermined polypeptide epitope (e.g., leucine zipper pair sequence, binding site of a second antibody, metal binding domain, epitope tag, etc.) recognized by a second reporter gene.

By "fluorescent label" is meant any molecule that can be detected via its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malachite green, stilbene, fluorescein, waterfall blue J, texas red, IAEDANS, EDANS, BODIPY FL, LC red 640, cy5, cy5.5, LC red 705, oregon green, alexa-Fluor dye (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, waterfall blue, and R-Phycoerythrin (PE) (Molecular Probes of Uygur City, eugenine, oreg)), FITC, rhodamine and Texas red (Alexa Fluor 5, cyco 7, pierPTH, 37, pierPTH). Suitable optical dyes (including fluorophores) are described in Richard p.haugland, molecular Probes Handbook, handbook of molecular probes.

Suitable protein fluorescent labels also include, but are not limited to, green fluorescent proteins, including Renilla, ptilosarcus of GFP, or the Aequorea species (Chalfie et al, 1994, science [ science ]]263:802-805), EGFP (Clontech laboratories,accession number U55762), blue fluorescent protein (BFP, quantum Biotechnology Co (Quantum Biotechnologies, inc.), michaelv Dadazu 1801, montreal, quebec, canada, layer 8 (postal code: H3H 1J 9) (1801de Maisonneuve Blvd.West,8th Floor,Montreal,Quebec,Canada H3H 1J9); stauber,1998, biotechnology [ Biotechnology ]]24:462-471; heim et al, 1996, curr.biol. [ contemporary biology.)]6:178-182), enhanced yellow fluorescent protein (EYFP, crotal laboratories Inc.), luciferase (Ichiki et al, 1993, J.Immunol. [ J.Immunol. ]]150:5408-5417), beta-galactosidase (Nolan et al, 1988, proc. Natl. Acad. Sci. U.S.A. [ Proc. Natl. Acad. Sci. USA.)]85:2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 98/2677, WO 99/49019, U.S. Pat. No. 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).

Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA binding proteins (Landschulz et al, 1988, science 240:1759) and have been found in a number of different proteins since then. Known leucine zippers include naturally occurring peptides and dimerized or trimerized derivatives thereof. Examples of leucine zipper domains suitable for use in the production of soluble oligomeric proteins are described in PCT application WO 94/10308, and leucine zippers derived from lung Surfactant Protein D (SPD) are described in Hoppe et al, 1994,FEBS Letters [ European Biochemical Association communication ] 344:191. The use of modified leucine zippers to allow stable trimerization of heterologous proteins fused thereto is described in Fanslow et al, 1994, semin. Immunol. J. Immunol. 6:267-78.

The polypeptide constructs of the invention may also comprise additional domains, for example, which aid in isolating the molecule or which relate to the adaptive pharmacokinetic profile of the molecule. The domains that aid in isolation of the construct may be selected from peptide motifs or assisted introduced moieties that may be captured in an isolation method (e.g., a separation column). Non-limiting examples of such additional domains include peptide motifs known as Myc-tags, HAT-tags, HA-tags, TAP-tags, GST-tags, chitin binding domains (CBD-tags), maltose binding proteins (MBP-tags), flag-tags, strep-tags, and variants thereof (e.g., strep II-tags) and His-tags. All constructs disclosed herein (characterized by the identified CDRs) can comprise a His-tag domain, which is commonly referred to as a repeat of consecutive His residues in the amino acid sequence of the molecule, e.g., a repeat of five His residues (SEQ ID NO: 638) or a repeat of six His residues (hexahistidine, SEQ ID NO: 639). The His-tag may be located, for example, at the N-terminus or C-terminus of the construct. In one embodiment, the hexahistidine tag (hhhhhhhh) is linked to the C-terminal end of the construct according to the present invention via a peptide bond.

It is also envisaged that the polypeptide construct of the invention comprises or consists of a polypeptide having an amino acid sequence selected from the group consisting of those depicted in SEQ ID nos. 22 and 24 and linked at its N-terminus or its C-terminus to a protein purification tag, preferably via a peptide bond (amide bond). Ligation of a protein purification tag at the C-terminus of the polypeptide is preferred. It is envisaged that the protein purification tag is a short peptide. For example, the short peptide may be 2-30 amino acids, 4-25 amino acids, 5-20 amino acids, or 6-19 amino acids in length. Examples of protein purification tags include, but are not limited to, AU1 epitopes (e.g., SEQ ID NO: 644), an AU5 epitope (e.g., as depicted in SEQ ID NO: 645), a T7-tag (e.g., as depicted in SEQ ID NO: 646), a V5-tag (e.g., as depicted in SEQ ID NO: 647), a B-tag (e.g., as depicted in SEQ ID NO: 656), an E2 epitope (e.g., as depicted in SEQ ID NO: 649), a FLAG epitope/FLAG tag (e.g., as depicted in SEQ ID NO: 650), a Glu-Glu tag (e.g., as depicted in SEQ ID NO:651 or 652), an HA tag, a histidine affinity tag (e.g., as depicted in SEQ ID NO: 653), an HSV epitope (e.g., as depicted in SEQ ID NO: 654), a KT3 epitope (e.g., as depicted in SEQ ID NO: 655), a Myc epitope (e.g., as depicted in SEQ ID NO: 656), a poly-arginine tag (5-6 His residues), a poly-aspartic acid tag (5-16 Asp residues), a poly-histidine tag (2-10 His residues, typically 6 His residues, see e.g., as depicted in SEQ ID NO: 650), a poly-tag (e.g., as depicted in SEQ ID NO: 6), a poly-L-alanine tag (e.g., as depicted in SEQ ID NO: 662), a tag (e.g., as depicted in SEQ ID NO: 662, or a tag Universal tags (e.g., depicted in SEQ ID NO: 663), VSV-G (e.g., depicted in SEQ ID NO: 664), protein C (e.g., depicted in SEQ ID NO: 665), and protein A. A histidine tag is preferred, in particular a 6 XHis tag (SEQ ID NO: 639). Thus, it is further contemplated that the constructs of the invention consist of the following polypeptides: the polypeptide has an amino acid sequence selected from the group consisting of those depicted in SEQ ID nos. 22 and 24 and is linked at its C-terminal end via a peptide bond to a 6 xhis tag.

T cells or T lymphocytes are a class of lymphocytes (which are themselves a class of leukocytes) that play a central role in cell-mediated immunity. There are several T cell subsets, 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 consist of two distinct protein chains. In 95% of T cells, TCRs consist of alpha (α) and beta (β) chains. When the TCR is conjugated to an antigenic peptide and MHC (peptide/MHC complex), 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.

The polypeptide constructs of the invention comprise a domain that binds to CD3 on the surface of a T cell. "CD3" (cluster 3) is a T cell co-receptor consisting of four chains. In mammals, the CD3 protein complex contains a cd3γ (gamma) chain, a cd3δ (delta) chain, and two cd3ε (eprosaurus) chains. These four chains associate with the T Cell Receptor (TCR) and the so-called zeta (truncated cone) chains to form a "T cell receptor complex" and generate activation signals in T lymphocytes. The cd3γ (gamma), cd3δ (delta), and cd3ε (eprosaurus) chains are highly related cell surface proteins of the immunoglobulin superfamily and each contain a single extracellular immunoglobulin domain. The intracellular tail of the CD3 molecule contains a single conserved motif, called the immune receptor tyrosine-based activation motif (ITAM), necessary for the signaling capacity of TCRs. The CD3 epsilon molecule is a polypeptide that is encoded in humans by the CD3 epsilon gene located on chromosome 11. In the context of the present invention, CD3 is understood as a protein complex and a T cell co-receptor involved in activating cytotoxic T cells (cd8+ primitive T cells) and T helper cells (cd4+ primitive T cells). It is usually composed of four different chains. Particularly in mammals, the complex contains a CD3 gamma chain, a CD3 delta chain and two CD3 epsilon chains. These chains associate with the T Cell Receptor (TCR) and zeta chain (zeta-chain), generating activation signals in T lymphocytes. The TCR, zeta chain and CD3 molecules together constitute the TCR complex.

Redirecting lysis of target cells by recruiting T cells from a construct that binds to CD3 on the T cells and to a target protein on the target cells typically involves cytolytic synapse formation and delivery of perforin and granzyme. The conjugated T cells are capable of continuous target cell lysis and are not affected by immune escape mechanisms that interfere with peptide antigen processing and presentation or clonal T cell differentiation; see, for example, WO 2007/042261.

Cytotoxicity mediated by CLDN6xCD3 constructs can be measured in a variety of ways. "half maximal effective concentration" (EC 50) is typically used as a measure of the efficacy of a biologically active molecule (e.g., a construct of the invention). It can be expressed in units of moles. In the present case of measuring cytotoxicity, EC50 value refers to the concentration of construct that induces a cytotoxic response (target cytolysis) halfway between baseline and maximum. The effector cells in the cytotoxicity assay may be, for example, stimulated enriched (human) CD8 positive T cells or unstimulated (human) Peripheral Blood Mononuclear Cells (PBMCs). Lower EC50 values are typically expected when stimulated/enriched cd8+ T cells are used as effector cells compared to unstimulated PBMCs. If the target cells are of macaque origin or expressed or transfected with macaque CLDN6, the effector cells should also be of macaque origin, such as a macaque T cell line, e.g., 4119LnPx. The target cells should express CLDN6 on the cell surface, such as human or cynomolgus CLDN6. Preferably, the target cell should express at least one or more extracellular loops of CLDN6, such as CLDN6 loop 1 and/or loop 2, on the cell surface. The target cell may be a cell line (e.g., CHO) stably or transiently transfected with CLDN6 (e.g., human or cynomolgus CLDN 6). Alternatively, the target cell may be a CLDN6 positive naturally expressing cell line, such as a human cancer cell line. Generally, lower EC50 values are expected when target cells are used that express higher levels of CLDN6 on the cell surface than target cells with lower target expression rates.

The ratio of effector cells to target cells (E: T) in a cytotoxicity assay is typically about 10:1, but may also vary. The cytotoxic activity of CLDN6xCD3 constructs can be measured in a chromium 51 release assay (e.g., having an incubation time of about 18 hours) or in a FACS-based cytotoxicity assay (e.g., having an incubation time of about 48 hours). Modification of the incubation time (cytotoxic response) is also contemplated. Other methods of 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.

According to one embodiment, the cytotoxic activity mediated by the CLDN6xCD3 construct of the invention is measured in a cell-based cytotoxicity assay. It can also be measured in a 51-chromium release assay. It is contemplated that the constructs of the invention have EC50 values of 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.

The EC50 values given above can be measured in different assays and under different conditions. For example, when human PBMC are used as effector cells and CLDN6 transfected cells such as CHO cells are used as target cells, it is contemplated that the CLDN6xCD3 construct has an EC50 value of 500pM, 400pM, 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. When human PBMC are used as effector cells, and when the target cell is, for example, a CLDN6 positive cell line, it is contemplated that the EC50 value of the CLDN6xCD3 construct is ∈300pM, +.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.

According to one embodiment, the CLDN6xCD3 polypeptide/polypeptide construct of the invention does not induce/mediate lysis or substantially does not induce/mediate lysis of cells that do not express CLDN6 on their surface (CLDN 6 negative cells), such as CHO cells. The terms "non-induced lysis", "substantially non-induced lysis", "non-mediated lysis" or "substantially non-mediated lysis" mean that the construct of the invention does not induce or mediate lysis of more than 30%, preferably not more than 20%, more preferably not more than 10%, particularly preferably not more than 9%, 8%, 7%, 6% or 5% of CLDN6 negative cells, whereby lysis of target cells expressing CLDN6 (e.g. cells transformed or transfected with CLDN6 or naturally expressed cell lines such as human cancer cell lines) is set to 100%. This generally applies to constructs with concentrations up to 500 nM. Cytolytic measurements are conventional techniques. Furthermore, the specification teaches specific instructions on how to measure cell lysis.

The difference in cytotoxic activity between the monomer and dimer isoforms of a single CLDN6xCD3 polypeptide/polypeptide construct is referred to as the "potency gap". This potency gap may be calculated, for example, as the ratio between EC50 values for monomeric and dimeric forms of the molecule. In one method of determining this gap, 18 hours of 51-chromium release assay or 48 hours of FACS-based cytotoxicity assay were performed with purified construct monomers and dimers as described below. Effector cells are stimulated enriched human cd8+ T cells or unstimulated human PBMCs. The target cells were hu CLDN6 transfected CHO cells. The ratio of effector cells to target cells (E: T) was 10:1. The potency gap of the CLDN6xCD3 construct of the invention is preferably ∈5, more preferably ∈4, even more preferably ∈3, even more preferably ∈2, and most preferably ∈1.

The domains of the polypeptide constructs of the invention are preferably trans-species specific for members of primate mammalian interest (e.g., cynomolgus monkey). The trans-species specific CD3 binding domain is described, for example, in WO 2008/119567. According to one embodiment, in addition to binding to human CD3, the domain will bind to primate CD3, including, but not limited to, new continental primates such as common marmoset (Callithrix jacchus), tamarix faku (saminius oredinicus) or Saimiri (Saimiri sciureus), old continental primates such as baboons and macaque, gibbons, gorillas and non-human subfamilies (hominina). It is envisaged that the domain that binds to human CD3 on the surface of T cells also binds at least cynomolgus CD 3. A preferred macaque is cynomolgus monkey (Macaca fascicularis). Macaque (Macaca mulatta) is also contemplated. One construct of the invention comprises a domain that binds to human CLDN6 on the surface of target cells and another domain that binds to human CD3 and at least cynomolgus CD3 on the surface of T cells.

In one embodiment, the construct according to the invention has an affinity difference [ KD ma CD3: KD hu CD3] (as determined by e.g. BiaCore or Scatchard analysis) for binding to cynomolgus CD3 to human CD3 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.

One domain of the constructs of the invention binds to CD 3. More preferably, it binds to CD3 on the surface of T cells. Furthermore, it is envisaged that the domain binds to human CD3, preferably to human CD3 on the surface of T cells. It is also contemplated that the domain binds to CD3 epsilon. More preferably, it binds to human CD3 epsilon, e.g., to human CD3 epsilon on the surface of T cells. The preferred amino acid sequence of the extracellular domain of human CD3 epsilon is depicted in SEQ ID NO: 442.

In one embodiment of the invention, the domain of the construct binds to human CD3 epsilon (or human CD3 epsilon on the surface of T cells) and to common marmoset (Callithrix jacchus) or Saimiri sciureus (Saimiri) CD3 epsilon. It is also contemplated that the domain binds to an extracellular epitope of CD3 epsilon, preferably to an extracellular epitope of human CD3 epsilon. It is also contemplated that the domains bind to extracellular epitopes of the human and macaque (Macaca) CD3 epsilon chain. A preferred epitope of CD3 epsilon is contained within amino acid residues 1-27 of the extracellular domain of human CD3 epsilon (see SEQ ID NO: 443). Even more particularly, the epitope comprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu. Common marmosets are new continental primates belonging to the family marmosiaceae (Calstrichidae), whereas Pinus marmosets are new continental primates belonging to the family Cebidae (Cebidae). Conjugates with such characteristics are described in detail in WO 2008/119567.

Antibodies or bispecific constructs directed against (human) CD3 or selectively and preferably specifically directed against CD3 epsilon are known in the art and their CDR, VH and VL sequences may be the basis of the binding domains of the polypeptide constructs of the invention. For example, kung et al reported in 1979 the development of OKT3 (Ortho Kung T3), the first mAb to recognize CD3 (particularly the epsilon chain of CD 3) on human T cells. OKT3 (muromonab) is the first murine monoclonal antibody to be used in human therapy. Newer anti-CD 3 monoclonal antibodies include oxylizumab (TRX 4), teprizumab (teplizumab) (MGA 031), fur Lei Lushan antibody (foralumab), and visilizumab (visilizumab), all targeting the epsilon chain of CD 3. Bispecific constructs directed against (cancer) targets and CD3 are also being developed and (pre) clinically tested, and their CD3 binding domain (CDR, VH, VL) can be used as the basis for the second binding domain of the constructs of the invention. Examples include, but are not limited to, bordetention, sottuzumab (MT 110, AMG 110), cetuximab (Catumaxomab), rituximab (Duvortuxizumab), ertuximab (Ertumaxomab), lo Mo Suozhu mab (Mosunet uzumab), FBTA05 (Bi 20, TPBs 05), CEA-TCB (RG 7802, RO 6958688), AFM11, and MGD006 (S80880). Other examples of CD3 binding domains are disclosed, for example, in US 7,994,289B2, US 7,728,114 B2, US 7,381,803 B1, US 6,706,265 B1.

For the polypeptide construct used according to the invention, it is envisaged that the domain that binds to CD3 on the surface of a T cell comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3, wherein the sequence of CDR-L1 is depicted in SEQ ID NO:673, the sequence of CDR-L2 is depicted in SEQ ID NO:674 and the sequence of CDR-L3 is depicted in SEQ ID NO: 675; or a VL region comprising CDR-L1, CDR-L2 and CDR-L3, the CDR-L1 sequence as depicted in SEQ ID NO:673, CDR-L2 as depicted in SEQ ID NO:674 and CDR-L3 as depicted in SEQ ID NO:675, wherein one or more of these CDRs has at least one amino acid residue modification.

For the polypeptide construct used according to the invention, it is also envisaged that the domain that binds to CD3 on the surface of a T cell comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein the sequence of CDR-H1 is as depicted in SEQ ID NO. 670, the sequence of CDR-H2 is as depicted in SEQ ID NO. 671 and the sequence of CDR-H3 is as depicted in SEQ ID NO. 672; or a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein the sequence of CDR-H1 is as depicted in SEQ ID NO:670, the sequence of CDR-H2 is as depicted in SEQ ID NO:671 and the sequence of CDR-H3 is as depicted in SEQ ID NO:672, wherein one or more of these CDRs has at least one amino acid residue modification.

For the polypeptide construct used according to the invention, it is further contemplated that the domain that binds CD3 comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 and a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein the sequence of CDR-L1 is as depicted in SEQ ID NO:673, the sequence of CDR-L2 is as depicted in SEQ ID NO:674, the sequence of CDR-L3 is as depicted in SEQ ID NO:675, wherein the sequence of CDR-H1 is as depicted in SEQ ID NO:670, the sequence of CDR-H2 is as depicted in SEQ ID NO:671 and the sequence of CDR-H3 is as depicted in SEQ ID NO: 672; or a domain that binds CD3 comprises a VL region comprising CDR-L1, CDR-L2 and CDR-L3 and a VH region comprising CDR-H1, CDR-H2 and CDR-H3, wherein the sequence of CDR-L1 is as depicted in SEQ ID NO:673, the sequence of CDR-L2 is as depicted in SEQ ID NO:674 and the sequence of CDR-L3 is as depicted in SEQ ID NO:675, wherein the sequence of CDR-H1 is as depicted in SEQ ID NO:670, the sequence of CDR-H2 is as depicted in SEQ ID NO:671 and the sequence of CDR-H3 is as depicted in SEQ ID NO:672, wherein one or more of these CDRs has at least one amino acid residue modification.

For the polypeptide constructs used according to the invention, it is envisaged that the domain that binds to CD3 on the surface of a T cell comprises the VL region depicted in SEQ ID NO:677, or wherein the VL region comprises at least one amino acid residue modification.

For the polypeptide constructs used according to the invention, it is envisaged that the domain that binds to CD3 on the surface of a T cell comprises the VH region depicted in SEQ ID NO:676, or wherein the VH region comprises at least one amino acid residue modification.

More preferably, the polypeptide construct used according to the invention is characterized by a domain that binds to CD3 on the surface of a T cell, which domain comprises a VL region and a VH region selected from the group consisting of the VL region depicted in SEQ ID NO:677 and the VH region depicted in SEQ ID NO: 676; or wherein the VL region or the VH region comprises at least one amino acid residue modification.

A preferred embodiment of the above polypeptide construct for use according to the invention is characterized in that the domain that binds to CD3 on the surface of a T cell comprises the amino acid sequence depicted in SEQ ID No. 678 or wherein the domain that binds to CD3 on the surface of a T cell comprises the amino acid sequence depicted in SEQ ID No. 678 comprises at least one amino acid residue modification.

For polypeptide constructs used according to the invention, it is envisaged that the domain that binds to CD3 on the surface of a T cell comprises a VL region (e.g.the VL region depicted in SEQ ID NO: 540) or a VH region (e.g.the VL region depicted in SEQ ID NO:522 or 533), or the CDRs depicted in SEQ ID NO:444 to 506, in particular the CDRs depicted in SEQ ID NO:480 to 482 and 504 to 506, or the scFv depicted in SEQ ID NO:551 or 562 or in any of SEQ ID NO:542-561, for example.

Amino acid sequence modifications of the polypeptides/polypeptide constructs described herein are also contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of a polypeptide construct. Amino acid sequence variants of the polypeptide/polypeptide construct are prepared by peptide synthesis or by introducing appropriate nucleotide changes into a nucleic acid molecule encoding the polypeptide/polypeptide construct. All amino acid sequence modifications described below should result in polypeptide constructs that retain the desired biological activity of the unmodified parent molecule (e.g., binding CLDN6 and CD3, inducing cytotoxicity against CLDN 6-positive target cells).

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. Basically there are four different classes of amino acids, determined by different side chains: (1) non-polar and neutral (uncharged): ala, gly, ile, leu, met, phe, pro, val; (2) polar and neutral (uncharged): asn, cys (only slightly polar), gln, ser, thr, trp (only slightly polar), tyr; (3) acidic and polar (negatively charged): asp and Glu; (4) basic and polar (positively charged): arg, his, lys.

Hydrophobic amino acids may be separated 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, amino acids with aliphatic side chains are alanine, isoleucine, leucine (also norleucine), proline and valine. The side chain of alanine is very short, meaning that it is not particularly hydrophobic, and proline has unusual geometry, making it a special role in proteins. Methionine is conveniently generally considered to be in the same class as isoleucine, leucine and valine, although it also contains a sulfur atom. The unifying subject matter is that these amino acids mainly contain non-reactive and flexible side chains. The amino acids alanine, cysteine, glycine, proline, serine and threonine are usually grouped together and therefore all are very small. Gly and Pro may affect chain orientation.

Amino acid modifications include, for example, deletions of residues, insertions of residues, and/or substitutions of residues within the amino acid sequence of the polypeptide/polypeptide construct. Any combination of deletions, insertions, and/or substitutions is performed to obtain the final construct, provided that the final construct possesses the desired characteristics, e.g., biological activity of the unmodified parent molecule (e.g., binding CLDN6 and CD3, inducing cytotoxicity against CLDN6 positive target cells). Amino acid changes may also alter post-translational processes of the construct, such as altering the number or position of glycosylation sites.

For example, 1, 2, 3, 4, 5 or 6 amino acids (of course, depending on their respective lengths) may be inserted, deleted and/or substituted in each CDR, whereas 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 Framework Region (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 intra-sequence insertions of single or multiple amino acid residues. An insertional variant of a construct of the invention includes fusion of a polypeptide that increases or extends the serum half-life of the construct to the N-terminus or C-terminus of the construct. It is also conceivable that such an insertion occurs within the construct, for example between the first domain and the second domain.

The sites of most interest for amino acid modifications (particularly for amino acid substitutions) include hypervariable regions, particularly the individual CDRs of the heavy and/or light chains, but FR variations in the heavy and/or light chains are also contemplated. Substitutions may be conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids may be substituted in the CDR, 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 substituted in the Framework Region (FR), depending on the length of the CDR or FR, respectively. For example, if the CDR sequence covers 6 amino acids, it is contemplated that 1, 2 or 3 of these amino acids are substituted. Similarly, if the CDR sequence covers 15 amino acids, it is contemplated that 1, 2, 3, 4, 5 or 6 of these amino acids are substituted.

A useful method for identifying certain residues or regions within a construct that is the preferred site of mutagenesis is known as "alanine scanning mutagenesis" and is described, for example, in Cunningham B.C. and Wells J.A. (Science [ Science ] 6.2.1989; 244 (4908): 1081-5). Here, residues or groups of residues within the construct (e.g., charged residues such as Arg, his, lys, asp and Glu) are identified and replaced with neutral or nonpolar amino acids (most preferably alanine or polyalanine) to affect the interaction of the individual amino acids with the target protein epitope. Alanine scanning is a technique used to determine 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 function, but still mimics the secondary structure preference of many other amino acids. Where conservation of the size of the mutated residue is desired, sometimes a large amino acid (e.g., valine or leucine) may be used. The 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 accomplished by site-directed mutagenesis or randomly by creating a PCR library. Furthermore, calculation methods have been developed to estimate thermodynamic parameters based on theoretical alanine substitution. The data may be tested by IR, NMR spectroscopy, mathematical methods, bioassays, and the like.

Those amino acid positions that exhibit functional sensitivity to substitution (as determined, for example, by alanine scanning) are then precisely identified by introducing additional or other variants at or for the substitution site. Thus, although the site or region for introducing the amino acid sequence change is predetermined, the nature of the mutation itself need not 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 construct variants screened for the optimal combination of desired activities. Techniques for substitution mutation at a predetermined site in DNA having a known sequence are well known, for example, M13 primer mutagenesis and PCR mutagenesis. Mutant screening is performed, for example, using assays for antigen (e.g., CLDN6 or CD 3) binding activity and/or cytotoxic activity.

Generally, if an amino acid is substituted in one or more or all CDRs of a heavy and/or light chain, then it is envisaged that the "substituted" sequence obtained at that time 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 CDRs. For example, a CDR having a total of 5 amino acids and containing 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 containing one amino acid substitution is 90% identical to the "original" or "parent" CDR sequence. Thus, the substituted CDRs of the constructs of the invention can have a different degree of identity to their original sequences, e.g., CDRL1 can have 80% homology and CDRL3 can have 90% homology. The same considerations apply to the framework regions and the entire VH and VL regions.

A "variant CDR" is a CDR that has a particular sequence homology, similarity, or identity to a parent CDR of the 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 individual variant CDRs is at least 60% and more typically has an increasing homology, similarity or identity of 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% of the parent sequences described herein. The same applies to "variant VH" and "variant VL". According to one embodiment, sequence variations within "variant VH" and/or "variant VL" do not extend to CDRs. Thus, the present invention relates to a construct as defined herein comprising VH and VL sequences having certain sequence homology (see above) to the specific sequences defined herein ("parent" VH and VL), wherein these CDR sequences are 100% identical to the specific CDR sequences defined herein ("parent" CDRs).

Preferred substitutions (or alternatives) are conservative substitutions. However, any substitution (including non-conservative substitutions or from one or more of the "exemplary substitutions" listed in table 1 below) is contemplated, provided that the construct retains its ability to bind to CLDN6 via the first domain and to CD3 or CD3 epsilon via the second domain, and/or provided that 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% with the original or parent sequence.

Conservative substitutions (also referred to as conservative mutations or conservative substitutions) are amino acid substitutions that change a given amino acid to a different amino acid that has similar biochemical properties (e.g., charge, hydrophobicity, size). Conservative substitutions in proteins typically 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 a change in biological activity, then more substantial changes as further described herein with reference to amino acids can be introduced and the products screened for desired characteristics.

Table 1: amino acid substitution (aa = amino acid)

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Substantial modification of the biological properties of the constructs of the invention is accomplished by selecting substitutions that differ significantly in maintaining the following effects: (a) the structure of the polypeptide backbone surrounding the substituents, e.g., in a folded 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 typically entail exchanging a member of one of the amino acid classes defined above (e.g., polar, neutral, acidic, basic, aliphatic, aromatic, small … …) for another class. Any cysteine residue that does not participate in maintaining the proper conformation of the construct may generally be substituted with serine to improve the oxidative stability of the construct.

Sequence identity, homology and/or similarity of amino acid sequences are determined by 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. [ applied math. Progress ]2:482, the sequence and Wunsch (J Mol Biol. [ journal of molecular biology ]1970 month 3; 48 (3): 443-53) sequence identity alignment algorithm, the similarity search method of Pearson and Lipman (Proc Natl Acad Sci USA. [ Proc. Natl. Acad. Sci. USA ]1988 month 4; 85 (8): 2444-8), computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wiconk genetics software package (Wisconsin Genetics Software Package), the genetic computer group (Genetics Computer Group,575Science Drive,Madison,Wis.)) of Wiconjex et al, massa science, inc. 575, by Devereux et al (Nucleic Acids Res.) [ 35 ] [ 1981 ] best fitting the sequence of nucleic acid sequence (95.) [ 35 ] 4 ] 1981 month 11. It is contemplated that percent identity is calculated by FastDB based on the following parameters: mismatch penalty 1; gap penalty of 1; gap size penalty of 0.33; the connection penalty is 30. See also "Current Methods in Sequence Comparison and Analysis [ current methods of sequence comparison and analysis ]", macromolecule Sequencing and Synthesis [ macromolecular sequencing and synthesis ], selected Methods and Applications [ methods of selection and use ], pages 127-149 (1988), alan R.List, inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a set of related sequences using progressive alignment. It may also draw a tree graph showing the cluster relationships used to create the alignment. PILEUP is simplified using a progressive alignment method of Feng and Doolittle (J Mol Evol. [ J. Mol. Evolution ]1987;25 (4): 351-60); the method is similar to that described by Higgins and Sharp (Comput Appl Biosci. [ computer application in bioscience ] month 4 in 1989; 5 (2): 151-3). Useful PILEUP parameters include a default slot weight of 3.00, a default slot length weight of 0.10, and weighted end slots.

Another example of a useful algorithm is the BLAST algorithm, described in the following: altschul et al (J Mol Biol. [ journal of molecular biology ]1990, 10/5; 215 (3): 403-10.); altschul et al (Nucleic Acids Res. [ nucleic acids research ]1997, 9, 1; 25 (17): 3389-402); karlin and Altschul (Proc Natl Acad Sci U S A. [ Proc. Natl. Acad. Sci. USA ]1993, 6/15; 90 (12): 5873-7). A particularly useful BLAST program is the WU-BLAST-2 program obtained from Altschul et al (Methods Enzymol. [ Methods of enzymology ]1996; 266:460-80). WU-Blast-2 uses a number of search parameters, most of which are set to default values. The adjustable parameters are set to the following values: overlap interval=1, overlap fraction=0.125, word threshold (T) =ii. HSP S and HSP S2 parameters are dynamic values and are established by the program itself based on the composition of the particular sequence and the composition of the respective database from which the sequence of interest is searched; however, these values can be adjusted to increase sensitivity.

Another useful algorithm is vacancy BLAST, as reported by Altschul et al (Nucleic Acids Res. [ nucleic acids research ]1997, 9, 1; 25 (17): 3389-402). Null BLAST uses BLOSUM-62 substitution scores; the threshold T parameter is set to 9; triggering a double-click method of non-vacancy extension, and charging the vacancy length of k by 10+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). The gap comparison is triggered by a score corresponding to about 22 bits.

In agreement therewith, the term "percent (%) of nucleic acid sequence identity/homology/similarity" in relation to nucleic acid sequences encoding the 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 construct. One method of aligning two sequences and thereby determining their homology uses the BLASTN module of WU-Blast2 (set as default parameter), with overlap spans and overlap scores set at 1 and 0.125, respectively. Generally, the nucleotide sequence encoding each variant CDR is at least 60% nucleic acid sequence homology, similarity or identity with the nucleotide sequences described herein, and more typically has an increasing homology, similarity or identity of 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%. Again, the same applies to nucleic acid sequences encoding "variant VH" and/or "variant VL".

In one embodiment, the percentage identity of the polypeptide/polypeptide constructs according to the invention or domains comprising the paratope (antigen binding (epitope binding) structures (binding domains)) of these constructs to the human germline is ≡70% or ≡75%, more preferably ≡80% or ≡85%, even more preferably ≡90% and most preferably ≡91%,. Gtoreq.92%,. Gtoreq.93%,. Gtoreq.94%,. Gtoreq.95% or even ≡96%. Identity with human antibody germline gene products is considered an important feature in reducing the risk of therapeutic proteins eliciting an immune response against drugs in patients during treatment. Hwang W.Y. and Foote J. (Methods [ method ] 5 month 2005; 36 (1): 3-10) demonstrate that the reduction of the non-human portion of the drug construct results in a reduced risk of induction of anti-drug antibodies in patients during treatment. By comparing countless clinically evaluated antibody drugs with corresponding immunogenicity data, the following trends were shown: humanization of the variable region of the antibody/construct resulted in lower protein immunogenicity (average 5.1% of patients) than antibodies/constructs carrying unchanged non-human variable regions (average 23.59% of patients). Thus, therapeutic agents based on variable regions and proteins in the form of polypeptides/polypeptide constructs need to have a higher degree of identity to human sequences. To determine germline identity, the V region of VL can be aligned with the amino acid sequences of the human germline V and J segments (http:// www2.Mrc-lmb. Cam. Ac. Uk/vbase /) using Vector NTI software and the amino acid sequences calculated by dividing the same amino acid residues by the total number of amino acid residues of VL (in percent). The same can be done for the VH segment (http:// ww2.Mrc-lmb. Cam. Ac. Uk/vbase /), except that VH CDR3 can be excluded due to the high diversity of VH CDR3 and the lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase sequence identity with human antibody germline genes.

In another embodiment, the polypeptide/polypeptide construct of the invention exhibits high monomer yields under standard research scale conditions, such as in a standard two-step purification process. It is envisaged that the monomer yield of the construct according to the invention is ≡0.25mg/L Supernatant (SN), preferably ≡0.5mg/L SN, more preferably ≡1mg/L SN, even more preferably ≡2mg/LSN, and most preferably ≡3mg/L SN. The yield of the construct named "CL-1x I2C-6His" is shown as 4.1mg/L supernatant and the yield of the construct named "CL-1x I2C-scFc" is shown as 36.5mg/L supernatant.

Likewise, the yield of isoforms of the dimeric polypeptide construct (i.e., the percentage of monomers (monomer + dimer)) of the construct may be determined. The productivity of the monomer and dimer constructs and the calculated percent monomer can be obtained, for example, in a SEC purification step on culture supernatants from standard research scale production in roller bottles. According to one embodiment, the percentage of monomers of the construct of the invention is not less than 80%, more preferably not less than 85%, even more preferably not less than 90%, and most preferably not less than 95%.

According to one embodiment, the polypeptide/polypeptide construct of the invention has a plasma stability (ratio of EC50 with plasma to EC50 without plasma) of 5 or 4, more preferably 3.5 or 3, even more preferably 2.5 or 2, and most preferably 1.5 or 1. The plasma stability of the construct may be tested by incubating the purified construct in human plasma at 37 ℃ for 24 to 96 hours, for example, at a concentration of 2-20 μg/ml, followed by EC50 determination in 18h 51-chromium release or 48h FACS cytotoxicity assays (e.g., as described in the examples section). The effector cells in the cytotoxicity assay may be stimulated enriched human CD8 positive T cells (preferred) or unstimulated human PBMCs. The target cell may for example be a CHO cell transfected with human CLDN 6. The ratio of effector cells to target cells (E: T) may be 10:1. The initial concentration of the construct in the cytotoxicity assay may be 0.01-0.1 μg/ml. The human plasma pool used for this purpose was derived from healthy donor blood collected from EDTA-coated syringes. Cellular components were removed by centrifugation and the upper plasma phase was collected and subsequently pooled. As a control, the non-incubated construct is immediately diluted in an appropriate medium such as RPMI-1640 prior to the cytotoxicity assay. Plasma stability was calculated as the ratio of EC50 (after plasma incubation) to EC50 (control/unhatched).

Furthermore, it is envisaged that the monomer to dimer conversion of the constructs of the invention is low. The conversion can be measured under different conditions and analyzed by high performance size exclusion chromatography. See example 8. For example, incubation of monomeric isoforms of the constructs may be performed in a universal formulation buffer in an incubator and at 37 ℃ for 7 days, e.g., at a concentration of 100 μg/ml or 250 μg/ml, followed by high performance SEC to determine the percentage of the original monomeric construct that has been converted to the dimeric construct. Under these conditions, it is contemplated that the polypeptide/polypeptide construct of the invention exhibits 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%.

Likewise, it is contemplated that the polypeptide/polypeptide construct of the invention exhibits very low dimer conversion after several freeze/thaw cycles. For example, construct monomers are adjusted to a concentration of 250 μg/ml in, for example, a universal formulation buffer, and subjected to three freeze/thaw cycles (30 min at-80 ℃ C., followed by 30min thawing at room temperature) followed by high performance SEC to determine the percentage of the original monomer construct that has been converted to dimer construct. It is envisaged that the percentage of dimer of the construct after, for example, three freeze/thaw cycles 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%.

According to one embodiment, the polypeptide/polypeptide construct of the invention shows a favourable thermostability with an aggregation temperature of 45 ℃ or more or 46 ℃ or more, more preferably 47 ℃ or 48 ℃ or more, even more preferably 49 ℃ or 50 ℃ or more, and most preferably 51 ℃ or more. The thermal stability parameter can be determined from the antibody aggregation temperature as follows: an antibody solution at a concentration of 250 μg/ml was transferred to a disposable cuvette and placed in a Dynamic Light Scattering (DLS) device. The sample was heated from 40 ℃ to 70 ℃ at a heating rate of 0.5 ℃/min, and the radius measured was constantly taken. The aggregation temperature of the antibodies was calculated using the radius increase indicating melting of the proteins and aggregates.

Alternatively, the temperature melting curve may be determined by Differential Scanning Calorimetry (DSC) to determine the intrinsic biophysical protein stability of the construct. These experiments can be performed using a MicroKel LLC VP-DSC apparatus. The energy uptake of the construct-containing samples was recorded from 20 ℃ to 90 ℃ compared to the sample containing formulation buffer alone. The construct is adjusted to a final concentration of 250 μg/ml, for example in SEC running buffer. To record the corresponding melting curve, the entire sample temperature was stepped up. The energy absorption of the sample and the formulated buffer reference was recorded at each temperature. The difference in energy uptake Cp (kilocalories/mole/. Degree.C.) of the sample minus the reference is plotted against the corresponding temperature. The melting temperature is defined as the temperature at which the energy uptake is the first maximum.

It is also contemplated that the polypeptide/polypeptide construct of the present invention has a haze of 0.2 or less than 0.15, preferably 0.10 or less than 0.08, more preferably 0.06 or less than 0.05, and most preferably 0.04 or less than 0.03. Turbidity can be measured by OD340 at a construct concentration of 2.5mg/ml and incubation for 16h at 5 ℃.

The change in potency of the target x CD3 construct can be measured as a change in pre-incubation of the construct on the target cells in the absence of T cells. If the construct is internalized, it is expected that it will undergo lysosomal degradation. Thus, the effective concentration is expected to decrease over time, and thus the apparent efficacy should also decrease. Some target effects have been observed for which this is a known phenomenon. It is contemplated that the constructs of the invention are not internalized by the target cell or do not undergo significant internalization of the target cell. The internalization rate can be determined, for example, as follows: t cells were counted and diluted in assay medium to a concentration of 1x 105/ml. Target positive target cells are counted and plated, for example, at 2500 cells/well (cpw). Constructs were serially diluted, for example, at an initial concentration of 100nM at 1:2. Constructs were added to culture assay plates to allow incubation for 0, 1 or 2 hours prior to T cell addition. T cells were then plated at 25000cpw (E: t=10:1) and the assay incubated at 37 ℃ for 48 hours. For example using Steady- The system (25. Mu.l/well) analyzes target cell viability. Preferably, after 2 hours of (pre) incubation of the construct with the target cells, the internalization rate (e.g., measured as a decrease in cytotoxicity) is ∈20%, more preferably ∈15%, even more preferably ∈10%, and most preferably ∈5%.

Furthermore, for the polypeptide constructs of the invention, it is contemplated that the shed or soluble target does not significantly impair its efficacy or biological activity. This can be measured, for example, in a cytotoxicity assay, wherein the soluble target is added to the assay at increasing concentrations (e.g., at 0nM-0.3nM-0.7nM-1nM-3nM-7nM-12 nM). Exemplary E: T values are 10:1. The EC50 value of the tested constructs should not increase significantly in the presence of the soluble target.

The EC50 values of the polypeptides/polypeptide constructs of the invention can be compared in an in vitro cytotoxicity assay using cells expressing CLDN6 (e.g., CHO cells expressing CLDN6, using it as a target) and cells expressing CLDN9 (e.g., CHO cells expressing CLDN9, using it as a target), the latter serving as a negative control. The selectivity and specificity of the polypeptide/polypeptide constructs of the invention can be determined using T cells (e.g., PBMCs) as effector cells and CHO cells as described above as targets. The cytotoxic effect of the polypeptides/polypeptide constructs of the invention can be determined. According to the invention, the polypeptide/polypeptide construct described herein is at least 500-fold, at least 1000-fold, at least 2000-fold, and preferably at least 3000-fold more potent against CLDN 6-positive target cells than against CLDN 9-positive target cells, wherein the targets preferably belong to the same cell source, e.g., CHO cells, which are transfected or transformed and expressed with genes encoding CLDN6 and CLDN9, respectively. Of course, other cell types expressing CLDN6 can be used, as well as control cells that do not express CLDN6 but express CLDN9 or CLDN4 or do not express a CLDN family member at all. These cells may be cell lines that naturally express the molecule of interest, or they may have been genetically modified to express CLDN6 and/or other CLDN molecules, the latter being controls. These cells can be used in methods for determining T cell dependent cytotoxicity associated with the polypeptides/polypeptide constructs of the invention.

In another embodiment, the polypeptide construct according to the invention is stable at acidic pH. The more tolerant the construct will behave at non-physiological pH (e.g., pH 5.5) (pH required to run, for example, cation exchange chromatography), the higher the recovery of the construct relative to the total amount of protein loaded from the ion exchange column. The recovery of the construct from an ion (e.g., cation) exchange column at pH 5.5 is preferably no less than 30%, more preferably no less than 40%, more preferably no less than 50%, even more preferably no less than 60%, even more preferably no less than 70%, even more preferably no less than 80%, and most preferably no less than 95%. The percentage represents the area under the curve (=auc) of the main peak.

Furthermore, it is contemplated that the polypeptides/polypeptide constructs of the 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 examples 13 or 14. In one embodiment, the construct of the present invention has a tumor growth inhibition T/C [% ] of 70, 60, 50, 40, 30, 20, 10, 5, 4, 3 or 2. It is also contemplated that certain parameters of these studies (such as the number of tumor cells injected, the injection site, the number of human T cells transplanted, the number of constructs to be administered, and the time line) be modified or adjusted while still obtaining meaningful and reproducible results.

The invention further provides a polynucleotide/nucleic acid molecule encoding a polypeptide construct of the invention. Nucleic acid molecules are biopolymers made up of nucleotides. Polynucleotides are biopolymers composed of 13 or more nucleotide monomers covalently bonded in a chain. DNA (e.g., cDNA) and RNA (e.g., mRNA) are examples of polynucleotides/nucleic acid molecules that have different biological functions. A nucleotide is an organic molecule that serves as a monomer or subunit of a nucleic acid molecule, such as DNA or RNA. The nucleic acid molecules or polynucleotides of the invention may be double-stranded or single-stranded, linear or circular. It is contemplated that the nucleic acid molecule or polynucleotide is contained in a vector. Furthermore, it is envisaged that such vectors are contained in host cells. The host cell is capable of expressing the construct, for example, after transformation or transfection with the vector or polynucleotide/nucleic acid molecule of the invention. For this purpose, a polynucleotide or nucleic acid molecule is operably linked to a control sequence.

The genetic code is a set of rules that translate information encoded within genetic material (nucleic acids) into proteins. Biological decoding in living cells is accomplished by ligating ribosomes of amino acids in the order specified by the mRNA, carrying the amino acids using tRNA molecules and reading the mRNA three nucleotides at a time. The code defines how the sequence of these nucleotide triplets (called codons) specifies which amino acids will be added next during protein synthesis. With some exceptions, a trinucleotide codon in a nucleic acid sequence designates a single amino acid. Since most genes are encoded using exactly the same code, this particular code is often referred to as the canonical or standard genetic code.

The degeneracy of the codons is a redundancy of the genetic code, expressed by the diversity of the three base pair codon combinations of the specified amino acids. Degeneracy occurs because there are more codons than can encode amino acids. Codons encoding an amino acid may be different at any of their three positions; however, typically this difference is in the second or third position. For example, codons GAA and GAG both specify glutamate and exhibit redundancy; however, no other amino acids are specified and therefore no ambiguity is manifested. The genetic code of different organisms may favor the use of one of several codons encoding the same amino acid over the other-that is, one will find that it is expected to have a greater frequency than ever. For example, leucine is specified by six different codons, some of which are rarely used. A codon usage table detailing the frequency of genomic codon usage of most organisms can be obtained. Recombinant gene technology typically exploits this effect by implementing a technique called codon optimization, in which those codons are used to design polynucleotides that are favored by the respective host cell, such as a cell of human hamster origin, an Escherichia coli (Escherichia coli) cell, or a saccharomyces cerevisiae (Saccharomyces cerevisiae) cell, for example, to increase protein expression. Thus, it is contemplated that the polynucleotides/nucleic acid molecules of the present disclosure are codon optimized. However, any codon encoding the desired amino acid may be used to design the polynucleotide/nucleic acid molecule encoding the construct of the invention.

According to one embodiment, the polynucleotide/nucleic acid molecule of the invention encoding the polypeptide construct of the invention is in the form of one single molecule or in the form of two or more separate molecules. If the construct of the invention is a single stranded 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 envisaged that different components of the polypeptide construct (such as different domains, e.g. paratope (antigen binding (epitope binding) structures comprising domains that bind CLDN6, paratope (antigen binding (epitope binding) structures) comprising domains that bind CD3, and/or other 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.

The same applies to vectors comprising the polynucleotides/nucleic acid molecules of the invention. If the construct of the invention is a single stranded construct, a vector may comprise the polynucleotide encoding the construct at a single location (as a single open reading frame, ORF). A vector may also comprise two or more polynucleotides/nucleic acid molecules at separate locations (with separate ORFs), each of which encodes a different component of the construct of the invention. It is contemplated that the vector comprising the polynucleotide/nucleic acid molecule of the invention is in the form of one single vector or in the form of two or more separate vectors. In one embodiment, and for the purpose of expressing the construct in a host cell, the host cell of the invention shall comprise the polynucleotide/nucleic acid molecule encoding the construct or the vector comprising such polynucleotide/nucleic acid molecule in its entirety, meaning that all components of the construct, whether encoded as one single molecule or encoded at separate molecules/positions, will assemble and together after translation to form the biologically active construct of the invention.

The invention also provides vectors comprising the polynucleotides/nucleic acid molecules of the invention. Vectors are nucleic acid molecules that act as vehicles for the transfer of (foreign) genetic material into cells (typically in order to ensure replication and/or expression of the genetic material). The term "vector" encompasses, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes. Some vectors are specifically designed for cloning (cloning vectors), others are designed for protein expression (expression vectors). So-called transcription vectors are mainly used for amplifying their inserts. DNA manipulation is typically performed on E.coli vectors containing the elements necessary for their maintenance in E.coli. However, the vectors may also have elements that allow them to be maintained in cells of another organism, such as yeast, plants or mammals, and these vectors are referred to as shuttle vectors. Insertion of a vector into a target or host cell is commonly referred to as transformation (for bacterial cells) and transfection (for eukaryotic cells), while insertion of a viral vector is commonly referred to as transduction.

Generally, an engineered vector comprises an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is typically a nucleotide sequence (typically a DNA sequence) comprising an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. While the genetic code determines the polypeptide sequence of a given coding region, other genomic regions may affect the timing and location of the production of these polypeptides. Thus, modern vectors may encompass additional features in addition to the transgene 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.

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

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if the DNA of a pre-sequence or secretion leader is expressed as a pre-protein involved in the secretion of a polypeptide, the DNA of the pre-sequence or secretion leader is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. Generally, "operably linked" means that the nucleotide sequences being linked are contiguous, and in the case of secretory leader sequences, contiguous and in reading phase. However, the enhancers do not have to be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

"transfection" is the process of deliberately introducing a nucleic acid molecule or polynucleotide (including a vector) into a target cell. The term is mainly used for non-viral methods in eukaryotic cells. Transduction is often used to describe viral-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening a transient pore or "hole" in the cell membrane to allow uptake of the substance. Transfection may be performed using biological particles (e.g., viral transfection, also known as viral transduction), chemical-based methods (e.g., using calcium phosphate, lipofection, fugene, cationic polymers, nanoparticles), or physical treatments (e.g., electroporation, microinjection, gene gun, cell extrusion, magnetic transfection, hydrostatic pressure, puncture transfection (impalefection), ultrasound, optical transfection, heat shock).

The term "transformation" is used to describe the nonviral transfer of a nucleic acid molecule or polynucleotide (including vectors) into bacteria, and into nonanimal eukaryotic cells (including plant cells). Thus, transformation is a genetic alteration of a bacterial or non-animal eukaryotic cell resulting from direct uptake from its surroundings through one or more cell membranes and subsequent incorporation of exogenous genetic material (nucleic acid molecules). The transformation may be achieved by human means. In order for transformation to occur, the cells or bacteria must be competent, which may occur as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced artificially.

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

As used herein, the term "host cell" or "recipient cell" is intended to include any single cell or cell culture that may or may not be a vector, an exogenous nucleic acid molecule and/or a recipient of a polynucleotide encoding a construct of the invention, and/or a recipient of the construct itself. The corresponding substances are introduced into the cells by transformation, transfection, etc. (see above). The term "host cell" is also intended to include progeny or potential progeny of a single cell. Because certain changes may occur in subsequent generations due to natural, unexpected, or intentional mutations or due to environmental influences, such progeny may not, in fact, be identical (in morphology or genomic or total DNA complement) to the parent cell, but are still included within the scope of the terms used herein. Suitable host cells include prokaryotic or eukaryotic cells, and include, but are not limited to, bacteria (e.g., E.coli), yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, e.g., hamster, murine, rat, macaque, or human.

In addition to prokaryotes, eukaryotic microbes (such as filamentous fungi or yeast) are suitable cloning or expression hosts for the constructs of the invention. Saccharomyces cerevisiae (Saccharomyces cerevisiae) or Saccharomyces cerevisiae are the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species and strains are commonly available and useful herein, such as schizosaccharomyces pombe (Schizosaccharomyces pombe), kluyveromyces (kluyveromyces) hosts, such as kluyveromyces lactis (k.lactis), kluyveromyces fragilis (k.fragilis) (ATCC 12424), kluyveromyces bulgaricus (k.bulgarisus) (ATCC 16045), kluyveromyces weicki (k.winkeramii) (ATCC 24178), kluyveromyces valus Lu Wei yeast (k.watii) (ATCC 56500), kluyveromyces drosophila (k.drosophila) (ATCC 36906), kluyveromyces thermotolens (k.thermals) and kluyveromyces marxianus (k.marxianus); yarrowia (EP 402 226); pichia pastoris (EP 183 070); candida (Candida); trichoderma reesei (EP 244 234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces western (Schwanniomyces occidentalis); and filamentous fungi such as Neurospora (Neurospora), penicillium (Penicillium), curvularia (Tolypocladium) and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (A. Nidulans) and Aspergillus niger (A. Niger).

Suitable host cells for expressing the glycosylation construct are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains and variants from hosts such as spodoptera frugiperda (Spodoptera frugiperda) (caterpillars), aedes aegypti (Aedes aegypti) (mosquitoes), aedes albopictus (mosquitoes), drosophila melanogaster (Drosophila melanogaster) (drosophila) and Bombyx mori (silk moths) have been identified, as well as corresponding permissive insect host cells. A variety of viral strains for transfection are publicly available, for example the L-1 variant of the NPV of Spodoptera frugiperda (Autographa californica) and the Bm-5 strain of the NPV of Bombyx mori, and according to the invention such viruses may be used as the viruses herein, in particular for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, arabidopsis and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell culture are known to those skilled in the art. See, e.g., hiatt et al, nature [ Nature (1989) 342:76-78; owen et al (1992) Bio/Technology [ Bio/Technology ]10:790-794; artsaaenko et al (1995) The Plant J [ J.Phytophyte ]8:745-750 and Fecker et al (1996) Plant Mol Biol [ Plant molecular biology ]32:979-986.

However, interest in vertebrate cells is greatest, and propagation of vertebrate cells in culture (cell culture) has become a routine procedure. An example of a useful mammalian host cell line is the monkey kidney CV1 line transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293 cells or subclones for 293 cells grown in suspension culture, graham et al, J. Gen Virol [ J. Virol. General J. Virology ]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 [ national academy of sciences of the united states of america ]77:4216 (1980)); mouse sertoli cells (e.g., TM4, mather, biol. Reprod. [ reproduction Biol. ]23:243-251 (1980)); monkey kidney cells (e.g., CVI ATCC CCL 70); african green monkey kidney cells (e.g., VERO-76, ATCC CRL 1587); human cervical cancer cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); brulo rat hepatocytes (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human hepatocytes (e.g., hep G2,1413, 8065); mouse mammary tumors (e.g., MMT 060562, ATCC CCL-51); TRI cells (Mather et al, annals N.Y Acad. Sci. [ New York academy of sciences (1982) 383:44-68); MRC 5 cells; FS4 cells; and human liver cancer cell lines (e.g., hep G2).

In another embodiment, the invention provides a process for producing a construct of the invention, comprising culturing a host cell of the invention under conditions that allow expression of the construct of the invention and recovering the produced construct from the culture.

As used herein, the term "culture" refers to the in vitro maintenance, differentiation, growth, proliferation and/or propagation of cells in a culture medium under suitable conditions. Cells are grown and maintained in a cell growth medium at an appropriate temperature and gas mixture. Culture conditions vary widely for each cell type. Typical growth conditions are a temperature of about 37 ℃, a CO2 concentration of about 5% and a humidity of about 95%. The formulation of the growth medium may vary, for example, in terms of pH, carbon source (e.g., glucose) concentration, nature and concentration of the growth factors, and the presence of other nutrients (e.g., amino acids or vitamins). The growth factors used to supplement the culture medium are typically derived from serum from animal blood, such as Fetal Bovine Serum (FBS), niu Xiaoniu serum (FCS), horse serum, and pig serum. Cells may be grown in suspension or as adherent cultures. There are also cell lines that have been modified to survive in suspension culture so that they can grow at higher densities than the adherent conditions would allow.

The term "expression" includes any step involved in the production of constructs of the 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 constructs from cell cultures. The "recovery" or "purification" process can separate the proteinaceous and non-proteinaceous parts of the cell culture and ultimately separate the desired construct from all other polypeptides and proteins. The separation step generally takes advantage of 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, whereas analytical purification produces relatively small amounts of protein for various research or analytical purposes.

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

In the case of secretion of the construct into the culture medium, the supernatant of such an expression system is typically first concentrated using a commercially available protein concentration filter (e.g., ultrafiltration unit).

Constructs of the invention prepared from host cells may be recovered or purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. Other techniques for protein purification, such as fractionation on ion exchange columns, mixed mode ion exchange, HIC, ethanol precipitation, size exclusion chromatography, reverse phase HPLC, chromatography on silica, chromatography on heparin agarose, chromatography on anion or cation exchange resins (e.g. polyaspartic acid columns), immunoaffinity (e.g. protein a/G/L) chromatography, chromatofocusing, SDS-PAGE, ultracentrifugation and ammonium sulfate precipitation are also available depending on the construct to be recovered.

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.

Furthermore, the invention provides a pharmaceutical composition or formulation comprising a construct of the invention or a construct produced according to the process of the invention.

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 invention comprise a preferably therapeutically effective amount of one or more constructs of the invention. Preferably, the pharmaceutical composition further comprises one or more (pharmaceutically effective) suitable formulations of 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 dosages and concentrations employed. Pharmaceutical compositions of the invention include, but are not limited to, liquid, frozen and lyophilized compositions.

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

Certain embodiments provide pharmaceutical compositions comprising a construct of the invention and one or more additional excipients, such as those excipients illustratively described in this section and elsewhere herein. Excipients may be used in the present invention for a variety of purposes such as adjusting the physical, chemical or biological properties of the formulation, such as adjusting the viscosity and/or the process of the present invention to improve effectiveness and/or stabilize such formulations and methods to prevent degradation and spoilage, for example, due to pressure occurring during manufacture, transportation, storage, pre-use, administration and thereafter. Excipients are generally used at their lowest effective concentration.

In certain embodiments, the pharmaceutical compositions may contain formulated substances (see Remington's Pharmaceutical Sciences [ pharmaceutical encyclopedia of the r.i., 18 th edition, 1990,Mack Publishing Company [ mark publication company ]) in order to alter, maintain, or preserve certain characteristics of the composition (e.g., pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorptivity, or permeability). In such embodiments, suitable formulations may include, but are not limited to:

Amino acids

Antimicrobial agents, such as antibacterial and antifungal agents

Antioxidant(s)

Buffers, buffer systems and buffers for maintaining the composition at physiological pH or at a slightly lower pH (typically in the range of about 5 to about 8 or 9)

Nonaqueous solvents, vegetable oils and injectable organic esters

The aqueous carrier comprises water, an alcohol/aqueous solution, an emulsion or a suspension comprising saline and a buffer medium

Biodegradable polymers, e.g. polyesters

Accumulation-increasing agent

Chelating agent

Isotonic and absorption delaying agent

Complexing agent

Filler (filler)

Carbohydrates

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

Coloring and flavoring agents

Sulfur-containing reducing agent

Diluents (diluent)

Emulsifying agent

Hydrophilic polymers

Salt-forming counterions

Preservative agent

Metal complex

Solvents and cosolvents

Sugar and sugar alcohol

Suspending agent

Surfactants or wetting agents

Stability enhancer

Tension enhancer

Parenteral delivery vehicle

Intravenous delivery vehicle

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

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

(a) As with the constructs described herein,

(b) At least one of the buffers is used for the preparation of a liquid,

(c) At least one sugar, and

(d) At least one surfactant;

wherein the pH of the pharmaceutical composition is in the range of 3.5 to 6.

In the above-described composition, 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 optionally the construct comprises a third domain comprising two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain, wherein the two polypeptide monomers are fused to each other via a peptide linker;

in the compositions described above, it is further contemplated that the at least one buffer is present in a concentration range of 5mM to 200mM, more preferably in a concentration range of 10mM to 50 mM. It is also contemplated that the at least one sugar is selected from the group consisting of: monosaccharides, disaccharides, cyclic polysaccharides, sugar alcohols, linear branched glucans or linear unbranched glucans. It is also envisaged 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 envisaged that the at least one sugar is present in a concentration in the range of 1% to 15% (m/V), preferably in a concentration in the range of 9% to 12% (m/V). It is further contemplated that the construct is present in a concentration range of 0.1mg/ml to 8mg/ml, preferably 0.2-2.5mg/ml, more preferably 0.25-1.0 mg/ml.

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, polyoxyethylene (polyoxythynyl), 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 envisaged that the pH of the composition is in the range 4.0 to 5.0, preferably 4.2. It is also contemplated that the pharmaceutical composition has an osmolarity of 150 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 excipient or excipients are present in a concentration range of 0.1% to 15% (w/V).

The present invention also provides a pharmaceutical composition comprising (a) an antibody construct as described herein, preferably in a concentration range of 0.1mg/ml to 8mg/ml, preferably 0.2mg/ml to 2.5mg/ml, more preferably 0.25mg/ml to 1.0 mg/ml; (b) 10mM glutamate or acetate; (c) 9% (m/V) sucrose or 6% (m/V) sucrose and 6% (m/V) hydroxypropyl-beta-cyclodextrin; (d) 0.01% (m/V) polysorbate 80; wherein the liquid pharmaceutical composition has a pH of 4.2.

It is contemplated that in addition to the constructs of the invention defined herein, the compositions of the invention may comprise additional bioactive agents, depending on the intended use of the composition. Such agents may be drugs acting on the gastrointestinal system, drugs acting as cytostatics, drugs preventing hyperuricemia, drugs inhibiting immune responses, drugs regulating inflammatory responses, drugs acting on the circulatory system and/or agents known in the art such as cytokines. It is also envisaged to use the polypeptide construct of the invention in co-therapy, i.e. in combination with another anticancer drug.

In this context, it is envisaged that the pharmaceutical composition of the invention (comprising a construct comprising a domain that binds CLDN6 on the surface of a target cell and another domain that binds CD3 on the surface of a T cell, as described in more detail herein above) additionally comprises an agent (preferably an antibody or 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 4-1 BB). The invention also relates to a combination and an agent, preferably an antibody or a polypeptide construct, comprising a domain of a paratope (antigen binding) structure that binds to CLDN6 on the surface of a target cell and another domain of a paratope (antigen binding) structure that binds to CD3 on the surface of a T cell, which is bound to a protein of the immune checkpoint pathway, such as PD-1 or CTLA-4, or to a protein of a co-stimulatory immune checkpoint receptor, such as 4-1 BB.

In certain embodiments, the optimal pharmaceutical composition will be determined, for example, based on the intended route of administration, the form of delivery, and the dosage desired. See, e.g., remington's Pharmaceutical Sciences [ Leimden pharmaceutical Specification ], supra. In certain embodiments, such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the constructs of the invention. In certain embodiments, the primary vehicle or carrier in the pharmaceutical composition may be aqueous or non-aqueous in nature. For example, a suitable vehicle or carrier may be water for injection or a physiological saline solution, possibly supplemented with other substances common in compositions for parenteral administration. In certain embodiments, compositions comprising constructs of the invention may be prepared for storage by mixing selected ingredients of the desired purity with optional formulation (Remington's Pharmaceutical Sciences [ Remington pharmaceutical book ], supra) in the form of a lyophilized cake or aqueous solution. Further, in certain embodiments, constructs of the invention may be formulated as a lyophilizate using suitable excipients.

When parenteral administration is contemplated, the therapeutic compositions for use in the present invention can be provided in the form of a pyrogen-free, parenterally acceptable aqueous solution comprising the desired construct of the present invention in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the construct of the invention is formulated as a sterile isotonic solution for suitable storage. In certain embodiments, the formulation may include formulating the desired molecule with an agent that may provide controlled or sustained release of the product, which may be delivered via depot injection, or may promote a sustained duration in the circulation. In certain embodiments, implantable drug delivery devices may be used to introduce a desired construct.

Additional pharmaceutical compositions will be apparent to those skilled in the art, including formulations involving inclusion of the constructs of the 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 construct may also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, in a colloidal drug delivery system, or in a macroemulsion. Such techniques are disclosed in Remington' sPharmaceutical Sciences [ Leimden pharmaceutical Specification ], supra.

Pharmaceutical compositions for in vivo administration are typically provided in sterile formulations. Sterilization may be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using the method may be performed before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Parenteral compositions are typically placed in a container (e.g., an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle) having a sterile access port.

Another aspect of the invention includes self-buffering formulations comprising the constructs of the invention, which may be used as pharmaceutical compositions, as described in international patent application WO 2006/138181. Regarding protein stabilization and formulation materials and methods useful in this regard, a variety of publications are available, such as Arawaka t et al, pharm Res [ pharmaceutical research ]1991, month 3; 8 (3) 285-91; kendrick et al, "Physical stabilization of proteins in aqueous solution [ physical stability of protein in aqueous solution ]", rational Design of Stable Protein Formulations: theory and Practice [ rational design of stable protein formulation: theory and practice Carpenter and Manning editions Pharmaceutical Biotechnology [ pharmaceutical biotechnology ].13:61-84 (2002); and Randolph and Jones, pharm Biotechnol 2002;13:159-75, see in particular the section relating to excipients and methods for self-buffering protein formulations, in particular with respect to protein pharmaceutical products and processes for veterinary and/or human medical use.

According to certain embodiments of the invention, salts may be used, for example, to adjust the ionic strength and/or isotonicity of a composition or formulation and/or to improve the solubility and/or physical stability of a construct or other component of a composition according to the invention. The ions can stabilize the natural state of the protein by binding to charged residues on the protein surface and by shielding charged and polar groups in the protein and reducing the strength of their electrostatic interactions, attraction and repulsion interactions. The ions may also stabilize the denatured state of the protein by specifically binding to the denatured peptide bond (- -CONH) of the protein. In addition, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions and thereby prevent or reduce protein aggregation and insolubilization.

The effect of ionic species on proteins varies significantly. Several classification ratings have been developed for the ions and their effects on proteins that can be used to formulate pharmaceutical compositions according to the invention. One example is the Hofmeister series, which rates ionic and polar nonionic solutes by their effect on the conformational stability of proteins in solution. The stable solute is referred to as "lyophile". Unstable solutes are known as "chaotropic". High concentrations of nucleophile are typically used to precipitate proteins from solution ("salting out"). Chaotropic agents are commonly used to denature and/or solubilize proteins ("saline"). The relative effectiveness of ion pairs "salting-in" and "salting-out" defines their positions in the Hofmeister series.

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

Polyols are lyophile 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 tonicity of the formulation and to prevent freeze-thaw stress during transportation or to prevent preparation of the pellets during manufacture. Polyols may also be used as cryoprotectants in the context of the present invention.

Certain embodiments of a formulation or composition comprising a construct of the invention may comprise a surfactant. Proteins may be prone to adsorption on surfaces and to denaturation and aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These deleterious interactions are generally inversely proportional to protein concentration and are typically exacerbated by physical oscillations (such as those generated during product transportation and handling). Surfactants are routinely used to prevent, minimize or reduce surface adsorption. Surfactants are also commonly used to control protein conformational stability. The use of surfactants in this regard is protein specific in that a particular surfactant typically stabilizes some proteins and destabilizes others.

Certain embodiments of a formulation or composition comprising a construct of the invention may comprise one or more antioxidants. By maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light, detrimental oxidation of proteins in the pharmaceutical formulation can be prevented to some extent. Antioxidant excipients may also be used to prevent oxidative degradation of the protein. It is contemplated that the antioxidants used in therapeutic protein formulations according to the present invention may be water soluble and retain their activity throughout the shelf life of the product (composition comprising the construct). Antioxidants may also destroy proteins and should therefore be selected in a way that eliminates or substantially reduces the likelihood that the antioxidants would destroy constructs or other proteins in the formulation, among other things.

Certain embodiments of a formulation or composition comprising a construct of the invention may comprise one or more preservatives. For example, preservatives are necessary when developing multi-dose parenteral formulations that involve more than one extraction from the same container. Its main function is to inhibit microbial growth and to ensure sterility of the product throughout its shelf-life or lifetime. Despite the long history of use of preservatives with small molecule parenteral drugs, developing protein formulations that include preservatives can be challenging. Preservatives generally have an unstable effect (aggregation) on proteins, and this has been a major factor limiting their use in multi-dose protein formulations. To date, most protein drugs are formulated for single use only. However, when multi-dose formulations are possible, they have the added advantage of patient convenience and increased marketability. A good example is human growth hormone (hGH), where the development of preservative formulations has led to the commercialization of more convenient, multi-use injection pen displays. Several aspects need to be considered during the formulation and development of preservative dosage forms. The effective preservative concentration in the pharmaceutical product must be optimized. This requires testing a given preservative in a dosage form in a concentration range that imparts antimicrobial effectiveness without compromising protein stability.

As can be expected, the development of liquid formulations containing preservatives is more challenging than freeze-dried formulations. The freeze-dried product may be lyophilized without a preservative and reconstituted with a diluent containing a preservative at the time of use. This shortens the time of contact of the preservative with the construct, thereby significantly minimizing the associated stability risks. In the case of liquid formulations, preservative effectiveness and stability should be maintained throughout the product shelf life. It is important to note that preservative effectiveness should be demonstrated in the final formulation containing the active drug and all excipient components. Once the pharmaceutical composition is formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored in a ready-to-use form or in a form that is reconstituted prior to administration (e.g., lyophilized form).

The biological activity of the pharmaceutical compositions defined herein can be determined, for example, by in vivo 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 of a pharmaceutical composition or formulation of the invention using, for example, standardized NCI response criteria. The success or in vivo efficacy of a therapy using a pharmaceutical composition of the invention refers to the effectiveness of the composition for its intended use, i.e., the ability of the composition to elicit its desired effect, i.e., deplete pathological cells (e.g., tumor cells). In vivo efficacy can be monitored by established standard methods for each disease entity including, but not limited to, white blood cell count, differential, 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-ray, nuclear magnetic resonance tomography, positron emission tomography scanning, lymph node biopsy/histology and other established standard methods may be used.

Another major challenge in developing a drug (e.g., a pharmaceutical composition of the present invention) is the predictable modulation of pharmacokinetic properties. For this purpose, a pharmacokinetic profile of the drug candidate, i.e. a profile of pharmacokinetic parameters affecting the ability of a particular drug to treat a given condition, may be established. Pharmacokinetic parameters of a drug that affect the ability of the drug to treat a disease entity include, but are not limited to: half-life, distribution capacity, liver first pass metabolism and serum binding extent. The efficacy of a given agent may be affected by each of the parameters mentioned above.

"half-life" is the time required to reduce the amount to half of its initial value. Medical science refers to the half-life of a substance or drug in the human body. In the medical context, half-life may refer to the time it takes a substance/drug to lose half of its activity (e.g., pharmacological, physiological, or radiological activity). Half-life may also describe the time it takes for the concentration of a drug or substance (e.g., a construct of the invention) in plasma/serum to reach half its steady state value ("serum half-life"). Typically, clearance or removal of the administered substance/drug refers to body cleansing through biological processes such as metabolism, excretion, and the like (also involving kidney and liver function). "first pass metabolism" is the phenomenon of metabolism of a drug, thereby decreasing the concentration of the drug before it reaches the circulation. It is the part of drug loss during absorption. Thus, "liver first pass metabolism" means the tendency of a drug to be metabolized upon first contact with the liver (i.e., during its first pass through the liver). "distribution volume" (VD) means the extent to which a drug is distributed in body tissue rather than plasma, with higher VD indicating a greater tissue distribution. The retention of the drug may occur in various compartments throughout the body, such as the intracellular and extracellular spaces, tissues and organs, and the like. By "degree of serum binding" is meant the propensity of a drug to interact with and bind to a serum protein (e.g., albumin), resulting in a decrease or loss of biological activity of the drug.

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

One embodiment provides a construct of the invention (or a construct produced according to the process of the invention) for use as a medicament, in particular for use in the prevention, treatment or alleviation of a disease, preferably a neoplasm. Another embodiment provides the use of a construct of the invention (or a construct produced according to the process of the invention) in the manufacture of a medicament for the prevention, treatment or alleviation of a disease, preferably a neoplasm. It is also contemplated to provide a method for preventing, treating or alleviating a disease, preferably a neoplasm, comprising the step of administering to a subject in need thereof a construct of the invention (or a construct produced according to the process of the invention). The terms "subject in need thereof", "patient" or "in need of treatment" include those already suffering from the disease, as well as those in which the disease is to be prevented. These terms also include human and other mammalian subjects receiving prophylactic or therapeutic treatment.

The polypeptides/polypeptide constructs of the invention and formulations/pharmaceutical compositions described herein are useful for treating, alleviating and/or preventing a medical condition as described herein in a patient in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic (prophlic) or preventative (predictive) measures. Treatment includes administering or administering the polypeptide/polypeptide construct/pharmaceutical composition to the body, isolated tissue or cells of a patient or subject in need thereof suffering from, having symptoms of, or having a predisposition to such a disease/disorder as described herein, with the aim of healing, moderating, reducing, altering, remediating, alleviating, ameliorating or affecting the disease, symptoms of the disease, or the predisposition to the disease. As used herein, the term "alleviating" refers to any improvement in the disease state of a patient by administering a polypeptide construct according to the invention to such patient or a subject in need thereof. Such an improvement may be considered to slow or stop the progression of the disease in the patient, and/or a decrease in the severity of the disease symptoms, an increase in the frequency or duration of disease-free periods, or prevention of damage or disability due to the disease. As used herein, the term "preventing" means avoiding the occurrence or recurrence of a disease as specified herein by administration of a construct according to the invention to a subject in need thereof.

The term "disease" refers to any condition that would benefit from treatment with the constructs or pharmaceutical compositions described herein. This includes chronic and acute disorders 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 CLDN6 positive, i.e. it is characterized by expression or overexpression of CLDN 6. Overexpression of CLDN6 means an increase of at least 10%, in particular at least 25%, at least 50%, at least 100%, at least 250%, at least 500%, at least 750%, at least 1000%, or even more. Expression was only found in diseased tissue, whereas no or significantly no expression was detected in the corresponding healthy tissue. According to the invention, diseases associated with cells expressing CLDN6 include cancer diseases. Furthermore, according to the present invention, cancer diseases are preferably those in which the cancer cells express CLDN 6.

"neoplasms" are abnormal growth of tissue, usually but not always forming a tumor. When a tumor is also formed, it is often referred to as a "tumor". A neoplasm or tumor may be benign, potentially malignant (precancerous), or malignant (cancerous). Malignant neoplasms/tumors are often referred to as cancers. They typically invade and destroy surrounding tissues and may form metastases, i.e. they spread to other parts of the body, tissues or organs. A "primary tumor" is a tumor that grows at the anatomical site where the tumor begins to progress and continues to produce cancerous tumors. Most cancers develop in their primary site, but then continue to metastasize or spread to other parts of the body (e.g., tissues and organs). These additional tumors are "secondary tumors". Most cancers continue to be invoked after their primary site, even after they have spread to other parts of the body.

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

The term "minimal residual disease" (MRD) refers to evidence of the presence of small amounts of residual cancer cells remaining in a patient after cancer treatment, for example, when the patient is in remission (no disease symptoms or signs). Very small amounts of residual cancer cells are generally not detectable by conventional means, as standard tests for assessing or detecting cancer are not sensitive enough to detect MRD. Today, molecular biological tests that are very sensitive to MRD are available, such as flow cytometry, PCR, and next generation sequencing. These tests can measure the minimum level of cancer cells in a tissue sample, sometimes as low as one in a million normal cells. In the context of the present invention, it is contemplated that the term "prevention", "treatment" or "alleviation" of cancer also encompasses "prevention, treatment or alleviation of MRD", whether or not MRD is detected.

In one embodiment of the invention, the neoplasm, cancer or tumor is selected from the group consisting of, but not limited to, germ cell cancer, ovarian cancer, and lung cancer.

According to one embodiment of the invention, the ovarian cancer is an ovarian epithelial cancer selected from the group consisting of mucinous endometrioid cancer, clear cell cancer and undifferentiated ovarian cancer; ovarian stromal tumors, including granulocytoma, granulosa tumor, and Sertoli-Leydig tumor; ovarian germ cell tumors, including teratoma, asexual cell tumor ovarian germ cell carcinoma, endoplasmic sinus tumor (yolk sac tumor) and choriocarcinoma tumors; ovarian sarcoma, kukenbo tumor, or ovarian cyst. According to another embodiment, the ovarian cancer is recurrent or recurrent ovarian cancer, or platinum (platinum) and/or standard chemotherapy refractory ovarian cancer. Treatment efficacy may be determined by measuring CA-125. CA-125 is a protein present in blood. A large amount of CA-125 may be indicative of ovarian cancer, fallopian tube cancer, while a reduced amount may be indicative of the efficacy of the selected treatment. Genetic factors that may lead to the development of ovarian cancer are mutations in one of two genes, termed breast cancer gene 1 (BRCA 1) and breast cancer gene 2 (BRCA 2). Women with BRCA1 mutations are at 35% to 70% higher risk of ovarian cancer. Women with BRCA2 mutations are at 10% to 30% higher risk (www.cancercenter.com/cancer-types/ovarian-cancer/risk-factors). However, most women diagnosed with ovarian cancer do not have these mutations.

According to another embodiment of the present invention, the lung cancer is non-small cell lung cancer, which may be further selected from the group consisting of squamous cell carcinoma, large cell carcinoma and adenocarcinoma. A subset of adenocarcinomas can be defined by specific mutations in genes encoding Epidermal Growth Factor Receptor (EGFR) and components of the downstream mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3K) signaling pathways. Genetic abnormalities potentially relevant to therapeutic decisions include translocation involving Anaplastic Lymphoma Kinase (ALK) -tyrosine kinase receptors that are sensitive to ALK inhibitors, and amplification of MET (mesenchymal epithelial transforming factor) encoding hepatocyte growth factor receptors. MET amplification is associated with secondary resistance to EGFR tyrosine kinase inhibitors.

The constructs of the invention are generally designed for a particular route and method of administration, a particular dosage and frequency of administration, a particular treatment for a particular disease, a range of bioavailability and persistence, and the like. The materials of the composition are preferably formulated at a concentration acceptable for the site of application. Formulations and compositions may therefore be designed according to the present invention for delivery by any suitable route of administration. In the context of the present invention, routes of administration include, but are not limited to, topical routes, enteral routes, and parenteral routes.

If the pharmaceutical composition has been lyophilized, the lyophilized material is first reconstituted in an appropriate liquid prior to administration. The lyophilized material may be reconstituted in, for example, bacteriostatic water for injection (BWFI), physiological saline, phosphate Buffered Saline (PBS), or the same formulation as the protein prior to lyophilization. The pharmaceutical compositions and constructs of the invention are particularly useful for parenteral administration, such as intravenous delivery, e.g., by injection or infusion. The pharmaceutical composition may be administered using a medical device. Examples of medical devices for administration of pharmaceutical compositions are described in U.S. patent No. 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.

The compositions of the invention may be administered to a subject at a suitable dose, which may be determined, for example, in a dose escalation study. As mentioned above, the constructs of the invention exhibiting the interspecies specificities described herein may also be advantageously used in preclinical testing of non-chimpanzee primates. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage of any one patient depends on many factors including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health and other drugs being administered simultaneously.

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

A therapeutically effective amount of a construct of the invention preferably results in a decrease in severity of disease symptoms, an increase in the frequency or duration of disease-free symptoms periods, or prevention of damage or disability due to the disease. In the treatment of a CLDN 6-expressing tumor, a therapeutically effective amount of a construct 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 an untreated patient. The ability of a compound to inhibit tumor growth can be evaluated in an animal model that predicts efficacy in human tumors.

In another embodiment, the invention provides a kit comprising a construct of the invention, a construct produced according to a process of the invention, a polynucleotide of the invention, a vector of the invention and/or a host cell of the invention. In the context of the present invention, the term "kit" means that two or more components, one of which corresponds to a construct, pharmaceutical composition, polynucleotide, vector or host cell of the present invention, are packaged together in a container, receptacle or other. Thus, a kit may be described as a set of products and/or appliances sufficient to achieve a particular goal, which may be sold as a single unit.

It is envisaged that a further component of the kit of the invention is an agent (preferably an antibody or 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 4-1 BB). These agents are described in more detail herein above. According to one embodiment, the kit comprises a construct of the invention and an antibody or construct that binds to PD-1. anti-PD-1 binding proteins useful for this purpose are described in detail, for example, in PCT/US 2019/013205. In certain embodiments, the kit allows for simultaneous and/or sequential administration of the components.

The kit may comprise one or more receptacles (such as vials, ampoules, containers, syringes, vials, bags) of any suitable shape, size and material (preferably waterproof, e.g. plastic or glass) containing a dose of a construct or pharmaceutical composition of the invention suitable for administration (see above). The kit may additionally contain instructions (e.g., in the form of a pamphlet or instruction manual), means for administering the constructs or pharmaceutical compositions of the invention (e.g., syringe, pump, infuser, etc.), means for reconstituting the constructs of the invention, and/or means for diluting the constructs of the invention.

The invention also provides a kit for a single dose administration unit. Kits of the invention may also contain a first receptacle comprising a dried/lyophilized construct or pharmaceutical composition and a second receptacle comprising an aqueous formulation. In certain embodiments of the present invention, kits are provided that contain single and multi-chamber prefilled syringes.

The invention refers to the following items:

i) A polypeptide or polypeptide construct comprising or consisting of:

a domain (comprising paratope) that binds to human CLDN6 (SEQ ID NO: 1) on the surface of a target cell, and

A domain that binds human CD3 (comprising a paratope), and

a domain that extends the half-life of the polypeptide.

ii) the polypeptide or polypeptide construct according to item i), wherein the polypeptide construct is a T cell activation construct.

iii) The polypeptide or polypeptide construct according to any one of items i) and ii), wherein the polypeptide construct is a T cell activating polypeptide as determined by a T cell activating assay selected from the group comprising determining the amount of expression of CD69, determining the amount of expression of CD25, determining the amount of secreted IL-2, and determining the cytotoxic activity of these T cells.

iv) the polypeptide or polypeptide construct according to any one of items i) to iii), wherein the domain that extends the half-life of the polypeptide comprises or consists of two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain.

v) the polypeptide or polypeptide construct according to any one of items i) to iv), wherein the antigen binding (epitope binding) domain that binds CLDN6 comprises or consists of a paratope that binds to an epitope within human CLDN6 corresponding to amino acids 29-81 of SEQ ID No.1 (UniProt entry P56747).

vi) the polypeptide or polypeptide construct according to any one of items i) to v), wherein the first antigen binding (epitope binding) domain that binds CLDN6 comprises or consists of a paratope that binds to an epitope within human CLDN6 corresponding to amino acids 138-160 of SEQ ID No.1 (unit prot entry P56747).

vii) the polypeptide or polypeptide construct according to any of clauses i) to vi), wherein the domain comprising or consisting of a paratope that binds to CLDN6 binds to amino acids 29-39 of SEQ ID NO:1 in extracellular loop 1 (ECL 1) of CLDN6 depicted in SEQ ID NO:9 and/or amino acids 151-160 of SEQ ID NO:1 in extracellular loop 2 (ECL 2) of CLDN6 depicted in SEQ ID NO:10 on the surface of target cells; it is clear that the conjugates do not require direct chemical interactions within the sequences depicted in SEQ ID NOs 9 and 10, but that at least one or more amino acids of these sequences are in direct contact with one and typically multiple amino acids of the binding domain, e.g. by hydrogen bonding. The general principle of interaction between a binding domain (i.e., paratope) and a target domain (i.e., epitope) is known in the art (see Janeway et al Immunobiology, 9 th edition, 2016).

viii) a polypeptide or polypeptide construct according to any one of items i) to vii), wherein the domain comprising or consisting of a paratope that binds to CD3 binds to extracellular epitopes of the CD3 epsilon chain of humans and macaques.

ix) the polypeptide or polypeptide construct according to any one of clauses i) to viii), wherein the paratope that binds CLDN6 binds the same epitope on CLDN6 as a polypeptide construct or antibody or derivative or fragment thereof comprising a domain comprising a paratope that binds CLDN6 on the surface of a target cell, wherein the paratope comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 of a Variable Heavy (VH) chain and/or complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of a Variable Light (VL) chain, selected from the group depicted in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are very preferred:

o) a VH region comprising or consisting of CDR-H1 depicted in SEQ ID NO. 209, CDR-H2 depicted in SEQ ID NO. 210 and CDR-H3 depicted in SEQ ID NO. 211, and CDR-L1 depicted in SEQ ID NO. 212, CDR-L2 depicted in SEQ ID NO. 213 and CDR-L3 depicted in SEQ ID NO. 214;

p) a VH region comprising or consisting of CDR-H1 as depicted in SEQ ID NO. 223, CDR-H2 as depicted in SEQ ID NO. 224 and CDR-H3 as depicted in SEQ ID NO. 225, and a VL region comprising or consisting of CDR-L1 as depicted in SEQ ID NO. 226, CDR-L2 as depicted in SEQ ID NO. 227 and CDR-L3 as depicted in SEQ ID NO. 228;

q) a VH region comprising or consisting of CDR-H1 as depicted in SEQ ID NO. 237, CDR-H2 as depicted in SEQ ID NO. 238 and CDR-H3 as depicted in SEQ ID NO. 239, and CDR-L1 as depicted in SEQ ID NO. 240, CDR-L2 as depicted in SEQ ID NO. 241 and CDR-L3 as depicted in SEQ ID NO. 242;

r) a VH region comprising or consisting of CDR-H1 depicted in SEQ ID NO:251, CDR-H2 depicted in SEQ ID NO:252 and CDR-H3 depicted in SEQ ID NO:253, and a VL region comprising or consisting of CDR-L1 depicted in SEQ ID NO:254, CDR-L2 depicted in SEQ ID NO:255 and CDR-L3 depicted in SEQ ID NO:256,

s) a VH region comprising or consisting of CDR-H1 depicted in SEQ ID NO. 265, CDR-H2 depicted in SEQ ID NO. 266 and CDR-H3 depicted in SEQ ID NO. 267, and CDR-L1 depicted in SEQ ID NO. 268, CDR-L2 depicted in SEQ ID NO. 269 and CDR-L3 depicted in SEQ ID NO. 270.

x) the polypeptide or polypeptide construct according to any one of items i) to ix), wherein the domain that binds to human CD3 epsilon (comprising or consisting of paratope) also binds to common marmoset or squirrel monkey CD3 epsilon.

xi) the polypeptide or polypeptide construct according to any one of items i) to x), wherein

a) The polypeptide or construct is a single-stranded construct,

b) The domain that binds CLDN6 (including the paratope) is in the form of an scFv,

c) The domain that binds CD3 (comprising the paratope) is in the form of an scFv,

d) These domains (containing paratopes) are linked via a linker, and/or

e) The polypeptide or polypeptide construct comprises a domain that provides an extended serum half-life.

xii) a polypeptide or polypeptide construct according to any one of items i) to xi), wherein the domain that binds to CLDN6 (comprising or consisting of a paratope) does not bind to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9 and/or CLDN18.1/CLDN 18.2.

xiii) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain binding to CLDN6 (comprising or consisting of paratopes) comprises a VH region comprising CDR-H1, CDR-H2 and CDR-H3 and a VL region comprising CDR-L1, CDR-L2 and CDR-L3, selected from the group depicted in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are very preferred:

xiv) a polypeptide or polypeptide construct according to any of the preceding items, wherein the domain comprising or consisting of the paratope binding to CLDN6 comprises a VH region having an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:39, SEQ ID NO:53, SEQ ID NO:67, SEQ ID NO:81, SEQ ID NO:95, SEQ ID NO:109, SEQ ID NO:123, SEQ ID NO:137, SEQ ID NO:151, SEQ ID NO:165, SEQ ID NO:179, SEQ ID NO:193, SEQ ID NO:207, SEQ ID NO:221, SEQ ID NO:235, SEQ ID NO:249, or the sequence depicted in SEQ ID NO:263,

Wherein said VH region amino acid sequence may have one or more modifications of one or several amino acid residues in the framework and/or hypervariable regions, provided that said domain comprising said modified VH region selectively binds CLDN6, and

optionally wherein the domain is part of a polypeptide or polypeptide construct that activates T cells and retains the ability to induce T cell dependent cytotoxicity,

further optionally, wherein the domain is part of a polypeptide or polypeptide construct that activates a T cell and retains the ability to induce more than 1000-fold more T cell-dependent cytotoxicity than the same cell type that expresses CLDN9 but does not express CLDN6, and

still further optionally, wherein the domain is part of a polypeptide or polypeptide construct that is incapable of activating T cells and inducing T cell-dependent cytotoxicity in CLDN 6-negative cells of the same cell type, preferably when tested in an in vitro cytotoxicity assay.

xv) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain comprising (comprising or consisting of) the paratope binding to CLDN6 comprises a VL region having an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:82, SEQ ID NO:96, SEQ ID NO:110, SEQ ID NO:124, SEQ ID NO:138, SEQ ID NO:152, SEQ ID NO:166, SEQ ID NO:180, SEQ ID NO:194, SEQ ID NO:208, SEQ ID NO:222, SEQ ID NO:236, SEQ ID NO:250, or SEQ ID NO:264,

Wherein the VL region amino acid sequence may have one or more modifications of one or more amino acid residues in the framework and/or hypervariable regions, provided that the domain comprising the modified VL region selectively binds CLDN6, and

optionally wherein the domain is a polypeptide or part of a polypeptide construct that activates a T cell and retains the ability to induce T cell-dependent cytotoxicity in a target cell,

further optionally, wherein the domain is part of a polypeptide or polypeptide construct that activates T cells and retains the ability to induce more than 500-fold T cell-dependent cytotoxicity than a control cell that does not express CLDN6, wherein the cells optionally express CLDN9, but

xvi) a polypeptide or polypeptide construct according to any one of the preceding claims, wherein the domain that binds CLDN6 (comprising or consisting of paratopes) comprises a pair of VH and VL regions having the amino acid sequences depicted in: SEQ ID NO 11+12, SEQ ID NO 25+26, SEQ ID NO 39+40, SEQ ID NO 53+54, SEQ ID NO 67+68, SEQ ID NO 81+82, SEQ ID NO 95+96, SEQ ID NO 109+110, SEQ ID NO 123+124, SEQ ID NO 137+138, SEQ ID NO 151+152, SEQ ID NO 165+166, SEQ ID NO 179+180, SEQ ID NO 193+194, SEQ ID NO 207+208, SEQ ID NO 221+222, SEQ ID NO 235+236, SEQ ID NO 249+250, or SEQ ID NO 263+264.

xvii) a polypeptide or polypeptide construct according to any one of the preceding claims, wherein the domain that binds to CLDN6 (comprising or consisting of paratopes) comprises the amino acid sequence depicted in: SEQ ID NO 19, SEQ ID NO 22, SEQ ID NO 33, SEQ ID NO 36, SEQ ID NO 47, SEQ ID NO 50, SEQ ID NO 61, SEQ ID NO 64, SEQ ID NO 75, SEQ ID NO 78, SEQ ID NO 89, SEQ ID NO 92, SEQ ID NO 103, SEQ ID NO 106, SEQ ID NO 117, SEQ ID NO 120, SEQ ID NO 131, SEQ ID NO 134, SEQ ID NO 145, SEQ ID NO 148, SEQ ID NO 159, SEQ ID NO 162, SEQ ID NO 173, SEQ ID NO 176, SEQ ID NO 187, SEQ ID NO 190, SEQ ID NO 201, SEQ ID NO 204, SEQ ID NO 215, SEQ ID NO 218, SEQ ID NO 229, SEQ ID NO 232, SEQ ID NO 243, SEQ ID NO 246, SEQ ID NO 257, or SEQ ID NO 260, SEQ ID NO 271 or SEQ ID NO 274.

xviii) a polypeptide or polypeptide construct according to any one of the preceding items, comprising or consisting of a polypeptide having an amino acid sequence selected from the group of those depicted in: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, 22, 63, 64, 65, 66, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92, 93, 94, 103, 104, 105, 106, 108, 150, 138, and 125, and SEQ ID NO. 121, 125, and 125, and SEQ ID NO. 15, 108, and 105, and SEQ ID NO. 15, and ID NO. 15, 108, 125, and ID NO. 13, 125, and ID NO. 15, SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, 173, 174, 175, 176, 177, and 178, 187, 188, 189, 190, 191, and 192, 201, 202, 203, 204, 205, and 206, 215, 216, 217, 218, 219, and 220, 229, 230, 231, 232, 233, 234, 243, 244, 247, 246, and 95, or any of the polypeptides having a sequence identical to that of at least one of SEQ ID No. 93, 95, or 93, or that of at least one of the polypeptides having the amino acid sequence of SEQ ID No. 93, or of at least one of SEQ ID No. 93, or of any of SEQ ID No. 95, or of any of SEQ ID No. 138, 219, and 220, 229, 230, 232, 233, and 234, 243, 244, 246, or any of SEQ ID No. 95, or of any of SEQ ID No. 215, or any of SEQ ID No. 108, or any of SEQ ID No. 215, or any of SEQ ID No. 108, or any of SEQ ID No. or any of which has an amino acid sequence of or any of sequence of SEQ ID No. or, or any of that has any of or, or,.

xix) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the cytotoxicity induced by the domain comprising or consisting of the paratope binding to CLDN6, as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID No. 1, is at least 100-fold, at least 250-fold, at least 500-fold or at least 1000-fold lower than the T-cell dependent cytotoxicity measured in an in vitro assay using cells expressing CLDN6 depicted in SEQ ID No. 1, the mutant comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L.

xx) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the cytotoxicity induced by the domain comprising or consisting of the paratope that binds to CLDN6, as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID NO:1, is at least 100-fold, at least 250-fold, at least 500-fold or at least 1000-fold lower than the T-cell dependent cytotoxicity measured in an in vitro assay using cells expressing CLDN6 depicted in SEQ ID NO:1, the mutant comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L, wherein the construct is capable of activating T cells and inducing T cell-dependent cytotoxicity in target cells expressing CLDN6, and wherein the construct has a heavy chain CDR3 sequence comprising: x1LIVX2APX3 (SEQ ID NO. 667), wherein X1 is A or N; x2 is V or E; and X3 is V or A.

xxi) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct is a single-stranded construct.

xxii) a polypeptide or polypeptide construct according to one of the preceding items, wherein the half-life extending structure comprising or consisting of two polypeptide monomers comprises a hinge, a CH2 domain and a CH3 domain, comprising in amino to carboxyl order:

hinge-CH 2-CH 3-linker-hinge-CH 2-CH3.

xxiii) a polypeptide or polypeptide construct according to one of the preceding items, wherein the CH2 domain comprises a intra-domain cysteine disulfide bridge.

xxiv) a polypeptide or polypeptide construct according to one of the preceding items, wherein

(a) An antigen binding (epitope binding) domain comprising a paratope that binds CLDN6 comprises or consists of two antibody variable domains, and an antigen binding (epitope binding) domain comprising a paratope that binds CD3 comprises or consists of two antibody variable domains;

(b) An antigen binding (epitope binding) domain comprising a paratope that binds CLDN6 comprises or consists of one antibody variable domain and an antigen binding (epitope binding) domain comprising a paratope that binds CD3 comprises or consists of two antibody variable domains;

(c) An antigen binding (epitope binding) domain comprising a paratope that binds CLDN6 comprises or consists of two antibody variable domains and an antigen binding (epitope binding) domain comprising a paratope that binds CD3 comprises or consists of one antibody variable domain; or (b)

(d) An antigen binding (epitope binding) domain comprising a paratope that binds CLDN6 comprises or consists of one antibody variable domain and an antigen binding (epitope binding) domain comprising a paratope that binds CD3 comprises or consists of one antibody variable domain.

xxv) a polypeptide or polypeptide construct according to one of the preceding items, wherein the antigen binding (epitope binding) domain comprising or consisting of the paratope that binds CLDN6 and the antigen binding (epitope binding) domain comprising or consisting of the paratope that binds CD3 are fused to the other domain by a peptide linker.

xxvi) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the polypeptide or polypeptide construct comprises or consists of the following in amino-to-carboxyl order, or in carboxyl-to-amino order:

(a) An antigen binding (epitope binding) domain (comprising paratope) that binds CLDN 6;

(b) A peptide linker, preferably having an amino acid sequence selected from the group consisting of SEQ ID NOs 563-575;

(c) An antigen binding (epitope binding) domain (comprising paratope) that binds to CD 3.

xxvii) the polypeptide or polypeptide construct according to clause xxvi), wherein the polypeptide or polypeptide construct is in amino-to-carboxyl order, or in carboxyl-to-amino order, or between an antigen binding (epitope binding) domain comprising a paratope that binds to CLDN6 and an antigen binding (epitope binding) domain comprising a paratope that binds to CD3, further comprising:

(a) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs 563-575;

(b) A first polypeptide monomer of a third domain;

(c) A peptide linker having an amino acid sequence selected from the group consisting of SEQ ID NOs 563-575; and

(d) A second polypeptide monomer of the third domain.

xxviii) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct is depicted in any of SEQ ID NOs 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, 105, 108, 119, 122, 133, 136, 147, 150, 161, 164, 175, 178, 189, 192, 203, 206, 217, 220, 231, 234, 245, 148, 259, 262, 273, 276, 287, 290, 301, 304, 315, 318, 329, 332, 343, 346, 357, 360, 371, 374, 385, 388, 399, 402, 413, 416, 427 and 430, in particular 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, more particularly 21, 24, 35, 38, 49, 52, 77 and 80, and even more particularly 21, 35, 49 and 77.

xxix) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratope) that binds to CD3 comprising a VH domain comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671 and/or 672.

xxx) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratopes) that binds CD3 comprising a VL domain comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 673, 674 and/or 675.

xxxi) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratopes) that binds CD3 comprising VH and VL domains comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671, 672, 673, 674 and/or 675.

xxxii) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratope) that binds to CD3 comprising the VH domain depicted in SEQ ID No. 676.

xxxiii) a polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratope) that binds to CD3 comprising the VL domain depicted in SEQ ID NO: 677.

xxxiv) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratope) that binds to CD3 comprising a VH domain depicted in SEQ ID No. 676 and a VL domain depicted in SEQ ID No. 677.

xxxv) a polypeptide or polypeptide construct according to one of the preceding items, wherein the construct comprises a domain (comprising paratope) that binds to CD3 comprising the scFv domain depicted in SEQ ID No. 678.

xxxvi) a polynucleotide encoding a polypeptide or polypeptide construct according to any of the preceding items.

xxxvii) a vector comprising a polynucleotide according to item xxxvi).

xxxviii) a host cell transformed or transfected with a polynucleotide according to clause xxxvi) or a vector according to clause xxxvii).

xil) a process for producing a polypeptide or polypeptide construct according to any one of clauses i) to xxxv), the process comprising culturing a host cell according to clause xxxviii under conditions allowing expression of the polypeptide construct and recovering the produced polypeptide or polypeptide construct from the culture.

xl) a pharmaceutical composition comprising a polypeptide or polypeptide construct according to any one of clauses i) to xxxv) or produced according to the process of clause xxxix).

xli) a polypeptide or polypeptide construct according to any one of items i) to xxxv) or produced according to the process of item xxxix), for use as a medicament, in particular for the prevention, treatment or alleviation of a disease, preferably a neoplasm.

xlii) a polypeptide or polypeptide construct according to item xli) for use as a medicament, in particular for the prevention, treatment or amelioration of a disease, wherein the disease or neoplasm is selected from the group consisting of: germ cell cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; uterine cancer; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma.

xliii) a polypeptide or polypeptide construct according to clause xli), wherein the lung cancer is non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma.

xliv) a kit comprising a polypeptide or polypeptide construct according to any one of clauses i) to xxxv), a polypeptide or polypeptide construct produced according to the process of clause xil), a polynucleotide according to clause xxxvi), a vector according to clause xxxvii), and/or a host cell according to clause xxxviii).

xlv) a method for treating or ameliorating a proliferative disease, a neoplastic disease, a cancer or an immune disorder, the method comprising the step of administering to a subject in need thereof a polypeptide or polypeptide construct according to any one of items i) to xxxv) or produced according to the process of item xil), wherein the disease is preferably selected from the group consisting of: germ cell cancer; ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; uterine cancer, more particularly ovarian serous cyst adenocarcinoma, uterine carcinoma sarcoma, endometrial carcinoma of the uterine body; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma.

xlvi) a polypeptide or polypeptide construct comprising

A domain that binds to human CLDN6 (SEQ ID NO: 1), and

domain binding to human CD3, and

a domain that extends the half-life of the polypeptide.

xlvii) the polypeptide or polypeptide construct according to clause xlvi), wherein the polypeptide construct is a T cell activating construct.

xlviii) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlvii), wherein the polypeptide construct is a T-cell activating polypeptide as determined in a T-cell activating assay selected from the group consisting of determining the amount of expression of CD69, determining the amount of expression of CD25, determining the amount of secreted IL-2, and determining the cytotoxic activity of these T-cells.

xlix) a polypeptide or polypeptide construct according to any one of clauses xlvi) to xlviii), wherein the domain that extends the half-life of the polypeptide comprises two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain.

xlx) the polypeptide or polypeptide construct according to any one of clauses xlvi) and xlix), wherein the antigen binding domain that binds CLDN6 comprises binding to an epitope within human CLDN6 corresponding to amino acids 29-81 of SEQ ID No.1 (unit entry P56747).

xlxi) the polypeptide or polypeptide construct according to any one of clauses xlvi) and xlx), wherein the first antigen binding domain that binds CLDN6 comprises binding to an epitope within human CLDN6 corresponding to amino acids 138-160 of SEQ ID No.1 (UniProt entry P56747).

xlxii) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxi), wherein the domain binds to CLDN6, i.e. amino acids 29-39 of SEQ ID NO:1 in extracellular loop 1 (ECL 1) of CLDN6 depicted in SEQ ID NO:9 and/or amino acids 151-160 of SEQ ID NO:1 in extracellular loop 2 (ECL 2) of CLDN6 depicted in SEQ ID NO: 10.

xlxiii) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxii, wherein the domain that binds to CD3 binds to extracellular epitopes of the human and cynomolgus CD3 epsilon chain.

xlxiv) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxiii), wherein the CLDN6 binding domain binds to the same epitope on CLDN6 as a polypeptide construct or antibody or derivative or fragment thereof comprising a CLDN6 binding domain, wherein the domain comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 of a Variable Heavy (VH) chain and/or complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of a Variable Light (VL) chain selected from the group depicted in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are very preferred:

xlxv) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxiv), wherein the domain that binds to human CD3 epsilon also binds to common marmoset or squirrel monkey CD3 epsilon.

xlxvi) a polypeptide or polypeptide construct according to any one of items xlvi) and xlxv), wherein

a) The polypeptide is a single-stranded construct,

b) The domain that binds to CLDN6 is in the form of an scFv,

c) The domain that binds to CD3 is in the form of an scFv,

d) The domains are linked via a linker, and/or

xlxvii) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxvi), wherein the domain that binds to CLDN6 does not bind to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9 and/or CLDN18.1/CLDN 18.2.

xlxviii) a polypeptide or polypeptide construct according to any one of clauses xlvi) and xlxviii), wherein the domain that binds CLDN6 comprises VH regions comprising CDR-H1, CDR-H2 and CDR-H3 and VL regions comprising CDR-L1, CDR-L2 and CDR-L3, selected from the group depicted in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) are very preferred:

xlxix) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds to CLDN6 comprises a VH region with an amino acid sequence selected from the group consisting of SEQ ID No. 11, SEQ ID No. 25, SEQ ID No. 39, SEQ ID No. 53, SEQ ID No. 67, SEQ ID No. 81, SEQ ID No. 95, SEQ ID No. 109, SEQ ID No. 123, SEQ ID No. 137, SEQ ID No. 151, SEQ ID No. 165, SEQ ID No. 179, SEQ ID No. 193, SEQ ID No. 207, SEQ ID No. 221, SEQ ID No. 235, SEQ ID No. 249, or the sequence depicted in SEQ ID No. 263,

xlxix) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds to CLDN6 comprises a VL region having an amino acid sequence selected from the group consisting of SEQ ID No. 12, SEQ ID No. 26, SEQ ID No. 40, SEQ ID No. 54, SEQ ID No. 68, SEQ ID No. 82, SEQ ID No. 96, SEQ ID No. 110, SEQ ID No. 124, SEQ ID No. 138, SEQ ID No. 152, SEQ ID No. 166, SEQ ID No. 180, SEQ ID No. 194, SEQ ID No. 208, SEQ ID No. 222, SEQ ID No. 236, SEQ ID No. 250, or the sequence depicted in SEQ ID No. 264,

xlxx) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds CLDN6 comprises a pair of VH and VL regions having the amino acid sequences depicted in: SEQ ID NO 11+12, SEQ ID NO 25+26, SEQ ID NO 39+40, SEQ ID NO 53+54, SEQ ID NO 67+68, SEQ ID NO 81+82, SEQ ID NO 95+96, SEQ ID NO 109+110, SEQ ID NO 123+124, SEQ ID NO 137+138, SEQ ID NO 151+152, SEQ ID NO 165+166, SEQ ID NO 179+180, SEQ ID NO 193+194, SEQ ID NO 207+208, SEQ ID NO 221+222, SEQ ID NO 235+236, SEQ ID NO 249+250, or SEQ ID NO 263+264.

xlxxi) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds CLDN6 comprises an amino acid sequence depicted in: SEQ ID NO 19, SEQ ID NO 22, SEQ ID NO 33, SEQ ID NO 36, SEQ ID NO 47, SEQ ID NO 50, SEQ ID NO 61, SEQ ID NO 64, SEQ ID NO 75, SEQ ID NO 78, SEQ ID NO 89, SEQ ID NO 92, SEQ ID NO 103, SEQ ID NO 106, SEQ ID NO 117, SEQ ID NO 120, SEQ ID NO 131, SEQ ID NO 134, SEQ ID NO 145, SEQ ID NO 148, SEQ ID NO 159, SEQ ID NO 162, SEQ ID NO 173, SEQ ID NO 176, SEQ ID NO 187, SEQ ID NO 190, SEQ ID NO 201, SEQ ID NO 204, SEQ ID NO 215, SEQ ID NO 218, SEQ ID NO 229, SEQ ID NO 232, SEQ ID NO 243, SEQ ID NO 246, SEQ ID NO 257, or SEQ ID NO 260, SEQ ID NO 271 or SEQ ID NO 274.

xlxxii) a polypeptide or polypeptide construct according to any one of the preceding items, comprising a polypeptide having an amino acid sequence selected from the group of those depicted in: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, 22, 63, 64, 65, 66, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92, 93, 94, 103, 104, 105, 106, 108, 150, 138, and 125, and SEQ ID NO. 121, 125, and 125, and SEQ ID NO. 15, 108, and 105, and SEQ ID NO. 15, and ID NO. 15, 108, 125, and ID NO. 13, 125, and ID NO. 15, SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, 173, 174, 175, 176, 177, and 178, 187, 188, 189, 190, 191, and 192, 201, 202, 203, 204, 205, and 206, 215, 216, 217, 218, 219, and 220, 229, 230, 231, 232, 233, and 234, 243, 244, 245, 246, 275, 95, or 95, and 95, or a polypeptide having at least one of the amino acid sequences of the polypeptides of SEQ ID NO:93, 95, or 93, or the amino acid sequences of the polypeptides of SEQ ID NO:93, or of the polypeptides of SEQ ID NO: 95, or of the polypeptides of the aspects of those aspects of those aspects.

xlxxiii) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds to CLDN6 induces at least 100-fold, at least 250-fold, at least 500-fold less cytotoxicity as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID NO:1 comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L.

xlxxiv) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the domain that binds to CLDN6 induces at least 100-fold, at least 250-fold, at least 500-fold less cytotoxicity as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID NO:1 comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L, wherein the construct is capable of activating T cells and inducing cytotoxicity in target cells expressing CLDN6, and wherein the construct has a heavy chain CDR3 sequence comprising: x1LIVX2APX3 (SEQ ID NO. 667), wherein X1 is A or N; x2 is V or E; and X3 is V or A.

xlxxv) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the construct is a single-stranded construct.

xlxxvi) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the half-life extending domain comprising two polypeptide monomers comprises a hinge, a CH2 domain and a CH3 domain, comprising in amino to carboxyl order:

hinge-CH 2-CH 3-linker-hinge-CH 2-CH3.

xlxxvii) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the CH2 domain comprises a intra-domain cysteine disulfide bridge.

xlxxviii) a polypeptide or polypeptide construct according to any one of the preceding items, wherein

(i) The antigen binding (epitope binding) domain that binds CLDN6 comprises two antibody variable domains and the antigen binding (epitope binding) domain that binds CD3 comprises two antibody variable domains;

(ii) The antigen binding (epitope binding) domain that binds CLDN6 comprises one antibody variable domain and the antigen binding (epitope binding) domain that binds CD3 comprises two antibody variable domains;

(iii) The antigen binding (epitope binding) domain that binds CLDN6 comprises two antibody variable domains and the antigen binding (epitope binding) domain that binds CD3 comprises one antibody variable domain; or (b)

(iv) The antigen binding (epitope binding) domain that binds CLDN6 comprises an antibody variable domain and the antigen binding (epitope binding) domain that binds CD3 comprises an antibody variable domain.

xlxxix) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the CLDN 6-binding antigen binding (epitope binding) domain and the CD 3-binding antigen binding (epitope binding) domain are fused to another domain by a peptide linker.

xlxxx) the polypeptide or polypeptide construct according to any one of the preceding items, wherein the polypeptide or polypeptide construct comprises in amino-to-carboxyl order, or in carboxyl-to-amino order:

(a) An antigen binding (epitope binding) domain that binds CLDN 6;

(b) Peptide linkers, in particular peptide linkers having an amino acid sequence selected from the group consisting of SEQ ID NOs 563-575;

(c) An antigen binding (epitope binding) domain that binds to CD 3.

xlxxxi) a polypeptide or polypeptide construct according to any of the preceding items, wherein the polypeptide or polypeptide construct is in amino-to-carboxyl order, or in carboxyl-to-amino order, or between an antigen binding (epitope binding) domain that binds CLDN6 and an antigen binding (epitope binding) domain that binds CD3, further comprising:

(b) A first polypeptide monomer of a third domain;

(d) A second polypeptide monomer of the third domain.

xlxxxii) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct is depicted in any of SEQ ID NOs 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, 105, 108, 119, 122, 133, 136, 147, 150, 161, 164, 175, 178, 189, 192, 203, 206, 217, 220, 231, 234, 245, 148, 259, 262, 273, 276, 287, 290, 301, 304, 315, 318, 329, 332, 343, 346, 357, 360, 371, 374, 385, 388, 399, 402, 413, 416, 427 and 430, in particular 21, 24, 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, more particularly 21, 24, 35, 38, 49, 52, 77 and 80, and even more particularly 21, 35, 49 and 77.

xlxxxiii) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising a VH domain comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671 and/or 672.

xlxxxiv) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising a VL domain comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 673, 674 and/or 675.

xlxxxv) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising VH and VL domains comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671, 672, 673, 674 and/or 675.

xlxxxvi) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising the VH domain depicted in SEQ ID No. 676.

xlxxxvii) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising the VL domain depicted in SEQ ID No. 677.

xlxxxviii) a polypeptide or polypeptide construct according to any of the preceding items, wherein the construct comprises a domain that binds CD3 comprising a VH domain depicted in SEQ ID No. 676 and a VL domain depicted in SEQ ID No. 677.

xlxxxix) a polypeptide or polypeptide construct according to any one of the preceding items, wherein the construct comprises a domain that binds CD3 comprising the scFv domain depicted in SEQ ID No. 678.

xc) a polynucleotide encoding a polypeptide or polypeptide construct according to any of the preceding items.

ixc) a vector comprising a polynucleotide according to item xc).

iiivc) a host cell transformed or transfected with a polynucleotide according to item xc) or a vector according to item ixc).

iivc) a process for producing a polypeptide or polypeptide construct according to any of the preceding items, the process comprising culturing a host cell according to item iivc) under conditions allowing expression of the polypeptide construct and recovering the produced polypeptide or polypeptide construct from the culture.

ivc) a pharmaceutical composition comprising a polypeptide or polypeptide construct according to any one of the preceding items or produced according to the process of the ivc).

vc) a polypeptide or polypeptide construct according to any of the preceding items or produced according to the process of claim iivc), for use as a medicament, in particular for the prevention, treatment or alleviation of a disease, preferably a neoplasm.

vic) a polypeptide or polypeptide construct according to claim vc) for use as a medicament, in particular for the prevention, treatment or alleviation of a disease, wherein the disease or neoplasm is selected from the group consisting of: germ cell cancer; ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratoma; uterine cancer; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma.

viii) a polypeptide or construct according to claim vc), wherein the lung cancer is non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma.

viii) a kit comprising a polypeptide or polypeptide construct according to any one of the preceding items, a polypeptide or polypeptide construct produced according to the process of claim vc, a polynucleotide, a vector, and/or a host cell according to the preceding items.

ic) a method for treating or ameliorating a proliferative disease, a neoplastic disease, a cancer or an immune disorder, the method comprising the step of administering to a subject in need thereof a polypeptide or polypeptide construct according to any one of the preceding items or produced according to the process of the preceding items, wherein the disease is preferably selected from the group consisting of: germ cell cancer; ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratocarcinoma; uterine cancer, more particularly ovarian serous cyst adenocarcinoma, uterine carcinoma sarcoma, endometrial carcinoma of the uterine body; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma.

Whenever the term "construct" is used in the figures, the term refers to the polypeptide/polypeptide construct of the invention used or the control indicated thereby.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes one or more of such different agents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that may modify or replace the methods described herein.

The term "at least" preceding a series of elements should be understood to refer to each element in the series unless otherwise indicated. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

The term "and/or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by the term.

As used herein, the term "about" or "approximately" means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range. It also includes specific values, for example, "about 50" includes the value "50".

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term "comprising" when used herein may be substituted with the term "containing" or "including" or sometimes with the term "having" when used herein.

As used herein, "consisting of … …" excludes any element, step or component not specified in the claim elements. As used herein, "consisting essentially of … …" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claims.

In each of the examples herein, any of the terms "comprising," "consisting essentially of … …," and "consisting of … …" may be substituted with either of the other two terms.

The above description and the following examples provide exemplary arrangements, but the invention is not limited to the specific methods, techniques, protocols, materials, reagents, substances, etc. described herein and may thus vary. The terminology used herein is for the purpose of describing particular embodiments only. The terms used herein are not intended to limit the scope of the invention, which is defined solely by the claims. Various aspects of the invention are provided in the independent claims. Some optional features of the invention are provided in the dependent claims.

All publications and patents (including all patents, patent applications, scientific publications, manufacturer's specifications, descriptions, etc.) cited throughout this specification, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that the material incorporated by reference conflicts or otherwise does not coincide with the present specification, the present specification will replace any such material.

A better understanding of the present invention and its advantages will be obtained from the following examples, which are set forth to illustrate only. These examples are not intended to be construed as limiting the scope of the invention in any way.

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Drawings

Figure 1 shows the results of epitope mapping analysis. The top row provides structural representations of CLDN4 (dashed line) and CLDN6 (continuous line) and chimeric proteins, indicating that the various regions of CLDN6 have been replaced by corresponding CLDN4 regions. The second and third lines above show the use of isotype control and anti-CLDN 4-Ab and anti-CLDN 6-Ab (5 μg/ml isotype control (R&DIC 003P) +5 μg/ml anti-CLDN 4-ab (clone 382321R)&Dmab 4219)). The two FACS results on the left show that cells expressing neither CLDN4 nor CLDN6 are not recognized by antibodies that immunospecifically detect either CLDN-4 or CLDN-6. The bottom four rows show FACS analysis results using four different polypeptides/polypeptide constructs of the invention as primary binders (5 μg/ml CLDN 6-polypeptide/polypeptide construct) or PBS with 2% FCS as control, 4 primary candidates: 343, 35, 21 and 105 (from top to bottom). As secondary conjugates, 1:50-hu Fcy-PE (Jacks. Imm. Res.) 109-116-98 was used. The E1A and E2B regions are particularly important for the binding of some polypeptides/polypeptide constructs of the invention to CLDN 6. The numerals 0, 20, 40, 60, 80, and 100 are depicted on the ordinate axis (x-axis). The value 10 is indicated on the abscissa (y-axis) ¹ 、10 ² 、10 ³ 、10 ⁴ And 10 ⁵ 。

FIG. 2 shows the experimental results of human pan T cells incubated with target cells at a ratio of 10:1 and the indicated concentrations of polypeptide/polypeptide construct. After 48 hours, cell viability was measured and percent cytotoxicity was calculated using the Cell Titer-glo assay. The graph shows representative data for duplicate samples (> 2 independent experiments were run). Data were analyzed using GraphPad Prism. Similarly, experiments were performed using polypeptide constructs based on prior art antibodies (A3E-20; disclosed in WO 2009/087978). The CLDN6 constructs in SEQ ID NOs 21, 24, 217, 147, 119 and 90 are more potent than A3E-20 based polypeptide constructs; the CLDN6xCD3 polypeptide/polypeptide construct according to the invention has > 3000-fold selectivity for CLDN6 compared to CLDN9. Fig. 2A = PA-1 cells; fig. 2 b=cho-CLDN 6; fig. 2 c=cho-CLDN 9.

FIG. 3 shows the equivalent activity of constructs with different CD3 binding moieties (CLDN 6XI2C and CLDN6XI2E molecules according to the invention (SEQ ID NOS: 21 and 24)) that selectively bind to CLDN6; CLDN 6-dependent cytotoxic activity. Human pan T cells were incubated with the target cells at a ratio of 10:1 and the indicated concentrations of the polypeptide construct. After 48 hours, cell viability was measured and percent cytotoxicity was calculated using the Cell Titer-glo assay. The graph shows representative data for duplicate samples (> 2 independent experiments were run). Data were analyzed using GraphPad Prism. The CLDN6xI2C molecule and CLDN6xI2E molecule according to the invention (SEQ ID NOs: 21 and 24) have the same cytotoxic activity in vitro; both molecules showed specificity to kill CLDN6 expressing cells (fig. 3A) and 3B)).

Figure 4 shows the results of human PBMCs incubated with different target cells at a 5:1 ratio and indicated concentrations of polypeptide/polypeptide constructs. After 48 hours, cell viability was measured and percent cytotoxicity was calculated using a flow cytometry-based assay. The graph shows representative data from three PBMC donors (> 2 independent experiments were run). Data were analyzed using GraphPad Prism. Black line, CLDN6xI2C (SEQ ID NO: 24); gray line, CLDN6xI2E (SEQ ID NO: 21). The numbers (# 150, #156, # 158) refer to three different donors of human PBMC (fig. 4A to 4F)). The cell lines used are shown above the graph (FIG. 4A: COV-362, FIG. 4B: LCLC-97TIM1, FIG. 4C: NCI-H322, FIG. 4D: NCI-H1435, FIG. 4E: OV-90 and FIG. 4F: OVCAR-3).

FIG. 5 shows the results of incubation of CLDN6 HLE BiTE (I2C) (SEQ ID NO: 24) with CHO-CLDN6 and CHO-CLDN9 cells at different concentration ranges. CHO-CLDN6 and CHO-On cell binding was assessed by flow cytometry. CHO-CLDN6 (left): dotted line = mouse isotype control (BD Biosciences), solid line = anti-CLDN 6 antibody (Creative Biolabs), TAB-510LC, based on AE3-20 clones); CHO-CLDN9 (right): dotted line = rat isotype control (BD bioscience company), solid line = anti-CLDN 9 antibody (Ai Bokang company (Abcam), ab187116, YD4E9 clone); second antibody (BV 421 channel, BD bioscience company)

Figures 6 to 8 show that CLDN6 HLE polypeptide/polypeptide constructs have cytotoxic activity against different types of target cells that show low levels of CLDN6 expression by IHC. Fig. 6A: CHO-CLDN6: dashed line = mouse isotype control (BD biosciences), solid line = anti-CLDN 6 antibody (creative bioscience, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory (Jackson ImmunoResearch)); fig. 6B: OVCAR-3: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 6C: COV-362: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative bioscience, TAB-510LC, based on AE3-20 clone); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 6D: NCI-H322: dotted line = mouse isotype control (BD biosciences), solid line = anti-CLDN 6 antibody (creative bioscience, TAB-510LC, clone based on AE 3-20); secondary antibody (BV 421 channel, BD biosciences); fig. 7A and 7B: PA-1: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 7C and 7D: OVCAR-3: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 7E and 7F: OAW28: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 7G and 7H: LCLC-97TM1: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory); fig. 7I and 7J: NCI-H1435: dotted line = mouse isotype control (BD bioscience company), solid line = anti-CLDN 6 antibody (creative biology laboratory, TAB-510LC, clone based on AE 3-20); secondary antibodies (APC pathway, jackson immunoresearch laboratory);

FIG. 9-antitumor efficacy against established OVCAR-3 xenograft tumors

Figure 10 shows a marker of the junction characteristics of T cell activating polypeptide/polypeptide constructs observed in exploratory toxicology studies.

FIG. 11 demonstrates that a CLDN6 HLE polypeptide construct according to the invention (SEQ ID NO: 24) has an extended half-life in non-human primates.

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EXAMPLE 1 CLDN6 Binder Generation

Hybridoma production

Pooled lymphocytes from spleen and lymph nodes were enriched for igg+b cells, followed by electrocytofusion with mouse P3 myeloma cells. The fused hybridoma cells were cultured in Hyaluronic Acid (HA) selection medium for 2-5 days, then plated onto 96-well plates and cultured for 2 weeks to produce depleted supernatant (ESN).

Screening and sequence analysis

Binding hybridomas on 837 human CLDN6 cells were identified using fluorescence microanalytical technology (FMAT) in a preliminary screen. A second, advanced primary screen by flow cytometry also demonstrated 776 of these hybridomas bind 293T of transiently expressed cynomolgus CLDN 6. To further characterize 837 hybridomas that bind CLDN6, ESN was tested by antibody-dependent cellular cytotoxicity (ADCC) activity on PA-1 cells to screen a list of 288 hybridomas with optimal killing activity. Most of these hybridomas showed ADCC killing of OVCAR3 cells expressing CLDN 6. The ESN of these hybridomas were further characterized for cross-reactive binding to 293T cells transiently expressing human CLDN3, CLDN4, CLDN8, or CLDN9 on the cell surface.

Overall, 20 hybridomas identified based on ADCC killing criteria in PA-1 and OVCAR3 cell lines showed no cross-reactivity with CLDN3, CLDN4 and CLDN 8. These hybridomas were sequenced, followed by recombinant mAb generation and amplification. These antibodies include, inter alia, three clones that exhibit acceptable sequence and binding properties and are selected for conversion to scFv and polypeptide/polypeptide constructs.

anti-CLDN 6 polypeptide construct generation

The assembly of anti-CLDN 6 targeted polypeptide/polypeptide construct conjugates is performed by genetic synthesis. In more detail: VH against CLDN6 heavy chain and VL against CLDN6 light chain DNA were derived from hybridoma clones. Amino acid position 44 in VH and position 100 in VL (Kabat numbering) are changed to cysteines, which results in additional disulfide bonds that stabilize the target conjugate. The linker (e.g., consisting of three repeats of four glycine) and serine (G4S 1) 3-linker can be inserted between VL and VH, resulting in a single chain fragment variable (scFv). A human IgG heavy chain signal peptide comprising an initiation codon embedded in the Kozak consensus sequence may be added to the N-terminus of the anti-CLDN 6 conjugate. By binding to the anti-CD 3 epsilon specific scFv conjugate, the assembled anti-CLDN 6 target conjugate is converted to a recombinant bispecific single chain conjugate form that is human in sequence and cross-reacts with human and cynomolgus CD3 epsilon. In these constructs, the anti-CD 3 epsilon scFv is fused to a single chain Fc (scFc) half-life extending (HLE) moiety having a C-terminal stop codon attached in-frame. In mammalian expression vectors, human anti-CLDN 6 binders bind to anti-CD 3 epsilon binders and scFc. Some human anti-CLDN 6 HLE polypeptide/polypeptide constructs have the domain arrangement VL CLDN6- (G4S 1) 3-VH CLDN6-S1G4S1-VHCD 3-peptide linker (G4S 1) 3-VLCD 3-peptide linker (G4) -Fc- (G4S 1) 6-Fc; other have the domain arrangement VH (CLDN 6) - (G4S) 3-VL (CLDN 6) -peptide linker (SG 4S) -VH (CD 3) - (G4S) 3-VL (CD 3) -peptide linker (G4) -Fc- (G4S) 6-Fc.

Exemplary sequence optimization of anti-CLDN 6 conjugates

Based on reasonable design, a sequence optimization method is performed. Furthermore, using a clone hit pool of 899 clones that bound CLDN6, LC shuffling was performed to further improve the binding and protein properties of selected polypeptide construct clones. For this library, a human light chain V kappa variable region library was constructed using PCR primers and ligated with phagemid pComb3H5BHis containing the VH of the selected polypeptide construct clone CLDN6 (WO 99/25818; sacl Spel digestion). The resulting combinatorial Ig light chain variable antibody library was then transformed into e.coli TG1, plated on agar, and individual clones were screened in flow cytometry measurements for binding to human CLDN6 and human CLDN 9. For this purpose, a periplasmic cell extract containing individual colonies of scFv molecules was incubated on CLDN 6-transfected CHO cells or CLDN 9-transfected CHO cells and binding was detected by mouse anti-Flag antibodies and R-phycoerythrin conjugated goat anti-mouse IgG or anti-mouse fcγ -Alexa488 secondary antibodies. Samples were measured on a FACSCanto II (BD bioscience, heidburg, FRG).

In the screening method, clones were identified in combination with the rational design optimization method described above and target binding polypeptide/polypeptide constructs depicted in, for example, SEQ ID NOs 21, 24, 35, 38, 49, 52, 63, 66, 77 and 80 were produced.

Evaluation of affinity in vitro for CLDN6 bispecific constructs

The cell-based affinity of CLDN6 bispecific constructs was determined by nonlinear regression (single site specific binding) analysis. CHO cells expressing human CLDN6 were incubated with reduced concentrations of CLDN6 bispecific construct (400 nM, steps 1:2, 11) for 16h at 4 ℃. Bound CLDN6 bispecific constructs were detected with Alexa Fluor 488 conjugated AffiniPure Fab fragment goat anti-human IgG (h+l; jackson immunoresearch laboratory).

The immobilized cells were stained with DRAQ5, far red fluorescent live cell permeabilizing DNA dye and the signal detected by Fluorescence Activated Cell Sorting (FACS). Each equilibrium dissociation constant (Kd) value was calculated using a single site specific binding evaluation tool in GraphPad Prism software (GraphPad).

Cytotoxic Activity

Human Peripheral Blood Mononuclear Cells (PBMCs) were prepared from enriched lymphocyte preparations (buffy coat, by-products of blood pool collection for transfusion) by Ficoll density gradient centrifugation. Buffy coats are provided by local blood banks and PBMCs are prepared on the same day after blood collection. After Ficoll density centrifugation and extensive washing with Dulbecco's PBS (Ji Boke Co. (Gibco)), the sample was washed with erythrocyte lysis buffer (155 mM NH) ₄ Cl、10mM KHCO ₃ 100 μm EDTA) and removing the remaining erythrocytes from PBMCs. After centrifugation of PBMCs at 100x g, platelets were removed via supernatant. The remaining lymphocytes mainly include B and T lymphocytes, NK cells and monocytes. PBMC were maintained in RPMI medium (Ji Boke Co.) containing 10% FCS (Ji Boke Co.) at 37deg.C/5% CO ₂ And (5) culturing.

Depletion of CD14+ and CD56+ cells

To deplete CD14+ cells, NK cell human CD56 microbeads (MACS, # 130-050-401) were depleted using human CD14 microbeads (MACS, #130-050-201, methawk Biotechnology Co., ltd.). PBMCs were counted and centrifuged at 300x g for 10 minutes at room temperature. The supernatant was discarded and the cell pellet was resuspended in MACS isolation buffer [ 80. Mu.L/107 cells; PBS (Invitrogen), # 20012-043), 0.5% (v/v) FBS (Ji Boke, # 10270-106), 2mM EDTA (Sigma-Aldrich), # E-6511). CD14 microbeads and CD56 microbeads (20. Mu.L/107 cells) were added and incubated at 4℃to 8℃for 15min. Cells were washed with MACS isolation buffer (1-2 mL/107 cells). After centrifugation (see above), the supernatant was discarded and the cells resuspended in MACS separation buffer (500. Mu.L/108 cells). CD14/CD56 negative cells were then isolated using LS columns (Methaemal and Biotechnology Co., ltd. # 130-042-401). PBMC w/o CD14+/CD56+ cells were cultured in an incubator at 37℃until needed in RPMI complete medium (i.e. RPMI1640 (cypress Co. # FG 1215) supplemented with 10% FBS (cypress Co., # S0115), 1x nonessential amino acids (cypress Co., # K0293), 10mM Hepes buffer (cypress Co., # L1613), 1mM sodium pyruvate (cypress Co., # L0473) and 100U/mL penicillin/streptomycin (cypress Co., # A2213).

Target cell markers

For analysis of cell lysis in flow cytometry assays, the fluorescent membrane dye DiOC18 (DiO) (Molecular Probes, # V22886) was used to label human CLDN 6-or cynomolgus CLDN 6-transfected CHO cells (as target cells) and distinguish them from effector cells. Briefly, cells were harvested, washed once with PBS, and adjusted to 106 cells/mL in PBS containing 2% (v/v) FBS and membrane dye DiO (5. Mu.L/106 cells). After incubation at 37 ℃ for 3min, the cells were washed twice in complete RPMI medium and the cell number was adjusted to 1.25x 105 cells/mL. Cell viability was determined using an NC-250 cell counter (chemometric).

Flow cytometry-based analysis

This assay was designed to quantify lysis of cynomolgus monkey or human CLDN 6-transfected CHO cells in the presence of serial dilutions of CLDN6 bispecific constructs. Equal volumes of DiO labeled target cells and effector cells (i.e., PBMC without CD14+ cells) were mixed to give a 10:1E: T cell ratio. Mu.l of this suspension was transferred to each well of a 96-well plate. Serial dilutions of 20 μl CLDN6xCD3 bispecific construct and bispecific negative control (CD 3 based bispecific construct recognizing unrelated target antigen) or RPMI complete medium (as additional negative control) were added. Bispecific antibody mediated cytotoxicity reactions were performed in a 7% CO2 humidified incubator for 48 hours. Cells were then transferred to a new 96-well plate and loss of target cell membrane integrity was monitored by addition of Propidium Iodide (PI) at a final concentration of 1 μg/mL. Samples were measured by flow cytometry on an iQue Plus instrument and analyzed by Forecyt software (all from Intellicyt). Target cells were identified as DiO positive cells. PI negative target cells are classified as viable target cells. The percent cytotoxicity was calculated according to the following formula:

n=number of events

The percentage of cytotoxicity was plotted against the corresponding bispecific construct concentration using GraphPad Prism 5 software (graphic software company, san diego). The dose response curves were analyzed using a four parameter logistic regression model for evaluating sigmoidal dose response curves with a fixed ramp and EC50 values were calculated.

Bispecific binding and cross-species reaction

To confirm binding to human CLDN6 and CD3 and cynomolgus CLDN6 and CD3, the polypeptide/polypeptide constructs of the invention were tested by flow cytometry using

Chinese Hamster Ovary (CHO) cells transfected with human CLDN6, human CLDN6 subtype (I143V) and cynomolgus CLDN6, respectively,

human CLDN 6-positive human cell line PA-1,

human T cell leukemia cell line HPB-all expressing CD3 (German collection of microorganisms and cell strains (DSMZ), and

t cell line LnPx 4119 expressing cynomolgus monkey CD3

To confirm that it does not bind to CHO, CLDN1, -3, -4, -8, -17 bispecific constructs of the invention were tested by flow cytometry using CHO cells transfected with human CLDN1, -3, -4, -8 and-17. For flow cytometry, 200,000 cells of each cell line were incubated with 50 μl of purified bispecific construct at a concentration of 5 μg/ml at 4 ℃ for 60min. Cells were washed twice in PBS/2% FCS and then incubated with internal mouse antibodies (2 μg/ml) specific for the CD3 binding portion of the bispecific construct for 30 min at 4 ℃. After washing, bound mouse antibodies were detected with goat anti-mouse Fcγ -PE (Jackson immunoresearch laboratory; 1:100) at 4℃for 30 min. The samples were measured by flow cytometry. Untransfected CHO cells (DSMZ) were used as negative control.

Generation of CHO cells expressing CLDN6 mutations

To generate control CHO cells expressing hu-CLDN6, hu-CLDN9, hu-CLDN4 (SEQ ID NOS: 1, 8 and 7), the respective coding sequences were cloned into plasmids designated pEF-DHFR (pEF-DHFR is described in Raum et al Cancer Immunol Immunother [ cancer immunotreatment ]50 (2001) 141-150). All cloning procedures were performed according to standard protocols (Sambrook, molecular Cloning; A Laboratory Manual [ molecular cloning, A laboratory Manual ], third edition, cold spring harbor laboratory Press, cold spring harbor, N.Y. (2001)). For each construct, the corresponding plasmid was transfected into DHFR-deficient CHO cells for eukaryotic expression as described by Kaufman r.j. (1990) Methods Enzymol [ Methods of enzymology ]185,537-566. Expression of the above polypeptide/polypeptide constructs on CHO cells was verified in FACS assays using antibodies against CLDN4, CLDN6 (R & D mouse anti-human CLDN6 monoclonal antibody MAB 3656) and CLDN9 (rat anti-human CLDN9 monoclonal antibody ABIN 1720917) at a concentration of 5 μg/ml, respectively. As a negative control, cells were incubated with isotype control antibody (BD 553454/R & D MAB0041/R & DMAB 0061) in place of the primary antibody. Bound monoclonal antibodies were detected with a secondary anti-mouse/anti-rat/anti-human IgG Fc-gamma-PE (Jackson immunoresearch laboratory 115-116-071/112-116-071/109-116-098). These samples were measured by flow cytometry.

Example 2 epitope mapping of anti-CLDN 6 constructs

For epitope mapping, E1 of CLDN6 (extracellular loop 1; ECL 1) was split into three subdomains (E1A, E B and E1C), and E2 (extracellular loop 2; ECL 2) was split into two subdomains (E2A and E2B). The amino acid sequences of the respective epitope regions (loop/domain or subdomain) of human CLDN6 (E1, E1A, E1B, E1C, E, E2A and E2B) were exchanged for the corresponding sequences of human CLDN 4. Expression of all chimeric constructs in CHO cells was verified via FACS analysis. CHO cells transfected with the construct described in example 1 were stained with purified CLDN6xCD3 polypeptide construct at a concentration of 20 μg/ml. The bound construct was detected with anti-human IgG Fc-gamma-PE (Jackson immunoresearch laboratory; 1:100). Antibodies were diluted in PBS/2% FCS. As a negative control, cells were incubated with PBS/2% FCS followed by incubation with anti-human IgG Fc-gamma-PE. These samples were measured by flow cytometry. The results of the epitope mapping analysis are shown in figure 1. When a loss of FACS signal is observed in cells expressing a certain CLDN6 chimeric or mutated, it is assumed that the respective CLDN6xCD3 polypeptide construct binds to an epitope (loop/domain/subdomain) or to a specific amino acid exchanged in the CLDN6 chimeric or mutated polypeptide construct. In other words, the epitope region or amino acid is necessary for binding to the CLDN6xCD3 polypeptide construct being analyzed. In addition to control antibodies used to demonstrate proper expression of the respective targets, the following CLDN6xCD3 polypeptide/polypeptide constructs were specifically tested in epitope mapping assays. As shown in FIG. 1, clDN6xCD3 polypeptide/polypeptide constructs according to the present invention (e.g., SEQ ID NOs: 21, 35, 49, 77, 203, etc.) require that regions E1A and/or E2B bind selectively to clDN 6. Thus and likewise, exchange of these subdomains with the CLDN4 corresponding sequences results in loss of FACS signal.

Example 3-Biacore-based determination of affinity for human and cynomolgus CD3 and FcRn

Biacore analysis experiments were performed using recombinant human/cynomolgus CD3-ECD (ecd=extracellular domain) fusion proteins with chicken albumin to determine target binding of the constructs of the invention.

EXAMPLE 4 CLDN6xCD3 HLE polypeptide/polypeptide constructs and antibody-based Comparison of polypeptide constructs of AE3-20

The cytotoxic activity and the specificity towards CLDN6 of CLDN6xCD3 (variant I2C), CLDN6xCD3 (variant I2E) and other CLDN6xCD3 (variant I2C) polypeptide/polypeptide constructs according to the invention as well as polypeptide constructs based on monoclonal antibody AE3-20 (disclosed in WO 2009/087978; depicted in SEQ ID NO: 441) depicted in SEQ ID nos. 21, 24 are compared.

Additional comparisons of cytotoxicity in ovarian cancer cell lines and non-small cell lung cancer cell lines were made for CLDN6xI2C and CLDN6xI2E (as depicted in SEQ ID NOs: 21 and 24). For binding analysis of AE3-20 polypeptide constructs by flow cytometry CHO cells transfected with human CLDN6, hu CLDN4 or human CLDN9 were used. For flow cytometry, corresponding CHO cells were incubated with AE3-20 polypeptide constructs (5 μg/ml) or following monoclonal antibodies (as positive controls for cell surface expression): anti-CLDN 6 (R & D Systems), MAB3656, anti-CLDN 4 (R & D Systems, MAB 4219) and anti-CLDN 9 (origold, AM26751 PU-N). Binding of the AE-320 polypeptide construct or positive control antibody was detected using a mouse anti-human IgG Fc antibody that binds R-Phycoerythrin (PE), a goat anti-mouse Fc gamma specific antibody that binds PE (Jackson immunoresearch laboratory 115-116-071), or a goat anti-rat Fc gamma specific antibody that binds PE (Jackson immunoresearch laboratory 112-116-071). As negative control, the corresponding isotype control antibody was used.

Polypeptides targeting the CLDN6 epitope cluster E1A/E2B or E1A/(E2B) show unexpectedly higher potency compared to BiTE molecules targeting E1A/e2a+b or E2A/(E2B) (AE-320 epitope). Polypeptides targeting CLDN6 epitope cluster E1A/E2B or E1A/(E2B) were more potent than BiTE molecules targeting E2A/(E2B) (AE-3-20 epitope), although candidates were within a similar affinity range. Surprisingly, the epitope cluster E2A should be avoided to obtain sufficient potency while maintaining selectivity for CLDN 9. The polypeptides studied herein (in particular those depicted in SEQ ID NOs: 21, 24, 35, 38, 49, 52, 63, 66, 77 and 80, more in particular in SEQ ID NOs: 21 and 24) also have advantageous protein properties, in particular achieving advantageous stability of the monomer conversion after storage in a 1mg/ml solution and after repeated freeze/thaw cycles, minimum turbidity at higher protein concentrations in overnight solutions, and thermostability, as well as very good affinity while maintaining advantageous efficacy on the CLDN6+ cell line.

Efficacy and specificity of CLDN6xCD3 constructs

In a T cell dependent cytotoxicity (TDCC) assay, human pan T cells (alcells) are incubated with target cells at a ratio of 10:1 and the indicated concentrations of polypeptide/polypeptide construct . After 48 hours, use of Cell Titer-The assay (Promega) measures cell viability and calculates percent cytotoxicity. The graph in fig. 2 shows representative data for duplicate samples (run>2 independent experiments). Data were analyzed using GraphPad Prism. Similarly, experiments were performed using polypeptide constructs based on the prior art antibody disclosed in WO 2009/087978 (AE 3-20; chinese exo-pharmaceutical (Chugai)). The CLDN6 polypeptide/polypeptide constructs of SEQ ID nos. 21 and 24 are more potent than A3E-20 based polypeptide constructs; the CLDN6xCD3 polypeptide/polypeptide construct has a sequence specific for CLDN6 as compared to CLDN9>Selectivity 3000 times.

Comparison of CLDN6 HLE polypeptide/polypeptide construct-with AE3-20 based construct

TDCC measurement: the CLDN6 expressing ovarian cancer cell line PA-1 and CHO cells transfected to stably express CLDN6 or CLDN9 were used as target cells to evaluate the cytotoxicity of CLDN6 constructs of the invention in vitro. Cells were plated in 384 well microplates (PerkinElmer) in medium containing 10% fetal bovine serum and human pan T cells (oscierles) from two donors were added to target cells at a ratio of 10:1. The CLDN6 construct of the invention was added in a 22-point dose range (60 nM as the highest concentration) and 5-fold dilution. After incubation at 37℃for 48 hours in a 5% CO2 humidity cabinet, cell Titer ℃was used according to the manufacturer's instructions The assay (Promega) evaluates cell viability. The luminescence signal was measured using PerkinElmer Envision. Data were analyzed in GraphPad Prism using nonlinear regression-variable slope (four parameters). The graph shows the mean and standard deviation of the dose response curves for the duplicate samples.

Activity of polypeptide/polypeptide constructs that selectively bind CLDN6 with different CD3 binding moieties

Human pan T cells were incubated with the target cells at a ratio of 10:1 and the indicated concentrations of the polypeptide construct. After 48 hours, cell viability was measured and percent cytotoxicity was calculated using the Cell Titer-glo assay. The graph in fig. 3 shows representative data for duplicate samples (> 2 independent experiments were run). Data were analyzed using GraphPad Prism. The CLDN6xI2C molecule and CLDN6xI2E molecule according to the invention (SEQ ID NOs: 21 and 24) are used; CLDN 6-dependent cytotoxic activity (figure 3).

The CLDN6xI2C molecule and CLDN6xI2E molecule according to the invention (SEQ ID NOs: 21 and 24) have the same cytotoxic activity in vitro and both molecules show specificity to kill cells expressing CLDN 6. In another set of experiments, human PBMCs were incubated with target cells at a 5:1 ratio and the indicated concentrations of polypeptide/polypeptide constructs. After 48 hours, cell viability was measured and percent cytotoxicity was calculated using a flow cytometry-based assay. The graph shows representative data from three PBMC donors (> 2 independent experiments were run).

Data were analyzed using GraphPad Prism. Black line, CLDN6xI2C (SEQ ID NO: 24); gray line, CLDN6xI2E (SEQ ID NO: 21). The numbers (# 150, #156, # 158) refer to three different donors of human PBMC (fig. 4A to 4F)). The cell lines used are shown above the graph (FIG. 4A: COV-362, FIG. 4B: LCLC-97TIM1, FIG. 4C: NCI-H322, FIG. 4D: NCI-H1435, FIG. 4E: OV-90 and FIG. 4F: OVCAR-3).

Example 5-CLDN6 HLE polypeptide/polypeptide construct has selectivity for CLDN6 over CLDN9

A range of concentrations of CLDN6 HLE BiTE (I2C) (SEQ ID NO: 24) was incubated with CHO-CLDN6 and CHO-CLDN9 cells (FIGS. 5A and 5B)). CHO-CLDN6 and CHO-CLDN9 cells were assessed for binding by flow cytometry (fig. 5). Cell-on-cell binding was assessed by flow cytometry, using a secondary antibody conjugated to APC. FIG. 5C) shows that the polypeptide/polypeptide construct of the invention binds selectively and specifically to CLDN6, but does not bind to CLDN9 expressed by CHO cells. Data was analyzed using FlowJo software (FlowJo, LLC). Alignment of CLDN6 and CLDN9 was performed using Geneious software.

Example 6-CLDN6 HLE polypeptide/polypeptide construct pairs are visualized by IHCCells showing low levels of CLDN6 expression have Cytotoxic Activity

TDCC assays were performed on various cancer cell lines and CHO cells expressing CLDN6 and CLDN9 (fig. 6). Antibody binding site (ABC) EC50 values are shown in the following table. It is apparent that the polypeptide/polypeptide constructs of the invention recognize and kill cells expressing lower and higher numbers of CLDN6 sites per cell (see table 2).

Table 2: TDCC EC in the context of cytotoxicity assays in various cell lines ₅₀ pM

* ABC = antibody binding site. By flow cytometryThe CLDN6 cell surface expression was assessed (Agilent) inc. Cytotoxic activity was assessed in the TDCC assay. Human T cells were incubated with different polypeptides according to the invention (see FIG. 7E) (SEQ ID NOS: 21, 35, 49, 77, 203) at a ratio of 10:1 and at the concentrations indicated. After 48 hours, cell Titer _ was used>The assay (Promega) evaluates cell viability. Data were analyzed in GraphPad Prism. In vitro cytotoxicity was determined to select polypeptide/polypeptide constructs for subsequent in vivo studies. As shown in the following table, the CLDN6 HLE polypeptide/polypeptide constructs (SEQ ID NOS: 21 and 24) have potent cytotoxic activity (FIGS. 6 and 7).

Example 7-CLDN6 HLE polypeptide/polypeptide construct induces T cell activation

Human pan T cells were incubated with target cells at a ratio of 10:1 and at indicated concentrations of an exemplary polypeptide/polypeptide construct according to the invention (SEQ ID NO: 21). After 48 hours, T cell activation (up-regulation of CD25, CD69 and PD-1) was assessed by flow cytometry. Using Cell Titer-The assay evaluates cell viability. These data, showing the expression of these biomarkers, clearly demonstrate that the polypeptide/polypeptide constructs induced T cell activity (fig. 8A-8E)).

Example 8-antitumor efficacy against established OVCAR-3 xenograft tumors

Female NSG mice (10 per group) were inoculated subcutaneously with 5e6 cells/mouse. The T cells used were activated human pan T cells, 2e7 cells per mouse. Once the tumor had formed a size of 200mm3, and then the human T cells were intraperitoneally injected, mice were treated once a week with vehicle alone or with CLDN6xCD3 HLE (SEQ ID NO: 21) (fig. 9A) to 9C)). Fig. 9A) to 9C) show the measurement of tumor volume over time (days on x-axis), the measurement of body weight over time, and pharmacokinetic data at different concentrations (serum concentration over time).

EXAMPLE 9 tolerance<CLDN6xCD3 HLE at 100mg/kg dose

A study was conducted with the aim of assessing tolerance of the CLDN6xCD3 HLE (SEQ ID NO: 21) according to the present invention in exploratory PK/tox studies of non-human primate (NHP). For this purpose, flexible and staggered doses are administered to each group; 15mg/kg, 45mg/kg, 100mg/kg (1 animal). The results show that signs of T cell dependent activation were observed at all doses. The polypeptide constructs of the invention are clinically tolerated up to 100. Mu.g/kg. The results are shown in fig. 10. The dosage of the medicine to the liver (bile duct) is more than or equal to 45 mug/kg; and at 45. Mu.g/kg or more for the skin (intravenous administration site, anus, breast, back and hind limb) (not found in all sites of all animals); and microscopic examination of the mucosal epithelium of the oesophagus and interstitial lung at > 100. Mu.g/kg was observed to be treatment-related (mild to severe). This study showed CLDN6 as a safe target for polypeptide constructs.

Example 10-conjugation of T cell activating polypeptide/polypeptide constructs was observed in exploratory toxicology studiesFeatures of the character Sign mark

A dose-dependent decrease in CLDN6 was observed in absolute counts of cd3+ T lymphocytes at day 1 and 4 hours after dosing on day 8. The magnitude of the drop observed on day 8 is generally different from that on day 1. Transient lymphocyte redistribution after administration was observed in all groups, with a transient decrease in total T cell count. Figure 10 shows that T cell activating polypeptide/polypeptide constructs induce increased activity, e.g., CD69 expression observed in medium and high dose groups. T cell activation was assessed by flow cytometry as described above. Further, the absolute number of CD3+ T lymphocyte counts and the percentage of CD3+ T lymphocyte counts were also analyzed, and transient cytokine release (IFN-. Gamma., TNF-. Alpha., MCP-1, IL-1β, IL-1rα, IL-2, IL-5, and IL-6) could also be shown to be consistent with the activity of the T cell activating polypeptide/polypeptide construct. Further, the CLDN6 HLE polypeptide construct (SEQ ID NO: 24) according to the invention has an extended half-life in non-human primates. The level of exposure of the T cell activating polypeptide/polypeptide construct in the non-human primate serum was assessed in samples from toxicology studies. This construct demonstrated dose-dependent exposure and prolonged half-life (8.39 days) (fig. 11).

EXAMPLE 11 in vitro and in vivo cytotoxicity Studies

Using the method described in example 6, cytotoxicity of different HLE polypeptide/polypeptide constructs of the invention in various cell lines was analyzed. As shown in table 3, the polypeptides/polypeptide constructs of the invention are cytotoxic in various cell lines.

₅₀ Table 3: TDCC EC pM for cytotoxicity determination of different polypeptides of the invention in various cell lines

Example 12-advanced subcutaneous stage in female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice A was evaluated in NSCLC modelAnti-tumor activity of MG 794.

When tumor growth was compared to vehicle treated mice of group 2 between day 17 and day 27 using the mixed effect model followed by the Dunnett post hoc test, treatment of NCI-H1435-peak-1-1 tumor bearing mice with CLDN6xCD3 HLE (SEQ ID NO: 21) resulted in statistically significant TGI at doses of 0.5, 0.15 and 0.05mg/kg with p-values <0.001. Tumor regrowth was observed after day 22 until tumor growth measurement ended on day 27. The increase in target negative cell population and lower numbers of human lymphocytes detected in the explanted tumor compared to the beginning of the study may explain tumor regrowth after day 22. At doses of 0.5, 0.15 and 0.05mg/kg CLDN6xCD3 HLE, TGI achieved at day 22 was 91%, 67% and 82%, respectively.

/>

Claims

1. A polypeptide or polypeptide construct comprising

● A domain that binds to human CLDN6 (SEQ ID NO: 1), and

● A domain that binds to human CD3, and

● A domain that extends the half-life of the polypeptide, wherein the domain that binds to human CLDN6 binds to amino acids 29-39 of SEQ ID No. 1 in extracellular loop 1 (ECL 1) of CLDN6 depicted in SEQ ID No. 9 and/or amino acids 151-160 of SEQ ID No. 1 in extracellular loop 2 (ECL 2) of CLDN6 depicted in SEQ ID No. 10.

2. The polypeptide or polypeptide construct of claim 1, wherein the polypeptide construct is a T cell activating construct.

3. The polypeptide or polypeptide construct according to any one of claims 1 and 2, wherein the polypeptide construct is a T cell activating polypeptide as determined in a T cell activating assay selected from the group comprising determining the amount of expression of CD69, determining the amount of expression of CD25, determining the amount of secreted IL-2, and determining the cytotoxic activity of these T cells.

4. A polypeptide or polypeptide construct according to any one of claims 1 to 3, wherein the domain that extends the half-life of the polypeptide comprises two polypeptide monomers, each comprising a hinge, a CH2 domain and a CH3 domain.

5. The polypeptide or polypeptide construct according to any one of claims 1 to 4, wherein the domain that binds to CD3 binds to extracellular epitopes of the human and cynomolgus CD3 epsilon chain.

6. The polypeptide or polypeptide construct according to any one of claims 1 to 5, wherein the domain that binds CLDN6 binds the same epitope on CLDN6 as a polypeptide construct or antibody or derivative or fragment thereof comprising a domain that binds CLDN6, wherein the domain comprises complementarity determining regions CDR-H1, CDR-H2 and CDR-H3 of a Variable Heavy (VH) chain selected from the group depicted in a) to s) below and/or complementarity determining regions CDR-L1, CDR-L2 and CDR-L3 of a Variable Light (VL) chain: a) To d), n) and s) are preferred, a) to c), e) and s) being particularly preferred:

7. The polypeptide or polypeptide construct according to any one of claims 1 to 6, wherein the domain that binds to human CD3 epsilon also binds to CD3 epsilon in common marmoset or cynomolgus monkey.

8. The polypeptide or polypeptide construct according to any one of claims 1 to 7, wherein

a) The polypeptide is a single-stranded construct,

b) The domain that binds to CLDN6 is in the form of an scFv,

c) The domain that binds to CD3 is in the form of an scFv,

d) The domains are linked via a linker, and/or

9. The polypeptide or polypeptide construct according to any one of claims 1 to 8 wherein the domain that binds to CLDN6 does not bind to CLDN1, CLDN2, CLDN3, CLDN4, CLDN9 and/or CLDN18.1/CLDN 18.2.

10. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the domain that binds CLDN6 comprises VH regions comprising CDR-H1, CDR-H2 and CDR-H3 and VL regions comprising CDR-L1, CDR-L2 and CDR-L3 selected from the group depicted in a) to s) below: a) To d), n) and s) are preferred, a) to c), e) and s) being particularly preferred:

11. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the domain that binds CLDN6 comprises a VH region with an amino acid sequence selected from the group consisting of SEQ ID No. 11, SEQ ID No. 25, SEQ ID No. 39, SEQ ID No. 53, SEQ ID No. 67, SEQ ID No. 81, SEQ ID No. 95, SEQ ID No. 109, SEQ ID No. 123, SEQ ID No. 137, SEQ ID No. 151, SEQ ID No. 165, SEQ ID No. 179, SEQ ID No. 193, SEQ ID No. 207, SEQ ID No. 221, SEQ ID No. 235, SEQ ID No. 249, or the sequence depicted in SEQ ID No. 263,

12. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the domain that binds to CLDN6 comprises a VL region having an amino acid sequence selected from the group consisting of SEQ ID No. 12, SEQ ID No. 26, SEQ ID No. 40, SEQ ID No. 54, SEQ ID No. 68, SEQ ID No. 82, SEQ ID No. 96, SEQ ID No. 110, SEQ ID No. 124, SEQ ID No. 138, SEQ ID No. 152, SEQ ID No. 166, SEQ ID No. 180, SEQ ID No. 194, SEQ ID No. 208, SEQ ID No. 222, SEQ ID No. 236, SEQ ID No. 250, or the sequence depicted in SEQ ID No. 264,

13. A polypeptide or polypeptide construct according to any one of the preceding claims wherein the domain that binds CLDN6 comprises a pair of VH and VL regions having the amino acid sequences depicted in: SEQ ID NO 11+12, SEQ ID NO 25+26, SEQ ID NO 39+40, SEQ ID NO 53+54, SEQ ID NO 67+68, SEQ ID NO 81+82, SEQ ID NO 95+96, SEQ ID NO 109+110, SEQ ID NO 123+124, SEQ ID NO 137+138, SEQ ID NO 151+152, SEQ ID NO 165+166, SEQ ID NO 179+180, SEQ ID NO 193+194, SEQ ID NO 207+208, SEQ ID NO 221+222, SEQ ID NO 235+236, SEQ ID NO 249+250, or SEQ ID NO 263+264.

14. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the domain that binds to CLDN6 comprises the amino acid sequence depicted in: SEQ ID NO 19, SEQ ID NO 22, SEQ ID NO 33, SEQ ID NO 36, SEQ ID NO 47, SEQ ID NO 50, SEQ ID NO 61, SEQ ID NO 64, SEQ ID NO 75, SEQ ID NO 78, SEQ ID NO 89, SEQ ID NO 92, SEQ ID NO 103, SEQ ID NO 106, SEQ ID NO 117, SEQ ID NO 120, SEQ ID NO 131, SEQ ID NO 134, SEQ ID NO 145, SEQ ID NO 148, SEQ ID NO 159, SEQ ID NO 162, SEQ ID NO 173, SEQ ID NO 176, SEQ ID NO 187, SEQ ID NO 190, SEQ ID NO 201, SEQ ID NO 204, SEQ ID NO 215, SEQ ID NO 218, SEQ ID NO 229, SEQ ID NO 232, SEQ ID NO 243, SEQ ID NO 246, SEQ ID NO 257, or SEQ ID NO 260, SEQ ID NO 271 or SEQ ID NO 274.

15. The polypeptide or polypeptide construct according to any one of the preceding claims, comprising an amino acid sequence selected from the group of those depicted in: SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, 22, 63, 64, 65, 66, 75, 76, 77, 78, 79, 80, 89, 90, 91, 92, 93, 94, 103, 104, 105, 106, 108, 150, 138, and 125, and SEQ ID NO. 121, 125, and 125, and SEQ ID NO. 15, 108, and 105, and SEQ ID NO. 15, and ID NO. 15, 108, 125, and ID NO. 13, 125, and ID NO. 15, SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164, 173, 174, 175, 176, 177, and 178, 187, 188, 189, 190, 191, and 192, 201, 202, 203, 204, 205, and 206, 215, 216, 217, 218, 219, and 220, 229, 230, 231, 232, 233, 234, 243, 244, 247, 246, and 95, or any of the polypeptides having a sequence identical to that of at least one of SEQ ID No. 93, 95, or 93, or that of at least one of the polypeptides having the amino acid sequence of SEQ ID No. 93, or of at least one of SEQ ID No. 93, or of any of SEQ ID No. 95, or of any of SEQ ID No. 138, 219, and 220, 229, 230, 232, 233, and 234, 243, 244, 246, or any of SEQ ID No. 95, or of any of SEQ ID No. 215, or any of SEQ ID No. 108, or any of SEQ ID No. 215, or any of SEQ ID No. 108, or any of SEQ ID No. or any of which has an amino acid sequence of or any of sequence of SEQ ID No. or, or any of that has any of or, or,.

16. A polypeptide or polypeptide construct comprising CDR-H1 depicted in SEQ ID No. 13, CDR-H2 depicted in SEQ ID No. 14 and CDR-H3 depicted in SEQ ID No. 15, and a VL region comprising CDR-L1 depicted in SEQ ID No. 16, CDR-L2 depicted in SEQ ID No. 17 and CDR-L3 depicted in SEQ ID No. 18.

17. A polypeptide or polypeptide construct comprising a VH region and a VL region having the amino acid sequences depicted in SEQ ID No. 11+12.

18. A polypeptide or polypeptide construct comprising the amino acid sequence depicted in SEQ ID No. 19.

19. A polypeptide or polypeptide construct comprising the amino acid sequence depicted in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 or SEQ ID No. 24.

20. A polypeptide or polypeptide construct comprising a domain that binds to human CLDN6 (SEQ ID NO: 1), and a domain that binds to human CD3, and a domain that extends the half-life of the polypeptide, wherein the domain that binds to CLDN6 comprises a VH region comprising CDR-H1 depicted in SEQ ID NO:13, CDR-H2 depicted in SEQ ID NO:14, and CDR-H3 depicted in SEQ ID NO:15, and a VL region comprising CDR-L1 depicted in SEQ ID NO:16, CDR-L2 depicted in SEQ ID NO:17, and CDR-L3 depicted in SEQ ID NO: 18.

21. A polypeptide or polypeptide construct according to any of the preceding claims wherein the domain that binds CLDN6 comprises a VH region and a VL region with the amino acid sequences depicted in SEQ ID No. 11+12.

22. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the domain that binds to CLDN6 comprises the amino acid sequence depicted in SEQ ID No. 19.

23. The polypeptide or polypeptide construct according to any of the preceding claims, comprising the amino acid sequence depicted in SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24.

24. The polypeptide or polypeptide construct according to any one of the preceding claims, wherein the cytotoxicity induced by the domain that binds to CLDN6 is at least 100-fold, at least 250-fold, at least 500-fold lower than the cytotoxicity measured in an in vitro assay using cells expressing CLDN6 depicted in SEQ ID No. 1, as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID No. 1, the mutant comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L.

25. The polypeptide or polypeptide construct according to any one of the preceding claims, wherein the cytotoxicity induced by the domain that binds to CLDN6 is at least 100-fold, at least 250-fold, at least 500-fold lower than the cytotoxicity measured in an in vitro assay using cells expressing CLDN6 depicted in SEQ ID No. 1, as determined in an in vitro assay using cells expressing the wild-type CLDN6 mutant depicted in SEQ ID No. 1, the mutant comprising at least one or more of the following mutations: M29X, wherein X is preferably L; R145X, wherein X is preferably Q; and/or Q156X, wherein X is preferably L, wherein the construct is capable of activating T cells and inducing cytotoxicity in target cells expressing CLDN6, and wherein the construct has a heavy chain CDR3 sequence comprising: x1LIVX2APX3 (SEQ ID NO. 667), wherein X1 is A or N; x2 is V or E; and X3 is V or A.

26. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct is a single-stranded construct.

27. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the half-life extending domain comprising two polypeptide monomers comprises a hinge, a CH2 domain and a CH3 domain, comprising in amino to carboxyl order:

hinge-CH 2-CH 3-linker-hinge-CH 2-CH3.

28. Polypeptide or polypeptide construct according to one of the preceding claims, wherein the CH2 domain comprises a intra-domain cysteine disulfide bridge.

29. The polypeptide or polypeptide construct according to any of the preceding claims, wherein

30. Polypeptide or polypeptide construct according to one of the preceding claims, wherein the antigen binding (epitope binding) domain that binds to CLDN6 and the antigen binding (epitope binding) domain that binds to CD3 are fused to another domain via a peptide linker.

31. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the polypeptide or polypeptide construct comprises in amino-to-carboxyl order, or in carboxyl-to-amino order:

(a) An antigen binding (epitope binding) domain that binds CLDN 6;

(c) An antigen binding (epitope binding) domain that binds to CD 3.

32. The polypeptide or polypeptide construct according to claim 31, wherein the polypeptide or polypeptide construct further comprises, in amino-to-carboxyl order, or in carboxyl-to-amino order, or between the antigen binding (epitope binding) domain that binds CLDN6 and the antigen binding (epitope binding) domain that binds CD 3:

(b) A first polypeptide monomer of a third domain;

(d) A second polypeptide monomer of the third domain.

33. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct is depicted in any of SEQ ID NOs 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, 105, 108, 119, 122, 133, 136, 147, 150, 161, 164, 175, 178, 189, 192, 203, 206, 217, 220, 231, 234, 245, 148, 259, 262, 273, 276, 287, 290, 301, 304, 315, 318, 329, 332, 343, 346, 357, 360, 371, 374, 385, 388, 399, 402, 413, 416, 427 and 430, in particular 35, 38, 49, 52, 63, 66, 77, 80, 91, 94, more particularly 35, 38, 49, 52, 77 and 80, and even more particularly 35, 49 and 77.

34. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct comprises a domain that binds to CD3 comprising a VH domain comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671 and/or 672.

35. Polypeptide or polypeptide construct according to one of the preceding claims, wherein the construct comprises a domain that binds to CD3 comprising at least one, two or all VL domains comprising CDR sequences depicted in SEQ ID NOs 673, 674 and/or 675.

36. A polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct comprises a domain that binds CD3 comprising VH and VL domains comprising at least one, two or all of the CDR sequences depicted in SEQ ID NOs 670, 671, 672, 673, 674 and/or 675.

37. A polypeptide or polypeptide construct according to any preceding claim wherein the construct comprises a domain that binds to CD3 comprising the VH domain depicted in SEQ ID No. 676.

38. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct comprises a domain that binds to CD3 comprising the VL domain depicted in SEQ ID No. 677.

39. A polypeptide or polypeptide construct according to any preceding claim wherein the construct comprises a domain that binds CD3 comprising a VH domain depicted in SEQ ID No. 676 and a VL domain depicted in SEQ ID No. 677.

40. The polypeptide or polypeptide construct according to any of the preceding claims, wherein the construct comprises a domain that binds to CD3 comprising the scFv domain depicted in SEQ ID No. 678.

41. A polynucleotide encoding the polypeptide or polypeptide construct according to any one of the preceding claims.

42. A vector comprising the polynucleotide of claim 41.

43. A host cell transformed or transfected with the polynucleotide of claim 41 or the vector of claim 42.

44. A process for producing a polypeptide or polypeptide construct according to any one of claims 1 to 40, the process comprising: culturing the host cell of claim 43 under conditions allowing expression of the polypeptide construct and recovering the produced polypeptide or polypeptide construct from the culture.

45. A pharmaceutical composition comprising a polypeptide or polypeptide construct according to any one of claims 1 to 40 or produced according to the process of claim 44.

46. A polypeptide or polypeptide construct according to any one of claims 1 to 40 or produced according to the process of claim 44 for use as a medicament, in particular for the prevention, treatment or alleviation of a disease, preferably a neoplasm.

47. The polypeptide or polypeptide construct according to claim 40 for use as a medicament, in particular for the prevention, treatment or alleviation of a disease, wherein the disease or neoplasm is selected from the group consisting of: germ cell cancer; ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratoma; uterine cancer; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma, further wherein the disease is a pediatric neoplasm selected from the group consisting of wilms' tumor, extracranial rhabdoid tumor, or connective tissue-promoting proliferative small round cell tumor.

48. The polypeptide or polypeptide construct according to claim 40 wherein the lung cancer is non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma.

49. A kit comprising a polypeptide or polypeptide construct according to any one of claims 1 to 40, a polypeptide or polypeptide construct produced according to the process of claim 44, a polynucleotide according to claim 41, a vector according to claim 42, and/or a host cell according to claim 43.

50. A method for treating or ameliorating a proliferative disease, a neoplastic disease, a cancer or an immunological disorder, the method comprising the step of administering to a subject in need thereof a polypeptide or polypeptide construct according to any one of claims 1 to 40 or produced according to the process of claim 41, wherein the disease is preferably selected from the group consisting of: germ cell cancer; ovarian cancer, particularly ovarian adenocarcinoma and ovarian teratoma; uterine cancer, more particularly ovarian serous cyst adenocarcinoma, uterine carcinoma sarcoma, endometrial carcinoma of the uterine body; and lung cancer, including Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC), particularly squamous cell lung cancer and adenocarcinoma; or a pediatric neoplasm selected from wilms' tumor, extracranial rhabdoid tumor, or connective tissue-promoting proliferative small round cell tumor.