Antibody-drug conjugates based on humanized CLDN18.2 antibodies

BACKGROUND

Tight junctions are multiprotein complexes connecting adjacent epithelial or endothelial cells to form a barrier, preventing molecules from passing in between the cells, and helping to maintain the cell and tissue polarity. Tight junctions consist of three main groups of transmembrane proteins: claudins and occludin, cytoplasmic plaque proteins, and cingulin. They also contain cytoskeletal and signaling proteins, e.g. actin, myosin II, and PKCij. These proteins interact to maintain tight junction structure (Yu and Turner 2008).

Claudins form a family of 23 proteins (Hewitt, Agarwal, and Morin 2006). Claudin 18 is human protein encoded by the CLDN18 gene, which forms tight junction strands in epithelial cells. The human CLDN18 can be alternatively spliced with two alternative first exons, resulting in two protein isoforms, CLDN18.1 (or Claudin 18.1) and CLDN18.2 (or Claudin 18.2). CLDN18.2 was first disclosed as Zsig28 protein in W02000/015659. The two isoforms differ in the N-terminal 69 amino acids, encompassing the first extracellular loop. The first extracellular domain spans from amino acid 28 to amino acid 80. Within this stretch there are 8 amino acid differences between CLDN18.1 and CLDN18.2. The two different isoforms are expressed in different tissues, with CLDN18.1 being predominantly expressed in lung tissue whereas CLDN18.2 displays stomach specificity (Niimi et al. 2001). CLDN18.2 expression in normal stomach is restricted to the differentiated short-lived cells of stomach epithelium. CLDN18.2 expression has further been identified in various tumor tissues. For example, CLDN18.2 has been found to be expressed in pancreatic, esophageal, ovarian, and lung tumors, correlating with distinct histologic subtypes (Sahin et al. 2008).

In view of its restricted expression pattern in normal tissues, and its ectopic expression in human cancers, CLDN18.2 is an attractive pan-cancer target for antibody therapy of epithelial tumors. A number of studies have been made towards such an antibody therapy. W02004/047863 identified the splice variants of CLDN18 and screened antibodies against different peptides derived from CLDN18.2: peptide DQWSTQDLYN (SEQ ID NO: 68), N-terminal extracellular of CLDN18.2, independent of glycosylation; peptide NNPVTAVFNYQ (SEQ ID NO: 69), N- terminal extracellular of CLDN18.2, mainly unglycosylated; and peptide STQDLYNNPVTAVF (SEQ ID NO: 70), N-terminal extracellular domain of CLDN18.2, unglycosylated. It also disclosed polyclonal rabbit antibodies screened with a pan-CLDN18 peptide TNFWMSTANMYTG (SEQ ID NO: 71) in the C-terminal extracellular domain common to both CLDN18.1 and CLDN18.2 isoforms. WO2005/113587 discloses antibodies against specific epitopes on CLDN18.2 defined by the following peptide sequences: ALMIVGIVLGAIGLLV (SEQ ID NO: 72) and RIGSMEDSAKANMTLTSGIMFIVS (SEQ ID NO: 73). W0200/7059997 discloses CLDN18.2 specific monoclonal antibodies obtained by immunization with the peptide

MEED TELL WVLLLWVPGSTGDAAQPARRARRTKLGTELGSTPVWWNSADGRMDQ WSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRAAIQH SGGRSRRARTKTHLRRGSE (SEQ ID NO: 74), including the first extracellular domain of CLDN18.2 with N- and C-terminal extensions. Antibodies obtained by this immunization mediate cell killing by complement dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody IMAB362, also known as Claudiximab or Zolbetuximab, is disclosed in W02007/059997 and WO2016/165762. IMAB362 is an IgGl antibody derived from a murine monoclonal antibody and has been chimerized to display the human IgGl constant region for clinical use. WO2008/145338 also discloses antibodies binding to overlapping peptides within the first extracellular domain (MDQWSTQDLYNNPVT (SEQ ID NO: 75), LYNNPVTAVFNYQGL (SEQ ID NO: 76), VFNYQGLWRSCVRES (SEQ ID NO: 77), QGLWRSCVRESSGFT (SEQ ID NO: 78), and RSCVRESSGFTECRG (SEQ ID NO: 79)). In an effort to produce antibodies targeting the C-terminal portion of CLDN18.2 for diagnostic purposes to detect CLDN18.2 expression in cells of cancer tissue sections, WO2013/167259 discloses antibodies binding to C-terminal epitopes of CLDN18.2. The sequences of the two epitopes are TEDEVQSYPSKHDYV (SEQ ID NO: 80) and EVQSYPSKHDYV (SEQ ID NO: 81). WO2013/174509 presents combinations of anti- CLDN18.2 antibodies with agents stabilizing y6 T cells or with agents stabilizing or increasing the expression of CLDN18.2. Antibodies may be conjugated to a therapeutic moiety such as a cytotoxin, a drug (e.g. an immunosuppressant) or a radioisotope. WO2014075788 discloses a method of treatment a cancer disease using a bispecific antibody binding CLDN18.2 and CD3. WO2014/127906 discloses combination agents stabilizing or increasing the expression of CLDN18.2. WO2016/166122 discloses anti-CLDN18.2 monoclonal antibodies that can be highly efficiently internalized upon CLDN18.2 binding and therefore, are suitable for antibody - drug conjugate (ADC) development. Furthermore, the conjugation of such antibodies to the drugs DM4 and MMAE using cleavable SPDB or Valine-Citrulline linkers, respectively, is disclosed. However, despite all the antibodies disclosed in the patent applications, only the chimeric IMAB362, disclosed in W02007/059997 and WO2016/165762, is currently tested in clinical trial. In addition to these antibodies and ADCs, W02016/008405, W02018/006882, CN111440245 disclose chimeric antigen receptor (CAR) based on anti-CLDN18.2 monoclonal antibodies. Antibodies of WO2018/006882 have been humanized and their sequence is disclosed in in the Supplementary Materials section associated with Jiang et al. (2018). CAR T-cells based on the humanized antibody are currently tested in a phase I clinical trial (ClinicalTrials.gov Identifier: NCT03159819) in patients with advanced gastric adenocarcinoma and pancreatic adenocarcinoma. CN109762067 discloses other anti- CLDN18.2 monoclonal antibodies mediating cell killing by CDC and ADCC. WO2019/173420 discloses anti-CLDN18.2 humanized monoclonal antibodies with ADCC activity. WO2019/175617 discloses anti-CLDN18.2 monoclonal antibodies binding to a different epitope than IMAB362. WO2019/219089 discloses monoclonal antibodies binding to a mutant of CLDN 18.2. Other antibodies binding to CLDN 18.2 have been disclosed in WO2019/242505, W02020/038404, W02020/043044, W02020/063988, W02020/082209, W02020/018852, W02020/023679, WO2020/135674, W02020/135201, WO2020/139956, W02020/025792, W02020160560, CN111808194 and W02020200196.

Chimeric antibodies, having mouse variable regions grafted on human constant domains, are often still immunogenic and this may result in enhanced clearance of the antibody and other safety implications (Sauerborn 2014). Therefore, further modification of the antibody sequence is required to reduce patient immune response and improve its therapeutic activity. Humanization is a process by which xenogeneic antibody sequences are modified to reduce this immunogenicity (Saldanha 2014). However, humanization of an antibody often also leads to loss of affinity. IMAB362, currently the clinically most advanced anti-CLDN18.2 antibody, is a chimeric antibody. Therefore, there is still a need for better anti-CLDN18.2 antibodies.

As already mentioned above, IMAB362 has already been developed as an antibody-drug conjugate (ADC) (disclosed in WO2016/165762), where the antibody has been conjugated to the MMAE or DM4 drugs. The DM4 drug was coupled to IMAB362 via SPBD (N- succinimidyl-3-(2 pyridyldithio)butyrate), an amino and sulfhydryl reactive heterobifunctional protein crosslinker which reacts via an N-hydroxysuccinimide (NHS) ester with primary amines (as found in lysine side chains or the N-terminus of proteins) of the antibody. The valine- citrulline-MMAE drug was coupled to thiolated IMAB362. In that case, IMAB362 was initially thiolated with the heterobifunctional linker 2-IT (2-iminothilane) which reacts with the free amines of lysine residues. The valine-citrulline is a linker cleavable by cathepsins. All the caveats listed above related to IMAB362 also apply to an ADC based on the same antibody.

The instant invention is directed to provide ADCs based on improved humanized IMAB362 antibodies addressing the needs of better ADCs based on better anti-CLDN18.2 antibodies, with, surprisingly, higher cytotoxic activity on cells expressing CLDN18.2 than an ADC based on IMAB362.

DESCRIPTION OF THE INVENTION

DEFINITIONS

"Antibodies" or “antibody”, also called "immunoglobulins" (Ig), generally comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, and are therefore multimeric proteins, or comprise an equivalent Ig homologue thereof (e.g., a camelid antibody comprising only a heavy chain, single-domain antibodies (sdAb) or nanobodies which can either be derived from a heavy or a light chain). The term “antibodies” includes antibody-based binding proteins, modified antibody formats retaining its target binding capacity. The term “antibodies” also includes full length functional mutants, variants, or derivatives thereof (including, but not limited to, murine, chimeric, humanized and fully human antibodies) which retain the essential epitope binding features of an Ig molecule, and includes dual specific, bispecific, multispecific, and dual variable domain Igs. Ig molecules can be of any class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), or subclass (e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2) and allotype. Ig molecules may also be mutated e.g. to enhance or reduce affinity for Fey receptors or the neonatal Fc receptor (FcRn).

An "antibody fragment", as used herein, relates to a molecule comprising at least one polypeptide chain derived from an antibody that is not full length and exhibits target binding, including, but not limited to (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL) and constant heavy 1 (CHI) domains; (ii) a F(ab') ₂ fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region (reduction of a F(ab') ₂ fragment result in two Fab’ fragment with a free sulfhydryl group); (iii) a heavy chain portion of a Fab (Fa) fragment, which consists of the VH and CHI domains; (iv) a variable fragment (Fv) fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment, which comprises a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain Fv fragment (scFv); (viii) a diabody, which is a bivalent, bispecific antibody in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with the complementarity domains of another chain and creating two antigen binding sites; (ix) a linear antibody, which comprises a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementarity light chain polypeptides, form a pair of antigen binding regions; (x) DualVariable Domain Immunoglobulin (xi) other non-full length portions of immunoglobulin heavy and/or light chains, or mutants, variants, or derivatives thereof, alone or in any combination.

An "antibody -based binding protein", as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin, or non-antibody derived components. Such antibody-based proteins include, but are not limited to (i) Fc-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and/or VL, and/or CH domains are combined and/or assembled in a fashion not normally found in naturally occurring antibodies or antibody fragments.

The term "modified antibody format", as used herein, encompasses polyalkylene oxidemodified scFv, monobodies, diabodies, camelid antibodies, domain antibodies, bi- or trispecific antibodies, IgA, or two IgG structures joined by a J chain and a secretory component, shark antibodies, new world primate framework and non-new world primate CDR, IgG4 antibodies with hinge region removed, IgG with two additional binding sites engineered into the CH3 domains, antibodies with altered Fc region to enhance or reduce affinity for Fc gamma receptors, dimerized constructs comprising CH3, VL, and VH, and the like. The Kabat numbering scheme (Martin and Allemn 2014) has been applied to the disclosed antibodies.

The term “Antibody-Drug conjugate” or "ADC" refers to an antibody or antibody fragment to which toxins (or drugs) have been linked. In an ADC, toxins are conjugated to the antibody or antibody fragment by cleavable or non-cleavable linkers. Cleavable linker may be designed to be cleaved extracellularly in the tumor environment or intracellularly within the cytosol. Cleavable linkers exploit differential conditions of reducing power or enzymatic degradation that can be present either outside or inside the target cell. Non-cleavable linkers require the ADC to be internalized, the antibody-linker component needs to be degraded by lysosomal proteases for the toxins to be released. Conjugation of the linker to the antibody may also vary. Conjugation may rely on the presence of lysine and cysteine residues within the polypeptide structure of the antibody as the point of conjugation. Reactive groups on the linker can e.g. be conjugated to the side chain of lysine residues through amide or amidine bond formation. Conjugation via cysteine residues requires a partial reduction of the antibody. Alternatively, site-specific enzymatic conjugation can be used. This requires enzymes that react with the antibody and can induce site- or amino acid sequence-specific modifications. Peptide sequences recognized by these enzymes may have to be inserted into the genetically engineered antibodies or fragments to be conjugated. Enzymes which have been used for such purpose are sortase, transglutaminase, galactosyltransferase, sialyltransferase and tubulin-tyrosine ligase. An overview of ADC linker conjugation and toxins can be found in Ponziani et al, 2020 (Ponziani et al. 2020). An overview of conjugation of toxins to antibody fragments can be found in Aguiar et al, 2018 (Aguiar et al. 2018). The type of linker and the method of conjugation used to conjugate the toxin to the antibody or antibody fragment may determine the drug-to-antibody ratio (DAR).

The term “toxin” refers to a cytotoxic and/or cytostatic agent that can be based on a synthetic, plant, fungal, or bacterial molecule. Cytotoxic or cytostatic means that they inhibit the growth of and/or inhibit the replication of and/or kill cells, particularly malignant cells typically due to their increased turnover. In a preferred embodiment, the toxin is selected from the group consisting of anthracyclines and derivatives thereof. Anthracy clines are antibiotic compounds that exhibit cytotoxic activity, and may kill cells by different mechanisms, including intercalation of the drug molecules into the DNA of the cell or DNA severing activity thereby inhibiting DNA-dependent nucleic acid synthesis, generation of free radicals by the drug which react with cellular macromolecules to cause damage to the cells, DNA alkylation and/or interactions of the drug molecules with the cell membrane. Anthracyclines include doxorubicin, epirubicin, idarubicin, daunomycin, nemorubicin, and derivatives thereof. A well-known and preferred anthracy cline derivative is PNU- 159682, or in short PNU, CAS No. 202350-68-3. It is a highly potent metabolite of nemorubicin having outstanding cytotoxicity. Anthracycline derivatives are understood as including also the toxin as a result of conjugation to specific ligands, where due to the conjugation chemistry used, some atoms of the original toxin may be missing (Broggini 2008; Quintieri et al. 2005), or the toxin as a result of lysosomal degradation, where a fragment of the linker may remain attached to the anthracycline molecule. The term “anthracyclines” as used herein refers to anthracyclines and anthracycline derivatives.

The term “selectively binds to CLDN18.2” or “selective binding to CLDN18.2” as referred to herein refers to an antibody exhibiting binding to CLDN18.2, while exhibiting no (specific) binding to CLDN18.1. Hence, the antibodies selectively binding to CLDN18.2 do not exhibit cross-reactivity to CLDN 18.1.

Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group, which preferably consists only of these embodiments.

Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated.

Technical terms are used by their common sense. If a specific meaning is conveyed to certain terms, definitions of terms will be given in the following in the context of which the terms are used.

DESCRIPTION

The inventors have surprisingly identified novel antibody-drug conjugates (ADCs) involving anti-CLDN18.2 antibodies and a toxin as further described herein. These antibodies bind to CLDN18.2 with a higher affinity than the IMAB362 antibody. Therefore, in one embodiment, the invention provides an ADC comprising an antibody or fragment thereof binding to CLDN18.2, which comprises the heavy chain complementarity determining regions (HCDR) HCDR1, HCDR2 and HCDR3 consensus sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively and the light chain complementary regions (LCDR) LCDR1, LCDR2 and LCDR3 consensus sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively and a toxin. The respective consensus sequences can be found in Table 1. It is understood that any antibody or fragment thereof based on any combination of CDRs derived from the consensus sequences and binding to CLDN18.2 is included.

In a preferred embodiment, the antibody or functional fragment thereof binds to CLDN18.2 but not to CLDN18.1. Hence, the ADCs provided herein specifically bind CLDN18.2. In one embodiment, the toxin is an anthracy cline.

The inventors engineered novel ADCs based on the humanized anti CLDN18.2 antibodies from above. These ADCs surprisingly exhibit better cytotoxic activity on tumor cells compared to corresponding ADCs based on IMAB362. The heavy and light chain sequences of IMAB362 are e.g. shown herein as SEQ ID NO: 47 (heavy chain) and SEQ ID NO: 48 (light chain). The original sequence of IMAB362, published in W02007/059997, has the same heavy chain sequence as SEQ ID NO: 47, wherein the C-terminal amino-acid is lysine (K). Substation of lysine (K) to an arginine (R) as used herein does not affect the binding affinity of the antibody to CLDN18.2.

In one embodiment, the ADC of the invention has the general formula A - (L-T) _n , wherein a. A is an antibody or fragment thereof binding to CLDN 18.2 comprising the HCDR1 , HCDR2 and HCDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, or comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively b. L is a linker, and c. T is a toxin, wherein the toxin is an anthracycline.

In one embodiment, n is an integer between >1 and < 10. The invention also relates to a pharmaceutically acceptable salt or ester of the ADC.

In one embodiment, A is an antibody or fragment thereof binding to CLDN18.2 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

The respective consensus sequences can be found in Table 1. It is understood that any ADC comprising an antibody or fragment thereof based on any combination of CDRs derived from the consensus sequences and binding to CLDN18.2 is part of the invention.

Table 1 : isolated antibody CDR consensus sequences

The linker L of the ADC of the invention may comprise one or more linker elements. In one embodiment, the linker L of the ADC of the invention comprises at least one non-cleavable linker element. A non-cleavable linker element may be defined as a linker element that is only subjected to lysosomal degradation, that is not the substrate of specific enzymes and/or that is stable in plasma and cytosol.

The non-cleavable linker element may be selected from the group consisting of: a. ethylenediamine (EDA), b . N -formyl -N,N’ -dimethyl ethyl enedi amine, c. di ethylamine (DEA), d. a piperazine-derived compound of the following formula: wherein the wavy lines indicate attachments to the toxin and another linker element, e. the compound of the following formula: wherein the wavy lines indicate attachments to the toxin and another linker element, f. the compound of the following formula: wherein the wavy line indicates attachment to the toxin and [Ab] indicates the antibody or fragment thereof, g. a maleimidocaproyl compound of the following formula: wherein the wavy line indicates attachment to another linker element and [Ab] indicates the antibody or fragment thereof, h. the compound of the following formula: wherein the wavy line indicates attachment to a toxin and [Ab] indicates the antibody or fragment thereof, and wherein the non-cleavable linker element is conjugated to the toxin by means of an amide bond or an ether bond.

The non-cleavable linker element may be directly covalently attached to the antibody (and thereby form the linker) or it may be attached via other linker elements such as oligopeptide linker elements. Alternatively, or additionally, cleavable linker elements may be present in the linker.

The non-cleavable linker element may be linked to the antibody via amino acids of the antibody sequence that have side chains with available nucleophilic groups such as s-NH ₂ of lysine and the sulfhydryl SH group of cysteine. Maleimide chemistry allows linkage to the cysteine side- chain while acylation chemistry is usually used for linkage to the lysine side-chain. Ample information on such linkages can be found e.g. in Jain et al, 2015 (Jain et al. 2015). Linkage of a non-cleavable linker element to an oligopeptide linker element may be carried out by carbodiimide crosslinking chemistry. Guidance for such crosslinking chemistry may be found in the Thermo Scientific Crosslinking Technical Handbook (2012) ("Crosslinking Technical Handbook" 2012).

The non-cleavable linker element may also be directly attached to an anthracy cline. In one embodiment, the non-cleavable linker element is attached to the anthracycline of formula I by means of an amide bond to C ₁₃ or an ether bond to Ci ₄ , wherein Ri is hydrogen atom, hydroxy or methoxy group and R ₂ is a Ci-C ₅ alkoxy group.

It is understood that a combination of one or more linker elements may be used to form the linker in order to link the antibody to the toxin, including enzyme-cleavable linker elements.

In another element, the linker further comprises an oligopeptide linker element and/or enzyme- cleavable linker element and/or a spacer element.

The oligopeptide linker element is understood as being an oligopeptide that is present in addition to the peptidic chain forming the antibodies or fragment thereof. The oligopeptide linker element may be directly attached to the C -termini of the heavy and/or light chains forming the antibody or the fragments thereof. In one embodiment, the DNA coding sequence of the oligopeptide linker element may be part of the DNA encoding the respective heavy and/or light chains forming the antibody or fragment thereof.

In another embodiment, the oligopeptide linker element may be the result of peptide ligation used to link two or more oligopeptide linker elements. Ligation may be catalyzed by peptide ligases such as sortases (i.e. Sortase A), asparaginyl endoproteases (i.e. Butelase 1), trypsin related enzymes (i.e. Trypsiligase) or subtili sin-derived variants (i.e. Peptiligase) (Nuijens et al. 2019). The oligopeptide linker elements may thus include peptide ligase recognition motifs.

In one embodiment, the oligopeptide linker element comprises a sortase recognition motif oligopeptide selected from: - LPXTG _m -, -LPXAG _m -, -LPXSG _m -, -LAXTG _m -, -LPXTG _m -, - LPXTA _m -, -NPQTG _m - or -NPQTN _m -, with G _m being an oligoglycine with m being an integer between >1 and < 21, A _m being an oligoalanine with m being an integer between > 1 and < 21, N _m being an oligoasparagine with m being an integer between > 1 and < 21 and X being any conceivable amino acid. Preferably, m is 2 or 3, especially 2. In a preferred embodiment, the sortase recognition motif oligopeptide is -LPQTGG- or -LPETGG-. The sortase recognition motif oligopeptide may be present at the C-termini of the heavy and/or light chains of the antibody or of fragments thereof, preferably at the C-termini of the light chains.

In a further preferred embodiment, the oligopeptide linker of the ADC comprises the sequence SEQ ID NO: 143. The sequence SEQ ID NO: 143 may be attached to the C-termini of the antibody heavy chains or to the C-termini of the antibody light chains, or to the C-termini of the antibody heavy and light chains.

In another embodiment, an enzyme-cleavable linker element is present in the linker. The enzyme-cleavable linker element may comprise a val-cit-PAB linker according to the compound of the following formula:

wherein the wavy lines indicate attachments to other linker elements or the antibody or the toxin. The enzyme-cleavable linker element may be attached to another linker element or the antibody or the toxin by known crosslinker chemistry e.g. as described above for the non- cleavable linker elements.

In yet another embodiment, the linker further comprises a spacer element. The term spacer element, in the context of the invention, is to be understood as a spacer added to the linker to avoid steric hindrance and/or to allow proper conjugation of the toxin to the antibody or fragment thereof. In one embodiment, the spacer element comprises a peptidic flexible oligopeptide. Flexible linker elements can be applied when the linked components require a certain degree of movement or interaction. Flexible oligopeptides are generally composed of small, non-polar (e.g. G) or polar (e.g. S or T) amino acids. The small size of these amino acids provides flexibility and allows for mobility of the connected functional components. The incorporation of S or T can maintain stability of the linker in aqueous solutions by forming hydrogen bonds with water molecules, and therefore reduces unfavorable interactions between the linker and protein moieties. Further guidance on peptidic flexible oligopeptides may be found in Chen et al, 2013 (Chen, Zaro, and Shen 2013).

Preferably, the spacer element comprises a peptidic flexible oligopeptide consisting of G and S, more preferably the peptidic flexible oligopeptide is (GGGGS) ₀ with o being 1, 2, 3, 4 or 5.

The invention also provides an ADC of the following structure: a. A - ([oligopeptide linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: i. [LPXTGG]-[ethylenediamine], and b. A - ([oligopeptide linker element - enzyme cleavable linker element - non- cleavable linker element] - T) _n and preferably wherein the linker is selected from: iii. [LPXTGG]-[vc-PAB]-[N-formyl-N,N’-dimethylethylenediamine], and i v . [LPXT GG] - [vc-P AB ] - [piperazine] ; c. A - ([spacer element - oligopeptide linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: v. [GGGGS]-[LPXTGG]-[ethylenediamine], and d. A - ([spacer element - oligopeptide linker element - enzyme cleavable linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: vii. [GGGGS]-[LPXTGG]-[vc-PAB]-[N-formyl-N,N’- dimethylethylenediamine], and viii. [GGGGS]-[LPXTGG]-[vc-PAB]-[piperazine], wherein A is an antibody or fragment thereof binding to CLDN18.2 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, or comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, and wherein T is an anthracycline. It is understood that the toxin may be conjugated via the linker to the C -termini of the antibody heavy and/or light chains, or at the C-termini of the antibody fragments.

In a preferred embodiment, the non-cleavable linker element is ethylenediamine and the oligopeptide linker element is LPXTGG wherein X is Q or E, preferably wherein X is Q.

In one embodiment, a. (L-T) is covalently linked to both light chains of the antibody, b. (L-T) is covalently linked to both heavy chains of the antibody, or c. (L-T) is covalently linked to both light chains and both heavy chains of the antibody.

In one embodiment, (L-T) a. is linked to the C-terminus of the antibody light chain or antibody heavy chain, or b. is linked to an amino acid side chain of the antibody light chain or antibody heavy chain.

In case the toxin is linked to the C-terminus of the antibody light chain and/or antibody heavy chain, an oligopeptide linker element and an optional spacer element may be part of the amino acid sequence when the antibody is recombinantly expressed with such C-terminal tag. In case the toxin is linked to an amino-acid side chain of the antibody amino acid sequence, the linker element may be linked by maleimide chemistry or acylation chemistry, depending on the amino acid side chain of choice.

Surprisingly the ADCs of the invention have a higher cytotoxic activity in cells expressing CLDN18.2 (see Figure 5 to 8 and Example 6) or in patient-derived tumor xenograft models (see Figure 9 and 11 and Example 7) than an ADC based on IMAB362.

In one embodiment, the anthracycline has the following formula (I):

wherein Ri is a hydrogen atom, a hydroxy or methoxy group, and wherein R ₂ is a C1-C5 alkoxy group. In one embodiment the anthracycline is attached to the linker via resulting in loss of C14 and the hydroxyl group via or C ₁₄ resulting in loss of the hydroxyl group. It is understood that linking the toxin (via CJJ or C14) to an antibody will not affect the cytotoxic activity of the toxin.

Further information on the synthesis of PNU- 159682 and its use as toxin in ADCs may be found in Holte D et al 2020 (Holte et al. 2020).

PNU- 159682 may be linked to the antibody by non-cleavable or enzyme-cleavable linkers as shown below.

The linker may be a maleimide acetal linker:

Such a linker was used in an PNU-159682-maleimide acetal-Ab ADC shown below:

Such a PNU-159682 maleimide acetal-Ab ADC has been disclosed in US 10,435,471, column 90. The PNU-159682 maleimide acetal compound has been disclosed as compound 51 in W02010/009124 and may be prepared as disclosed in Example 3d (paragraphs [0576] to [0578]), based on the compound prepared in Example 2 (paragraphs [0542] to [0550]) of the same application.

PNU-159682 may also be linked to the antibody by a val-cit-PAB enzyme-cleavable linkers to form a PNU-159682-val-cit-PAB-Ab ADC as shown below:

Such an ADC has been disclosed in US 10,435,471, column 91-92. The PNU-159682-val-cit- PAB compound is disclosed as compound 55 in W02010/009124 and may be prepared as shown in Example 3b (paragraph [0567]-[0573 ] and Figure 7d) of the same application. PNU- 159682 may also be linked to the antibody via an enzyme cleavable linker val-cit-PAB and an additional non-cleavable linker element as shown below:

Such an ADC has been disclosed in US 10,435,471, column 91-92 and Yu SF et Clin Cancer

Research 2015 (Yu et al. 2015). The PNU-159682-val-cit-PAB + non-cleavable linker compound may be prepared as follows: wherein MC-val-cit-PAB is commercially available (MedChemExpress Cat. No.: HY-78738) and Boc is a tert-butyloxycarbonyl protecting group.

PNU- 159682 may also be linked to the antibody via a non-cleavable mal eimide linker as shown below:

Such ADC has been disclosed in US 10,435,471, column 93. The PNU-159682-maleimid compound is disclosed as compound 55 in W02010/009124 and its preparation in Example 3a (paragraphs [0564] to [0566] of the same application. A combination of non-cleavable, enzyme-cleavable and oligopeptide linker elements has also been used to link PNU-159682 to an antibody. Such ADC is shown below:

Such a compound is disclosed in Stefan et al. (Stefan et al. 2017). Such an ADC may be synthesized as disclosed above for the PNU-159682-val-cit-PAB + non-cleavable linker ADC, substituting MC-Val-Cit-PAB by Fmoc-Gly3-Val-Cit-PAB (commercially available from MedChemExpress Cat No.: HY-136106), and the resulting linker-toxin compound may be conjugated to an antibody as disclosed in WO2016/102679, page 34, 2 ^nd paragraph.

PNU- 159682 may also be linked to an antibody via a non-cleavable EDA linker element combined with an oligopeptide linker element (-GGGGG-) as shown below:

Such a compound is disclosed in WO2016/102679, Figure 3 A. It may be prepared as disclosed in the scheme on Figure 3B and page 33, last paragraph to page 34, 1 ^st paragraph of WO2016/102679 and the resulting linker-toxin compound may be conjugated to an antibody as disclosed in WO2016/102679, page 34, 2 ^nd paragraph. The oligo peptide linker element used above may also be (-GGG-) or preferably (-GG-).

Antibody binding or binding affinity is generally expressed in terms of equilibrium association or dissociation constants (K _a or K _d , respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k _off and k _on , respectively). Thus, equivalent affinities may correspond to different rate constants, so long as the ratio of the rate constants remains the same. Binding affinities and/or rate constants can be determined using techniques well known in the art or described herein, such as ELISA, flow cytometry (FC) titration, isothermal titration calorimetry (ITC), Biacore (SPR), biolayer inferometry or fluorescent polarization. In some cases, due to the nature of the antigen, the K _a or K _d of antibodies may be difficult to measure. This is especially true for integral membrane proteins such as Claudins (Hashimoto et al. 2018). In such cases, the integral membrane protein may be expressed as proteoliposomes or lipoparticles. Such lipoparticles may be immobilized on plastic and used in ELISA assay to determine the binding affinity of antibodies to the immobilized antigen. Instead of K _a or K _d values, half maximal effective concentration (EC50) values may thus be calculated for each tested antibody or functional fragment thereof, reflecting its binding affinity to the antigen. Example 3 below and Figure 2 exemplify ELISA assay binding affinity curves of antibodies with CDRs comprised in the consensus sequences of Table 1. Therefore, binding can be determined as in Example 4, where binding is quantified using EC50 values (Table 4 in Example 4) and the upper curve values (Figure 4). The EC50 values and upper curves values (maxMFI) show surprisingly that the humanized antibodies of the present invention have a higher binding affinity, i.e. they exhibit increased binding to CLDN18.2 than the IMAB362 antibody. Maximum mean fluorescent intensity (maxMFI) can also be used to quantify the binding of antibodies. When comparing two antibodies binding to the same target, a higher maxMFI is indicative of a higher affinity and/or of a lower off rate. MaxMFI can be determined as shown in Example 4 and maxMFI values for the antibodies of the invention are shown in Table 4, when binding is measured by FC on HEK293T cells expressing CLDN18.2 or PA-TU- 8988S-High cells.

Accordingly, preferably the antibodies of the invention or fragments thereof, bind with a higher affinity to CLDN18.2 than the IMAB362 antibody. In turn, Figure ID shows that all tested antibodies do not bind to HEK293T cells expressing CLDN18.1, and accordingly, all tested antibodies selectively bind to CLDN18.2. Further, in a preferred embodiment, such antibodies or fragments thereof are humanized.

The cytotoxic activity of ADCs can be characterized by EC50 values retrieved from an ADC cytotoxic assay. Example 6 and Table 5 below relate to the calculation of EC50 values of the ADCs of the invention using cytotoxic assays with cells expressing CLDN18.2.

As shown in Example 4 and Table 4, the binding affinity measured by flow cytometry, reflected by the binding Maximum MFI and EC50, of all hGBA antibodies is similar between each other but is higher than the binding affinity of reference antibody IMAB362.

Table 5 and Example 6 show that the EC50 values for the cytotoxic activity of the ADCs of the invention are lower than the EC50 value for the cytotoxic activity of an ADC based on IMAB362.

EC50 values were measured on the HEK293T or A549 cells overexpressing CLDN18.2 and on the PA-TU-8988S-High cell lines. Surprisingly, the EC50 for cytotoxic activity of the ADC of the invention is multiple fold lower than the EC50 for cytotoxic activity of a corresponding ADC based on IMAB362. Importantly, the ADC of the invention shows a higher cytotoxic activity compared to the cytotoxic activity of a corresponding ADC based on IMAB362 than could have been expected from the EC50 binding values of the respective antibodies (see also Figures 5 to 8). In one embodiment, the antibody or fragment thereof binds to CLDN18.2 and comprises: a. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 18, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; b. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 19, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; c. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 20, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 30, respectively; d. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 21, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26, SEQ ID NO: 5 and SEQ ID NO: 30, respectively; e. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 18, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 31, respectively; f. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 14 and SEQ ID NO: 22, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; g. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 15 and SEQ ID NO: 23, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 27, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; h. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 23, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; or i. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 24, respectively and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 28, SEQ ID NO: 5 and SEQ ID NO: 31 respectively.

In a preferred embodiment, the antibody is humanized. As described above, these humanized antibodies bind with higher affinity to CLDN18.2 than the IMAB362 antibody, as for example shown by EC50 and maxMFI values. Further, the provided antibodies selectively bind to CLDN18.2.

In another preferred embodiment, the ADCs based on the humanized antibodies have a higher cytotoxic activity on CLDN18.2-expressing cells than the corresponding ADC based on IMAB362 as for example shown by the EC50 values for cytotoxic activity.

In yet another embodiment, the antibody or fragment thereof binds CLDN18.2 and comprises: a. a VH sequence of SEQ ID NO: 32; b. a VH sequence of SEQ ID NO: 34; c. a VH sequence of SEQ ID NO: 35; d. a VH sequence of SEQ ID NO: 37; e. a VH sequence of SEQ ID NO: 39; f. a VH sequence of SEQ ID NO : 41 ; g. a VH sequence of SEQ ID NO: 42; h. a VH sequence of SEQ ID NO: 44; or i. a VH sequence of SEQ ID NO: 45; and j. a VL sequence of SEQ ID NO: 33; k. a VL sequence of SEQ ID NO: 36; l. a VL sequence of SEQ ID NO: 38; m. a VL sequence of SEQ ID NO: 40; n. a VL sequence of SEQ ID NO: 43; or o. a VL sequence of SEQ ID NO: 46.

In a preferred embodiment, the antibody is humanized. As described above, these humanized antibodies bind with higher affinity to CLDN18.2 than the IMAB362 antibody, as for example shown by EC50 and maxMFI values. Likewise, the ADCs of the invention have a higher cytotoxic activity on CLDN18.2-expressing cells than corresponding ADCs based on IMAB362, as shown by the EC50 values for cytotoxic activity. Further, the provided antibodies selectively bind to CLDN18.2. In a preferred embodiment, the antibody or functional fragment thereof binds to CLDN18.2 but not to CLDN18.1.

In another embodiment, the antibody or fragment thereof binds CLDN18.2 and comprises: a. a VH sequence of SEQ ID NO: 32 and a VL sequence of SEQ ID NO: 33; b. a VH sequence of SEQ ID NO: 34 and a VL sequence of SEQ ID NO: 33; c. a VH sequence of SEQ ID NO: 35 and a VL sequence of SEQ ID NO: 36 d. a VH sequence of SEQ ID NO: 37 and a VL sequence of SEQ ID NO: 38; e. a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40; f. a VH sequence of SEQ ID NO: 41 and a VL sequence of SEQ ID NO: 33; g. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; h. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 33; or i. a VH sequence of SEQ ID NO: 45 and a VL sequence of SEQ ID NO: 46.

In a preferred embodiment, the antibody is humanized. Again, as described above, these humanized antibodies bind with higher affinity to CLDN18.2 than the IMAB362 antibody, as for example shown by EC50 and maxMFI values. Further, the provided antibodies selectively bind to CLDN18.2. Likewise, the ADCs of the invention have a higher cytotoxic activity on CLDN18.2-expressing cells than corresponding ADCs based on IMAB362, as shown by the EC50 values for cytotoxic activity.

In a further embodiment, the antibody or fragment thereof binds to CLDN18.2 and consists of: a. the heavy chain sequence of SEQ ID NO: 49 and light chain sequence of SEQ ID NO: 50; b. the heavy chain sequence of SEQ ID NO: 51 and light chain sequence of SEQ ID NO: 50; c. the heavy chain sequence of SEQ ID NO: 52 and light chain sequence of SEQ ID NO: 53; d. the heavy chain sequence of SEQ ID NO: 54 and light chain sequence of SEQ ID NO: 55; e. the heavy chain sequence of SEQ ID NO: 56 and light chain sequence of SEQ ID NO: 57; f. the heavy chain sequence of SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 50; g. the heavy chain sequence of SEQ ID NO: 59 and light chain sequence of SEQ ID NO: 60; h. the heavy chain sequence of SEQ ID NO: 61 and light chain sequence of SEQ ID NO: 50; or i. the heavy chain sequence of SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 63.

In a preferred embodiment, the antibody is humanized. Again, as described above, these humanized antibodies bind with higher affinity to CLDN18.2 than the IMAB362 antibody, as for example shown by EC50 and maxMFI values. Further, the provided antibodies selectively bind to CLDN18.2. Likewise, the ADCs of the invention have a higher cytotoxic activity on CLDN18.2-expressing cells than corresponding ADCs based on IMAB362, as shown by the EC50 values for cytotoxic activity.

In another embodiment, the antibody or fragment thereof binds to CLDN18.2and is humanized. Humanization of monoclonal antibodies has been well-established. The Handbook of Therapeutic Antibodies, Second Edition, gives ample information on humanization of monoclonal antibodies (Saldanha 2014), bioinformatics tools for analysis of such antibodies (Martin and Allemn 2014) or development and manufacture of therapeutic antibodies (Jacobi et al. 2014). When used as human therapeutics, humanized antibodies and related ADCs have a lower risk, compared to chimeric antibodies or ADCs based on chimeric antibodies, of inducing anti-drug antibodies, which would limit the therapeutic benefit and increase the risk of side effects of the antibody of the invention especially after repeated administration. In one embodiment, the antibody does not bind to CLDN 18.1. Hence, it does not exhibit crossreactivity.

In another embodiment, the antibody or functional fragment thereof binds to CLDN18.2 and consists of the heavy chain sequence of SEQ ID NO: 58 and the light chain sequence of SEQ ID NO: 50.

In yet another embodiment, the antibody has an amino acid sequence with at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity or at least 98% identity to the amino acid sequence of an antibody described herein. Preferably, the antibody binds with higher affinity to CLDN18.2 than the IMAB362 antibody, as for example shown by EC50 and maxMFI values and/or selectively binds to CLDN18.2. In one embodiment, the antibody is humanized.

In one embodiment, the antibody or fragment thereof binding to CLDN18.2 competes for binding with an antibody or fragment thereof as described herein. In a preferred embodiment, the antibody or fragment thereof competes for binding with an antibody consisting of the heavy chain sequence of SEQ ID NO: 58 and the light chain sequence of SEQ ID NO: 50. In one embodiment, the antibody is humanized. In a further preferred embodiment, the antibody exhibits a binding affinity that is identical or increased as compared to the binding affinity of IMAB362. In another preferred embodiment, the antibody exhibits a binding affinity that is identical or increased as compared to the binding affinity of an antibody consisting of the heavy chain sequence of SEQ ID NO: 58 and the light chain sequence of SEQ ID NO: 50. The binding affinity may be measured by any suitable means. For example, the binding of the antibody may be measured as EC50 value or maxMFI by flow cytometry titration on HEK295T cells or PA- TU-8988-High cell expressing CLDN18.2.

In another embodiment, the Fc domain of the antibody (or antibody fragment when present) may comprise modifications or mutations, such as the modifications or mutations listed in Table 2 below. Such a modification or mutation may be introduced to modulate the effector activity of the Fc domain of the antibody. Modification of antibodies may also include peptide tags added to the C -terminal end of the antibody HC and/or LC chain. Such tags may be used e.g. for protein purification or protein conjugation. In another embodiment, the humanized antibody or fragment thereof that binds CLDN18.2 is in the format selected from an IgAl, IgA2, IgD, IgE, IgGl, IgG2, IgG3, IgG4, synthetic IgG, IgM, F(ab) ₂ , Fv, scFv, IgGACH2, F(ab’) ₂ , scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (SCFV) ₂ , a non-depleting IgG, a diabody, a bivalent antibody or Fc-engineered versions thereof.

In a preferred embodiment, the antibody is an IgGl type of antibody. The Fc region of immunoglobulins interacts with multiple Fey receptors (FcyR) and complement proteins (e.g. Clq), and mediates immune effector functions, such as elimination of targeted cells via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC). For therapeutic approaches, it may be beneficial to enhance or silence Fc related effector functions. The type of immunoglobulin (IgA, IgD, IgE, IgG, IgM) may be selected according to the desired effector function of the antibody related to the Fc domain given their known activities. One may also employ a synthetic immunoglobulin, such as an immunoglobulin with the IgG2 amino acids 118 to 260 and the IgG4 amino acids 261 to 447 or an IgG2 variant with point mutations from IgG4 (e.g. H268Q/V309L/A30S/P331S). Such synthetic immunoglobulins reduce effector functions of the antibody. Fc-engineered immunoglobulins may also be employed to modulate antibody effector function. Table 2 shows examples of such Fc engineering. Expression in production cell lines with altered fucosylation may also impact FcyR binding in order to modulate pharmacokinetics of the antibody.

Table 2: Examples of modifications to modulate antibody effector function. Unless otherwise noted, the mutations are on the IgGl subclass (Wang, Mathieu, and Brezski 2018).

In vivo half-life of antibodies may also be modulated. The Fc domain plays a central role in the stability and serum half-life on antibodies. For therapeutic approaches, antibody half-life may be reduced by using an antibody fragment missing the Fc domain or with truncated Fc domains, such as F(ab) ₂ , Fv, scFv, IgGACH2, F(ab’) ₂ , scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc or (scFv) ₂ . The antibodies may also be in the form of diabodies or bivalent antibodies. Diabodies or bivalent antibodies may be used to increase the affinity to the target allowing lower dosage. Functional fragments missing the Fc domain or with truncated Fc domains may also be used in the development of other therapeutic approaches. The VH and VL domains used in the scFv fragment may be the ones of the antibodies listed in Table 3. BiTEs typically consist of the fusion of two scFv of two different antibodies. One scFv domain may be of the isolated antibodies binding CLDN18.2 listed in Table 3, while the other scFv domain is from an antibody that binds e.g. to CD3, CD16, NKG2D, NKp46, CD2, CD28 or CD25. Ample guidance on BiTEs antibody formats and other bispecific antibody formats used for T- cell redirecting may be found in the review by Diego Ellerman (2019).

In another embodiment, the antibody or fragment thereof is a humanized antibody or fragment thereof that binds to CLDN18.2, the antibody having the constant light chain region (CL) of SEQ ID NO: 65 and preferably the constant heavy chain region CHI and Fc region of SEQ ID NO: 66 with reduced FcyR binding having the L234A/L235A mutations in the constant heavy chain region CH2. More preferably, the antibody has the constant heavy chain region CHI and Fc region of SEQ ID NO: 67 having the L234A/L235AZP329G mutations in the constant heavy chain region CHI and Fc region with even further reduced FcyR binding.

In another embodiment, the humanized antibody or fragment thereof binds to CLDN18.2 with a VH sequence of SEQ ID NO: 41 associated to the constant heavy chain region CHI and Fc region of SEQ ID NO: 66 and the VL sequence of SEQ ID NO: 33 associated to having the constant light chain region (CL) of SEQ ID NO: 65.

In yet another embodiment, the antibody or fragment thereof binds to CLDN18.2, wherein the antibody or fragment thereof does not bind to CLDN 18.1. Hence, the antibody does not exhibit cross-reactivity or cross-binding to CLDN18.1. Binding of an antibody to a target protein can be tested by flow cytometry on cells expressing the target protein. Specific binding of a tested antibody to its target protein can be visualized on a histogram plot. Such plot results in a peak with high fluorescent signal when the antibody specifically binds to the expressed target protein, and in a peak with low fluorescent signal when the antibody does not, or only very weakly bind to the expressed target protein. Such histogram can be seen in Figure 1, showing binding of antibodies of the invention to CLDN18.2 but not to CLDN18.1 expressed in HEK293T cells. The degree of binding can also be expressed in a bar graph showing the maximal mean fluorescent intensity (maxMFI) measured by flow cytometry, with high maxMFI reflecting strong binding and low/no maxMFI reflecting non-binding. Examples of such binding assays can be found in Example 4.

In another embodiment, the ADC is bound to another moiety. This moiety may include radioisotopes, fluorescent tags, histological markers, cytotoxins or cytokines. Binding of the moiety may be facilitated by linkers known in the art.

In yet another embodiment, the antibody or fragment binds to CLDN18.2, wherein the antibody or fragment thereof exhibits stronger binding to CLDN18.2 than antibody IMAB362. Preferably, the antibody or fragment binds to CLDN18.2, wherein the antibody or fragment thereof binds with a higher affinity to CLDN18.2 than antibody IMAB362. Binding affinities and/or rate constants can be determined using techniques well known in the art or described herein, such as ELISA, flow cytometry titration, isothermal titration calorimetry (ITC), Biacore (SPR), biolayer inferometry or fluorescent polarization. The inventors have determined the affinity of the antibodies to CLDN18.2 by ELISA as shown for example in Example 3 or by FC titration experiments as shown in Example 4. In ELISA on lipoparticles containing CLDN18.2, all the humanized antibodies hGBA-1 to hGBA-9 have a higher maximum binding values (expressed in MFI) than IMAB362. In FC titration experiments on HEK293T cells overexpressing CLDN18.2 or PA-TU-8988S cells endogenously expressing CLDN18.2, all the humanized antibodies hGBA-1 to hGBA-9 have higher maximum binding values (expressed in MFI units) and lower EC50 values (expressed in pg/ml) than the antibody IMAB362, indicative of higher affinity of the humanized antibodies of the present invention to CLDN18.2 than antibody IMAB362. In one embodiment, the antibodies have a measured EC50 value at least 10% lower, at least 20% lower, at least 40% lower, at least 50% lower or at least 75% lower than the EC50 value measured for antibody IMAB362. In one embodiment, the antibodies have a measured maxMFI value at least 10% higher, at least 20% higher, at least 40% higher, at least 50% higher or at least 75% higher than the maxMFI value measured for antibody IMAB362.

In another embodiment, the invention relates to a method of producing an ADC of the invention.

In one embodiment, the method comprises the following steps: a. providing A, an antibody or fragment thereof with one or more linker elements, b. providing one or more toxins T with one or more linker elements, and c. conjugating the antibody and the toxin resulting in the antibody-drug conjugate.

In one embodiment, the method comprises the following steps: a. providing A, an antibody or fragment thereof with an oligopeptide linker element preferably at its C-terminus, optionally preceded by a spacer element at the antibody light and/or heavy chains, b. providing one or more toxins T with a non-cleavable linker element optionally followed by an oligopeptide linker element, and c. conjugating the antibody and the toxin resulting in the antibody-drug conjugate.

It is understood that any antibody A herein disclosed may be provided with any oligopeptide linker and optional spacer element herein disclosed. Likewise, any anthracycline toxin T may be linked with any non-cleavable linker and oligopeptide herein disclosed. The type of conjugation may depend on the linker elements and/or on the method for preparing the ADC. The antibody A provided with an oligopeptide linker element comprising a sortase recognition motif may be conjugated with any anthracycline toxin T comprising an oligoglycine oligopeptide linker element via sortase-mediated transpeptidation (see Example 5). A representation of an ADC produced by this method can be found in Figure 13.

In a preferred embodiment, the ADC of the invention consists of:

• the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 58, and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of CM and of the hydroxyl group.

In another preferred embodiment, the ADC of the invention consists of: • the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 145, and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of C14 and of the hydroxyl group.

In yet another preferred embodiment, the ADC of the invention consists of:

• the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 146 and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of C ₁₄ and of the hydroxyl group.

The invention also relates to a pharmaceutical composition comprising the disclosed ADCs and an excipient.

Also provided are nucleic acid sequences encoding the antibodies or fragments thereof binding to CLDN18.2 for their use in the manufacturing of an ADC. The nucleic acid sequences may encode for the CDRs alone, for the VH and VL regions, or for the entire heavy and light chains of the antibodies. These nucleic acid sequences may be found in Table 3. The nucleic acid sequence may also encode for F(ab) ₂ , Fv, scFv, IgGACH2, F(ab’) ₂ , scFvCH3, Fab, VL, VH, scFv4, scFv3, scFv2, dsFv, Fv, scFv-Fc, (scFv) ₂ , a non-depleting IgG, a diabody, a bivalent antibody or Fc-engineered versions thereof. The encoded immunoglobin may be an IgAl, IgA2, IgD, IgE, IgGl, IdG2, IgG3, IgG4, synthetic IgG, IgM or mutated and Fc-engineered versions thereof. The nucleic acids may additionally comprise coding sequences for oligopeptide linker elements that are directly fused to the C -termini of the antibody heavy chains and or the antibody light chains.

In yet another embodiment, the antibody or fragment thereof is an antibody-based binding protein that binds to CLDN 18.2, e.g. a protein comprising at least a CLDN 18.2 binding domain of the disclosed antibodies and another protein domain not related to antibodies. In another embodiment, the antibody or fragment thereof is a modified humanized antibody format that binds to CLDN18.2. In a preferred embodiment, the antibody -based binding protein does not bind to CLDN 18.1.

Also provided are expression vectors comprising such nucleic acids or a degenerate nucleic acid as a result of codon degeneracy. The expression vectors may be expression vectors aimed for mammalian cells, bacteria, fungal or insect cell expression, and chosen for the type of host cell bearing the expression vector comprising the nucleic acid encoding the antibodies or functional fragments thereof. Ample guidance for the construction of such vectors may be found in Green and Sambrook (Green and Sambrook 2012). Preferred are expression vectors for mammalian cells, especially CHO cells.

Also provided are host cells comprising the expression vectors of encoding the antibodies or fragments thereof binding to CLDN18.2 or having the nucleic acids encoding the antibodies or fragments thereof binding to CLDN18.2 integrated into its genome. The host cell may be a mammalian cell or cell line, bacteria, fungal or insect cell. Preferred are mammalian cells, especially CHO cells.

In another embodiment, the invention relates to an ADC of the present invention for use in the treatment of a subject that is suffering from a neoplastic disease, or is at risk of developing a neoplastic disease, and/or for the treatment of a subject being diagnosed for a neoplastic disease. The disclosed ADCs may be used as monotherapy or preferably as combinations therapy with the established standard of care of the neoplastic disease.

In yet another embodiment, the invention provides for the use of an ADC of the present invention for the manufacture of a medicament for the treatment of the neoplastic disease. The neoplastic disease may be at least one disease selected from the group consisting of pancreatic, gastric, esophageal, ovarian and lung cancer. It is understood that the neoplastic disease to be treated is characterized by overexpression of CLDN18.2.

Another embodiment of the invention provides a method to treat a neoplastic disease, including pancreatic, gastric, esophageal, ovarian or lung cancer, with an ADC as provided herein, wherein the method comprises administering a therapeutically effective amount of the ADC. The method of treatment may be a monotherapy or preferably a combination therapy with the established standard of care of the neoplastic disease.

Also provided is a pharmaceutical composition comprising the ADC and a pharmaceutically acceptable carrier.

Preferably, patients suffering from pancreatic, gastric, esophageal, ovarian or lung cancer may be treated with an ADC provided herein.

DESCRIPTION OF DRAWINGS

Figure 1 : FACS binding assay of humanized antibodies and IMAB362. Binding of selected antibodies to huCLDN1.2 and huCLDN18.1 was tested in HEK293T cells stably expressing huCLDN18.2 or huCLDN18.1. Parental HEK293T cells not expressing the target protein were used as negative control. 1A: A: IMAB362, B: hGBA-1, C: hGBA-2, D: hGBA-3, E: hGBA- 4, F: hGBA-5, G: hGBA-6, H: hGBA-7, I: hGBA-8, J: GBA-9, K: secondary antibody alone, L: pan-CLDN18 antibody; IB: Bar graph showing the Mean Fluorescent Intensity (MFI) of the FACS binding data for each humanized antibody, compared to IMAB362, on parental HEK293T cells and HEK293T cells expressing huCLDN18.2 or huCLDN18.1.

Figure 2: 2A-D: ELISA binding assay on humanized antibodies, compared to IMAB362. The ELISA binding assay was performed on lipoparticles bearing CLDN18.2 or null-lipoparticles without CLDN 18.2.

Figure 3: Sorting of PA-TU-8988S cells for expression levels of CLDN18.2. 3 A: FACS profile of PA-TU-9888S stained with IMAB362. 3B: FACS profile of PA-TU-8988S cells sorted by FACS for medium and high expression of CLDN18.2.

Figure 4: FC titration assay on PA-TU-8988S-High cells (4A-D) and HEK-293T expressing huCLDN18.2 (4E-H). Figure 5: In-vitro cytotoxic assay on HEK-293T-CLDN18.2 cells of the ADCs were PNU is conjugated to the LC of the humanized antibodies hGBAl, hGBA2 and hGBA3 (A), hGBA4, hGBA5 and hGBA6 (B) and hGBA7, hGBA8 and hGBA9 (C). The cytotoxic activity of the ADCs is compared to the cytotoxic activity of IMAB362 conjugated to PNU is the same manner.

Figure 6: In-vitro cytotoxic assay on HEK-293T-CLDN18.1 cells of the ADCs were PNU is conjugated to the LC of the humanized antibodies hGBAl, hGBA2 and hGBA3 (A), hGBA4, hGBA5 and hGBA6 (B) and hGBA7, hGBA8 and hGBA9 (C). The cytotoxic activity of the ADCs is compared to the cytotoxic activity of IMAB362 conjugated to PNU is the same manner.

Figure 7: In-vitro cytotoxic assay on A549-CLDN18.2 cells of the ADCs were PNU is conjugated to the LC of the humanized antibodies hGBAl, hGBA2 and hGBA3 (A), hGBA4, 11GBA5 and hGBA6 (B) and hGBA7, 11GBA8 and hGBA9 (C). The cytotoxic activity of the ADCs is compared to the cytotoxic activity of IMAB362 conjugated to PNU is the same manner.

Figure 8: In-vitro cytotoxic assay on PATU-8988-S-High cells of the ADCs were PNU is conjugated to the LC of the humanized antibodies hGBAl, hGBA2 and hGBA3 (A), hGBA4, hGBA5 and hGBA6 (B) and hGBA7, hGBA8 and hGBA9 (C). The cytotoxic activity of the ADCs is compared to the cytotoxic activity of IMAB362 conjugated to PNU is the same manner.

Figure 9: In-vivo efficacy of ADC hGBA2-LC-G2-PNU (A) and hGBA6-LC-G2-PNU (B) in the gastric patient-derived tumor xenograft model GXA 3037, compared to the ADC IMAB362-LC-G2-PNU. Each ADC is tested either at 0.2 mg/kg/day or 0.6 mg/kg/day.

Figure 10: In-vivo efficacy of ADC hGB A6-LC-G2-PNU in the colon cancer patient-derived tumor xenograft model CXF 742, compared to the isotype control ADC AclO-LC-G2-PNU. Each ADC is tested at 2 mg/kg/day. Figure 11 : In-vivo efficacy of ADC hGB A6-LC-G2-PNU in the pancreatic cancer patient- derived tumor xenograft model PAXF 2175, compared to the ADC IMAB362-LC-G2-PNU. Each ADC is tested at 0.2 mg/kg/day.

Figure 12: In-vivo efficacy of ADC hGBA6-LC-G2-PNU in the lung cancer patient-derived tumor xenograft model LIXFC 2050, compared to the isotype control ADC AclO-LC-G2-PNU.

Each ADC is tested at 2 mg/kg/day.

Figure 13: Graphical representation of and ADC were PNU has been conjugated to the antibody LC via a spacer element -GGGGS-, an oligopeptide linker element -LPQTGG- and a non-cleavable linker element EDA, linked to the C ₁₃ of PNU.

EXAMPLES

Example 1 : Humanization of Fab fragments

Techniques to humanize monoclonal antibodies have been well-established. The Handbook of Therapeutic Antibodies, Second Edition, gives ample information on humanization of monoclonal antibodies (Saldanha 2014), bioinformatic tools for analysis such antibodies (Martin and Allemn 2014) or development and manufacture of therapeutic antibodies (Jacobi et al. 2014). In brief, the variable domain sequences of the parental IMAB362 antibody were analyzed to reveal the closest human germlines. Next, a structural analysis of the variable regions of IMAB362 was performed to reveal the best fitting Fv model, followed by structural analysis of CDR grafting by in-silico modeling. Based on these in-silico modeling, humanized VH and VL domains were designed. Combinations of the humanized VH and VL domains were cloned and produced as Fab and IgGl antibodies and screened for their binding by ELISA and AlphaLISA™ to CLDN18.2-expressing lipoparticles and by flow cytometry with CLDN18.1- and CLDN18.2-expressing pre-B cell Li l (Waldmeier et al. 2016) and HEK293T (ATCC CRL-3216) cell lines. After testing and comparison to IMAB362, one VH and VL combination was selected and a library was designed in scFv format, performing further humanization including the CDRs. The scFv library was further screened by ELISA and AlphaLISA™ to CLDN18.2-expressing lipoparticles and by flow cytometry with CLDN18.1- and CLDN18.2- expressing pre-B cell LI 1 cell lines. Humanization of IMAB362 thus resulted in the humanized antibodies hGBA-1, hGBA-2, hGBA-3, hGBA-4, hGBA-5, hGBA-6, hGBA-7, hGBA-8 and hGBA-9 antibodies (see Table 3), collectively named hGBA antibodies herein.

Table 3: nucleic acid and amino-acid sequences of selected antibodies

The antibodies described in further Examples 2 to 4 were modified to contain a LPXTGG tag (SEQ ID NO: 143) at the C-terminal end of the HC and/or a GGGGSLPXTGG tag (SEQ ID NO: 144) at the C-terminal end of the LC, where X is any of the 20 natural amino acids. The C-terminal lysine (K) on the HC was in this case replaced by an Arginine (R). The addition of the tags did not change the affinity and selectivity to CLDN18.2 of the antibodies.

Example 2: FACS binding analysis of humanized mAbs

The HEK293T (ATCC CRL-3216) cell line does not endogenously express CLDN18.1 or CLDN18.2. Therefore, in order to test antibody binding activity, CLDN18.1 and CLDN18.2 were overexpressed in the HEK293T cell line. Cells were co-transected by electroporation with a transposase expression construct (pcDNA3.1 -hy-mPB), a construct bearing transposable full - 1 ength huCLDN 18.1 (pPB -Puro-huCl dn 18.1 ) or huCLDN 18.2 (pPB -Puro-huCl dn 18.2) al ong with puromycin expression cassette and a construct carrying EGFP as transfection control (pEGFP-N3). Upon transfection, cells were allowed to recover for two days in growth media at 37°C in a humidified incubator in a 5% CO ₂ atmosphere. Transfection was verified by FC analysis of the EGFP expression. Cells expressing huCLDN18.1 or huCLDN18.2 were then selected by the addition of puromycin into culture at 1 pg/ml, and further expanded to allow the generation of frozen stocks in FCS with 10% DMSO. The expression of huCLDN18.2 in the transfected HEK293T cells was analyzed by FACS. In brief, HEK293T cells were trypsinized and collected by centrifugation, resuspended in PBS/2% FCS and stained for huCLDN18.2 using IMAB362 as primary antibody at 2 pg/ml on ice for 30 min and, upon washing in PBS/2% FCS, stained with PE-labelled anti-human Fcy-specific IgG goat antibody (eBioscience) as secondary antibody for 30 min on ice. Upon further wash, resuspended stained cells in ice-cold FACS buffer were analyzed using a FACSCalibur™ instrument (see Figure 1A). Un-transfected parental cells, not expressing CLDN18.2, were used as negative control. The expression of CLDN18.1 was analyzed in a similar fashion, using a proprietary pan- CLDN 18 antibody recognizing CLDN 18.1 and CLDN 18.2. Any pan-CLDN 18 antibody usable for flow cytometry measurement would also be adequate such as antibody anti Claudin- 18/CLDN18 (C-term) provided by OriGene Technologies (catalog number AP50944PU-N), CLDN18 (C-Term) Rabbit pAb from MyBioSource (catalog number MBS8555451) or the CLDN18 Antibody from ProSci (catalog number 63-847).

The HEK293T cells stably expressing huCLDN18.1 and huCLDN18.2 were consequently used to test the binding specificity of the humanized antibodies hGBA-1, hGBA-2, hGBA-3, hGBA- 4, hGBA-5, hGBA-6, hGBA-7, hGBA-8 and hGBA-9 to CLDN18.2 and not to huCLDN18.1. The cells were stained on ice for 30 min using the antibodies at 2pg/ml and, upon washing in FACS buffer (PBS/2% FCS), stained with PE-labelled anti-human Fcy-specific IgG goat antibody (eBioscience) as secondary antibody for 30 min on ice. Expression of CLDN18.1 in the HEK293T cells stably expressing huCLDN18.1 was verified with a pan-CLDN18 antibody (see Figure 1, panel L) and expression of CLDN18.2 in the HEK293T cells stably expressing huCLDN18.2 was verified with the IMAB362 (see Figure 1, panel A). Figure 1 shows that all humanized antibodies bind specifically to huCLDN18.2 expressed by HEK293T cells, and not to huCLDN 18.1. Furthermore, all humanized antibodies bind to huCLDN 18.2 stronger than the parental antibody IMAB362. Example 3: ELISA binding analysis of humanized mAbs

The binding affinity to CLDN 18.2 of the humanized antibodies (hGBA) was tested in an ELISA assay with lipoparticles bearing CLDN18.2 as source of antigen. CLDN18.2-lipoparticles and Null-lipoparticles (without antigen as a negative control) were used to coat 96-well plates at a final concentration of 10 U/ml. Upon washing with PBS/0.05% Tween-20 (PBS-T) and blocking with PBS-T/3% BSA for at least 1 h at 37°C, 1 :3 serial dilutions of hGBA and IMAB362 antibodies with a starting concentration of 2 pg/ml in PBS-T/1% BSA were added to the coated wells and incubated for at least 1 h at 37°C. The presence of bound antibodies was revealed through binding of an HRP-goat anti-human secondary antibody diluted in PBS-T/1% BSA, development with Sigma-Fast OPD as peroxidase substrate and the reaction was stopped by adding 2M H ₂ SO ₄ , followed by reading the OD at 490 nm on an ELISA plate reader. Representative binding curves are shown in Figure 2. Surprisingly, the binding curves in Figure 2 show that all humanized antibodies (hGBA-1 to hGBA-9) bind to CLDN18.2-lipoparticles with a higher affinity than IMAB362, shown by a higher maximal binding value.

Example 4: FC titration on HEK293T and PA-TU-8988 High cells

PA-TU-8988S cells (Creative Bioarray, catalog number CSC-C0326) expressing high levels of CLDN18.2 were selected by FACS. Herein, these cells are designated as PA-TU-8988S-High cells. Based on FACS staining with IMAB362, the PA-TU-8988S cell population expresses different levels of CLDN18.2, with a high and a medium level of expression (see Figure 3 A). In order to have a more homogenous cell population, the cells were sorted by FACS to select only cells with a higher CLDN18.2 expression. In brief, PA-TU-8988S cells suspended in FACS buffer (PBS, 2% FCS) were incubated on ice for 30 min with IMAB362 at 2 pg/ml. After wash in FACS buffer, the cells were incubated with the PE-labeled Fey specific IgG goat antihuman secondary antibody (eBioscience) on ice for 30 min. After wash, the stained cells were resuspended in FACS buffer, analyzed and sorted by a FACSAria™ instrument, separating medium expressing cells (Figure 3B) from high expressing cells (Figure 3B). After sorting, the collected PA-TU-8988S-High cells were resuspended in growth media, expanded in growth media and frozen aliquots were preserved in liquid N ₂ .

In order to quantify the affinity of the antibodies to CLDN18.2, 250 x 10 ³ cells/well of HEK293T cells overexpressing CLDN18.2 or PA-TU-8988-High cells were seeded in FC buffer (PBS/2% FCS) into 96-well plates and allowed to settle by centrifugation. IMAB362 and hGBA antibodies to be tested were diluted at 4 pg/ml, followed by 1 :4 serial dilutions and incubated with the platted cells for 30 min at 4°C. A PE-coupled secondary anti-human IgG antibody was added to the cells for additional 30 min at 4°C after washes with the FACS buffer, followed by further washes with FC buffer. The cells were then resuspended in 100 pl FC buffer and measured with a FACSCalibur™ cell analyzer (BD Biosciences, USA). The FC analysis (see Figure 4 and Table 4) shows that all hGBA antibodies have a stronger binding affinity to CLDN18.2 (reflected by a higher Max MFI for all tested new antibodies, see Table 4) than IMAB362, in both cell lines. The binding affinity of all hGBA antibodies is similar between each other but is significantly higher than the parental antibody IMAB362.

Table 4: Maximum MFI and EC50 (ug/ml) measured on all the hGBA and IMAB362 antibodies on the HEK293T cells lines overexpressing CLDN18 2 and on the PA-TU-8988S-High cell lines. Example 5: Conjugation of mAbs with glycine-modified toxin to form ADCs using sortase- mediated conjugation. Sortase A enzyme: Recombinant and affinity purified Sortase A enzyme from Staphylococcus aureus was produced in E. coli as disclosed in W02014140317A1.

Generation of glycine-modified toxins: the biglycine-modified ED A-anthracy cline derivative GG-EDA-PNU- 159682 was manufactured by Levena Biopharma, San Diego, USA. Here the toxin PNU- 159682 was synthesized to already include the non-cleavable linker EDA and an oligopeptide linker GG. The identity and the purity of the glycine-modified toxin was confirmed by mass-spectrometry and HPLC. The glycine-modified toxins exhibited > 95% purity, as determined by HPLC chromatography.

Sortase-mediated antibody conjugation: the above-mentioned toxins were conjugated to the light chain LPQTG-tagged anti-CLDN18.2 antibodies and comparative antibodies as per Table 3 by incubating light chain LPQTG-tagged mAbs at 20 pM with glycine-modified toxin at 100 pM and Sortase A at 4 pM in the conjugation buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM CaCl ₂ , 10% glycerol) for 3.5h at 25°C or overnight at 4°C. The reaction was stopped by passage through a rProtein A GraviTrap column (GE Healthcare). The column was washed with 36 column volumes of wash buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 10 % (v/v) Glycerol). Bound conjugate was eluted with 5 column volumes of elution buffer (0.1 M glycine pH 2.7, 50 mMNaCl, 10% (v/v) Glycerol), with 0.5 column volume fractions collected into tubes containing IM HEPES pH 8 to neutralize the acid. Protein containing fractions were pooled and formulated in Histidine buffer (15 mM Histidine, pH 6.5, 175 mM Sucrose, 0.02% Tween 20) using a Zeba Spin (Thermo Fisher) desalting column. Endotoxins were removed using Pierce High Capacity Endotoxin Removal Resin (Thermo Fisher) and sterile filtered through a 0.22 pm filter. The final concentration of the ADCs was measured by UV-visible spectroscopy.

ADC analytics: DAR was assessed by Reverse Phase Chromatography performed on a PLRP- S, 300 A, 2.1 x 150 mm, 3 pm column (Agilent) run at 0.7 ml/min at 60°C with a 9-minute linear gradient (25-40 %) followed by a 4-minute linear gradient (40-75 %) between 0.1% TFA/3% CH ₃ CN/H ₂ O and 0.1% TFA/CH ₃ CN. Samples were first reduced by incubation with 10% v/v 0.5 M DTT, pH 8.0 at 37°C for 15 minutes. All generated ADCs had a DAR LC = 2.

Example 6: in-vitro cytotoxicity assays of anti-CLDN18,2 antibody -based ADCs on

CLDN18,2-expressing cells In Example 6 and following Example 7, an ADC of the formula [antibody]-LC-PNU is an ADC where the antibody is conjugated at the light chain with the toxin PNU- 159682 and has a DAR = 2. All these ADCs also have an -GGGGSLPQTGG- oligopeptide linker and ethylenediamine (EDA) non-cleavable linker. The structure of an ADC of the formula [antibody]-LC-PNU can be seen in Figure 13.

Cytotoxicity of anti-CLDN18.2 ADCs was investigated using A549 cells or HEK293T cells engineered to overexpress hCLDN18.2 (see Example 2) or PATU8988S-high cells (see Example 4) endogenously expressing hCLDN18.2 and compared to IMAB362-LC-G2-PNU. HEK293T cells engineered to overexpress hCLDN18.1 (from Example 2) were used as negative control.

In brief, 1000 cells/well of A549 cells or HEK293T cells and 10000 cells/well ofPATU8988S cells were platted in white clear bottom 96-well plates (Greiner) (excluding edge wells, which contained water) in 75 pl DMEM high glucose, 10% FCS, 100 lU/ml Pen/Step/Fungizone, 2mM L-Glutamine and were grown at 37°C in a humidified incubator at 7.5% CO ₂ atmosphere. After on day of incubation, each ADC was added to respective wells in an amount of 25 pl of 4-fold serial dilution in complete growth medium resulting in concentration of ADCs from 5000 to 0.076 ng/ml for A549 cells, from 1000 to 0.015 ng/ml for HEK293-T cells and from 20000 to 0.31 ng/ml for PATU8988S cells. After 4 additional days, plates were removed from the incubator and equilibrated to room temperature. After approximately 30 min, 50 pl of CellTiter- Glo® 2.0 Luminescent Solution (Promega) was added to each well. After shaking the plates at 450 rpm for 5 min followed by 10 min incubation without shaking, luminescence was measured on a Tecan Spark 10M plate reader with an integration time of 250 ms per well. Curves of luminescence versus ADC concentration (ng/ml) were fitted with the Graphpad Prism Software (see Figures 5, 6 and 8). The EC ₅₀ values, determined using built-in “log(inhibitor) vs. response - variable slope (four parameters)” EC ₅₀ determination function of the Prism Software, are reported in Table 5

Table 5: EC50 (ng/ml) values for tested ADCs

Overall, the ADCs of the invention have a high in vitro cytotoxic potential, with a higher cytotoxic activity than IMAB362-LC-G2-PNU.

Example 7: Analysis of in-vivo efficacy of ADC hGBA6 and hGBA2 in patient-derived tumor xenograft models

The following studies were performed at Charles River GmbH (Freiburg, Germany).

Table 6: Patient-derived tumor xenograft models used for evaluation of anti-CLDN18.2 ADC hGBA-2-LC-G2-PNU and hGBA-6-LC-G2-PNU The anti-CLDNl 8.2 ADCs hGB A-2-LC-G2-PNU and hGB A-6-LC-G2-PNU were investigated in the patient-derived tumor xenograft (PDX) models according to the following study protocol:

Table 7: Protocols used for evaluation of anti-CLDNl 8.2 hGBA-2-LC-G2-PNU, and hGBA-6- LC-G2-PNU ADCs in PDX models.

Mice were subcutaneously implanted unilaterally with PDX material. Mice allocated into groups when tumors reached randomization criteria and were treated with ADCs as indicated in Table 7 or vehicle for a total of 3 times. Tumor volumes were determined by caliper measurements and body weight was recorded twice weekly. Mice were euthanized on reaching a tumor burden of 2000 mm ³ , or on significant body weight loss (overall more than 30%, or more than 20% in two days).

Figures 9 to 12 show the relative tumor volume evolution over the studies in the different PDX models. Tumor xenografts established with patient-derived tumor material having CLDN18.2 expression responded significantly to treatment with the ADCs of the invention. The response (delayed tumor growth or tumor shrinkage) with the ADCs of the invention when administered at lower doses (0.2 mg/kg or 0.6 mg/kg) was better than the similar ADC based on the anti- CLDN18.2 antibody IMAB362 administered at the same doses and comparably good when administered at the higher dose of 2 mg/kg. EMBODIMENTS

The invention is inter alia described by the following embodiments:

1. An antibody-drug conjugate having the general formula A - (L-T) _n , wherein a. A is an antibody or fragment thereof binding to CLDN18.2 comprising the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively, b. L is a linker, c. T is a toxin, wherein the toxin is an anthracy cline derivative, wherein n is an integer between >1 and < 10; or a pharmaceutically acceptable salt thereof.

2. The antibody-drug conjugate of embodiment 1, wherein the linker L comprises at least one non-cleavable linker element.

3. The antibody-drug conjugate of embodiment 2, wherein the non-cleavable linker element is selected from the group consisting of a. ethylenediamine (EDA), b . N -formyl -N,N’ -dimethyl ethyl en edi amine, c. diethylamine (DEA), d. a piperazine-derived compound of the following formula: wherein the wavy lines indicate attachments to the toxin and another linker element, e. the compound of the following formula: wherein the wavy lines indicate attachments to the toxin and another linker element, f. the compound of the following formula: wherein the wavy line indicates attachment to the toxin and [Ab] indicates the antibody or fragment thereof, g. a maleimidocaproyl compound of the following formula: wherein the wavy line indicates attachment to another linker element and [Ab] indicates the antibody or fragment thereof, h. the compound of the following formula: wherein the wavy line indicates attachment to a toxin and [Ab] indicates the antibody or fragment thereof, and wherein the bifunctional linker element is conjugated to the toxin by means of an amide bond or an ether bond. The antibody-drug conjugate of embodiment 2 or embodiment 3, wherein the linker further comprises an oligopeptide linker element and/or enzyme-cleavable linker element and/or a spacer element. The antibody-drug conjugate of embodiment 4, wherein one oligopeptide linker element comprises a sortase recognition motif oligopeptide selected from: - LPXTG _m -, -LPXAG _m -, -LPXSG _m -, -LAXTG _m -, -LPXTA _m -, NPQTG _m or -NPQTN _m -, with G _m being an oligoglycine with m being an integer between >1 and < 21, A _m being an oligoalanine with m being an integer between > 1 and < 21, N _m being an oligoasparagine with m being an integer between > 1 and < 21 and X being any conceivable amino acid, preferably the sortase recognition motif oligopeptide being - LPQTGG- or -LPETGG-. The antibody-drug conjugate of embodiment 5, wherein the oligopeptide linker comprises: a. the sequence SEQ ID NO: 143, or b. the sequence SEQ ID NO: 144. The antibody-drug conjugate of any of embodiments 4 to 6, wherein the enzyme- cleavable linker element comprises a val-cit-PAB linker according to the compound of the following formula: wherein the wavy lines indicate attachments to other linker elements. The antibody-drug conjugate of any of embodiments 4 to 7, wherein the spacer element comprises a peptidic flexible oligopeptide, preferably wherein the peptidic flexible oligopeptide consists of G and S, more preferably wherein the peptidic flexible oligopeptide is (GGGGS) ₀ with o being 1, 2, 3, 4 or 5. 9. The antibody-drug conjugate of any of embodiments 1 to 8, wherein the antibody drug conjugate has the following structure: a. A - ([oligopeptide linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: i. [LPXTGG]-[ethylenediamine], and ii. b. A - ([oligopeptide linker element - enzyme cleavable linker element - non- cleavable linker element] ™ T) _n and preferably wherein the linker is selected from: i. [LPXTGG]-[vc-PAB]-[N-formyl-N,N’-dimethylethylenediamine], and ii . [LPXT GG] - [vc-P AB ] - [piperazi ne] ; c. A - ([spacer element - oligopeptide linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: i. [GGGGS]-[LPXTGG]-[ethylenediamine], and ii. d. A - ([spacer element - oligopeptide linker element - enzyme cleavable linker element - non-cleavable linker element] - T) _n and preferably wherein the linker is selected from: i. [GGGGS]-[LPXTGG]-[vc-PAB]-[N-formyl-N,N’- dimethylethylenediamine], and ii. [GGGGS]-[LPXTGG]-[vc-PAB]-[piperazine],

10. The antibody-drug conjugate of embodiment 9, wherein the non-cleavable linker element is ethylenediamine and wherein the oligopeptide linker element is LPXTGG wherein X is Q or E, preferably wherein X is Q.

11. The antibody-drug conjugate of any of embodiments 1 to 10, wherein a. (L-T) is covalently linked to both light chains of the antibody, b. (L-T) is covalently linked to both heavy chains of the antibody, or c. (L-T) is covalently linked to both light chains and both heavy chains of the antibody. 12. The antibody-drug conjugate of any of embodiments 1 to 11, wherein (L-T) a. is linked to the C-terminus of the antibody light chain or antibody heavy chain, or b. is liked to an amino acid side chain of the antibody light chain or antibody heavy chain.

13. The antibody-drug conjugate of any of embodiments 1 to 12, wherein the anthracycline derivative has the following formula (I), and is covalently linked to the non-cleavable linker element by the C ₁₃ resulting in the loss of the C _M and the hydroxyl group, or is covalently linked to the non-cleavable linker element by the hydroxyl group on Ci ₄ : and wherein Rx is a hydrogen atom, a hydroxy or methoxy group, and wherein R ₂ is a Ci-C ₅ alkoxy group.

14. The antibody-drug conjugate of any of embodiments 1 to 13, wherein the anthracycline derivative is a derivative of 3’-deamino-3”,4’-anhydro-[2”(S)- methoxy-3 ’ ’ (R)-oxy-4” -morpholinyl]doxorubicin (PNU- 159682).

15. The antibody-drug conjugate of any of embodiments 1 to 14, wherein A, the antibody or fragment thereof, comprises: a. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 18, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; b. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 10 and SEQ ID NO: 19, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; c. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 20, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; d. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 12 and SEQ ID NO: 21, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 26, SEQ ID NO: 5 and SEQ ID NO: 30, respectively; e. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 13 and SEQ ID NO: 18, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 31, respectively; f. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 14 and SEQ ID NO: 22, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; g. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 15 and SEQ ID NO: 23, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 27, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; h. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 7, SEQ ID NO: 16 and SEQ ID NO: 23, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 25, SEQ ID NO: 5 and SEQ ID NO: 29, respectively; or i. the HCDR1, HCDR2 and HCDR3 sequences of SEQ ID NO: 8, SEQ ID NO: 17 and SEQ ID NO: 24, respectively, and the LCDR1, LCDR2 and LCDR3 sequences of SEQ ID NO: 28, SEQ ID NO: 5 and SEQ ID NO: 31, respectively. The antibody-drug conjugate of any of embodiments 1 to 15, wherein A, the antibody or fragment thereof comprises: a. a VH sequence of SEQ ID NO: 32 and a VL sequence of SEQ ID NO: 33; b. a VH sequence of SEQ ID NO: 34 and a VL sequence of SEQ ID NO: 33; c. a VH sequence of SEQ ID NO: 35 and a VL sequence of SEQ ID NO: 36; d. a VH sequence of SEQ ID NO: 37 and a VL sequence of SEQ ID NO: 38; e. a VH sequence of SEQ ID NO: 39 and a VL sequence of SEQ ID NO: 40; f. a VH sequence of SEQ ID NO: 41 and a VL sequence of SEQ ID NO: 33; g. a VH sequence of SEQ ID NO: 42 and a VL sequence of SEQ ID NO: 43; h. a VH sequence of SEQ ID NO: 44 and a VL sequence of SEQ ID NO: 33; or i. a VH sequence of SEQ ID NO: 45 and a VL sequence of SEQ ID NO: 46. The antibody-drug conjugate of any of embodiments 1 to 16, wherein A, the antibody or fragment thereof, comprises: a. the heavy chain sequence of SEQ ID NO: 49 and light chain sequence of SEQ ID NO: 50; b. the heavy chain sequence of SEQ ID NO: 51 and light chain sequence of SEQ ID NO: 50; c. the heavy chain sequence of SEQ ID NO: 52 and light chain sequence of SEQ ID NO: 53; d. the heavy chain sequence of SEQ ID NO: 54 and light chain sequence of SEQ ID NO: 55; e. the heavy chain sequence of SEQ ID NO: 56 and light chain sequence of SEQ ID NO: 57; f. the heavy chain sequence of SEQ ID NO: 58 and light chain sequence of SEQ ID NO: 50; g. the heavy chain sequence of SEQ ID NO: 59 and light chain sequence of SEQ ID NO: 60; h. the heavy chain sequence of SEQ ID NO: 61 and light chain sequence of SEQ ID NO: 50; or i. the heavy chain sequence of SEQ ID NO: 62 and light chain sequence of SEQ ID NO: 63; or versions thereof with an engineered Fc domain. A method of producing an antibody-drug conjugate according to any of embodiments

1 to 17, wherein the method comprises the following steps: a. providing A, an antibody or fragment thereof with an oligopeptide linker element, optionally preceded by a spacer element at the antibody light and/or heavy chains, b. providing one or more toxins T with a non-cleavable linker element, and c. conjugating the antibody and the toxin resulting in the antibody-drug conjugate. n antibody-drug conjugate consisting of:

• the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 58, and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPXTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of CM and of the hydroxyl group. n antibody-drug conjugate consisting of:

• the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 145, and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPXTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of CM and of the hydroxyl group. n antibody-drug conjugate consisting of:

• the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 146 and two light chains of the amino acid sequence according to SEQ ID NO: 50, wherein the antibody binds to CLDN18.2,

• the linker [GGGGS]-[LPXTGG]-[ethylenediamine] at the C-terminus of the light chains, and

• the anthracycline-based small molecule toxin 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), linked covalently to the ethylenediamine of the linker at C _13; resulting in the loss of C14 and of the hydroxyl group.

22. A pharmaceutical composition comprising the antibody-drug conjugate of any of embodiments 1 to 23 and an excipient. 23. The antibody-drug conjugate of any of embodiments 1 to 23 for use in treatment.

24. The antibody-drug conjugate of any of embodiments 1 to 23 for use in the treatment of cancer.

25. The antibody-drug conjugate of embodiment 24, wherein the cancer is selected from pancreatic, gastric, esophageal, ovarian, and lung cancer.

SEQUENCES

SEQ ID NO: 1 GYXFTSYWIG X in 2nd position is T or S

SEQ ID NO: 2 GXIYPXXXXTXYX X in 2 ^nd position is N or I; X in 6 ^th position is S or G; X in 7 ^th position is A, E or D; X in 8 ^th position is A or S; X in 9 ^th position is Y or D; X in 11 ^th position is N or R; X in last position is A or S

SEQ ID NO: 3 XRXWRGNSFDX X in 1st position is A or T; X in 3rd position is L, M, I or Q; X in last position is A or Y

SEQ ID NO: 4 KSSQSXLNSGNQKNYLX X in 6th position is L or V; X in last position is T or A

SEQ ID NO: 5 WASTRES

SEQ ID NO: 6 QXDYSYPXT X in 2nd position is N or Q; X in L or F

SEQ ID NO : 7 GYSFTS YWIG

SEQ ID NO : 8 GYTFTS YWIG

SEQ ID NO : 9 GNIYPGASDTRYA

SEQ ID NO : 10 GNIYPGD ADTRYA

SEQ ID NO : 11 GIIYPGASDTNYA

SEQ ID NO : 12 GIIYPGD AYTRYS

SEQ ID NO : 13 GIIYPGAAYTRYA

SEQ ID NO : 14 GNIYPGAS YTR YS

SEQ ID NO : 15 GNIYPGEAYTRYS

SEQ ID NO : 16 GNIYPSES YTNYA

SEQ ID NO : 17 GIIYPS AAYTRYA

SEQ ID NO : 18 ARLWRGNSFD Y

SEQ ID NO : 19 ARMWRGNSFD Y

SEQ ID NO : 20 ARIWRGNSFD Y

SEQ ID NO : 21 TRLWRGNSFD A

SEQ ID NO : 22 TRQWRGNSFD Y

SEQ ID NO : 23 TRLWRGNSFD Y

SEQ ID NO : 24 TRMWRGNSFD Y

SEQ ID NO: 25 KSSQSLLNSGNQKNYLA SEQ ID NO: 26 KSSQSLLNSGNQKNYLT

SEQ ID NO: 27 KSSQSVLNSGNQKNYLT

SEQ ID NO: 28 KSSQSVLNSGNQKNYLA

SEQ ID NO : 29 QND YS YPFT

SEQ ID NO : 30 QND YS YPLT

SEQ ID NO : 31 QQD YS YPFT

SEQ ID NO: 32 hGBA-1 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGAS

DTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLWRGNSFDYWGQGT

LVTVSS

SEQ ID NO: 33 hGBA-1, hGBA-2, hGBA-6 , hGBA-8 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPFTFGQGTKVEIK

SEQ ID NO: 34 hGBA-2 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGDA

DTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARMWRGNSFDYWGQG

TL VTVSS

SEQ ID NO: 35 hGBA-3 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGASD

TNYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARIWRGNSFDYWGQGTL

VTVSS

SEQ ID NO: 36 hGBA-3 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKVEIK

SEQ ID NO: 37 hGBA-4 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDA

YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDAWGQGT

LVTVSS

SEQ ID NO: 38 hGBA-4 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKVEIK

SEQ ID NO: 39 hGBA-5 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGAA

YTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLWRGNSFDYWGQGT

LVTVSS SEQ ID NO: 40 hGBA-5 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYSYPFTFGQGTKVEIK

SEQ ID NO: 41 hGBA-6 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGNIYPGAS YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRQWRGNSFDYWGQGT

LVTVSS

SEQ ID NO: 42 hGBA-7 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGEA

YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDYWGQGT

LVTVSS

SEQ ID NO: 43 hGBA-7 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLTWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPFTFGQGTKVEIK

SEQ ID NO: 44 hGBA-8 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPSES YTNYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDYWGQGT

LVTVSS

SEQ ID NO: 45 hGBA-9 HC variable region

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGIIYPSAA

YTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRMWRGNSFDYWGQGT LVTVSS

SEQ ID NO: 46 hGBA-9, hGBA-10 LC variable region

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYSYPFTFGQGTKVEIK

SEQ ID NO: 47 IMAB362 HC full

QVQLQQPGAELVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSY

TNYNQKFKDKATLT VDKS S STAYMQLS SPTSED S AVYYCTRSWRGNSFD YWGQGTT

LTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT F

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP P

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO: 48 IMAB362 LC full

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA

STRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPFTFGSGTKLEIKR TV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO : 49 hGB A- 1 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGAS

DTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLWRGNSFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC P

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO: 50 hGBA-1, hGBA-2, hGBA-6, hGBA-8 LC full

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPFTFGQGTKVEIKR T

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO : 51 hGBA-2 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGDA

DTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARMWRGNSFDYWGQG

TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV H

TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT C

PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE V

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO: 52 hGBA-3 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGASD

TNYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARIWRGNSFDYWGQGTL

VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT F

PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP P

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH

NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO : 53 hGBA-3 LC full

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKVEIKR T

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO : 54 hGB A-4 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDA

YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDAWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K or R

SEQ ID NO : 55 hGB A-4 LC full

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPLTFGQGTKVEIKRT

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO : 56 hGB A-5 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGAA

YTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLWRGNSFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC P

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K or R

SEQ ID NO : 57 hGB A-5 LC full

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYSYPFTFGQGTKVEIKR T

VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ

DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO : 58 hGB A-6 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGNIYPGAS

YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRQWRGNSFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC P

PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK

GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV L

DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K or R

SEQ ID NO : 59 hGB A-7 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPGEA

YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K orR

SEQ ID NO : 60 hGB A- 7 LC full

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLTWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYSYPFTFGQGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO : 61 hGB A-8 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGNIYPSES YTNYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRLWRGNSFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV

HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K or R

SEQ ID NO : 62 hGB A-9 HC full

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGIIYPSAA YTRYAPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRMWRGNSFDYWGQGT

LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH T FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is

K or R

SEQ ID NO : 63 hGB A-9, LC full

DIVMTQSPDSLAVSLGERATINCKSSQSVLNSGNQKNYLAWYQQKPGQPPKLLIYWA

STRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQDYSYPFTFGQGTKVEIKR T VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 64

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL Q S SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALP APIEKTISKAKGQPREPQ VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO: 65

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVT EQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO: 66

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL Q S SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS F FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO: 67

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL Q S SGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREP

QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS F FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or R

SEQ ID NO : 68 DQWSTQDLYN

SEQ ID NO : 69 NNP VT AVFNYQ

SEQ ID NO : 70 STQDLYNNP VT AVE

SEQ ID NO : 71 TNFWMSTANMYTG

SEQ ID NO : 72 ALMI VGIVLGAIGLL V

SEQ ID NO: 73 RIGSMEDSAKANMTLTSGIMFIVS

SEQ ID NO: 74

METDTLLLWVLLLWVPGSTGDAAQPARRARRTKLGTELGSTPVWWNSADGRMDQ WSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRAAIQH SGGRSRRARTKTHLRRGSE

SEQ ID NO : 75 MDQWSTQDLYNNP VT

SEQ ID NO : 76 LYNNP VTAVFNYQGL

SEQ ID NO : 77 VFNYQGLWRSCVRES

SEQ ID NO: 78 QGLWRSCVRESSGFT

SEQ ID NO: 79 RSCVRESSGFTECRG

SEQ ID NO: 80 TEDEVQSYPSKHDYV SEQ ID NO: 81 EVQSYPSKHDYV

SEQ ID NO: 82 GYXFTSYWIX X in 2 ^nd position is T or S, X in the last position is G or N

SEQ ID NO: 83 GXIYPXXXXTXYX X in 2 ^nd position is N or I; X in 6 ^th position is S or G; X in 7 ^th position is A, E or D; X in 8 ^th position is A or S; X in 9 ^th position is Y or D; X in 11 ^th position is N or R; X in last position is A, N or S

SEQ ID NO: 84 XRXWRGNSFDX X in 1 ^st position is A or T; X in 3 ^rd position is L, M, I, S or Q; X in last position is A or Y

SEQ ID NO: 85 ggctatagctttacatcatattggattgga

SEQ ID NO: 86 gggaacatttaccctggggcatcggatacgcgatacgca

SEQ ID NO: 87 gcgagactttggcgggggaatagcttcgactac

SEQ ID NO: 88 aaaagctcccaaagcctattgaactcgggaaaccaaaagaattacttggca

SEQ ID NO: 89 tgggcaagcacccgagagagc

SEQ ID NO: 90 caaaacgactattcatacccattcaca

SEQ ID NO: 91 ggatattcatttacaagctactggatcgga

SEQ ID NO: 92 ggaaatatataccccggagacgcggacacgagatacgca

SEQ ID NO: 93 gcgcggatgtggcgcggcaatagctttgactac

SEQ ID NO: 94 gggatcatctatccgggggcatccgataccaactatgcg

SEQ ID NO: 95 gctaggatttggcgaggaaatagctttgattat

SEQ ID NO: 96 aagagctcgcaaagtttgctgaactccgggaaccaaaagaattacctggca

SEQ ID NO: 97 tgggcatcaacgcgggaaagc

SEQ ID NO: 98 caaaacgactactcctatccgctgacc

SEQ ID NO: 99 ggatactcatttacatcatactggatagga

SEQ ID NO: 100 gggattatataccccggcgacgcttacactcgatattcg

SEQ ID NO: 101 acgaggctatggagggggaatagctttgatgcc

SEQ ID NO: 102 aagagctcccaaagcctattgaactcgggaaatcaaaagaattatctgaca

SEQ ID NO: 103 tgggcctcgacaagggagagc

SEQ ID NO: 104 caaaatgactactcatacccgctgaca

SEQ ID NO: 105 ggatatagctttacgagctactggatcgga

SEQ ID NO: 106 gggataatataccccggagcggcatacacgagatatgcg SEQ ID NO: 107 gcgagactatggcgcgggaactcatttgattac

SEQ ID NO: 108 aaatcatcgcaatcattgctaaattcggggaaccaaaagaattatttggca

SEQ ID NO: 109 tgggcatccacgagagaatcg

SEQ ID NO: 110 caacaagattattcatacccatttaca

SEQ ID NO: 111 ggatatacatttacatcttactggatcgga

SEQ ID NO: 112 gggaacatttatcctggcgcgagctatacgcgctat

SEQ ID NO: 113 acccggcaatggaggggcaatagctttgactac

SEQ ID NO: 114 ggatattcctttacatcatactggatcggc

SEQ ID NO: 115 gggaacatatatcccggagaagcctatacgagatactcg

SEQ ID NO: 116 acgcgactatggaggggaaatagctttgactat

SEQ ID NO: 117 aagagctcccaatcagtcctgaactctgggaatcaaaagaattacctgaca

SEQ ID NO: 118 tgggcgagcacgagggagagc

SEQ ID NO: 119 caaaatgattattcataccccttcaca

SEQ ID NO: 120 ggatactcctttacatcatattggatcgga

SEQ ID NO: 121 ggaaacatatatccgagcgaatcatatacgaactacgcg

SEQ ID NO: 122 acgaggctatggagggggaatagcttcgactat

SEQ ID NO: 123 ggatatacattcacgagctactggatagga

SEQ ID NO: 124 ggaatcatatatccttccgcggcatatacgcgatatgcg

SEQ ID NO: 125 acgcggatgtggaggggaaatagctttgattac

SEQ ID NO: 126 aagagctcgcaatcggtcctgaatagcgggaaccaaaagaattatctggcc

SEQ ID NO: 127 caacaagactactcatacccatttaca

SEQ ID NO: 128 gaagtccaactggtccaatccggcgcggaggttaagaagcccggagaatcgctgaagatc tcatgcaaagggagcggct atagctttacatcatattggattggatgggtcaggcaaatgccggggaaggggctggaat ggatggggaacatttaccctggggcatc ggatacgcgatacgcacctagctttcaagggcaagtcacaatttcggcggacaagagcat ctcaacggcatacctgcaatggtcgagc ttgaaggcatctgatactgcaatgtactactgcgcgagactttggcgggggaatagcttc gactactgggggcagggtaccctggttac ggtctcgagc

SEQ ID NO: 129 gacattgtgatgacgcaaagccccgattcgctggctgtatcgctaggggagcgcgctacg atcaattgcaaaagctcccaa agcctattgaactcgggaaaccaaaagaattacttggcatggtatcaacaaaaaccgggg caaccgccgaagctgctgatctattggg caagcacccgagagagcggtgtcccggaccgatttagcgggagcggatcgggcaccgact tcacgctgacaataagctcattgcaa gccgaggatgtggcggtctattattgccaaaacgactattcatacccatcacattcgggc aaggtaccaaggtcgagatcaag SEQ ID NO: 130 gaagtccaactggtccaatctggagcggaagtcaagaagcctggggagagcctgaaaatt tcatgcaaggggagcggat attcatttacaagctactggatcggatgggtccggcaaatgccggggaagggcttggaat ggatgggaaatatataccccggagacgc ggacacgagatacgcaccgagctttcaagggcaggtcaccattagcgctgataaatcgat ttcaaccgcatatctgcaatggtcatcgc tgaaggcctccgacaccgcgatgtactattgcgcgcggatgtggcgcggcaatagctttg actactgggggcagggtaccctcgtcac ggtctcgagc

SEQ ID NO: 131 gaggtccaactggtccaaagcggcgcggaggtcaagaagccgggagaatccctgaagatt agctgcaaaggctccggct atagctttacatcatattggatcggatgggtcagacaaatgccgggaaagggacttgaat ggatggggatcatctatccgggggcatcc gataccaactatgcgccgagcttccaagggcaggtcacgatatccgcggataaatcgatt agcaccgcatatctgcaatggagctcgct gaaggcatccgacaccgcgatgtactactgcgctaggatttggcgaggaaatagctttga ttattgggggcagggtacccttgtcacgg tctcgagc

SEQ ID NO: 132 gacattgtcatgacgcaaagccccgactcgctggccgtctcactgggggagcgggcgaca atcaactgcaagagctcgc aaagtttgctgaactccgggaaccaaaagaattacctggcatggtatcaacaaaagccgg ggcaacccccgaagctgctgatatattg ggcatcaacgcgggaaagcggagtcccggatagatttagcggatctggatcggggaccga cttcacgctgacgatatctagccttcaa gccgaggatgtggctgtatattattgccaaaacgactactcctatccgctgaccttcggg caaggtaccaaggtcgagatcaag

SEQ ID NO: 133 gaagtccaactagtccaaagcggagccgaagtcaagaaaccgggggagagccttaagatc tcatgcaaggggagcgga tactcatttacatcatactggataggatgggtcagacaaatgcccggcaaggggctggaa tggatggggattatataccccggcgacg cttacactcgatattcgccatcattccaagggcaggtcacgatatcggccgataaatcga tatccacggcatacctgcaatggagctcac tgaaagcatctgatacggcaatgtattattgcacgaggctatggagggggaatagctttg atgcctgggggcagggtaccctggtcacg gtctcgagc

SEQ ID NO: 134 gacatagttatgacacaatcgccggatagcctcgcggtcagccttggagagcgggcgacg atcaactgcaagagctccca aagcctattgaactcgggaaatcaaaagaattatctgacatggtatcaacaaaagccggg gcaaccaccgaaactgctgatctattggg cctcgacaagggagagcggagtcccggaccgcttctctggatcgggaagcgggactgact tcacgctgaccataagctcgctgcaa gccgaggacgtcgccgtctattattgccaaaatgactactcatacccgctgacatttggc caaggtaccaaggtcgagatcaag

SEQ ID NO: 135 gaggtgcaactggtacaatccggggcggaagtgaagaagccgggggaatcgctgaagata agctgcaaaggctctggat atagctttacgagctactggatcggatgggtcaggcaaatgccggggaagggactggaat ggatggggataatataccccggagcg gcatacacgagatatgcgccgagcttccaagggcaagtgacaataagcgcggacaaatcg attagcacggcatatctgcaatggtcct cgctgaaggcgagcgataccgcaatgtactattgcgcgagactatggcgcgggaactcat ttgattactgggggcagggtaccctagt gacggtctcgagc

SEQ ID NO: 136 gacattgtcatgacgcaaagcccggatagcctggctgtatcgctgggggagagagcgacg atcaactgcaaatcatcgca atcattgctaaattcggggaaccaaaagaattatttggcatggtatcaacaaaagccggg gcaaccgccgaaactgctgatttactggg catccacgagagaatcgggagtcccggaccgatttagcggatctgggagcgggaccgatt tcacgctgaccattagctcgctgcaag cggaggatgtggcggtctattactgccaacaagattattcatacccatttacatttgggc aaggtaccaaggtcgagatcaag SEQ ID NO: 137 gaagtacaattggttcaatcgggggccgaagtcaagaagccgggggaatcgctgaagata tcctgcaaggggagcggat atacatttacatcttactggatcggatgggtcagacaaatgcccggaaaggggcttgaat ggatggggaacatttatcctggcgcgagc tatacgcgctatagcccgagcttccaagggcaggtcacgattagcgccgacaagagcatt tcgacggcatacctgcaatggagctcgc tgaaagcatcggatacggcaatgtattactgcacccggcaatggaggggcaatagctttg actactgggggcagggtaccctagtcac ggtctcgagc

SEQ ID NO: 138 gaagttcaattggtccaatctggagccgaagtcaagaagcccggagaatcgctgaagatt agctgcaaggggagcggata ttcctttacatcatactggatcggctgggtcagacaaatgcccggaaagggactggaatg gatggggaacatatatcccggagaagcc tatacgagatactcgccatcatttcaaggacaggtcaccataagcgcggacaagagcata agcaccgcatacctgcaatggagctcgc tgaaggcatcggacaccgccatgtattactgcacgcgactatggaggggaaatagctttg actattgggggcagggtaccttagtcacg gtctcgagc

SEQ ID NO: 139 gatatagtaatgactcaatcacccgatagcttggctgtgagcctgggagaaagagctaca atcaactgcaagagctcccaat cagtcctgaactctgggaatcaaaagaattacctgacatggtatcaacaaaagcccggac aaccgccgaagctgctgatctactgggc gagcacgagggagagcggagtcccggatcgattttctggctccgggagcggaaccgactt cacactgactattagctcgctgcaagc ggaggacgtcgccgtctactattgccaaaatgattattcataccccttcacatttgggca aggtaccaaggtcgagatcaag

SEQ ID NO: 140 gaggtgcaactagtgcaatcgggggccgaagtgaagaaacctggggaatcgctgaagata tcatgcaaggggagcggat actcctttacatcatattggatcggatgggtcaggcaaatgccggggaaggggctggaat ggatgggaaacatatatccgagcgaatc atatacgaactacgcgccgagctttcaaggacaagtcacgatatccgcggataaatcgat atcgaccgcatacctgcaatggagctcg ctgaaggcttccgacactgcgatgtattactgcacgaggctatggagggggaatagcttc gactattgggggcagggtaccctggtga cggtctcgagc

SEQ ID NO: 141 gaagtccaattagtccaatcgggggccgaggtcaagaagccgggggaatcgctcaagata agctgcaagggatcgggat atacattcacgagctactggataggatgggtcaggcaaatgccggggaaggggctggaat ggatgggaatcatatatccttccgcggc atatacgcgatatgcgccatcatttcaaggacaggtcacgataagcgccgacaagagcat tagcaccgcatacctgcaatggtcgagc cttaaggcatcggacaccgcgatgtactactgcacgcggatgtggaggggaaatagcttt gattactgggggcagggtaccctagtca cggtctcgagc

SEQ ID NO: 142 gacatcgtcatgacgcaaagcccggactcgctggcggtctcgctgggggagcgggccaca ataaattgcaagagctcgc aatcggtcctgaatagcgggaaccaaaagaattatctggcctggtatcaacaaaagccgg ggcaaccaccgaagctgctaatctattg ggcgagcacgagggagagcggagtccccgatcgatttagcggatcgggaagcgggaccga tttcacgctgacgatttcgagcctac aagccgaggatgtggcggtctattactgccaacaagactactcatacccatttacatttg gacaaggtaccaaggtcgagatcaag

SEQ ID NO : 143 LPXTGG

X is any of the 20 natural amino acids

SEQ ID NO : 144 GGGGSLPXTGG

X is any of the 20 natural amino acids SEQ ID NO: 145 hGBA-6 HC full LAL A

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGNIYPGAS YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRQWRGNSFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC P PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or

R

SEQ ID NO : 146 hGBA-6 HC full L AL APG

EVQLVQSGAEVKKPGESLKISCKGSGYTFTSYWIGWVRQMPGKGLEWMGNIYPGAS YTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCTRQWRGNSFDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT

FPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC P PCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGX X is K or

R

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CN109762067

US10,435,471

W02000/015659

W02004/047863

WO2005/113587

W02007/059997

WO2008/145338

W02010/009124

WO2013/167259

WO2013/174509

WO2014/075788

WO2014/127906

WO2016/102679

WO2016/166122

W02018/006882

WO2019/175617

WO2019/219089

W02016/008405

CN111440245

WO2019/242505

W02020/038404

W02020/043044

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