WO2025078686A1

WO2025078686A1 - Combination therapy involving antibody-drug conjugates against claudin18.2 and pd1/pd-l axis inhibitors for treatment of cancer

Info

Publication number: WO2025078686A1
Application number: PCT/EP2024/078871
Authority: WO
Inventors: Kati RÄSÄNEN; Ilona PROCHÁZKOVÁ; Michaela FOJTU; Lenka KYRYCH SADÍLKOVÁ; Ulrich Moebius
Original assignee: Sotio Biotech AS
Current assignee: Sotio Biotech AS
Priority date: 2023-10-12
Filing date: 2024-10-14
Publication date: 2025-04-17
Anticipated expiration: 2026-04-12

Abstract

The present invention relates to combination therapies involving an anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2, wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracycline derivative, and a PD-1/PD-L axis inhibitor.

Description

Combination therapy involving antibody-drug conjugates against Claudinl8.2 and PD- 1/PD-L axis inhibitors for treatment of cancer

Background of the invention

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 PKC(^. These proteins interact to maintain the tight junction structure (Yu and Turner, 2008).

Claudins form a family of 23 proteins (Hewitt et al., 2006). Claudin 18 is a 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). The amino acid sequence of human CLDN18.2 protein has the NCBI reference sequence: NP_001002026.1.

Several antibody-drug conjugates (ADC) targeting CLDN18.2 have been developed. MMAE was used as conjugated toxin for the ADC CMG901 (WO2022/078523, (Xu et al., 2023), RC118 (WO2022/237666), SYSA-1801 (WO2022/111616), ATG-022 (WO2021/259304), TORL-2-307-ADC (WO2021/011885) and JS107

(https://ncit.nci.nih.gOv/ncitbrowser/ConceptReport.j sp?dictionary=NCI%20Thesaurus&code =C 190687). The conjugated toxin has not been disclosed for the ADCs IB343, SHR-A1904, BA1301, SKB325 and TQB-2102. Growing evidence indicates that ADCs might increase the efficacy of immunotherapeutic agents (Fuentes-Antras et al., 2023). Antibodies targeting CLDN18.2 have been tested in mice in combination with immunotherapeutic agents such as PD-l/PD-L axis inhibitors (W02020/063988, WO2021/025177, W02022/203090). Likewise, ADCs targeting CLDN18.2 have been tested in phase 1 clinical trials in combination with PD-l/PD-L axis inhibitors: JS107 is tested in combination with Toripalimab (anti-PDl antibody) (NCT05502393 available at https://ClinicalTrials.gov ) and SOT102 is tested in combination with Nivolumab (anti-PDl antibody), where Nivolumab is also part of the standard of care for tested indications (NCT05525286 available at https://ClinicalTrials.gov). Results from these clinical trials related to ADCs supporting the benefit of this approach over standard treatments are still awaited. Furthermore, JS 107 is an antibody conjugated to MMAE, which is believed of having limited immunotherapeutic activity and has known of target toxicity such as neutropenia, peripheral neuropathy, anemia and skin toxicity (Nguyen et al., 2023). There is thus a need to improve therapies targeting CLDN18.2 for the treatment of cancer where CLDN18.2 is expressed (pancreatic, gastric, esophageal, ovarian, biliary tract or lung cancer). To the contrary of ADCs based on MMAE, an ADC based on trastuzumab conjugated to the anthracy cline derivative PNU- 159682 has been shown to exhibit immunotherapeutic properties by inducing immunogenic cell death and showed potential benefit of a combination with an anti-PD-1 antibody (D'Amico et al., 2019).

The inventors have now shown that an anti-CLDN18.2 ADC, where the antibody is conjugated to an anthracy cline derivative, in combination with a PD-l/PD-L axis inhibitor synergizes the effect of the ADC or PD-l/PD-L axis inhibitor alone and leads to a durable antitumor efficacy, while allowing sub-optimal dosing of the ADC and improving tumor infiltration and proliferation of immune cells. The combination thus has surprisingly immunotherapeutic activity and lower toxicity due to lower dosing of the anthracycline derivative.

Definitions, abbreviations and acronyms

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 purposes comprise sortase, transglutaminase, galactosyltransferase, sialyltransferase and tubulintyrosine ligase. An overview of ADC linker conjugation and toxins can be found in Fu et al (2022). 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 “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.

As used herein, “binding” refers to the attachment of a polypeptide to an antigen or an antigen epitope and can be quantified by measuring the “binding affinity”. “Binding affinity” refers to the apparent association constant K_A or to the apparent dissociation constant K_D. The K_A is the reciprocal of the dissociation constant (K_D). The antigen binding domain in the antibody described herein may have a binding affinity (K_D) of at most 10'⁵, 10'⁶, 10'⁷, 10'⁸, 10'⁹, 10'¹⁰ M, or lower for the target antigen or antigenic epitope. An increased binding affinity corresponds to a decreased K_D. Higher affinity binding of an antigen binding domain for a first antigen relative to a second antigen can be indicated by a higher K_A (or a smaller numerical value K_D) for binding the first antigen than the K_A (or numerical value K_D) for binding the second antigen. In such cases, the antigen binding domain has specificity for the first antigen (e.g., a first protein in a first conformation or mimic thereof) relative to the second antigen (e.g., the same first protein in a second conformation or mimic thereof; or a second protein). Differences in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000 or 100,000-fold.

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 of the invention

In a first embodiment, the invention relates to an anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2 for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti-CLDN18.2 ADC in combination with a PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracycline derivative.

The invention further relates to a PD-1-/PD-L axis inhibitor for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated with the PD-l/PD-L axis inhibitor in combination with an anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2, and wherein the ADC comprises an anti- CLDN18.2 antibody conjugated to an anthracycline derivative.

The invention also relates to an anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2 and a PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti- CLDN18.2 ADC and the PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti- CLDN18.2 antibody conjugated to an anthracycline derivative.

Figure 2 and Example 3 show that the combination of an anti-CLDN18.2 antibody-drug conjugate (ADC) conjugated to an anthracycline derivative and a PD-1-/PD-L axis inhibitor leads synergistically to antitumor efficacy and increased immune cell infiltration.

An anti-CLDN18.2 antibody specifically binding to CLDN18.2 does not bind to CLDN18.1.

Hence, such antibody does not exhibit cross-reactivity or cross-binding to CLDN 18.1. 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 binds to the expressed target protein. 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 specific binding and low/no maxMFI reflecting non-binding.

Programmed death protein 1 (PD-1) is a common immunosuppressive member on the surface of T cells and plays an imperative part in downregulating the immune system and advancing self-tolerance. Its ligands are programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2). PD-L1 is overexpressed on the surface of malignant tumor cells, and PD-L2 has been shown to be expressed in head and neck squamous carcinoma (HNSCC) and other solid tumors (Wang et al., 2023). PD-L1 and PD-L2 binds to PD-1, inhibits the proliferation of PD-1 -positive cells, and participates in the immune evasion of tumors leading to treatment failure. A PD-l/PD-L axis inhibitor inhibits either immunosuppression triggered by PD-1 or inhibition of proliferation of PD-1 positive cells when PD-1 is bound to PD-L1 or PD-L2, or immune evasion of tumors overexpressing PD-L1 or PD-L2. PD-l/PD-L axis inhibitors of the present invention are either antibodies binding PD-1 or antibodies binding PD- L1 or PD-L2, blocking the interaction of PD-1 with PD-L1. Among antibodies binding PD-L1 or PD-L2, preferred are antibodies binding PD-L1.

The toxin conjugated to the anti-CLDN18.2 antibody is selected from the group consisting of anthracyclines and derivatives thereof. Anthracyclines 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 anthracycline derivative is 3’-deamino-3”,4’-anhydro-[2”(S)-methoxy-3”(R)-oxy-4”- morpholinyl]doxorubicin (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). In some instances, the term anthracycline derivatives may be understood as anthracyclines resulting from lysosomal degradation, where fragments of the linker may remain attached to the anthracycline molecule. The term “anthracyclines” as used herein refers to anthracyclines and anthracycline derivatives. Treating cancer means inhibiting growth of a tumor or metastasis. The treatment may result in complete remission or in a stable disease where the tumor stops growing.

In another embodiment, the ADC used in the method of treatment has the general formula A- (L-T)_n, wherein A is an antibody, L is a linker and the toxin T is 3’-deamino-3”,4’-anhydro- [2”(S)-methoxy-3”(R)-oxy-4”-morpholinyl]doxorubicin (PNU- 159682), wherein n is an integer between > 1 and < 10. The drug-to-antibody ratio (DAR) of the ADC is 2 when n=2 or 4 when n=4. For the ADC tested in the present Examples 1-3, the ADC had a DAR=2.

Toxins may be conjugated to antibodies by various methods, involving for example stochastic conjugation via pre-existing amino acids in the antibody, such as lysines or reduced cysteines, site-specific conjugation via engineered reactive cysteine residues, disulfide re-bridging, unnatural amino acids, enzyme-assisted ligation, glycan remodeling and glycoconjuagtion and/or pClick technology (Fu et al., 2022).

In a preferred embodiment, the anti-CLDN18.2 ADC of the invention is the result of an enzyme-assisted ligation of the linker-toxin. The antibody A has been conjugated to the toxin T by means of a sortase enzyme, wherein the linker L at the C-terminus of the heavy and/or light chains of the antibody 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.

In a further preferred embodiment, the sortase recognition motif used for the enzyme-assisted ligation is the oligopeptide -LPQTGG- or -LPETGG-.

The linker L may further comprise a spacer element comprising a peptidic flexible oligopeptide between the C-terminus of the heavy and/or light chains and the antibody and the sortase recognition motif oligopeptide, preferably wherein the peptidic flexible oligopeptide consists of G and S, more preferably wherein the peptidic flexible oligopeptide is (GGGGS)_m with m being 1, 2, 3, 4 or 5.

Additionally, the Linker L may also comprise at least one non-cleavable linker element or one cleavable element in C-terminus of the sortase tag, preferably wherein the non-cleavable linker element is selected from the group consisting of: a. ethylenediamine, and b.

wherein the cleavable linker element is selected from the group consisting of: c. -[vc-PAB]-[N-formyl-N,N’ -dimethylethylenediamine], and d. -[vc-PAB]-[piperazine],

The toxin T may be conjugated to the heavy and/or light chains on the antibody A.

In one embodiment, the antibody A of the anti-CLDN18.2 ADC may be the antibodies derived from antibodies HB37A6 or HZ69H9 as disclosed in WO2021/254481; antibody 18D10- VH6/VL1 as disclosed in WO2022/078523; antibody RGCLN18.2 as disclosed in WO2022/237666; antibodies 4F11E2 HC N55E-LC S32A or 72C1B6A3 HC WT-LC S32A as disclosed in WO2021/088927; antibody SYJS001 as disclosed in WO2022/111616; mAbl902 humanized antibody with the VH11 variable heavy chain and VL11 variable light chain as disclosed in Table 8 of W02020/200196; antibodies lE9.2-hzl 1 or 2C6.9-hz21 as disclosed in W02020/135201; antibodies CLDQMIX-CA808.1 or CLDQ1-CA841 as disclosed in W02020/239005; antibodies Abl5, AblO or Abl7 as disclosed in WO2021/259304; antibody HuAb307-3 as disclosed in W02021/011885; antibodies JS012-Chi9-hu7, JS012-Chi2-hu2- v3-2 or JS012-Chi2-hu2-v3-2a as disclosed in WO2022/012559, Zolbetuximab (CAS number 1496553-00-4), antibody hGBA6 and hGBA6(LALA) as disclosed in WO2021/111003 or hClla as disclosed in W02021/130291. Depending on the type of conjugation, such antibodies may be modified to allow enzyme-assisted ligation, glyco-conjugation or other chemical modifications. Implementing such modification is known by a person skilled in the art.

In another embodiment, the antibody A of the anti-CLDN18.2 ADC comprises the HCDR1, HCDR2 and HCDR3 of sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 of sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively. CDR numbering is according to EU numbering. Sequences are disclosed in Table 1.

In yet another embodiment, the antibody A of the anti-CLDN18.2 ADC comprises the variable heavy chain VH of sequence SEQ ID NO: 7 and the variable light chain VL of sequence SEQ ID NO: 8. Any antibody may further be modified to alter their effector functions (see WO2022/136642, Table 2), e.g. via introduction of the L234A/L235A or L234A/L235A/P329G mutations in the IgGl heavy chain to reduce FcyR binding.

In a preferred embodiment, the anti-CLDN18.2 ADC consists of: 1. the antibody consisting of two heavy chains of the amino acid sequence according to

SEQ ID NO: 9, and two light chains of the amino acid sequence according to SEQ ID NO: 10, wherein the antibody binds to CLDN18.2,

2. the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and 3. 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_i3, resulting in the loss of C_i4 and of the hydroxyl group.

Positions C_i3 and C_i4 in PNU-159682 can be seen in formula (I):

An ADC from the preferred embodiment can be seen in Figure 1.

Table 1 : Sequences

In another preferred embodiment, the anti-CLDN18.2 ADC is SOT 102 as disclosed in Example 1.

In another embodiment, the invention relate to an anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, where the anti-CLDN18.2 ADC is administered to the patient to be treated at a dose between 0.016 mg/kg and 0.29 mg/kg every 2 weeks, preferably between 0.016 mg/kg and 0.22 mg/kg every 2 weeks, more preferably between 0.016 mg/kg and 0.16 mg/kg every 2 weeks, even more preferably between 0.032 and 0.16 mg/kg every 2 weeks. In a further embodiment, the invention relate to an anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, where the anti-CLDN18.2 ADC is administered to the patient to be treated at a dose of about: 0.016 mg/kg every 2 weeks, 0.032 mg/kg every 2 weeks, 0.064 mg/kg every 2 weeks, 0.107 mg/kg every 2 weeks, 0.16 mg/kg every 2 weeks, 0.22 mg/kg every 2 weeks, 0.29 mg/kg every 2 weeks or 0,38 mg/kg every 2 weeks; preferably about 0.064 mg/kg every 2 weeks or 0.107 mg/kg every 2 weeks, more preferably about 0.107 mg/kg every 2 weeks, especially where the anthracy cline is PNU- 159682.

The term about here means +/- 10%.

The ADC may be administered intravenously, subcutaneously or by either route, preferably intravenously.

In another embodiment, PD-l/PD-L axis inhibitor for use in combination with the anti- CLDN18.2 ADC is an anti-PD-1 antagonistic antibody or an anti-PD-L blocking antibody. An antagonistic anti-PD-1 antibody is an antagonist of the immune inhibitory checkpoint molecule PD-1. An anti-PD-L blocking antibody will interrupt the PD-1 - PD-L interaction.

In one embodiment, the PD-l/PD-L axis inhibitor to which the invention relates is an anti-PD-1 antibody such as Nivolumab, Pembrolizumab, Spartalizumab, Cemiplimab, Camrelizumab, Tislelizumab, MEDI-0680, Pidilizumab, Toripalimab, Dostarlimab, AGEN-2034, Sintilimab, BCD-100, Zimberelimab (GLS-010) or AMP -224; or an anti-PD-Ll antibody such as Avelumab, Atezolizumab, Durvalumab, CX-072, BMS-936559 (MDX 1105), Sugemalimab (WBP-3155 or CS1001), Cosibelimab (CK-301) or Envafolimab (KN035).

Nivolumab may be administered to the patient to be treated at a dose of 240 mg IV every 2 weeks or 360 mg IV every 3 weeks.

Pembrolizumab may be administered to the patient to be treated at a dose of 200 mg IV every 3 weeks or 400 mg IV every 6 weeks.

Spartalizumab may be administered to the patient to be treated at a dose of 400 mg IV every 4 weeks.

Cemiplimab may be administered to the patient to be treated at a dose of 350 mg IV every 3 weeks.

Camrelizumab may be administered to the patient to be treated at a dose of 200 mg IV every 3 weeks.

Tislelizumab may be administered to the patient to be treated at a dose of 200 mg IV every 3 weeks.

MEDI-0680 may be administered to the patient to be treated at a dose of 20 mg/kg IV every 2 weeks.

Pidilizumab may be administered to the patient to be treated at a dose of 3 mg/kg IV every 4 weeks.

Toripalimab may be administered to the patient to be treated at a dose of 240 mg IV every 3 weeks. Dostarlimab may be administered to the patient to be treated at a dose of 500 mg IV every 3 weeks.

AGEN-2034 may be administered to the patient to be treated at a dose of 300 mg IV every 3 weeks.

Sintilimab may be administered to the patient to be treated at a dose of 200 mg IV every 3 weeks.

BCD-100 may be administered to the patient to be treated at a dose of 1 mg/kg IV every 2 weeks or 3 mg/kg IV every 3 weeks.

Zimberelimab (GLS-010) may be administered to the patient to be treated at a dose of 240 mg IV every 2 weeks.

AMP-224 may be administered to the patient to be treated at a dose of 10 mg/kg IV every 2 weeks.

Avelumab may be administered to the patient to be treated at a dose of 800 mg IV every 2 weeks.

Atezolizumab may be administered to the patient to be treated at a dose of 840 mg IV every 2 weeks or 1200 mg IV every 4 weeks.

Durvalumab may be administered to the patient to be treated at a dose of 10 mg/kg IV every 2 weeks.

CX-072 may be administered to the patient to be treated at a dose of 800 mg/kg IV every 2 weeks,

BMS-936559 (MDX 1105) may be administered to the patient to be treated at a dose of 10 mg/kg IV every 2 weeks.

Sugemalimab (WBP-3155 or CS1001) may be administered to the patient to be treated at a dose of 1200 mg IV every 3 weeks.

Cosibelimab may be administered to the patient to be treated at a dose of 800 mg IV every 2 weeks.

Envafolimab (KN035) may be administered to the patient to be treated at a dose of 150 mg IV every week.

In a preferred embodiment, the anti-CLDN18.2 ADC is administered to the patient in combination with Nivolumab where Nivolumab is administered at a dose of 240 mg IV every 2 weeks.

In another preferred embodiment, the anti-CLDN18.2 ADC is administered to the patient in combination with Pembrolizumab where Pembrolizumab is administered at a dose of 200 mg IV every 3 weeks. In another embodiment, the anti-CLDN18.2 ADC within the combination with the PD-l/PD- L axis inhibitor may be administered to the patient in need at a dose of 20-80% of the single agent efficacious dose of the ADC, preferably 50% of the single agent efficacious dose. The efficacious dose is the dose showing durable antitumor efficacy when used as single agent, either in monotherapy (ADC alone) or when combined with the standard of care chemotherapy. The anti-CLDN18.2 ADC may be administered at a sub-optimal dose in combination with the PD-l/PD-L axis, as long as the effect of the PD-l/PD-L axis inhibitor synergizes with the anti- CLDN18.2 ADC to achieve durable antitumor efficacy, an effect which would not be achieved when each agent, either the ADC or the PD-l/PD-L axis inhibitor, would be administered alone. Suboptimal dosing of the ADC in a combination treatment while maintaining durable antitumor efficacy has the advantage of lowering the toxicity of the ADC.

In yet another embodiment, the anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor may be administered to the patient so that the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered on the same day. The ADC may be administered first, followed during the same day by the administration of the PD-l/PD-L inhibitor, or vice versa. Both administrations may be separated by a few hours. The timing of the administration may be at the discretion of the practicing physician.

Alternatively, the anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor may be administered to the patient so that the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered 1, 2, 3, 4, 5, 6 or 7 days apart.

In another embodiment, the anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor may be administered in multiple cycles. One cycle may be a period of 1 week, 2 weeks, three weeks or 4 weeks between two consecutive administrations. The anti-CLDN18.2 ADC and the PD- l/PD-L axis inhibitor may have to be administered with a different period between two consecutive administrations. The number of cycles may be at least 3 cycles and up to 20 cycles, preferably between 3 and 18 cycles.

In a preferred embodiment, the anti-CLDN18.2 ADC and Nivolumab may be administered with a period of two weeks between two cycles, for at least 3 cycles and up to 18 cycles, where the cancer to be treated is gastric cancer or pancreatic cancer. It is understood that the practicing physician may decide on the number of cycles of treatment, depending on the efficacy of the treatment. In another preferred embodiment, where the cancer is gastric cancer of pancreatic cancer, the anti-CLDN18.2 ADC and Pembrolizumab may be administered with a period of two weeks between two cycles, for at least 5 cycles.

In another embodiment of the invention, the anti-CLDN 18.2 ADC and the PD-l/PD-L inhibitor are used to treat cancer expressing or overexpressing CLDN18.2, comprising pancreatic, gastric, esophageal, ovarian, biliary tract or lung cancer. These cancer types are known to express or overexpress CLDN18.2. Preferably the cancer to be treated is gastric or pancreatic cancer, more preferably gastric cancer.

The invention further relates to the anti-CLDN18.2 ADC PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises at least one additional cytotoxic agent. In general, the method shall comprise standard of care for the specific cancer to be treated. The cytotoxic agent may be a combination of two or multiple cytotoxic agents. Cytotoxic agents comprise: platinum-based antineoplastic agents such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phananthriplatin, picoplatin or satraplatin, preferably cisplatin, carboplatin, oxaliplatin or nedaplatin; fluoropyrimidines compounds such as capecitabine, l-hexylcarbamoyl-5-fluorouracil (HCFU or Carmofur), doxifluridine, fluorouracil (5-FU), Tegafur or the Tegafur/gimeracil/oteracil combination;

- taxanes such as paclitaxel, nab-paclitaxel or docetaxel, irinotecan,

5 -fluorouracil (5-FU), leucovorin, gemcitabine, capecitabine,

FOLFIRI (5-FU + leucovorin + irinotecan),

FOLFOX (5-FU + leucovorin + oxaliplatin), or mFOLFIRINOX (5-FU + leucovorin + irinotecan + oxaliplatin)

In a preferred embodiment, the cancer to be treated in gastric cancer and the additional cytotoxic agent is: a. a platinum-fluoropyrimidine doublet, b. paclitaxel, docetaxel, irinotecan or FOLFIRI (5-FU + leucovorin + irinotecan), or c. FOLFOX (5-FU + leucovorin + oxaliplatin).

The platinum-fluoropyrimidine doublet may be the platinum compound oxaliplatin or cisplatin and the fluoropyrimidine 5-FU or capecitabin or the Tegafur/gimeracil/oteracil combination. In another embodiment, the cancer to be treated in pancreatic cancer and the additional cytotoxic agent is: a. mFOLFIRINOX (5-FU + leucovorin + irinotecan + oxaliplatin), b. gemcitabine and capeci tabine, or c. nab-paclitaxel and gemcitabine.

In another embodiment, the invention relates to the anti-CLDN18.2 ADC and a PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor, wherein the combination of the anti-CLDN18.2 ADC and the anti-PD-1 axis inhibitor exhibits less toxicity as compared to anti-CLDN18.2 ADC monotherapy. The anti-CLDN18.2 ADC monotherapy showed efficacy at 2 mg/kg in mice, which is double of the Img/kg dose used in combination with the PD-l/PD-L axis inhibitor (see for example WO2022/136642, Figures 21A, 21C, 22, 23B, 24). Therefore, the dose of the anti-CLDN18.2 ADC can be lowered while maintaining efficacy and will result in a treatment with less toxicity. Example 2 shows that the mice treated with the combination with the anti-CLDN18.2 dosed at 1 mg/kg did not show any weight loss during the treatment, and even gained weight, which is a clear sign of low toxicity.

In yet another embodiment, the invention relates to the anti-CLDN18.2 ADC and a PD-l/PD- L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti-CLDN18.2 ADC and the PD-l/PD- L axis inhibitor, wherein the combination of the anti-CLDN18.2 ADC and the anti-PD-l/PD- L axis inhibitor exhibits reduced side-effects as compared to anti-CLDN18.2 ADC monotherapy. Reducing side-effects can usually be achieved by lowering the dose of the drug to be administered. As it is shown in the examples below, the present combination the anti- CLDN18.2 ADC can be administered at U of the efficacious dose used in monotherapy (ADC alone) to achieve antitumor efficacy. The present combination thus allows to reduce the side- effects which may occur during monotherapy, as indirectly shown by the lack of weight loss during treatment, while still maintaining the antitumor efficacy of the ADC.

In a further embodiment, the invention relates to the anti-CLDN18.2 ADC and a PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor, wherein the PD-1 axis inhibitor enhances efficacy of the anti-CLDN18.2 ADC. As it can be seen in Example 3 and Figure 2E, the inventors have surprisingly found that the combination of the anti-CLDN18.2 ADC with the PD-1 antibody synergistically enhances the efficacy of the ADC alone, as, when administered at 1 mg/kg for the ADC alone or at 0.5 mg/kg for the anti -PD-1 antibody alone, the treatment does not result in tumor regression (see Figure 2C and 2D). Tumor regression is however achieved when the ADC dosed at 1 mg/kg is combined with the anti-PD-1 antibody dosed at 0.5 mg/kg.

In one embodiment, the provided combination treatment results in an increased immune cell infiltration in the tumor over a non-treated control, as seen e.g. in Figure 5

In one embodiment, the provided combination treatment results in an increased proliferation rate of CD8⁺ T-cells in the tumor as compared to the proliferation rate of CD8⁺ T-cells in the tumor observed for the corresponding monotherapies, as seen e.g. in Figure 6.

In one embodiment, the invention relates to a method of treatment of a patient suffering from a cancer, the method comprising administering to the patient to be treated an anti-CLDN18.2 ADC in combination with an PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracycline derivative, preferably where the patient is selected to express CLDN18.2 in the cancer by an FDA and/or EMA approved immunohistochemistry assay.

Figures

Figure 1: Representation of the ADC SOT 102

Figure 2: Tumor volume over the duration of the treatment.

In a MC38-hCLDN18.2 overexpressing syngeneic mouse model suboptimal dose of SOT102 led to durable anti-tumor efficacy when co-administered concomitantly with suboptimal dose of an anti-mouse PD-1 antibody (E). Animals were administered i.v. with the vehicle of the antibody or ADC (A), single-dose isotype-PNU (1 mg/kg) (B), single-dose SOT102 (Img/kg) (C) and i.p. dosing of anti-PD-1 antibody (0.5 mg/kg) at day 0, 3 and 6 (D). Individual tumor volumes over the duration of the study are shown (n=8). Figure 3: Relative body weight of each study group measured over the whole period of treatment.

No significant change in relative body weight was observed between each treatment study group over the duration of treatment.

Figure 4: Expression of CLDN 18.2 in the tumor by the end of the treatment period. Co-administration of SOT102 with anti-PD-1 antibody did not change CLND18.2 levels.

Figure 5: Evaluation of the presence of immune cell tumor infiltration by the end of the treatment period.

Co-administration of SOT102 with anti-PD-1 antibody led to the increase in CD45+ immune cell population.

Figure 6: Evaluation of dividing immune cells in the tumor at day 4 of the treatment period.

Samples (n=3) were harvested on day 4 and analyzed by flow cytometry. Co-administration of SOT102 with anti-PD-1 antibody led to an increase in particular of CD8+/Ki67+ immune cells. Figure 7: Anti-PD-1 antibody and SOT 102 ADC PK profiles.

Pharmacokinetic (PK) profiles were measured by ELISA for the SOT 102 ADC, Total SOT 102 (ADC and potential antibody having lost the drug) and the anti-PD-1 antibody following 3 administrations (DO, D3, D6).

Figure 8: Presence of dividing immune cells in the tumor in treatment endpoint tumor samples. (A): IHC of endpoint samples stained for Ki67 positivity (light gray: nucleus staining; black: Ki67 staining); (B): Ki67 staining quantified by the H-score determined with the HALO software.

Examples

1. Example 1: Generation of the anti-CLDN18.2 ADC (SOT102 ADC)

The hClla(LALA) antibody (heavy chain SEQ ID NO: 9 and light chain SEQ ID NO: 10), disclosed in WO2022/136642, was conjugated in a site-specific, sortase-mediated manner via a non-cleavable amide/peptide linker to a derivative of the highly potent anthracycline PNU- 159682 payload in a drug-antibody ratio (DAR) 2 light chain format as previously described (Stefan et al., 2017). The resulting ADC is SOT102. The structure of the ADC can be seen in Figure 1.

2. Example 2: In vivo syngeneic CDX model - methodology

The combination study of SOT102 with the mouse anti-PD-1 antibody mIgGle3 InvivoFit™ (InvivoGen, Toulouse, France, mPD-l-mabl5) was conducted with MC38-hCLDN18.2 cells using C57BL/6 syngeneic mouse model. Briefly, 5* 10⁵ cells were implanted in the right flank. After the mean tumor size reached 80-100 mm³, mice were randomized into 9 groups (n=8 mice per group). Dosing started on the day of randomization (Day 0). SOT 102 and isotype- PNU-159682 were administered once i.v. at 1 mg/kg concentration (Day 0). Anti-murine PD- 1 antibody was administered i.p. at 0.5 mg/kg concentration (Day 0, 3 and 6). Anti-PD-1 antibody and SOT 102 PK profiles were measured by a validated ELISA method.

In all studies tumor volumes and body weights were measured twice per week. Relative volumes of individual tumors (individual RTVs) for Day x was calculated by dividing the absolute individual tumor volume on Day x (T_x) by the absolute individual tumor volume of the same tumor on the day of randomization (To) multiplied by 100.

When the studies were terminated, the tumors were collected for flow cytometry from the selected groups and stained for respective markers:

• Tumor cells panel: mCD45, Annexin V, hCLDN18.2 and DAPI

• Tumor infiltrating lymphocytes (TILs) panel: Live/dead, mCD45, mCD3, mCD4, mCD8 and mKi67

Immunohistochemistry (IHC) staining of FFPE samples obtained from the studies was performed to detect Ki67⁺ cells (Ki67 Recombinant Rabbit Monoclonal Antibody, MAS- 14520, Invitrogen). Slides were scanned by Aperio AT2 (Leica Biosystems, Wetzlar, Germany) and H-score was determined in HALO software through analysis of intracellular staining of the whole sample.

3. Example 3: SOT102 in combination with anti-PD-1 leads to durable antitumor response.

The efficacy of SOT 102 was investigated in the human CLDN18.2 expressing MC38 syngeneic mouse model in combination with a mouse anti-murine PD-1 antibody. To demonstrate the combination effect, suboptimal dosing of test items was used. SOT 102 was administered once (Day 0) at dose 1 mg/mg i.v. and anti-PD-1 was administered three times (Day 0, 3 and 6) at dose 0.5 mg/kg i.p. In monotherapy groups, single dose of SOT102 alone at 1 mg/kg or DO, D3 and D6 dose of anti-PD-1 antibody alone at 0.5 mg/kg, tumor regressions were not observed (see Figure 2C, 2D), whereas the combination led to durable antitumor response that lasted up to day 40 (Figure 2E). The antibody or ADC vehicle solution (Vehicle, Figure 2A) and Isotype-PNU (Figure 2B) were used as treatment controls. Single and combination doses were well tolerated; body weights of the animals increased throughout the study (Figure 3).

Immunomodulatory responses to the treatments were evaluated from tumors analyzed by flow cytometry from day 4 (D4) samples. As shown in Figure 4, analysis of the CD45' cancer cells indicated that CLDN18.2 expression levels were not affected by any of the treatments. The analysis of the intra-tumoral immune cells between the different treatment arms revealed an increased immune cell infiltration, as shown by increased CD45⁺ cell population in both SOT 102 and SOT 102 + anti-PD-1 treatment groups compared to vehicle and isotype-PNU- 159682 groups (Figure 5). Further analysis of the immune cell subsets revealed that after combination treatment CD8⁺ T-cells exhibited a higher proliferation rate, as indicated by increase in Ki67 positivity (Figure 6). These results indicate that the combination of SOT102 and the anti-PD-1 antibody synergizes in the immunotherapeutic activity of each individual component, as the anthracy cline PNU- 159682 and the anti-PD-1 antibody are known to exhibit immunotherapeutic activity on their own, but not at the single agent doses used in these experiments (see Figure 2C, 2D and Figure 6).

PK analysis of the samples from the combination group revealed that the half-life of singledose SOT102 and total antibody was, 2.72 ± 0.39 and 2.93 ± 0.36 days, respectively (Figure 7).

Immunohistochemistry (IHC) staining of FFPE samples was performed on tumor samples at the endpoint of the studies (day 49, D49) to detect Ki67⁺ cells. An increase in Ki67⁺ cells was observed only in the samples from the combination treatment, and the increase was maintained over the duration of the treatment as Ki67⁺ cells were present in higher number at D4 (Figure 6, measured by flow cytometry) and D49 (Figure 8A, measured by IHC). Figure 8B shows that the increase in Ki67⁺ cells was only observed in the combination treatment and not in the SOT102 treatment alone underdosed at 1 mg/kg or the anti-PD-1 treatment alone. This increase in Ki67 positivity can be attributed mainly to the increase in dividing infiltrating immune cells and not in the increase in dividing tumor cells, as tumor regressions was observed in the combination study group. Embodiments

1. An anti-CLDN 18.2 antibody-drug conjugate (ADC) specifically binding to CLDN 18.2 for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti- CLDN 18.2 ADC in combination with an PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracy cline derivative.

2. A PD1/PD-L axis inhibitor for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated the PD- l/PD-L axis inhibitor in combination with an anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2, and wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracy cline derivative.

3. An anti-CLDN 18.2 antibody-drug conjugate (ADC) specifically binding to CLDN 18.2 and an PD-l/PD-L axis inhibitor for use in a method for treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti- CLDN 18.2 ADC and the PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti-CLDN18.2 antibody conjugated to an anthracy cline derivative.

4. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of embodiment 1 to 3, where the ADC has the general formula A-(L-T)_n, wherein A is an antibody, L is a linker and the toxin T is 3’-deamino-3”,4’-anhydro-[2”(S)-methoxy-3”(R)-oxy- 4”-morpholinyl]doxorubicin (PNU-159682), wherein n is an integer between > 1 and < 10.

5. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of embodiment 1 to 4, wherein the antibody A has been conjugated to the toxin T by means of a sortase enzyme, wherein the linker L at the C-terminus of the heavy and/or light chains of the antibody 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 the sortase recognition motif oligopeptide being - LPQTGG- or -LPETGG-. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of embodiment 1 to 5, wherein the linker L further comprises: a. a spacer element comprising a peptidic flexible oligopeptide between the C- terminus of the heavy and/or light chains and the antibody and the sortase recognition motif oligopeptide, preferably wherein the peptidic flexible oligopeptide consists of G and S, more preferably wherein the peptidic flexible oligopeptide is (GGGGS)_m with m being 1, 2, 3, 4 or 5, and b. at least one non-cleavable linker element or one cleavable element in C- terminus of the sortase tag, preferably wherein the non-cleavable linker element is selected from the group consisting of: i. ethylenediamine, and

wherein the cleavable linker element is selected from the group consisting of: iii. -[vc-PAB]-[N-formyl-N,N’-dimethylethylenediamine], and iv. -[vc-PAB]-[piperazine], The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of embodiments 1 to 6, wherein the toxin T is conjugated to the heavy and/or light chains on the antibody A. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of embodiments 1 to 7, wherein A is an anti-CLDN18.2 antibody or fragment thereof comprising the HCDR1, HCDR2 and HCDR3 sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 7 respectively. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, where the ADC consists of: a. the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 9, and two light chains of the amino acid sequence according to SEQ ID NO: 10, wherein the antibody binds to CLDN18.2, b. the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and c. 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_i3, resulting in the loss of C14 and of the hydroxyl group.

10. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, where the anti-CLDN18.2 ADC is administered to the patient to be treated at a dose between 0.016 mg/kg and 0.29 mg/kg every 2 weeks.

11. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, where the PD-l/PD-L inhibitor is an anti-PD-1 antagonistic antibody or an anti-PD-L blocking antibody.

12. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, where the anti-PD-1 antibody is Nivolumab, Pembrolizumab, Spartalizumab, Cemiplimab, Camrelizumab, Tislelizumab, MEDI-0680, SSI-361, Pidilizumab, Toripalimab, Dostarlimab, AGEN-2034, Sintilimab, BCD-100, Zimberelimab (GLS- 010) or AMP-224, or where the anti-PD-L antibody is Avelumab, Atezolizumab, Durvalumab, CX-072, BMS-936559 (MDX 1105), Sugemalimab (WBP-3155 or CS1001), Cosibelimab (CK-301) or KN035.

13. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, where 20-80% of the single agent efficacious dose of the ADC, preferably 50% of the dose is administered to the patient to be treated.

14. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding embodiments, a. where the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered at the same day or b. where the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered 1, 2, 3, 4, 5, 6 or 7 days apart.

15. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of preceding embodiments, where multiple cycles of the ADC and the PD-l/PD-L inhibitor are administered.

16. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of any preceding embodiments, where the cancer to be treated is pancreatic, gastric, esophageal, ovarian, biliary tract or lung cancer, preferably gastric or pancreatic cancer. 17. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of any preceding embodiments, where the treatment further comprises at least one cytotoxic agent.

18. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of embodiment 18, where the cancer to be treated is gastric cancer and where the cytotoxic agent is: a. a platinum-fluoropyrimidine doublet, b. paclitaxel, docetaxel, irinotecan or FOLFIRI (5 -fluorouracil (5-FU) - leucovorin - irinotecan) or c. FOLFOX (5-FU + oxaliplatin + leucovorin).

19. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of embodiment 18, where the cancer to be treated is pancreatic cancer and where the cytotoxic agent is: a. mFOLFIRINOX (oxaliplatin, irinotecan, leucovorin and 5 -fluorouracil (5-FU), b. gemcitabine and capeci tabine, or c. nab-paclitaxel and gemcitabine.

20. The anti-CLDN 18.2 ADC for use of any of embodiments any preceding embodiments, wherein the combination of the anti-CLDN18.2 ADC and the anti-PD-l/PD-L axis inhibitor exhibits less toxicity as compared to anti-CLDN18.2 ADC monotherapy.

21. The anti-CLDN18.2 ADC for use of any of embodiments 1-19, wherein the combination of the anti-CLDN18.2 ADC and the anti-PD-l/PD-L axis inhibitor exhibits reduced side-effects as compared to anti-CLDN18.2 ADC monotherapy.

22. The anti-CLDN18.2 ADC for use of any of embodiments 1-19, wherein the PD-l/PD- L axis inhibitor enhances efficacy of the anti-CLDN18.2 ADC.

Literature

BROGGINI, M. 2008. Nemorubicin. Top Curr Chem, 283, 191-206.

D'AMICO, L., MENZEL, U„ PRUMMER, M„ MULLER, P., BUCHI, M„ KASHYAP, A., HAESSLER, U„ YERMANOS, A., GEBLEUX, R., BRIENDL, M„ HELL, T., WOLTER, F. I., BEERLI, R. R., TRUXOVA, I., SPISEK, R., VLAJNIC, T., GRAWUNDER, U„ REDDY, S. & ZIPPELIUS, A. 2019. A novel anti-HER2 anthracy cline -based antibody-drug conjugate induces adaptive anti -tumor immunity and potentiates PD-1 blockade in breast cancer. J Immunother Cancer, 7, 16.

FU, Z., LI, S., HAN, S., SHI, C. & ZHANG, Y. 2022. Antibody drug conjugate: the "biological missile" for targeted cancer therapy. Signal Transduct Target Ther, 7, 93.

FUENTES-ANTRAS, J., GENTA, S„ VIJENTHIRA, A. & SIU, L. L. 2023. Antibody-drug conjugates: in search of partners of choice. Trends Cancer, 9, 339-354.

HEWITT, K. J., AGARWAL, R. & MORIN, P. J. 2006. The claudin gene family: expression in normal and neoplastic tissues. BMC Cancer, 6, 186. NGUYEN, T. D., BORDEAU, B. M. & BALTHASAR, J. P. 2023. Mechanisms of ADC Toxicity and Strategies to Increase ADC Tolerability. Cancers (Basel), 15.

NIIMI, T., NAGASHIMA, K., WARD, J. M„ MINOO, P., ZIMONJIC, D. B., POPESCU, N. C. & KIMURA, S. 2001. claudin-18, a novel downstream target gene for the T/EBP/NKX2.1 homeodomain transcription factor, encodes lung- and stomach-specific isoforms through alternative splicing. Mol Cell Biol, 21, 7380-90.

QUINTIERI, L., GERONI, C., FANTIN, M„ BATTAGLIA, R., ROSATO, A., SPEED, W., ZANOVELLO, P. & FLOREANI, M. 2005. Formation and antitumor activity of PNU- 159682, a major metabolite of nemorubicin in human liver microsomes. Clin Cancer Res, 11, 1608-17.

SAHIN, U„ KOSLOWSKI, M„ DHAENE, K., USENER, D., BRANDENBURG, G., SEITZ, G., HUBER, C. & TURECI, O. 2008. Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development. Clin Cancer Res, 14, 7624-34.

STEFAN, N„ GEBLEUX, R., WALDMEIER, L., HELL, T., ESCHER, M„ WOLTER, F. I., GRAWUNDER, U. & BEERLI, R. R. 2017. Highly Potent, Anthracycline-based Antibody- Drug Conjugates Generated by Enzymatic, Site-specific Conjugation. Mol Cancer Ther, 16, 879-892.

WANG, Y., DU, J., GAO, Z., SUN, H., MEI, M„ WANG, Y., REN, Y. & ZHOU, X. 2023. Evolving landscape of PD-L2: bring new light to checkpoint immunotherapy. Br J Cancer, 128, 1196- 1207.

XU, R.-H., WEI, X., ZHANG, D., QIU, M„ ZHANG, Y., ZHAO, H., CHEN, B. & YAN, J. 2023. A phase la dose-escalation, multicenter trial of anti-claudin 18.2 antibody drug conjugate CMG901 in patients with resistant/refractory solid tumors. Journal of Clinical Oncology, 41, 352-352.

YU, D. & TURNER, J. R. 2008. Stimulus-induced reorganization of tight junction structure: the role of membrane traffic. Biochim Biophys Acta, 1778, 709-16.

W02020/063988

WO202 1/025177

W02022/203090

W02000/015659

WO202 1/254481

WO2022/078523

WO2022/237666

WO202 1/088927

WO2022/111616

W02020/200196

W02020/135201

W02020/239005

W02021/011885

WO2022/012559

WO202/1111003

W02021/130291

Claims

1. An anti-CLDN18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2 for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated the anti-CLDN 18.2 ADC in combination with an PD-l/PD-L axis inhibitor, and wherein the ADC comprises an anti-CLDN 18.2 antibody conjugated to an anthracycline derivative.

2. A PD 1/PD-L axis inhibitor for use in a method of treating cancer in a patient, wherein the method comprises administering to the patient to be treated the PD-l/PD-L axis inhibitor in combination with an anti-CLDN 18.2 antibody-drug conjugate (ADC) specifically binding to CLDN18.2, and wherein the ADC comprises an anti-CLDN 18.2 antibody conjugated to an anthracycline derivative.

3. The anti-CLDN 18.2 ADC and the PD-l/PD-L axis inhibitor for use of claim 1 to 3, where the ADC has the general formula A-(L-T)_n, wherein A is an antibody, L is a linker and the toxin T is 3 ’ -deamino-3 ” ,4 ’ -anhydro-[2 ’ ’ (S)-methoxy-3 ’ ’ (R)-oxy-4 ’ ’ -morpholinyl] doxorubicin (PNU-159682), wherein n is an integer between > 1 and < 10.

4. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of claim 1 to 4, wherein the antibody A has been conjugated to the toxin T by means of a sortase enzyme, wherein the linker L at the C-terminus of the heavy and/or light chains of the antibody 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 the sortase recognition motif oligopeptide being -LPQTGG- or -LPETGG-.

5. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of claim 1 to 5, wherein the linker L further comprises: a. a spacer element comprising a peptidic flexible oligopeptide between the C-terminus of the heavy and/or light chains and the antibody and the sortase recognition motif oligopeptide, preferably wherein the peptidic flexible oligopeptide consists of G and S, more preferably wherein the peptidic flexible oligopeptide is (GGGGS)_m with m being 1, 2, 3, 4 or 5, and b. at least one non-cleavable linker element or one cleavable element in C-terminus of the sortase tag, preferably wherein the non-cleavable linker element is selected from the group consisting of: i. ethylenediamine, and ii.

wherein the cleavable linker element is selected from the group consisting of: iii. -[vc-PAB]-[N-formyl-N,N’-dimethylethylenediamine], and iv. -[vc-PAB]-[piperazine].

6. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of claims 1 to 6, wherein the toxin T is conjugated to the heavy and/or light chains on the antibody A.

7. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any of claims 1 to 7, wherein A is an anti-CLDN 18.2 antibody or fragment thereof comprising the HCDR1 , HCDR2 and HCDR3 sequences SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the LCDR1, LCDR2 and LCDR3 sequences SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 7 respectively.

8. The anti-CLDN 18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding claims, where the ADC consists of: a. the antibody consisting of two heavy chains of the amino acid sequence according to SEQ ID NO: 9, and two light chains of the amino acid sequence according to SEQ ID NO: 10, wherein the antibody binds to CLDN18.2, b. the linker [GGGGS]-[LPQTGG]-[ethylenediamine] at the C-terminus of the light chains, and c. 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 Ci₃, resulting in the loss of Ci₄ and of the hydroxyl group.

9. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding claims, where the anti-CLDN 18.2 ADC is administered to the patient to be treated at a dose between 0.016 mg/kg and 0.29 mg/kg every 2 weeks.

10. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding claims, where the PD-l/PD-L inhibitor is an anti-PD-1 antagonistic antibody or an anti-PD-L blocking antibody, optionally wherein the anti-PD- 1 antibody is Nivolumab, Pembrolizumab, Spartalizumab, Cemiplimab, Camrelizumab, Tislelizumab, MEDI-0680, SSI-361, Pidilizumab, Toripalimab, Dostarlimab, AGEN-2034, Sintilimab, BCD-100, Zimberelimab (GLS-010) or AMP-224, or where the anti-PD-L antibody is Avelumab, Atezolizumab, Durvalumab, CX-072, BMS- 936559 (MDX 1105), Sugemalimab (WBP-3155 or CS1001), Cosibelimab (CK-301) or KN035.

11. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding claims, where 20-80% of the single agent efficacious dose of the ADC, preferably 50% of the dose is administered to the patient to be treated.

12. The anti-CLDN18.2 ADC and the PD-l/PD-L axis inhibitor for use of any preceding claims, a. where the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered at the same day or b. where the 1^st dose of the ADC and the 1^st dose of the PD-l/PD-L inhibitor are administered 1, 2, 3, 4, 5, 6 or 7 days apart, optionally wherein multiple cycles of the ADC and the PD-l/PD-L inhibitor are administered.

13. The anti-CLDN 18.2 ADC and the PD-l/PD-L inhibitor for use of any preceding claims, where the cancer to be treated is pancreatic, gastric, esophageal, ovarian, biliary tract or lung cancer, preferably gastric or pancreatic cancer.

14. The anti-CLDN18.2 ADC and the PD-l/PD-L inhibitor for use of any preceding claims, where the treatment further comprises at least one cytotoxic agent, optionally wherein: a. the cancer to be treated is gastric cancer and where the cytotoxic agent is: i. a platinum -fluoropyrimidine doublet, ii. paclitaxel, docetaxel, irinotecan or LOLLIRI (5 -fluorouracil (5-LU) + leucovorin + irinotecan) or iii. LOLLOX (5-LU + leucovorin + oxaliplatin), or b. the cancer to be treated is pancreatic cancer and where the cytotoxic agent is: i. mFOLFIRINOX (5 -fluorouracil (5-FU) + leucovorin + irinotecan + oxaliplatin), ii. gemcitabine and capecitabine, or iii. nab-paclitaxel and gemcitabine.

15. The anti-CLDN18.2 ADC for use of any of claims any preceding claims, wherein the combination of the anti-CLDN18.2 ADC and the anti-PD-l/PD-L axis inhibitor exhibits less toxicity as compared to anti-CLDN18.2 ADC monotherapy.