WO2022128716A1

WO2022128716A1 - Pharmaceutical composition comprising a bispecific anti-muc1/egfr antibody-drug conjugate

Info

Publication number: WO2022128716A1
Application number: PCT/EP2021/084902
Authority: WO
Inventors: Matthias WINZER; Dirk Schiroky; Markus Weigandt; Senta VOSS
Original assignee: Merck Patent Gmbh
Priority date: 2020-12-17
Filing date: 2021-12-09
Publication date: 2022-06-23
Also published as: TW202241518A

Abstract

The present invention relates to a pharmaceutical composition comprising a bispecific anti-MUC1/EGFR antibody-drug conjugate (EGFR x MUC1 - Hemiasterlin bispecific antibody-drug conjugate (ADC)), a polyoxyethylene-polyoxypropylene block copolymer and a buffer, particularly a pharmaceutical preparation comprising the pharmaceutical composition in the form of an aqueous solution, a frozen solution or a lyophilizate, a method of making same, as well as medical uses thereof.

Description

PHARMACEUTICAL COMPOSITION COMPRISING A BISPECIFIC

ANTI-MUC1/EGFR ANTIBODY-DRUG CONJUGATE

The present invention relates to pharmaceutical composition comprising a bispecific anti-MUC1/EGFR antibody-drug conjugate (EGFR x MUC1 - Hemiasterlin bispecific antibody-drug conjugate (ADC)), particularly a pharmaceutical preparation comprising the pharmaceutical composition in the form of an aqueous solution, a frozen solution or a lyophilizate, a method of making same, as well as medical uses thereof.

Epidermal growth factor receptor (EGFR; also known as ErbB1 or HER1 ) is a transmembrane protein that is overexpressed in several epithelial cancers. Some EGFR mutations, including deletion mutations, point mutations, insertion mutations, and gene amplifications have been associated with cancer. Some EGFR mutations, as well as EGFR overexpression, are associated with poor prognosis and/or resistance to targeted EGFR inhibitors and other receptor tyrosine kinase inhibitors. Several novel pathways leading to escape from anti-EGFR therapy have recently been reported, highlighting the challenges of anti-EGFR therapy.

MUC1 , a Type I transmembrane glycoprotein, is expressed on many cancer cells, but also exhibits some expression in normal cells. In tumor cells, MUC1 co-localizes and interacts with EGFR, and their interaction blocks ligand-activated EGFR degradation. The bispecific antibody-drug conjugate of the pharmaceutical composition targets both MUC1 and EGFR. By targeting both MUC1 and EGFR with the same antibody, the presently disclosed antibody-drug conjugate not only enhance antibody internalization and tumor inhibition, they also enable higher specificity of binding to cancer cells, which may thereby reduce effects on normal cells.

The bispecific anti-MUCI/EGFR antibody-drug conjugates (ADCs) have therapeutic effects across a range of cancers, varying in tissue type, expression patterns for MLIC1 and EGFR, and EGFR mutational status. Moreover, they have demonstrated superior growth inhibition as compared to monospecific ADCs in various non-small cell lung cancer (NSCLC) patient- derived xenograft models.

The bispecific anti-MUC1/EGFR antibody-drug conjugate address both the lack of efficacy and the lack of tumor selectivity observed with some anti- EGFR therapeutics.

The bispecific anti-MUC1/EGFR antibody-drug conjugate comprises: (a) a bispecific antibody that binds to EGFR and MUC1 and (b) four hemiasterlin molecules. The bispecific antibody comprises: (i) a first polypeptide comprising a first engineered Fc domain and a single-chain Fv fragment (scFv), wherein the scFv binds to MUC1 , (ii) a second polypeptide comprising a second engineered Fc domain and a heavy chain of an Fab fragment, and (iii) a third polypeptide comprising a light chain of the Fab fragment; wherein the Fab fragment binds to EGFR. The first polypeptide and the second polypeptide are covalently linked by one or more disulfide bonds formed between the first engineered Fc domain and the second engineered Fc domain. The second polypeptide and the third polypeptide are covalently linked by one or more disulfide bonds formed between the heavy chain of the second polypeptide and the light chain of the third polypeptide. The antibodydrug conjugate is the only known bispecific ADC (simultaneous recognition of 2 antigens on 2 completely different targets or receptors) intended for clinical use.

An object of the invention is to provide viable pharmaceutical compositions of the bispecific anti-MUC1/EGFR antibody-drug conjugate. Unpredictabilities inherent in the art of formulating biologies, especially antibody- or antibody fragment-containing biologies, cause difficulties in the discovery of such viable pharmaceutical compositions, since most formulations of a given biopharmaceutical (if said formulation is arbitrarily chosen) are unstable over prolonged periods and/or under stressed conditions owing to the variety of degradation pathways open to biologies formulations, especially aqueous formulations. Degradation factors may, for instance, include one or more (typically two or more, and potentially three or more) of the following:

• Physical effects, such as: o Inadequate inhibition of aggregation of the relevant protein molecules; o Inadequate inhibition of precipitation; o Inadequate inhibition of adsorption of the relevant protein molecules at the interface of water and air or at the contact surface of any packaging material; o Inadequate regulation of osmotic pressure;

• Chemical effects, such as: o Inadequate regulation of oxidation; o Inadequate inhibition of photo-oxidation; o inadequate inhibition of hydrolysis of ester bonds leading to the formation of acid, aldehyde and peroxide products, thus affecting the stability of the antibody-drug conjugate; o Inadequate stabilisation and maintenance of pH; o Inadequate inhibition of protein fragmentation; o Inadequate inhibition of protein unfolding.

Any, some, or all of the above factors can lead to either an unviable drug product (which may be unsafe for use in medical treatments) or a drug product whose viability is variable and unpredictable, especially in view of the variable stresses (shaking, shearing, freeze-thawing, heat, light exposure) different batches of drug product may be exposed to during manufacture, transport, and storage.

Chemical compounds exhibit different physicochemical compared to biologies thus leading to further / other unpredictibilities for the formulation development. If such chemical compounds are conjugated with biologies, as it is the case for the bispecific anti-MUC1/EGFR antibody-drug conjugate used in the present invention, the formulation unpredictabilites are potentiated.

The anti-MUCI/EGFR antibody-drug conjugate comprises four hemiasterlin molecules each connected with the anti-MUCI/EGFR antibody with a SC239 linker as chemical compound. The SC239 linker- (hemiasterlin)payload is extremely hydrophobic and comprises with hemiasterlin one of the most hydrophobic payloads applied in ADCs (see Buecheler et al. Impact of Payload Hydrophobicity on Stability of Antibody- Drug-Conjugates, Molecular pharmaceutics, 2018). Thus, the good water solubility of the biologic part (anti-MUC1/EGFR antibody) strongly contrasts with the lipophilicity of chemical part (four linker-payloads) of the anti- MUCI/EGFR antibody-drug conjugate thereby increasing the challenge to provide a drug product the meets the requirements set forth herein.

The present invention preferably seeks to address one or more of the aforesaid stability issues and, in doing so, furnish viable pharmaceutical preparations.

Forced degradation studies and preformulated bispecific anti- MUCI/EGFR antibody-drug conjugate shipments in deep frozen state showed that the molecule is especially very prone to visible particle formation induced by dynamic stress such as shaking stress, shearing stress or by freeze/thawing stress (which is likely due to the segregation of water, excipients and drug during fractionated freezing and potentially due to upconcentration of the drug after separate freezing of water to ice).

The visible particle formation under dynamic stress and freeze/thawing stress is a challenging formulation issue. The formulation as a solid and virtually water-free drug product (freeze-dried lyophilizate) for stabilization against shaking during shelf life and storage of drug product cannot solve the issue since dynamic stresses cannot completely be prevented during manufacturing of drug product (pumping, mixing, filtration steps and lyophilisation) since freezing-stress is part of a lyophilisation process. Finally, clinical administration of bispecific anti-l\/IUC1/EGFR antibody-drug conjugate drug product means additional shear stress after reconstitution of the DP (e.g. push through thin 18 - 23g IV needles). Common formulation strategies for antibodies showed only partial stabilization and could not appropriately mitigate the particle formation.

While the antibody-drug conjugate is relatively stable under static storage at 2-8°C without any movement formation of particles is observed upon frozen storage.

For the clinical development and application of bispecific anti- MUC1/EGFR antibody-drug conjugate a particle free formulation is needed, which is stable over the entire lifetime of the drug including all drug product manufacturing steps (in particular after aseptic filtration as the last manufacturing step which is capable to remove particles >0.22pm), the whole drug product shelf life/storage and its clinical preparation and administration procedure such as dilution for infusion and clinical injection through thin needles.

Brief Description of the Figures

Figure 1A shows the structure of SC239, a linker-payload molecule used in the synthesis of Molecule 1 (a bispecific anti-MUC1/EGFR antibody-drug conjugate of the present disclosure). SC239 comprises a DBCO group, a Val- Cit-PAB cleavable linker, and 3-aminophenyl-hemiasterlin.

Figure 1 B shows a schematic depicting the structure of an antibody-drug conjugate of the present disclosure. In Molecule 1 , n (the number of SC239 moieties) is 4.

Figure 2 shows a schematic depicting Molecule 1 with its binding sites (MUC1-scFv and EGFR-Fab). The positions where the linker-payload (SC239) is conjugated to the antibody are shown as “o” together with the respective number in the amino acid sequence of the antibody. Unexpectedly it has been found that a pharmaceutical preparation, that meets all such requirements, could be provided if it comprises, beside the bispecific anti-MUC1/EGFR antibody, a polyoxyethylene-polyoxypropylene block copolymer and a buffer. Thus, one object of the invention is directed to a pharmaceutical composition comprising a bispecific anti-MUC1/EGFR antibody, a polyoxyethylene-polyoxypropylene block copolymer and a buffer. As used herein, “bispecific anti-MUC1/EGFR antibody-drug conjugate" is also designated in abbreviated form as “anti-MUC1/EGFR antibody-drug conjugate" or “antibody-drug conjugate".

The term "about", as used herein, refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term "about" generally refers to a range of numerical values (e.g., +/- 1-3% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term "about" may include numerical values that are rounded to the nearest significant figure.

As used herein, "a" or "an" shall mean one or more. As used herein when used in conjunction with the word "comprising," the words "a" or "an" mean one or more than one. As used herein "another" means at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

As used herein, “%” or “percent” shall mean percent by weight (% (w/w)), unless specified otherwise herein.

Bispecific anti-MUC1/EGFR antibody-drug conjugate

As used herein, the term “bispecific anti-MUC1/EGFR antibody-drug conjugate” refers to an antibody-drug conjugate comprising: (a) a bispecific antibody that binds to EGFR and MUC1 and (b) four hemiasterlin molecules. The bispecific antibody comprises: (i) a first polypeptide comprising a first engineered Fc domain and a single-chain Fv fragment (scFv), wherein the scFv binds to MUC1 , (ii) a second polypeptide comprising a second engineered Fc domain and a heavy chain of an Fab fragment, and (iii) a third polypeptide comprising a light chain of the Fab fragment; wherein the Fab fragment binds to EGFR. The first polypeptide and the second polypeptide are covalently linked by one or more disulfide bonds formed between the first engineered Fc domain and the second engineered Fc domain. The second polypeptide and the third polypeptide are covalently linked by one or more disulfide bonds formed between the heavy chain of the second polypeptide and the light chain of the third polypeptide.

As used herein, the term “bispecific anti-MUC1/EGFR antibody” within the antibody-drug conjugate comprise (i) a first polypeptide comprising a first engineered Fc domain and a single-chain Fv fragment (scFv), wherein the scFv binds to MUC1 ; (ii) a second polypeptide comprising a second engineered Fc domain and a heavy chain of an Fab fragment, and (iii) a third polypeptide comprising a light chain of the Fab fragment, wherein the Fab fragment binds to EGFR.

As used herein, the term “Fc domain” refers to a CH2 domain and a CH3 domain of an immunoglobulin. Thus, a homodimer or heterodimer of two Fc domains is an Fc fragment. Fc domains used in accordance with the disclosure may be engineered in the sense that they (1 ) comprise an engineered CH3 domain (as described herein) and/or (2) comprise one or more non-natural amino acids.

An Fc fragment comprises the carboxy-terminal portions of both heavy chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, the region which is also recognized by Fc receptors (FcR) found on certain types of cells.

As used herein, the term “scFv” is used in accordance with its common usage in the art to refer to a single chain in which the VH domain and the VL domain from an antibody are joined, typically via a linker.

As used herein, the term “Fab fragment” is used in accordance with its common usage in the art. Fab fragments typically comprise an entire light chain (VL and CL1 domains), the variable region domain of the heavy chain (VH), and the first constant domain of one heavy chain (CH1 ).

The first and second polypeptide each comprise at least one non-natural amino acid at a predetermined site or sites intended to be used for conjugation. Non-natural amino acid may be located, e.g., in an Fc domain, in the heavy chain of an Fab domain, or both. Non-limiting examples of suitable non-natural amino acids include p-acetyl-L-phenylalanine, O-methyl- L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L- phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p- iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxyphenylalanine, and p-azidomethyl- L-phenylalanine. (See, e.g., US Pat. No. 9,732,161.) In some embodiments, the non-natural amino acid is para-azidomethyl-L-phenylalanine (pAMF).

In the first polypeptide, the engineered Fc domain may be fused to the scFv, e.g., with a hinge region intervening between the CH2 domain of the engineered Fc domain and the VH domain of the scFv. In some embodiments, the first polypeptide has an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 1.

SEQ ID NO: 1 (anti-MUC1 scFvFc (AG SEED))

MQMQLVQSEAELKKPGASVKVSCKASGYSFTSHFMHWVRQAPGQGL EWMGWIDPVTGGTKYAQNFQGWVTMTRDTSIRTAYLELSRLRSDDTAMY YCAREARADRGQFDKWGQGTLVTVASGGGGSGGGGSGGGGSQSVLTQ PPSVeivltSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPALVIYYGSNR PSGIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDWVFGGGT KLTVLKPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPFRPEVHLLPPSREEMTK NQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAV

TSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISLSPGK

For example, the first polypeptide may have an amino acid sequence that is 100% identical to that set forth in SEQ ID NO: 11.

SEQ ID NO: 11 (anti-MUC1 scFvFc (AG SEED) with non-natural amino acids introduced at sites indicated by *)

MQMQLVQSEAELKKPGASVKVSCKASGYSFTSHFMHWVRQAPGQGL EWMGWIDPVTGGTKYAQNFQGWVTMTRDTSIRTAYLELSRLRSDDTAMY YCAREARADRGQFDKWGQGTLVTVASGGGGSGGGGSGGGGSQSVLTQ PPSVSVAPGKTARITCGGNNIGSKSVHWYQQKPGQAPALVIYYGSNRPS GIPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDWVFGGGTKL TVLKPKSSDKTHTCPPCPAPELLGGPSV*LFPPKPKDTLMISRTPEVTCW VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPFRPEVHLLPPSREEMTKNQ VSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTT*AVTSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKTISLSPGK

In the second polypeptide, the engineered Fc domain may be fused to the heavy chain of the Fab, e.g., with a hinge region intervening between the CH2 domain of the engineered Fc domain and the CH1 domain of the Fab fragment. In some embodiments, the second polypeptide has an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 2.

SEQ ID NO: 2 (anti-EGFR Fab(heavy)Fc (GA SEED))

MEVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGL

EWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCA

RALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLFSLSSVVTVPSSSL

GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF

PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR

EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ

PREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREK

YLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCSVMHEALHNHYTQK SLDRSPGK

For example, the second polypeptide may have an amino acid sequence that is 100% identical to that set forth in SEQ ID NO: 12.

SEQ ID NO: 12 ((anti-EGFR Fab(heavy)Fc (GA SEED) with non-natural amino acids introduced at sites indicated by *)

MEVQLVQSGAEVKKPGASVKVSCKASGFSLTNYGVHWMRQAPGQGL EWIGVIWSGGNTDYNTPFTSRVTITSDKSTSTAYMELSSLRSEDTAVYYCA RALTYYDYEFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL*SLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV*LF PPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPPSEELALNELVTLTCLVKGFYPSDIAVEWLQGSQELPREK YLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCSVMHEALHNHYTQK SLDRSPGK

In some embodiments, the third polypeptide has an amino acid sequence at least 99% identical to that set forth in SEQ ID NO: 3. In some embodiments, the third polypeptide has an amino acid sequence that is 100% identical to that set forth in SEQ ID NO: 3. SEQ ID NO: 3 (anti-EGFR Fab(light chain))

MDIQMTQSPSSLSASVGDRVTITCRASQSIGTNIHWYQQKPGKAPKLLI KYASESISGVPSRFSGSGYGTDFTLTISSLQPEDVATYYCQQNNNWPTTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH QGLSSPVTKSFNRGEC

Typically, the first polypeptide and second polypeptide are covalently linked by one or more disulfide bonds formed between the first engineered Fc domain and the second engineered Fc domain. The second polypeptide and the third polypeptide are also typically covalently linked by one or more disulfide bonds formed between the heavy chain of the second polypeptide and the light chain of the third polypeptide.

In some embodiments, the bispecific antibody is devised using a strandexchange engineered domains (SEED)-based CH3 heterodimer platform, as described, e.g., in US Pat. No. 8,891 ,912 and US Pat. No. 9,505,848. In this platform, each SEED-CH3 domain comprises alternating segments of human IgA and IgG sequences. The “AG SEED” refers to a CH3 domain that has an lgA1 sequence segment on the N-terminal end, while the “GA seed” refers to a CH3 that has an IgG 1 sequence segment on the N-terminal end. Each Fc heterodimer of a SEEDbody antibody comprises an AG SEED paired with a GA SEED.

Once constructs are designed for each chain (e.g., the first polypeptide, the second polypeptide, and the third polypeptide) of the bispecific anti- MUC1/EGFR antibody, constructs may also be mutagenized for the purpose of introducing non-natural amino acids (as discussed herein) at specific sites to be used as conjugation sites. These constructs may be expressed using any of a variety of expression systems known in the art.

In some embodiments, bispecific anti-MUC1/EGFR antibodies are produced using a cell-free system. Bispecific anti-MUC1/EGFR antibodies may have certain features reflecting how they were produced. For example, antibodies produced in a cell-free system may be aglycosylated and may lack effector functions.

Bispecific anti-MUCI/EGFR antibodies may optionally be purified before undergoing additional steps, such as conjugation.

Hemiasterlin molecules

Hemiasterlin is a tri-peptide isolated from marine sponges that binds to the vinca binding site on tubulin. Hemiasterlin may thereby inhibit tubulin polymerization, which can trigger mitotic arrest and apoptosis. As used herein, term “hemiasterlin molecule” refers to a hemiasterlin or a hemiasterlin derivative that retains at least some function of hemiasterlin (e.g., tubulin- binding). In some embodiments, the hemiasterlin derivative is 3- aminophenyl-hemiasterlin.

As described further herein, the number of hemiasterlin molecules per antibody-drug conjugate may be controlled by using a site-specific conjugation method in which hemiasterlin molecules are conjugated to nonnatural amino acids inserted at particular sites within a chain of the bispecific antibody. (See, e.g., International Pat. Publication WO 2019/055931.)

In some embodiments, each antibody-drug conjugate has four hemiasterlin molecules.

Conjugation and linkers

Conjugation reactions may be performed using functionalized linkerpayload molecule, wherein the linker is a cleavable linker. Copper-free click chemistry reactions may be used with certain functionalized groups. In a preferred embodiment, the antibody-drug conjugate is generated by reacting bispecific anti-MUCI/EGFR antibodies with the SC239 linker-payload molecule whose structure is depicted in Figure 1. SC239 comprises a 3- aminophenyl-hemiasterlin and a cleavable valine citrulline p-aminobenzyl carbamate (Val-Cit-PAB) linker functionalized with dibenzocyclooctyne (DBCO). (See, e.g., WO 2019/0055931 A1.) Preferably the bispecific anti-MUC1/EGFR antibody-drug conjugate is a bispecific antibody-drug conjugate consisting of:

• an aglycosylated bispecific protein scaffold as described above;

• protease cleavable bioconjugation linkers

• Hemiasterlin analogs (SC209)

While biparatopic ADC approaches for human use (simultaneous recognition of 2 epitopes of the same target or receptor) have been described in the scientific literature, the bispecific anti-MUC1/EGFR antibody-drug conjugate is the first bispecific ADC (simultaneous recognition of 2 antigens on 2 completely different targets or receptors) intended for clinical use.

A schematic illustration of the antibody-drug conjugate of a preferred embodiment is given in Figure 2. The average drug to antibody ratio (DAR) is appr. 4.

The pharmaceutical preparation can contain the antibody-drug conjugate in any amount up to 50 mg/ml, e.g. 0.5mg/ml, 1 mg/ml, 2mg/ml, 3mg/ml, 5mg/ml, 8mg/ml, 10mg/ml, 12.5mg/ml, 15mg/ml, 20mg/ml, 25mg/ml, 30mg/ml, 35mg/ml, 40mg/ml, 45mg/ml or 50mg/ml. According to an appropriate embodiment the antibody-drug conjugate is present in an amount from 1 mg/ml to 45mg/ml, from 2mg/ml to 40mg/ml, preferably from 3 to 30mg/ml or from 5 to 20mg/ml, more preferably from 5 to 15mg/ml and most preferably in an amount of about 10mg/mL

As used herein, the term “polyoxyethylene-polyoxypropylene block copolymer” refers to any block copolymer having at least one polyoxyethylene block and at least one polyoxypropylene block. Suitable polyoxyethylene-polyoxypropylene block copolymers are represented by the formula HO-(CH2-CH2O-)a-(CH2-CH(CH3)-O)b-(CH2-CH2-O)a-H, wherein a is an integer of from 10 to 200 and b is an integer of 10 to 100. Suitable polyoxyethylene-polyoxypropylene block copolymers include a variety of commercially available products sold under various trade names, including Synperonic PE Series (ICI); Pluronic™ Series (BASF), Lutrol (BASF). These polyoxyethylene-polyoxypropylene block copolymers are generically referred to as poloxamers. A preferred polyoxyethylene-polyoxypropylene block copolymer is poloxamer 188, having a polyoxypropylene molecular mass of 1800 g/mol and 80% polyoxyethylene content. Therefore, one embodiment of the invention is directed to the pharmaceutical composition, wherein the polyoxyethylene-polyoxypropylene copolymer is poloxamer 188.

The pharmaceutical composition preferably comprises polyoxyethylenepolyoxypropylene block copolymer at 5-300 mM, preferably 10-150 mM, or more preferably 25-75mM, and most preferably 30-60 mM or 60-75 mM.

Amounts and concentrations in relation to the polyoxyethylenepolyoxypropylene block copolymer relate to total amounts and concentrations of all inclided species, unless only a single antioxidant is stipulated.

As used herein, the term “buffer” refers to a mixture of an acid (usually a weak acid, e.g. citric acid, acetic acid) and its conjugate base (e.g. an citrate or acetate salt, for example, sodium acetate, sodium citrate, or histidine) or alternatively a mixture of a base (usually a weak base, e.g. histidine) and its conjugate acid (e.g. protonated histidine salt), which in aqueous solution have a “buffering effect” meaning that upon addition of a small quantity of strong acid or base the pH of the aqueous solution to only slightly changed.

According to suitable embodiments the buffer present in the pharmaceutical composition comprises of one or more citrate salt(s), acetate salt(s), histidine salt(s), succinate salt(s), malate salt(s), phosphate salt(s), tartrate salt(s), aconitate salts(s), adipate salt(s), bicarbonate salt(s), glutamate salt(s) or lactate salt(s) and/or the respective free acid or base thereof or a mixture of one or more of the various salts and/or the acid(s) or base(s) thereof. Accordingly, the present invention is further directed to the pharmaceutical composition, wherein the buffer comprises of one or more citrate salt(s), acetate salt(s), histidine salt(s), succinate salt(s), malate salt(s), phosphate salt(s), tartrate salt(s), aconitate salts(s), adipate salt(s), bicarbonate salt(s), glutamate salt(s) or lactate salt(s) and/or the respective free acid or base thereof or a mixture of one or more of the various salts and/or the acid(s) or base(s) thereof.

Preferably the buffer present in the pharmaceutical composition comprises one or more citrate salt(s) and/or the free acid thereof, acetate salt(s) and/or the free acid thereof or L-histidine and/or an acid-addition salt thereof, preferably L-histidine and/or an acid-addition salt thereof. Thus, the present invention is also directed to the pharmaceutical composition, wherein the buffer comprises one or more citrate salt(s) and/or the free acid thereof, acetate salt(s) and/or the free acid thereof or L-histidine and/or an acid-addition salt thereof, preferably L-histidine and/or an acid-addition salt thereof.

According to a preferred embodiment the pharmaceutical composition further comprises a sugar component. The sugar component can serve one or more functions within the pharmaceutical composition. For instance, they may serve as a lyoprotectant during freeze drying. A sugar component may impart tonicity, for instance, to bring the osmolality within a desired range (e.g. for isotonicity of a formulation for injection without prior dilution - e.g. an osmolality of 200-400 mOsmol/L, more preferably 250-350 mOsmol/L, most preferably 270-310 mOsmol/L).

Preferably, the sugar component is a non-reducing sugar component. The sugar component may also comprise one or more sugar(s) and/or sugar alcohol(s). In some embodiments, the sugar component comprises one or more sugar(s) or one or more sugar alcohol(s). Preferably, the sugar component consists of a single compound. In other embodiments, the sugar component comprises at most one sugar or at most one sugar alcohol. Preferably, the sugar component is selected from the group consisting of trehalose, sucrose, mannitol, and sorbitol, more preferably from trehalose or sucrose.

The sugar component can be a sugar, which may be selected from the group consisting of a monosaccharide, a disaccharide, a polysaccharide, and a complex carbohydrate. According to a preferred embodiment of the invention the sugar component is a disaccharide, such as trehalose or sucrose, preferably trehalose.

In some embodiments, the sugar component is a sugar alcohol, such as a (3-12C) sugar alcohol, a (3-6C) sugar alcohol, or a (5-6C) sugar alcohol selected from mannitol, sorbitol, arabitol, xylitol, ribitol, and inositol. In some embodiments, the sugar component is a sugar alcohol selected from mannitol or sorbitol.

The pharmaceutical composition preferably comprises 30-400 mM sugar component, 40-300 mM sugar component, 40-100 mM sugar component, 40-60 mM sugar component, 90-290 mM sugar component, 100-200 mM sugar component, 130-170 mM sugar component, 200-300 mM sugar component, 210-270 mM sugar component, about 240 mM sugar component, about 230 mM sugar component or about 234 mM sugar component.

The pharmaceutical composition preferably comprises a sugar component in a molar ratio of sugar component to antibody-drug conjugate of from 7300:1 to 50:1 , of from 5700:1 to 70:1 , of from 1000:1 to 100:1 , of from 1000:1 to 500:1 , of from 500:1 to 180:1 , of from 2900:1 to 2700:1 , preferably of from 4000:1 to 2000:1 , of from 3500:1 to 2500:1 , or of from 3100:1 to 2900:1.

The pharmaceutical composition preferably comprises 40-300 mM trehalose, 40-100 mM trehalose, 40-60 mM trehalose, 65-85 mM trehalose, 70-130 mM trehalose, 90-110 mM trehalose, 100-200 mM trehalose, 140- 180 mM trehalose, or 150-170 mM trehalose. In some embodiments, the pharmaceutical composition comprises trehalose at a concentration of about 50 mM, about 75 mM, about 100 mM or about 159 mM. The pharmaceutical composition preferably comprises trehalose in a molar ratio of trehalose to antibody-drug conjugate of from 7300:1 to 50:1 , of from 3700:1 to 70:1 , of from 1000:1 to 100:1 , of from 1000:1 to 500:1 , or of from 500:1 to 180:1. Most preferably the pharmaceutical composition comprises trehalose as the sole sugar component, most preferably at a concentration of 40-200 mM.

The pharmaceutical composition preferably comprises 50-300 mM sucrose, 150-290 mM sucrose, 220-280 mM sucrose, or 240-260 mM sucrose.

The pharmaceutical composition preferably comprises 40-300 mM mannitol, 40-100 mM mannitol, 40-60 mM mannitol, 80-120 mM mannitol, 100-200 mM mannitol, 210-290 mM mannitol, or 240-260 mM mannitol.

The pharmaceutical composition preferably comprises 40-300 mM sorbitol, 40-100 mM sorbitol, 40-60 mM sorbitol, 80-120 mM sorbitol, 100- 200 mM sorbitol, 210-290 mM sorbitol, or 240-260 mM sorbitol.

Amounts and concentrations in relation to the sugar component relate to total amounts and concentrations of all sugar components, unless only a single sugar component is stipulated. Concentrations of the or any particular sugar component can be replaced by molar ratios.

According to an advantageous embodiment the pharmaceutical composition further comprises an antioxidant. An antioxidant may inhibit oxidative degradation pathways open to the antibody-drug conjugate and/or other components within the pharmaceutical composition. For instance, an antioxidant may inhibit oxidation of the antibody-drug conjugate, for example by inhibiting oxidation of certain oxidizable amino acid residues within the antibody-drug conjugate, and/or inhibit oxidative deamination pathways. The antioxidant may be selected from the group consisting of an amino acid or peptide antioxidant, a mineral antioxidant, a vitamin antioxidant, a carotenoid antioxidant, a polyphenol antioxidant, an aromatic or phenolic antioxidant, a chelating agent antioxidant, a thiol antioxidant, and any combination thereof. Preferably, the antioxidant is a single compound.

According to a preferred embodiment, the antioxidant comprises an amino acid antioxidant selected from the group consisting of methionine, N- acetyl-l-cysteine, cysteine and glutathione. Thus, the invention is also directed to a pharmaceutical composition, wherein the antioxidant is methionine, N-acetyl-l-cysteine, cysteine or glutathione, preferably methionine.

The pharmaceutical composition preferably comprises 0.5-50 mM antioxidant, 1-30 mM antioxidant, 2-20 mM antioxidant, 3-10 mM antioxidant, or 4-6 mM antioxidant.

The pharmaceutical composition preferably comprises an antioxidant in a molar ratio of antioxidant to antibody-drug conjugate of from 910:1 to 1 :6, of from 365:1 to 3:1 , of from 182:1 to 5:1 , of from 150:1 to 7:1 , or of from 100:1 to 10:1.

The pharmaceutical composition preferably comprises 1-20 mM methionine, 2-12 mM methionine, 2-8 mM methionine, 4-6 mM methionine, or about 5 mM methionine.

The pharmaceutical composition preferably comprises methionine in a molar ratio of methionine to antibody-drug conjugate of from 910:1 to 1 :6, of from 365:1 to 3:1 , of from 182:1 to 5:1, of from 100:1 to 7:1 , or of from 50:1 to 10:1.

Amounts and concentrations in relation to the antioxidant relate to total amounts and concentrations of all antioxidants, unless only a single antioxidant is stipulated. Concentrations of the or any particular antioxidant can be replaced by molar ratios. An appropriate embodiment the pharmaceutical composition of the invention comprises, beside the antibody-drug conjugate, Poloxamer P188 as polyoxyethylene-polyoxypropylene block copolymer, histidine buffer as buffer, trehalose as disaccharide and methionine as antioxidant. Accordingly, the invention is also directed to a pharmaceutical composition, wherein the polyoxyethylene-polyoxypropylene block copolymer is Poloxamer P188, the buffer is a histidine buffer, the disaccharide is trehalose and the antioxidant is methionine.

The pharmaceutical composition can be provided in the form a pharmaceutical preparation an aqueous solution, a frozen aqueous solution or a lyophilizate. Therefore, the invention is as well directed to a pharmaceutical preparation comprising the pharmaceutical composition, wherein the pharmaceutical preparation is an aqueous solution, a frozen aqueous solution or a lyophilizate.

The term “pharmaceutical composition”, as used herein, only refers to the qualitative and/or quantitative composition of the ingredients contained therein and does not contain any information relating to its form of appearance, such as the case may be, a liquid solution or a dry mixture.

The term “pharmaceutical preparation” as used herein, refers to a pharmaceutical product, which comprises the pharmaceutical composition and which is dedicated to be administered to a person or animal in need thereof directly or after its conversion in a usable form, such as, for example by thawing or diluting. In the present invention the pharmaceutical preparation may be an aqueous solution, a frozen aqueous solution or a lyophilizate. Whereas an aqueous solution might be usable directly without any further steps (but may be also further processed e.g. by dilution with an appropriate diluent, such as, for example, physiologic saline), a frozen aqueous solution or lyophilizate is preferable converted to a liquid prior to its application to the person or animal in use thereof by thawing or diluting with an appropriate diluent, such as, for example, physiologic saline, respectively. "Aqueous" above and below is taken to mean water or mixtures of water with other solvents, in particular organic solvents. If one or more substances are dissolved in water or in the mixtures of water with other solvents, aqueous solutions are present. If the aqueous solution comprises one or more active compounds and if it is suitable for therapeutic or prophylactic use in humans or animals, an aqueous pharmaceutical composition is present. If the aqueous solution/aqueous pharmaceutical composition comprises organic solvents, the latter are preferably those which are suitable for parenteral use, such as dimethyl sulfoxide (DMSO), but in particular alcohols, such as, for example, ethanol, 1 ,2-propanediol, glycerol, polyethylene glycols and glycofurol.

If a liquid composition comprises exclusively organic solvents or the organic solvents present, naturally and in undiluted form, already have a certain water content (thus, for example, pure ethanol has a water content of about 4 per cent), an aqueous composition is not present. If, however, water or an aqueous solution (for example physiological saline solution) is added to a composition of this type, an aqueous composition in the sense of the present application arises.

Above and below, "water" means the solvent water or a solution with water as solvent, for example a solution of water comprising adjuvants, such as isotonic agents, such as, for example, sodium chloride, sodium phosphates or glucose. An example of a solution with water as the sole solvent is physiological saline solution.

The term “frozen aqueous solution” as used herein, is an aqueous solution which has been converted to a solid state of aggregation by applying temperatures below 0°Celsius.

It will be appreciated that pharmaceutical compositions of the invention may be provided, and preferably duly stored as in the form of an aqueous solution. However, the pharmaceutical composition of the invention may be provided, and preferably duly stored, as a frozen solution or lyophilizate. A frozen solution may be formed by applying to the liquid a temperature below 0°Celsius and thereby converting it to a solid state of aggregation. Preferably any such frozen solution will be thawed prior to use, administration, or even (potentially short-term) storage to afford a liquid or aqueous solution as defined herein. A lyophilised solution may be formed by freeze-drying a liquid (e.g. aqueous) solution as defined herein. Most preferably any such lyophilised solution will be reconstituted prior to use, administration, or even (potentially short-term) storage to afford a liquid or aqueous pharmaceutical solution as defined herein. Such reconstitution preferably comprises dissolving the lyophilised solution in water (for injection), preferably to afford a solution of a desired concentration of the fusion protein.

Freezing to a frozen solution or removal of the water by freeze-drying enables the stability of the aqueous conjugate solutions, which are already relatively stable per se, to be increased still further.

According to an advantageous embodiment the pharmaceutical preparation is an aqueous solution. Therefore, the invention is also directed to a pharmaceutical preparation, which is characterized by that it is an aqueous solution.

According to an appropriate embodiment the aqueous solution has a pH of 4.8 to 7.0, preferably of 5.0 to 6.0, more preferably of 5.3 to 5.7, and most preferred of about 5.5. Thus, the invention is further directed to a pharmaceutical preparation, which is characterized by that it has a pH of 4.8 to 7.0, preferably of 5.0 to 6.0, more preferably of 5.3 to 5.7, and most preferred of about 5.5.

Advantageously, the aqueous solution has a pH of 4.8 to 7.0 and comprises a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, and optionally an antioxidant. Hence, the invention is further directed to a pharmaceutical preparation, which is characterized by that it has a pH of 4.8 to 7.0 and comprises a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, and optionally an antioxidant. According to a preferred embodiment of the invention the pharmaceutical preparation comprises a histidine buffer as buffer, trehalose as disaccharide and methionine as antioxidant. Therefore, the inventions is also directed to a pharmaceutical preparation, wherein the buffer is a histidine buffer, the disaccharide is trehalose and the antioxidant is methionine.

In one embodiment the pharmaceutical preparation has a pH of 5.3-5.7 and comprises 5-15 mg/mL bispecific anti-MllC1/EGFR antibody-drug conjugate, 5-15 mM buffer, 0.4-2.5 % (w/v) poloxamer 188, 5-15 % (w/v) disaccharide, and optionally 2-8 mM antioxidant. Accordingly, the invention is also directed to a pharmaceutical preparation, which is characterized by that it has a pH of 4.8 to 7.0, preferably of 5.0 to 6.0, more preferably of 5.3 to 5.7, and most preferred of about 5.5.

In an advantageous embodiment the pharmaceutical preparation is a lyophilizate and is reconstitutable to an aqueous solution as described herein. Thus, the invention is as well directed to a pharmaceutical preparation, which is a lyophilizate and is reconstitutable to an aqueous solution.

The lyophilizate can be prepared by preparing an aqueous preparation comprising bispecific anti-MUC1/EGFR antibody-drug conjugate, a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, optionally an anioxidant, and, if desired, further pharmaceutical auxiliaries, and subsequently lyophilizing said aqueous preparation. Hence, the invention is also directed to a process for the preparation of the lyophilized pharmaceutical preparation, which is characterized in that an aqueous preparation comprising bispecific anti-MUC1/EGFR antibody-drug conjugate, a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, optionally an anioxidant, and, if desired, further pharmaceutical auxiliaries is prepared, and such aqueous preparation is subsequently lyophilized. In the following, the present invention will be described by reference to exemplary embodiments thereof, which shall not be regarded as limiting the invention.

PREPARATION ANTIBODY-DRUG CONJUGATE

Example 1. Sequence optimization and affinity maturation to develop an anti-MUC1 scFv sequence

An anti-MUC1 scFv (H02) was developed by affinity maturation of anti- MUC1 antibody HT186-D11 (see Thie H. et al. PLoS One 201, 6, 1, e15921) using ribosome display selection. For the scFv library, CDRs H1, H2, H3 and L3 (SEQ ID NOs: 4, 5, 6, and 9) were targeted.

SEQ ID NO: 4 (CDRH1 for HT186-D11 and 1993 H02)

GY[SP]F[TN][GDS][HN][YF]MH

SEQ ID NO: 5 (CDRH2 for HT186-D11 and 1993 H02)

WIDPVTG[GE]T[KR]YAQ[ND]FQG

SEQ ID NO: 6 (CDRH3 for HT186-D11 and 1993 H02)

E[VA][TR][GA][DS]RGQFDK

SEQ ID NO: 9 (CDRL3 for HT186-D11 and 1993 H02)

QVWDSSSDWV scFv ribosome display selections were then performed against a biotinylated synthetic VNTR peptide of MUC (APDTRPAPGSTAPPAC- biotin) (SEQ ID NO: 10).

Antibody variants were screened and characterized based, among other things, on binding to MUC1 -expressing cells (WISH, MDA-MB-468, and OVCAR-3 cancer cells, with HepG2 cells as MUC1 -negative controls) (see Example 4 for additional details), binding to a biotinylated synthetic VNTR peptide of MUC1 (APDTRPAPGSTAPPAC-biotin; SEQ ID NO: 10), association kinetics, stability in storage, and cell killing of MUC-1 positive cells by a drug conjugate of the antibody variant (ADC).

SEQ ID NO: 10 (VNTR peptide of MUC1 )

APDTRPAPGSTAPPAC

ADCs were generated by site-specific conjugation using a cell-free expression system and conjugation to SC239 (a cleavable linker- [hemiasterlin]-payload derivative, see Example 3 for details regarding SC239).

Based on these studies, antibody variant “1993-H02” (hereinafter H02) was chosen and developed as an scFv. A summary of the H02 sequence’s binding characteristics is provided in Table 1A; a summary of results from cell killing assays is provided in Table 1B.

Table 1A: H02 characterization for 1993-H02 with an HT186-D11 light chain

Table 1B: ADC cell killing assay results for 1993 H02 conjugated to

SC239 (DAR = 4)

NK = no killing

Example 2. Construction of bispecific anti-MUC1/EGFR antibodies

Bispecific anti-MUC1/EGFR antibodies were developed using a strandexchange engineered domains (SEED)-based CH3 heterodimer platform. (See, e.g., in US Pat. No. 8,891 ,912 and US Pat. No. 9,505,848).

A bispecific antibody (hereinafter “Molecule 10”) was designed as a heterodimer of:

- an anti-MUC1 scFv (H02) fused to a human lgG1 Fc (AG SEED); and

- an anti-EGFR Fab (derived from humanized cetuximab) fused to a human lgG1 Fc (GA SEED).

Expression constructs encoding the anti-MUC1 scFvFc (AG SEED), the heavy chain of the anti-EGFR Fab fused to the IgG 1 Fc (GA SEED), and the light chain of the anti-EGFR Fab were constructed. Upon protein expression and heterodimer formation, the resulting product is a bispecific anti-MUC1/EGFR antibody (H02/hC225 SEED, or “Molecule 10”):

For purposes of conjugation site optimization studies described in Example 3, similar methods were used to construct a similar bispecific anti- MUC1/EGFR antibody (D11/hC225). In D11/hC225, the anti-MUC1 arm was based on the HT186-D11 scFv (the parental sequence from which H02 was developed; see Example 1) fused to a human lgG1 Fc (AG SEED), and the anti-EGFR arm was based on the Fab of humanized cetuximab (hC225) fused to an lgG1 Fc (GA seed

Example 3. Synthesis of a Molecule 1, a bispecific anti-MUCI/EGFR antibody conjugated to 3-aminophenyl-hemiasterlin (“bispecific anti- MUC1/EGFR ADC”)

The XpressCF+™ (Sutro Biopharma) cell-free expression system and site-specific conjugation method (see, e.g., U.S. Pat. No. 9,732,161 and International Patent Publication No. WO 2019/055931 A1) was used to generate antibody-drug conjugates based on the bispecific anti- MUCI/EGFR antibody H02/hC225 SEED (Molecule 10) described in Example 1.

For initial experiments to optimize conjugation sites, the anti-MUC1 arm of D11/hC225 (AG SEED) and the heavy chain of the anti-EGFR arm of D11/hC225 (GA SEED) (see Example 1) were mutagenized by incorporating the non-natural amino acid para-azido methyl L-phenylalanine (pAMF) at TAG sites (amber stop codons). A series of mutants were generated for each arm (anti-MUC1 scFvFc (AG SEED) arm or anti-EGFR Fab(heavy chain)Fc (GA SEED) arm), each mutant having only one pAMF residue incorporated. The pAMF residues in each mutant arm were conjugated to a hemiasterlin derivative by copper-free click chemistry using SC239, which comprises a tubulin-targeting 3-aminophenyl hemiasterlin and a cleavable valine citrulline p-aminobenzyl carbamate (Val-Cit-PAB)

linker functionalized with dibenzocyclooctyne (DBCO). (See, e.g., WO 2019/0055931 A1.)

SC239 has the structure shown in Formula I:

DBCO cleavable linker 3-aminophenyl-hemiasterlin

Formula I

Conjugated anti-MUC1 scFvFc (AG) and anti-EGFR Fab(heavy chain)Fc (GA) arms were separately tested in vitro for binding to MUC1 and EGFR, respectively, and for MDA-MB-468 (human breast cancer) cell killing. Combinations of anti-MUC1 scFvFc (AG) and anti-EGFR Fab(heavy chain)Fc (GA) arms were also tested in vitro for binding to EGFR, binding to MUC1 , and MDA-MB-468 cell killing. Factors affecting manufacturability, such as protein expression, yield, and thermostability, were also taken into consideration.

Based on results from conjugation site optimization studies, the following conjugation sites were chosen. (All positions are numbered according to the EU index.)

- Heavy chain position F404 on the anti-MUC1 scFv;

- Heavy chain position F241 on the anti-MUC1 scFv; - Heavy chain position F241 on the anti-EGFR Fab; and

- Heavy chain position F180 on the anti-EGFR Fab

Using these conjugation sites, an ADC having the structure shown in Figure 1 B (with n =4) was synthesized using sequences for the H02/hC225 SEED bispecific antibody (Molecule 10) described in Example 2. This ADC (hereinafter “Molecule 1”) has a drug-antibody-ratio of approximately 4 and comprises a bispecific antibody having an anti-MUC1 scFvFc (AG SEED), an anti-EGFR Fab(heavy chain)Fc (GA SEED), and an anti-EGFR Fab (light chain), the H02/hC225 SEED bispecific antibody being conjugated at each of the above-mentioned conjugation sites to a 3-aminophenyl- hemiasterlin molecule via the Val-Cit-PAB cleavable linker.

Formulation examples

Formulations of Molecule 1 are checked by the following dynamic stress tests or freeze/thawing methods in order to trigger formation of visible particles:

Method A - shaking stress: 5 days at 200rpm on a horizontal shaker in sealed 1ml_ pharmaceutical clear glass vials with rubber stoppers including a 1ml_ fill of formulations containing Molecule 1

Method B - shearing stress: formulations containing drug are sheared in a manual pipet with piston in direct contact with the product solution (indirect pipetting via air displacement is not an appropriate method). 1 shearing cycle consists of 1 drawing of the sample into the pipet followed by expelling it from the pipet.

Method C - in-use stress: formulations containing drug are stressed by manual drawn and expelled through a 23g intravenous (IV) injection needle

Method D - freeze/thawing stress: drug formulations are frozen and stored in deep freezers at <-60°C and subsequently thawn at ambient conditions (e.g. 25°C) without movement at a lab bench. This stress test has been applied to formulation sample volumes of <1ml_. It has been confirmed later on for a selected stabilized formulation in volumes of up to 800ml_.

The methods are used to investigate on relevant stresses during the lifetime of Molecule 1 in drug product formulation covering manufacturing, storage and eventually the intravenous application.

Detection of visible particles

Visible particles are monitored by visual inspection using the following method, which was adjusted from the methods described in US and European pharmacopeias (USP<790> and EP 5.17.2):

• Inspection conducted in a dark room

• Visual inspection against a black and white background • Gently swirl and /or invert each container with drug

• Ensure that no air bubbles impact the inspection (let air bubbles lift for some seconds)

• Inspect at least 5 seconds with each of the backgrounds by using a cold light illumination (Schott KL1500LCD with intensity setting 5

[3200K] with light cone setting C). In addition, a Waldmann LX111 magnifying glass for lab purposes was used to identify very small particles that may appear in the first stress cycles. Formulations of Molecule 1 that fail adequate stabilization against formation of visible particles upon dynamic stress or freeze/thawing acc. to method A, B, C or D are listed in Table 1.

Formulations that appropriately stabilize Molecule 1 in lyophilized, liquid and frozen state against formation of visible particles upon dynamic stress or freeze/thawing acc. to method A, B, C or D are listed in Table 2.

Table 2

Further formulations that stabilize Molecule 1 at least in liquid state against formation of visible particles upon dynamic stress or freeze/thawing (method A, B, C or D) are listed in Table 3. Lyophilized or frozen dosage forms of these formulations listed in table 3 are not preferred due to the choice of excipients and the phenomenons decribed hereafter.

Table 3

Freeze-drying/lyophilization was done with a freezing-step to -40°C, a sublimation phase (primary drying) at -10 to -23°C shelve temperature and an evaporation phase (secondary drying) at up to +40°C shelve temperature. The chamber vacuum was kept at <100pbar.

Lyophilized formulations of Molecule 1 are most stabilized. Table 4 summarizes the observations over 10 draw/expel cycles with Method C - in-use stress in Formulation 3 (see Table 1) and Formulation 1 , 2, 6 and 7 (see Table 2). In the table no visible particles are indicated as NVP, small visible particles as SVP and large visible particles as LVP.

Table 4

Table 5 summarizes the observations over 10 draw/expel cycles with Method B - shearing stress in Formulation 3 (see Table 1) and Formulation 1 , 2, 6 and 7 (see Table 2). In the table no visible particles are indicated as

NVP, small visible particles as SVP and large visible particles as LVP.

Table 5

The results show that Polysorbate 20 slightly stabilizes against the particle formation whereas the effect of Poloxamer 188 is superior. Other Molecule 1 quality attributes do not change significantly over the shear stress applied. Particles formation is the most pronounced degradation route of Molecule 1.

Claims

36

Patent Claims A pharmaceutical composition comprising a bispecific anti-MUC1/EGFR antibody-drug conjugate, a polyoxyethylene-polyoxypropylene block copolymer and a buffer. The pharmaceutical composition according to claim 1 , wherein the polyoxyethylene-polyoxypropylene copolymer is poloxamer 188. The pharmaceutical composition according to claim 1 or 2, wherein the buffer comprises of one or more citrate salt(s), acetate salt(s), histidine salt(s), succinate salt(s), malate salt(s), phosphate salt(s), tartrate salt(s), aconitate salts(s), adipate salt(s), bicarbonate salt(s), glutamate salt(s) or lactate salt(s) and/or the respective free acid or base thereof or a mixture of one or more of the various salts and/or the acid(s) or base(s) thereof. The pharmaceutical composition according to claim 3, wherein the buffer comprises one or more citrate salt(s) and/or the free acid thereof, acetate salt(s) and/or the free acid thereof or L-histidine and/or an acid-addition salt thereof, preferably L-histidine and/or an acid-addition salt thereof. The pharmaceutical composition according any one of claims 1 to 4, further comprising a sugar component. The pharmaceutical composition according to claim 5, wherein the sugar component is a disaccharide, such as trehalose or sucrose, preferably trehalose. The pharmaceutical composition according to any one of claims 1 to 6, further comprising an antioxidant. 37

8. The pharmaceutical composition according to claim 7, wherein the antioxidant is methionine, N-acetyl-l-cysteine, cysteine or glutathione, preferably methionine.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the polyoxyethylene-polyoxypropylene block copolymer is Poloxamer P188, the buffer is a histidine buffer, the disaccharide is trehalose and the antioxidant is methionine.

10. A pharmaceutical preparation comprising the pharmaceutical composition according to any one of claims 1 to 9, wherein the pharmaceutical preparation is an aqueous solution, a frozen aqueous solution or a lyophilizate.

11 . The pharmaceutical preparation according to claim 10, which is characterized by that it is an aqueous solution.

12. The pharmaceutical preparation according to claim 11 , which is characterized by that it has a pH of 4.8 to 7.0, preferably of 5.0 to 6.0, more preferably of 5.3 to 5.7, and most preferred of about 5.5.

13. The pharmaceutical preparation according to claim 11 or 12, which is characterized by that it has a pH of 4.8 to 7.0 and comprises a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, and optionally an antioxidant.

14. The pharmaceutical preparation according to any one of claims 10 to 13, wherein the buffer is a histidine buffer, the disaccharide is trehalose and the antioxidant is methionine.

15. The pharmaceutical preparation according any one of claims 11 to 14, which is characterised by a pH of 5.3-5.7 and comprises 5-15 mg/mL bispecific anti-MUC1/EGFR antibody-drug conjugate, 5-15 mM buffer, 0.4-1 .2 % (w/v) poloxamer 188, 5-15 % (w/v) disaccharide, and optionally 2-8 mM antioxidant.

16. The pharmaceutical preparation according to claim 10, which is a lyophilizate and is reconstitutable to an aqueous solution according to any one of claims 11 to 15.

17. Process for the preparation of the lyophilized pharmaceutical preparation according to claim 16, characterized in that an aqueous preparation comprising bispecific anti-MUC1/EGFR antibody-drug conjugate, a polyoxyethylene-polyoxypropylene block copolymer, a buffer, a disaccharide, optionally an anioxidant, and, if desired, further pharmaceutical auxiliaries is prepared, and such aqueous preparation is subsequently lyophilized.

18. The pharmaceutical preparation according to one or more of Claims 11 to 16 for use in the treatment of cancer.