Formulation for antibody constructs

Abstract

The present invention relates to formulations which stabilize antibody constructs, more specifically, antibody constructs which do not have inter-chain covalent bond.

Description

FORMULATION FOR ANTIBODY CONSTRUCTSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority of European patent application EP 23212923.9 filed on 29 November 2023, the content of which is hereby incorporated by reference in its entirety for all purposes.FIELD OF THE INVENTION

[0002] The present invention relates to formulations which stabilize antibody constructs, more specifically, antibody constructs which do not have inter-chain covalent bond.BACKGROUND

[0003] Natural killer cells are cytotoxic, IFN-y and TNF-a producing innate lymphoid cells that are considered the first line of defense against virus-infected cells and cancer cells. The cytotoxic potential of NK cells can be utilized in cancer immunotherapy by redirecting NK cell lysis to tumor cells and stimulating the activating receptor CD16A, also known as FcyRIIIA, expressed on the surface of NK cells. NK cells are equipped with multiple activating and inhibitory receptors on their surface jointly regulating NK cell activation and triggering of effector functions. Several of these receptors play a pivotal role for NK cell mediated recognition, killing of cancer cells and cytokine secretion. CD16A activation promotes NK cell proliferation and memory-like cytotoxicity against cancer cells. Upon ligation, CD16A induces a potent series of signals resulting in cytokine production and cytotoxic effector activity via antibody dependent cellular cytotoxicity (ADCC). In this respect, tumor-specific monoclonal antibodies (mABs), such as rituximab, that recognize tumor- selective antigens, such as CD20, on the surface of tumor cells are described to induce NK cell-mediated anti-tumor activity via ADCC.

[0004] Moreover, directing NK cells for tumor cell lysis using bi- or multispecific antibodies is considered a potent immunotherapeutic approach and offers opportunities for increasing specificity, potency, and utilizing novel mechanisms of action. Aside from the natural IgG format, various formats of multispecific NK cell antibodies have been developed, such as BiKE and TandAb.

[0005] While the NK cell antibodies are a promising new method for treating cancer. There are several challenges that need to be tackled during manufacturing to deliver a drug to treat patients. For instance, there is a need to improve the stability of the antibody constructs in theirfinal formulations. After the final purification step in the downstream process, the antibody construct is formulated in a liquid form which allows freezing of the drug substance. For drug product manufacture the antibody construct could be filled after thawing in a liquid format or it could be lyophilized to prolong the shelf life. Lyophilized drug products are later reconstituted before usage in the clinical setting. That means, there is a need to stabilize the antibody construct in a formulation matrix under various conditions. Also, before freezing / lyophilization and after thawing, the antibody construct may be kept under different temperature ranges for some time. As changes of the antibody construct quality under higher temperature stress conditions are inevitably over time, it is desirable to keep such changes to the minimum.

[0006] Hence, to ensure high drug quality, one strives to achieve optimal conditions in the formulation matrix. The changes introduced in the formulation matrix with time should be as small as possible under various conditions.

[0007] One of the common approaches is to link the polypeptide chains together with covalent bonds. A common form of such covalent bond is the -S-S- disulfide bond, which also exists in multiple locations in the natural human antibodies (see hinge region of naturally occurring Ig antibodies). Since the covalent bonding is a very strong and stabilizing bond, the antibody construct with inter-chain covalent bonds is usually much more stable than those constructs without, and can withstand much harsher conditions. This allows much more freedom to formulate the antibody, such as using much lower / higher pH, temperature, longer storage time etc.

[0008] Further, Fc region is also known to play a major role in stabilizing the antibody structure. The Fc region in an Ig is a dimeric form composed of two copies of CH2 domains and two copies of CH3 domains. In each domain, there are native disulfide bond that are important for the structural stability. Thus, similarly, the antibody which comprises a Fc region is usually much more stable and can also withstand much harsher conditions than the antibody without a Fc region.

[0009] However, while inter-chain covalent bonding and / or Fc region is seen in many artificial antibody constructs, there exist also very promising antibody constructs which do not have interchain covalent bonding or Fc region. For instance, the bispecific, tetravalent chimeric antibody construct (TandAb) AFM13 is the first antibody that specifically recruits NK cells by binding exclusively to the iso form CD 16 A. AFM13 is formed by homodimerization of a single polypeptide in a head-to-tail fashion through noncovalent interactions of the immunoglobulinheavy (VH) and light (VL) variable chains of the constituting domains (cf. Figure 7). AFM13 has two binding sites for each antigen but no Fc domains. AFM13 specifically targets CD30 on Hodgkin's lymphoma cells, and recruits and activates NK cells by binding to CD16A. Preclinical and clinical data of AFM13 demonstrate a specific and efficient antitumor activity via the engagement of NK cells.

[0010] To date, it is still very challenging to stabilize in a formulation the antibody constructs where the multiple polypeptide chains in the antibody construct are only non-covalently linked with each other (such as in an TandAb antibody such as AFM13 mentioned above).SUMMARY OF THE INVENTION

[0011] The current invention relates to the problem of stabilizing antibody constructs that are in the so-called TandAb format. Generally, different mechanisms can lead to the destabilization of antibody constructs:

[0012] Sometimes, antibody constructs can undergo fragmentation, in which a polypeptide chain of the construct is broken down to smaller fragments. Fragmentation is more often observed at lower pH. Acidic pH can in principle promote fragmentation by hydrolysis of the polypeptide chain.

[0013] Polypeptide chains in the antibody construct may experience unfolding or partial unfolding to present key stretches of residues or aggregation-prone regions to achieve strong interactions between antibody constructs which leads to the aggregation of antibodies. This happens oftentimes when antibodies are denatured by heat or low pH. Aggregation can be measured by determining HMWS% in a sample.

[0014] Polypeptide chains of the antibody construct may form larger aggregates that form particles. Such particles can be detected optically. Sometimes, particle formation leads to an increase of turbitiy of the composition. Generally speaking, factors that can promote particle formation are, but are not limited to, temperature, antibody concentration, ionic strength, pH, etc.

[0015] The inventors of the present application sought to find conditions, in which all three types of destabilizing processes can be reduced to a minimum. Compared with the antibodies where multiple chains are bound together covalently or by a Fc region, the inventors were faced with the challenge that antibody constructs, which are in the TandAb format, where the multiple chains adhere with each other only non-covalently, have a much higher tendency to unfold, expose aggregation-prone regions and aggregate. While unfolding and refolding may sometimes be desired during upstream processing, where misfolded proteins can be refolded and thus(re-)activated, and where a certain degree of aggregation forming can be tolerated, conditions that favor unfolding and refolding are commonly not desired during downstream processing, where antibody constructs are stored for longer periods, in which the disadvantages of aggregation become more pronounced. Stabilizing a TandAb antibody construct is a new challenge and presents a different technical picture than stabilizing the antibody constructs with inter-chain covalent bonds or Fc domain.

[0016] A usual environment for a covalently linked antibody construct can be already too harsh for AFM13. Take pH for instance, too low pH are often believed to increase the charge of the polypeptide chains, thereby increase the repulsive force among them and increase the risk of unfolding, exposing the hydrophobic regions and then aggregation. As the covalent inter-chain bond and / or Fc region is a much higher binding force, antibody constructs with such structures are much less affected by harsh conditions, such as much lower pH conditions.

[0017] Hence, the conventional thinking is to keep the storage environment for AFM13 as mild as possible. Even the developer of AFM13 themselves believed previously that the AFM13 should be stored at physiological pH as reflected in WO2021 / 130383.

[0018] However, the inventors of the current application have surprisingly found out that, through a combination of different factors, which include the particular selection of the buffer agent and the selection of a low pH, aggregation, particle formation and fragmentation of the antibody can be kept to an acceptable minimum. The inventors of the present application have surprisingly found that the particular buffer agents histidine, glutamic acid, and aspartic acid, and an acidic pH range stabilize an TandAb antibody, such as AFM13. Contrary to the conventional belief, a very low pH (e.g., pH 3.5-5.2 or optimally at 4.0±0.2), when combined with a histidine buffer, glutamic acid buffer, and / or aspartic acid buffer, will significantly increase the stability of the formulation for antibody constructs with no inter-chain covalent bond. Such pH is even much lower than the usual pH range that one would apply even to antibodies with stable covalent interchain bond and / or Fc region.

[0019] The inventors have further surprisingly found that fragmentation of an TandAb antibody, such as AFM13, is not an issue when stored under aforementioned conditions. Generally, acidic pH can in principle promote fragmentation by hydrolysis of the polypeptide chain(s) of the antibody construct. However, this was not observed for AFM13 when stored under the aforementioned conditions.

[0020] The inventors of the current invention also surprisingly found out that the stabilizing effect of a low pH is not achieved with each and any buffer system. For instance, the test data in the examples show that citrate buffer at a low pH 4.5 did not bring the stabilizing effect that was observed with glutamic acid, histidine and aspartic acid at a similarly low pH. Among the specific buffers and low pH range, glutamic acid buffer with pH 4.0 showed the best stabilizing effect for AFM13.

[0021] The at least two-folded surprising finding, i.e., lower pH and the specific buffer types, significantly improved the stability of antibody formulations where the antibody construct is of the TandAb format. As the examples show, this invention not only leads to better properties of the formulation, but also reduced the extent of changes in properties with time under various temperatures.

[0022] Based on the above surprising findings, the current invention concerns at least the following:

[0023] The present invention relates to a pharmaceutical composition comprising: (a) an antibody construct; (b) a buffer agent comprising one or more of the following agents: glutamic acid, histidine, aspartic acid, and one or more salts thereof ; (c) a saccharide; and (d) a surfactant; wherein the pH of the pharmaceutical composition is 3.5 to 5.2, wherein the antibody construct comprises two polypeptide chains, wherein said antibody construct comprises (i) a first binding domain (A) specific for a first target and comprising the variable domains VH1 and VL1, and (ii) a second binding domain (B) specific for a second target and comprising the variable domains VH2 and VL2, wherein the variable domains are positioned within each of the two polypeptide chains from the N-terminus to the C-terminus in the order: VH1-L1-VL2-L2-VH2-L3-VL1, wherein LI is a linker, wherein L2 is a linker, and wherein L3 is a linker.

[0024] The present invention also relates to a solid pharmaceutical composition obtainable or obtained by lyophilisation of a liquid pharmaceutical composition of the invention.

[0025] The present invention also relates to a frozen pharmaceutical composition obtainable or obtained by freezing of a pharmaceutical composition of the invention.

[0026] The present invention also relates to a vial comprising a pharmaceutical composition of the invention.

[0027] The present invention also relates to a method of producing a lyophilized pharmaceutical composition, comprising lyophilization of a pharmaceutical composition of the invention.

[0028] The present invention also relates to a method of producing a frozen pharmaceutical composition, comprising freezing a pharmaceutical composition of the invention.

[0029] The present invention also relates to a method of producing a liquid pharmaceutical composition comprising reconstituting a solid pharmaceutical composition of the invention or a lyophilized pharmaceutical composition obtainable or obtained by a method of the invention with a liquid pharmaceutically acceptable carrier.

[0030] The present invention also relates to a method of treating a patient suffering from a CD30 positive cancer, the method comprising administering a pharmaceutical composition of the invention or a pharmaceutical composition obtained by reconstituting a solid pharmaceutical composition of the invention or produced by a method of the invention.

[0031] The present invention also relates to a pharmaceutical composition of the invention or a pharmaceutical composition obtained by reconstituting a solid pharmaceutical composition of the invention or produced by a method of the invention for use in a method for treating a patient suffering from a CD30 positive cancer.

[0032] The present invention also relates to a a use of a pharmaceutical composition of the invention, a solid pharmaceutical composition of the invention, a frozen pharmaceutical composition of the invention, a lyophilized pharmaceutical composition or frozen pharmaceutical composition produced by a method of the invention, or a liquid pharmaceutical composition produced by a method of the invention for the manufacture of a medicament for treating a CD30 positive cancer.BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1: Schematic illustration of Freeze-Thaw treatment.

[0034] Figure 2: Changes in turbidity (Example 1).

[0035] Figure 3: Storage stability - Physical aggregation by HP-SEC (Example 2).

[0036] Figure 1: HP-SEC data in Example 3.

[0037] Figure 2: Cumulative particle concentrations obtained from MFI of AFM13 samples at pH 4.0 and 4.5 (Example 4).

[0038] Figure 3: Species contents obtained from SE-HPLC analyses at 280 nm of AFM13 samples at pH 4.0 (upper panel) and 4.5 (lower panel).

[0039] Figure 4 The structure of AFM13.DETAILED DESCRIPTION1. Definitions

[0040] The singular forms “a”, “an”, and “the”, include both singular and plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a reagent” includes one or more of such different reagents.

[0041] It should also be understood that when describing a range of values, the characteristic being described could be an individual value found within the range including the endpoints. For example, “a pH of 3.5 to 5.2” includes 3.5 and 5.2 and any value in between such values.

[0042] The term “and / or” wherever used herein includes the meaning of “and”, “or” and “all or any other combination of the elements connected by said term.

[0043] The term “about”, “around” or “approximately” as used herein means within 5%, preferably within 1% of a given value or range (plus (+) or minus (-)). It includes, however, also the concrete number, e.g., about 20 includes 20.

[0044] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, element, or step or group of integers, element or steps but not the exclusion of any other integer, element, or step or group of integer, element or step.

[0045] When used herein “consisting of’ excludes any element, step, or ingredient not specified in the text. When used herein, “consisting essentially of’ does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0046] In each instance herein, any of the terms “comprising”, “consisting essentially of’ and “consisting of’ may be replaced with either of the other two terms. For example, when disclosure uses the term “comprising”, the disclosure also encompasses the replacement of the term “comprising” with the terms “consisting essentially of’ as well as “consisting of’ and vice versa. E.g., the term “comprising” is meant to provide explicit support also for its replacement with “consisting essentially of’ and / or “consisting of’, the term “consisting essentially of’ is meant to provide explicit support also for its replacement with “comprising” and / or “consisting of’, and the term “consisting of’ is meant to provide explicit support also for its replacement with “consisting essentially of’ and “comprising”. The possibility to replace terms with each other is not to be understood that these terms are synonymous. For the avoidance of doubt, theterm “comprising”, “consisting essentially of’ or “consisting of’ that is explicitly recited in the respective context is the preferred term, while its replacement with any one of the other two terms is less preferred.

[0047] An acid buffer mentioned herein refer to a buffer comprising said acid and also the salt form thereof. The mentioning of acid buffer and the salt form are interchangeable. For instance, “glutamic acid buffer” and “glutamate buffer” are used interchangeably.

[0048] As used herein, the term “formulation(s)” means a combination of at least one active agent with one or more other agents for one or more particular uses, such as storage, further processing, sale, and / or administration to a subject, such as, for example, administration to a subject of a specific agent in a specific amount, by a specific route, to treat a specific disease.

[0049] The term “antibody construct” refers to a molecule in which the structure and / or function is / are based on the structure and / or function of an antibody, e.g., of a full-length or whole immunoglobulin molecule and / or is / are drawn from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or fragment thereof. An antibody construct is hence capable of specifically binding to its specific target or antigen. Furthermore, the binding region of an antibody construct defined in the context of the invention comprises the minimum structural requirements of an antibody which allow for the target binding. This minimum requirement may e.g. be defined by the presence of at least the three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or the three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably of all six CDRs. An alternative approach to define the minimal structure requirements of an antibody is the definition of the epitope of the antibody within the structure of the specific target, respectively, the protein domain of the target protein composing the epitope region (epitope cluster) or by reference to a specific antibody competing with the epitope of the defined antibody. The antibodies, on which the constructs of the invention are based, include for example monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies.

[0050] The term “variable” refers to the portions of the antibody or immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the “variable domain(s)”). The pairing of a variable heavy chain (VH) and a variable light chain (VL) together forms a single antigen- binding site.

[0051] The terms “CDR”, and its plural “CDRs”, refer to the complementarity determining region of which three make up the binding character of a light chain variable region (CDR-L1,CDR-L2 and CDR-L3) and three make up the binding character of a heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). CDRs contain most of the residues responsible for specific interactions of the antibody with the antigen and hence contribute to the functional activity of an antibody molecule: they are the main determinants of antigen specificity. In defining the CDR sequences, the numbering in accordance with the so-called Kabat system is preferred.

[0052] As used herein, a “diabody” or “Db” refers to an antibody construct comprising two binding domains, which may be constructed using heavy and light chains with the individual CDR regions disclosed herein. Typically, a diabody comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Preferred linkers for this purpose include glycine serine linkers with about up to 12 amino acids, preferably up to about 10 amino acids. Preferred glycine serine linkers may be the linker as shown in SEQ ID NO: 18. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VH and VL domains of another fragment, thereby forming two antigenbinding sites. A diabody can be formed by two separate polypeptide chains, each comprising a VH and a VL.

[0053] Alternatively, two VH and two VL domains can be comprised in one single polypeptide chain. In such a case, the diabody can also be termed “single chain diabody” or “scDb”. Typically, a scDb comprises the two chains of a non-single chain diabody that are fused together, preferably via a linker. A preferred linker for this purpose is a glycine serine linker, which preferably comprises from about 5 to about 15 amino acids. Preferred glycine serine linkers may have one or more repeats of GGS, GGGS (SEQ ID NO: 22), or GGGGS (SEQ ID NO: 23). Such linker preferably comprises 3, 4, 5 or 6 repeats of GGS, preferably (GGS)s (SEQ ID NO 18) or (GGS)4 (SEQ ID NO: 24), preferably (GGS)s (SEQ ID NO 18). On the polypeptide chain, the variable domains of a scDb can be arranged (from N to C terminus) in a VL-VH-VL-VH or VH- VL-VH-VL order. Similarly, the spatial arrangement of the four domains in the tertiary / quaternary structure can be in a VL-VH-VL-VH or VH-VL-VH-VL order. The term diabody does not exclude the fusion of further binding domains to the diabody.

[0054] A “Tandab” or “tandem diabody” refers to an antigen-binding molecule constructed by linking at least four variable domains (two heavy chain variable domains (VH) and two light chain variable domains (VL) ) in a single gene construct enabling dimerization. In such tandem diabodies, the linker length is such that it prevents intramolecular pairing of the variable domains so that the molecule cannot fold back upon itself to form a monomeric single-chain molecule, but rather is forced to pair with the complementary domains of another chain. The variabledomains are also arranged such that the corresponding variable domains pair during this dimerization (Weichel et al., 2015, European Pharmaceutical Review, 20 (1) :27-32). Following expression, two polypeptide chains fold head-to-tail forming a functional non-covalent dimer. Tandem diabodies contain only antibody variable domains and therefore are contemplated to lack side effects or non-specific interactions that may be associated with an Fc moiety.

[0055] Exemplified in the examples is, among others, the bispecific and tetravalent CD30 / CD16A tandem diabody AFM13 having specificity for CD30 and CD16A (CD30 / CD16A tandem diabody), which has been described in Reusch, et al., 2014. This CD30 / CD16A tandem diabody specifically recruits NK cells by binding exclusively to the isoform CD16A. The CD30 and CD1 6A bispecific tandem diabody described herein is designed to allow specific targeting of CD30+ tumor cells by recruiting cytotoxic NK cells. In such tandem diabody, the linker length is such that it prevents intramolecular pairing of the variable domains so that the molecule cannot fold back upon itself to form a single-chain diabody, but rather is forced to pair with the complementary domains of another chain. The domains are also arranged such that the corresponding VH and VL domains pair during this dimerization. Following expression, two identical polypeptide chains fold head-to-tail forming a functional non-covalent homodimer.

[0056] The term “binding domain” or “domain which binds” characterizes in connection with the present invention a domain which is capable of (specifically) binding to / interacting with / recognizing a given target epitope or a given target site on the target molecules (antigens), e.g., CD16A on the surface of an immune effector cell, and a target cell surface antigen, respectively. The structure and / or function of the first binding domain (e.g. recognizing CD16A), and also the structure and / or function of the second binding domain (recognizing the target cell surface antigen, e.g. CD30), is / are preferably based on the structure and / or function of an antibody, e.g. of a full-length or whole immunoglobulin molecule and / or is / are drawn from the variable heavy chain (VH) and / or variable light chain (VL) domains of an antibody or fragment thereof.

[0057] The term “specifically binding”, “specific binding”, or “specific for” as used herein means that a binding domain preferentially binds or recognizes the target even when the target is present in a mixture with other molecules or other structures. Preferably, the binding domain exhibits preferential binding for its target molecule compared to other related / similar but nontarget molecules or compared to other non-target molecules. Preferably, such binding domain exhibits detectable binding to its target molecule while exhibiting little or no detectable binding to another related / similar but non-target molecule. Quantitatively, an antibody construct of the present disclosure that specifically binds to a target (e.g., CD16A or CD30), preferably does notshow reactivity of more than 30%, 20%, or 10%, preferably not more than 5%, with proteins or antigens other than said target.

[0058] Specific binding can be determined by any of a variety of measurements known to those skilled in the art including, for example, affinity (Ka or Kd), association rate (kon), dissociation rate (koff), avidity or a combination thereof. Methods and measurements include, for example, relative binding between a target molecule and a non-target molecule. Both quantitative and qualitative measurements can be employed for making such apparent or relative binding determinations. Specific examples of binding determinations include, for example, competitive binding assays, protein or Western blot methodology, ELISA, RIA, surface plasmon resonance, evanescent wave methodology, flow cytometry and / or confocal microscopy. Preferably, the binding affinity is about 10'12to 10'8M, 10'12to 10'9M, 10'12to 10'10M, 10-11 to 10-8 M, preferably of about 10-11 to 10-9 M, preferably measured by ELISA.

[0059] A “bispecific” antibody construct is an artificial hybrid antibody having distinct binding sides with different specificities. A bispecific antibody comprises specificities for two different antigens or targets, but no further specificity against a third or further antigen or target. Bispecific antibody constructs can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315- 321 (1990).

[0060] The binding domains and the variable domains (VH / VL) of the antibody construct of the present disclosure may comprise peptide linkers (spacer peptides). The term “linker” comprises an amino acid sequence by which the amino acid sequences of one (variable and / or binding) domain and another (variable and / or binding) domain of the antibody construct defined herein are linked with each other. The linker may have a length of about 5 to about 15 amino acids. The length of the linker is preferably determined as described by Rossmalen et al Biochemistry 2017, 56, 6565-6574, which also describes suitable linkers that are well known to the skilled person.

[0061] “ CD16A” or “CD 16a” refers to the activating receptor CD16A, also known as FcyRIIIA, expressed on the cell surface of NK cells. CD16A is an activating receptor triggering the cytotoxic activity of NK cells. The amino acid sequence of human CD16A is given as SEQ ID NO: 20. An antigen-binding site comprising heavy (VH) and light (VL) chain variable domains binding to CD 16 A, but not binding to CD16B, may be provided by an antigen-binding site which specifically binds to an epitope of CD16A which comprises amino acid residues of the C-terminal sequence SFFPPGYQ (positions 201-208 of SEQ ID NO: 21) and / or residues G147 and / or Y158 of CD16A which are not present in CD16B.

[0062] “ CD16B” refers to receptor CD16B, also known as FcyRIIIB, expressed on neutrophils and eosinophils. The receptor is glycosylphosphatidyl inositol (GPI) anchored and is understood to not trigger any kind of cytotoxic activity of CD16B positives immune cells. The amino acid sequence of human CD16B is given as SEQ ID NO: 25.

[0063] The term “target cell“ describes a cell or a group of cells, which is / are the target of the mode of action applied by the antibody construct of the invention. This cell / group of cells comprise, e.g., pathological cells, which are eliminated or inhibited by engaging these cells with the effector cell via the antibody construct of the invention. A preferred target cell is a cancer cell.

[0064] The term "target cell surface antigen" refers to an antigenic structure expressed by a cell and which is present at the cell surface such that it is accessible for an antibody construct as described herein. It may be a protein or the extracellular portion of a protein or a peptide that is presented on the cell surface in an MHC context (including HLA-A2, HLA-A11, HLA-A24, HLA-B44, HLA-C4) or a carbohydrate structure of a protein, such as a glycoprotein. It is preferably a tumor associated or tumor restricted antigen. Target cell surface antigens particularly envisaged in the context of the present invention are CD30, EGFR, CD123, CD19, CD20, and CD38, preferably CD30. It is envisaged that CD16A is not a target cell surface antigen of the present invention.

[0065] As used herein, the term “CD30” refers to the Cluster of Differentiation 30 protein, also known as “TNF-Receptor 8” or “TNFRSF8”. CD30 is an antigenic determinant expressed by activated, but not by resting, T and B cells. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD30 can be found as UniProt / Swiss-Prot Accession No. P28908 (entry version 217 of 8 November 2023) and the nucleotide sequence encoding of the human CD30 can be found at Accession No. NM_001243 (version NM_001243.5). As used herein, “CD30” includes proteins comprising mutations, e.g., point mutations, fragments, insertions, deletions and splice variants of full length wild-type CD30, preferably refers to a sequence that is at least 80%, 90%, 95% or 98% homologous to UniProt / Swiss-Prot Accession No. P28908 (entry version 217 of 8 November 2023).

[0066] As used herein, the term "excipient" is intended to mean a therapeutically inactive substance. Excipients can be included in a formulation for a wide variety of purposes including, for example, as a diluent, vehicle, buffer, stabilizer, tonicity agent, bulking agent, surfactant, cryoprotectant, lyoprotectant, anti-oxidant, metal ion source, chelating agent and / or preservative. Excipients include, for example, polyols such as sorbitol or mannitol; sugars such as sucrose, lactose, or dextrose; polymers such as polyethylene glycol; salts such as NaCl, KC1 or calcium phosphate, amino acids, for example, proline, glycine or methionine, surfactants, metal ions, buffer salts such as glutamate, acetate or aspartate, preservatives and polypeptides such as human serum albumin, as well as saline and water. Excipients can comprise sugars, for example sugar alcohols, reducing sugars, non-reducing sugars and sugar acids. Excipients are well known in the art and can be found described in, for example, Wang W., Int. J. Pharm. 185: 129-88 (1999) and Wang W., Int. J. Pharm. 203: 1-60 (2000).

[0067] As used herein, the term "surfactant" is intended to mean a substance that functions to reduce the surface tension of a liquid in which it is dissolved. Surfactants can be included in a formulation for a variety of purposes including, for example, to prevent or control aggregation, particle formation and / or surface adsorption in liquid formulations or to prevent or control these phenomena during the lyophilization and / or reconstitution process in lyophilized formulations. Surfactants include, for example, amphipathic organic compounds that exhibit partial solubility in both organic solvents and aqueous solutions. General characteristics of surfactants include their ability to reduce the surface tension of water, reduce the interfacial tension between oil and water and also form micelles. Surfactants of can include non-ionic and ionic surfactants. Surfactants are known in the art and can be found described in, for example, Randolph T.W. and Jones L.S., Surfactant-protein interactions. Pharm Biotechnol. 13: 159-75 (2002).

[0068] “Pharmaceutically acceptable carrier” means any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffers, e.g., phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, such as oil / water emulsions, various types of wetting agents, liposomes, dispersion media and coatings, which are compatible with pharmaceutical administration, in particular with parenteral administration. The use of such media and agents in pharmaceutical compositions is well known in the art, and the compositions comprising such carriers can be formulated by well-known conventional methods.

[0069] High molecule weight species (HMWS) refers to those molecules which are oligomers from the active agent that can be detected by the HP-SEC method. One can detect the HMWS by comparing with the molecular weight of the original active agent (i.e. the homodimer). Forinstance, when the active agent in the formulation is AFM13 which is a TandAb homodimer having a molecular weight of about 104 kDa, then the HMW species are the molecules formed from AFM13 and have a molecular weight of higher than about 104 kDa. The percentage of HMWS (i.e., HMWS%) can be measured by the HP-SEC method, preferably by the method as essentially described in Method 4 of the Examples.

[0070] “Turbidity”, as used herein, relates to the cloudiness or haziness of a fluid caused by large numbers of individual particles that are generally invisible to the naked eye. Turbidity can, e.g., be measured using a turbidimeter, preferably as essentially described in Example 4, under “Turbidity measurement - Measurement method”.

[0071] “Particle count” as used herein, relates to the number of particles that are present in a medium, such as a pharmaceutical composition. Particle count can be measured by Micro-Flow Imaging (MFI), preferably as essentially described in Method 3 of the Examples.

[0072] The term “treating” or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Treatment includes the application or administration of the formulation to the body, an isolated tissue, or cell from a patient who has a disease / disorder, a symptom of a disease / disorder, or a predisposition toward a disease / disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptom of the disease, or the predisposition toward the disease.

[0073] The term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts or doses effective for this use will depend on the condition to be treated (the indication), the delivered antibody construct, the therapeutic context and objectives, the severity of the disease, prior therapy, the patient's clinical history and response to the therapeutic agent, the route of administration, the size (body weight, body surface or organ size) and / or condition (the age and general health) of the patient, and the general state of the patient's own immune system. The proper dose can be adjusted according to the judgment of the attending physician such that it can be administered to the patient once or over a series of administrations, and in order to obtain the optimal therapeutic effect.

[0074] As used herein, the term “sequence identity” or “identity” denotes a property of sequences that measures their similarity or relationship. The term “sequence identity” or“identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[0075] For amino acid sequences, sequence identity and / or similarity is determined by using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity method of Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al., 1984, Nucl. Acid Res. 12:387-395, preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0076] An example of a useful algorithm is the BLAST algorithm, described in: Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1997, Nucleic Acids Res. 25:3389- 3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787. A particularly useful BLAST program is the WU-BL AST-2 program which was obtained from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=l, overlap fraction=0.125, word threshold (T)=ll. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

[0077] An additional useful algorithm is gapped BLAST as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits.2. The antibody construct

[0078] The antibody construct comprised in the pharmaceutical composition of the present disclosures comprises two polypeptide chains. The antibody construct comprises (i) a first binding domain (A), which is specific for a first target and comprises the variable domains VH1 and VL1, and (ii) a second binding domain (B) specific for a second target and comprising the variable domains VH2 and VL2. Generally, there are multiple possibilities how the variable domains can be positioned with the polypeptide clains, which include the following possibilities: a) VL1-L1-VH2-L2-VL2-L3-VH1 b) VH1-L1-VL2-L2-VH2-L3-VL1 c) VL2-L1-VH1-L2-VL2-L3-VH1 d) VH2-L1-VL1-L2-VH1-L3-VL2 e) VH1-L1-VH2-L2-VL2-L3-VL1, f) VL1-L1-VL2-L2-VH2-L3-VH1, g) VH2-L1-VH1-L2-VL1-L3-VL2, or h) VL2-L1-VL1-L2-VH1-L3-VH2.Here, LI, L2, and L3 are optional linkers. The preferred arrangement is VH1-L1-VL2-L2-VH2- L3-VL1, in which LI, L2 and L3 are linkers.

[0079] According to the present disclosure the two polypeptide chains comprised in the antibody construct may associate with each other non-covalently (i.e., without inter-chain covalent bond). The antibody construct may be in the form of a diabody, tandem diabody (TandAb), multibodies such as triabodies or tetrabodies.

[0080] The antibody construct can be a TandAb which comprises or consists of two polypeptide chains, wherein the two polypeptide chains associate with each other with non-covalent bond. Each polypeptide chain may comprise at least four variable domains: VH1, VL1, VH2 and VL2.

[0081] The present disclosure contemplates that each of the VL1, VH2, VL2, and VH1 of one of the two polypeptide chains may form an antibody variable region with the VH1, VL2, VH2, and VL1 of the other one of the two polypeptide chains.

[0082] The present disclosure contemplates that the VL1, VH2, VL2 and VH1 in the first polypeptide chain respectively may associate with the VH1, VL2, VH2 and VL1 in the secondpolypeptide chain non-covalently. Hence, the VL1, VH2, VL2 and VH1 of the first polypeptide chain associated with the VH1, VL2, VH2 and VL1 of the second polypeptide chain may form a first CD16A binding site, a first target antigen binding site, a second antigen binding site and a second CD16A binding site respectively.

[0083] The present disclosure contemplates that the two polypeptide chains in the antibody construct may comprise identical amino acid sequences that define the VH1-L1-VL2-L2-VH2- L3-VL1 elements. Preferably, both polypeptide chains are identical.

[0084] Further, the antibody construct can be bispecific. On the one hand, the antibody construct may bind to CD 16 A, but does not bind to CD16B of the NK cell. Hence, the first target is preferably CD 16 A. On the other hand, the antibody construct may also bind to a second target, which is preferably not CD 16 A. The second target may be a cancer cell surface antigen, such as CD30, EGFR, CD123, CD19, CD20, and CD38, preferably CD30. Preferably, the first and / or second targets disclosed herein are the human version of said targets. Hence, the first variable region (VH1 and VL1) may be specific for CD16A and the second variable region (VH2 and VL2) may be specific for a target cell antigen. Preferably, the antibody construct is bispecific and binds specifically to both human CD16A and human CD30.

[0085] As contemplated by the present disclosure, the heavy chain variable domain specific for CD16A (VH1) preferably comprises:(a) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1;(b) a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; and(c) a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable domain specific for CD16A (VL1) preferably comprises:(a) a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4;(b) a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and(c) a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0086] In the antibody construct of the disclosure, VH1 may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14. In the antibody construct of the disclosure, VL1 may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. For example, VH1 and VL1 may comprise amino acid sequences having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 14 and 15. VH1 may comprise the amino acid sequence set forth in SEQ ID NO: 14. VL1 may comprise the amino acid sequence set forth in SEQ ID NO: 15. Preferably, VH1 comprises the amino acid sequence shown in SEQ ID NO: 14 and VL1 comprises the amino acid sequence shown in SEQ ID NO: 15. For example, VH1 may consist of the amino acid sequence shown in SEQ ID NO: 14 and VL1 may consist of the amino acid sequence shown in SEQ ID NO: 15.

[0087] A heavy chain variable domain specific for CD16A (VH1) may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14 and optionally, contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 1, 2 and 3, respectively.

[0088] A light chain variable domain specific for CD16A (VL1) may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15, and optionally, contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 4, 5 and 6, respectively.

[0089] As contemplated by the present disclosure, the heavy chain variable domain specific for CD30 (VH2) preferably comprises(a) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7;(b) a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8; and(c) a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 9; and a light chain variable domain specific for CD30 (VL2) preferably comprises:(a) a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10;(b) a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11; and(c) a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 12.

[0090] In the antibody construct of the disclosure, VH2 preferably comprises an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16. In the antibody construct of the disclosure, VL2 preferably comprises an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. For example, VH2 and VL2 may comprises amino acid sequences having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NOs: 16 and 17 respectively. VH2 may comprise the amino acid sequence set forth in SEQ ID NO: 16. VL2 may comprise the amino acid sequence set forth in SEQ ID NO: 17. Preferably, VH2 comprises the amino acid sequence shown in SEQ ID NO: 16 and VL2 comprises the amino acid sequence shown in SEQ ID NO: 17. For example, VH2 may consist of the amino acid sequence shown in SEQ ID NO: 16 and VL2 may consist of the amino acid sequence shown in SEQ ID NO: 17.

[0091] A heavy chain variable domain specific for CD30 (VH2) may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16 and optionally, contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 7, 8 and 9, respectively.

[0092] A light chain variable domain specific for CD30 (VL2) may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17, and optionally, contains the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 10, 11 and 12, respectively.

[0093] The number of variable domains in each polypeptide chain can be four and these four variable domains are as described above.

[0094] Further, the variable domains in the antibody construct may be linked, preferably one after another, by peptide linkers. In the case where the number of variable domains in each polypeptide is four, the linkers are preferably LI, L2 and L3. Each of the linkers may consists of 15 or less amino acid residues, preferably 12 or less residues.

[0095] The linker length of LI, L2 and L3 can be the same or different. The possible linker length of LI can be less than 12, 10, 8, 7, 6, or 5 amino acid residues. The linker LI can also consist of 3-13, 4-12, 5-11, 6-10 or 9 amino acid residues. The same options apply also to the linkers L2 and L3.

[0096] Preferably, all the linkers LI, L2 and L3 have 9 amino acid residues.

[0097] Each of the linkers LI, L2, and L3 may be a glycine serine linker, such as a linker as shown in anyone of SEQ ID NOs: 18 and 22-24. The sequence of the linker LI can be SEQ ID NO: 18 or can be a sequence which has 1, 2, 3, or 4 amino acid residues which are different / extra / less compared with SEQ ID NO: 18. The sequence of the linker L2 can be SEQ ID NO: 18 or can be a sequence which has 1, 2, 3, or 4 amino acid residues which are different / extra / less compared with SEQ ID NO: 18. The sequence of the linker L3 can be SEQ ID NO: 18 or can be a sequence which has 1, 2, 3, or 4 amino acid residues which are different / extra / less compared with SEQ ID NO: 18. LI may be a glycine- serine linker, preferably a linker as shown in SEQ ID NO: 18. L2 may be a glycine-serine linker, preferably a linker as shown in SEQ ID NO: 18. L3 may be a glycine-serine linker, preferably a linker as shown in SEQ ID NO: 18.

[0098] The linkers can be positioned within each of the two polypeptide chains from the N- terminus to the C-terminus in the order of: a) VL1-L1-VH2-L2-VL2-L3-VH1 b) VH1-L1-VL2-L2-VH2-L3-VL1 c) VL2-L1-VH1-L2-VL2-L3-VH1 d) VH2-L1-VL1-L2-VH1-L3-VL2 e) VH1-L1-VH2-L2-VL2-L3-VL1, f) VL1-L1-VL2-L2-VH2-L3-VH1, g) VH2-L1-VH1-L2-VL1-L3-VL2, or h) VL2-L1-VL1-L2-VH1-L3-VH2.

[0099] The polypeptide chains in the antibody construct may be the same or different. One polypeptide chain may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Likewise, the second polypeptide chain may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. Accordingly, each of the two polypeptide chains may comprise an amino acid sequence having at least 80%, at least 85%, at least 87%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. In the case where two identical polypeptide chainsassociate with each other non-covalently, the antibody construct is then a homodimer. Preferably, each of the two polypeptide chains comprises the sequence shown in SEQ ID NO: 13. For example, each of the two polypeptide chains may consist of the sequence shown in SEQ ID NO: 13.

[0100] Preferably, the antibody construct of the current invention is AFM13, which is a homodimer formed by two identical polypeptide chains, wherein each polypeptide chain has the sequence of SEQ ID NO: 13. The two identical polypeptide chains associate with each other non-covalently.

[0101] The present disclosure contemplates that an antibody construct comprised in the pharmaceutical composition of the disclosure may be a variant of an antibody construct disclosed herein, such as AFM13. Above mentioned variant preferably retains the biological activity of the original antibody, i.e., binding to a first target, such as CD 16 A, and a second target, such as the target cell surface antigen. For example, it may be desirable to make changes in properties of the antibody construct which are not detrimental to the antibody construct’s biological function, e.g., adding or exchanging purification tags of the antibody construct in view of e.g., the different purification methods. Amino acid sequence variants of the antibody constructs can be prepared by introducing appropriate nucleotide changes into the antibody constructs nucleic acid, or by peptide synthesis. All of the below described amino acid sequence modifications should result in an antibody construct which still retains the desired biological activity (i.e., binding to the first target, such as CD 16 A, and the second target, such as the target cell surface antigen disclosed herein).

[0102] Amino acid modifications may include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the antibody constructs. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired biological activity. The amino acid modifications also may alter post-translational processes of the antibody constructs, such as changing the number or position of glycosylation sites.

[0103] 1, 2 or 3 amino acids may be inserted, substituted or deleted in each of the CDRs. Similarly, 1, 2, 3, 4, 5, or 6 amino acids may be inserted, substituted or deleted in the framework region. The antibody construct may also have amino- and / or carboxyl-terminal fusions. For instance, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues may be added to the amino- and / or carboxyl-terminus. The antibody construct may also be fused to an enzyme, to another molecule or to another polypeptide at the N- terminus or to the C-terminus of the antibody construct.

[0104] Preferred substitutions (or replacements) are conservative substitutions. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 2, or as further described below in reference to amino acid classes, may be introduced and the products screened for a desired characteristic.Table 2: Amino acid substitutions

[0105] Modifications in the biological properties of the antibody construct of the present disclosure may be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, and / or (c) the bulk of the side chain. Naturally occurring residues are divided into various classes based on common side-chain properties: (1) hydrophobic: norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.3. Liquid formulation

[0106] A pharmaceutical composition of the current disclosure, sometimes also referred to as “liquid formulation” or simply “formulation”, may be a liquid formulation that, without further change of the agents or purification, is ready to be administered to the patient, to be stored as a final product, and / or to be lyophilized.

[0107] The pharmaceutical composition of the current disclosure comprises at least the following: (a) an antibody construct; (b) a buffer agent; (c) a saccharide; and (d) a surfactant, wherein the pH of the formulation is 3.5-5.2.

[0108] The pharmaceutical composition has a pH of about 5.2 or lower, such as about 5.2 or lower, about 5.0 or lower, about 4.8 or lower, about 4.6 or lower, about 4.4 or lower, about 4.2 or lower, or about 4.0. The pharmaceutical composition has a pH of about 3.5 or higher, such as about 3.5 or higher, about 3.6 or higher, about 3.7 or higher, about 3.8 or higher, about 3.9 or higher, or about 4.0. The pH of the formulation may be in a range of about 3.5 to about 5.2, about 3.6 to about 5.0, about 3.7 to about 4.8, about 3.8 to about 4.6, about 3.9 to about 4.4, about 3.9 to about 4.2, or about 4.0. Preferably, the pH of the pharmaceutical composition is about 3.9 to about 4.4, or about 3.9 to about 4.2.

[0109] More specifically, the pH of the pharmaceutical composition can be about 3.9, about 4.0, about 4.1 or about 4.2, and preferably about 4.0.

[0110] The antibody construct in the current formulation is / are as described elsewhere herin. The antibody construct is preferably present in the pharmaceutical composition with a therapeutically effective amount. By "therapeutically effective amount" is meant an amount of said antibody construct that elicits the desired therapeutic effect. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, ED50 / LD50.

[0111] Specifically, the concentration of the antibody construct may be up to about 50 mg / ml, up to about 45 mg / ml, up to about 40 mg / ml, up to about 35 mg / ml, or up to about 30 mg / ml, up to about 25 mg / ml, up to about 20 mg / ml, up to about 18 mg / ml, up to about 16 mg / ml or up to about 14 mg / ml. The concentration of the antibody construct may be at least about 3 mg / ml, at least about 4 mg / ml, at least about 5 mg / ml, at least about 6 mg / ml, at least about 7 mg / ml, at least 8 mg / ml, or at least about 9 mg / ml. More specifically, the concentration of the antibodyconstruct may be in the range of about from about 3 mg / ml to about 50 mg / ml, from about 4 mg / ml to about 40 mg / ml, or from about 5 mg / ml to about 30 mg / ml, from about 6 mg / ml to about 25 mg / ml, from about 7 mg / ml to about 20 mg / ml, or about 8 mg / ml to about 15 mg / ml. For example, the concentration of the antibody construct may be about 8 to about 12 mg / ml. For example, the concentration of the antibody construct may be about 10 mg / ml. Preferably, the concentration of the antibody construct refers to the active concentration of the antibody construct.

[0112] The concentration of the antibody construct mentioned above can preferably be measured by ELISA, preferably in an ELISA as essentially described in the following. Briefly, the active concentration of AFM13 can be evaluated using a sandwich-ELISA: To ensure that only functional AFM13 can be detected, both antigens are integrated in the assay setup. AFM13 binds to an immobilised human CD30-Fc through its anti-CD30 moiety. The detection of the AFM13 utilises the CD16A specificity of the TandAb via biotinylated CD16A-Fc that is visualised by a biotin-streptavidin affinity. The streptavidin is coupled to alkaline phosphatase. Upon addition of alkaline phosphatase yellow substrate (pNPP) the colouration of the solution is measured at 405 nm. The colouration is proportional to the amount of CD30xCD16A TandAb bound and is quantified by comparison with a standard curve.

[0113] It is contemplated by the present disclosure that the buffer agent comprised in the pharmaceutical composition comprises glutamic acid, histidine, aspartic acid, and / or one or more salts thereof. The buffer agent may consist of glutamic acid, histidine, aspartic acid, and / or one or more salts thereof. For example, the buffer agent may comprise histidine and / or one or more salts thereof. The buffer agent may consist of histidine and / or one or more salts thereof. Here, the group of glutamic acid, aspartic acid, and / or one or more salts thereof is preferred. Thus, the buffer agent may comprise glutamic acid, aspartic acid, and / or one or more salts thereof. The buffer agent may consist of glutamic acid, aspartic acid, and / or one or more salts thereof. For example, the buffer agent may comprise aspartic acid and / or one or more salts thereof. The buffer agent may consist of aspartic acid and / or one or more salts thereof. Most preferred, however, is glutamic acid and / or one or more salts thereof. Hence, the buffer agent may comprise glutamic acid and / or one or more salts thereof. The buffer agent may consist of glutamic acid and / or one or more salts thereof.

[0114] The concentration of the buffer can be in the range of about 3 to about 50 rnM, about 5 to about 25 mM, about 10 to about 20 rnM, about 12 to about 17 mM, such as about 15 mM. Preferably the buffer agent is glutamic acid at about 12 to about 17 mM.

[0115] The saccharide in the pharmaceutical composition may comprise monosaccharides; disaccharides and / or other carbohydrate (such as glucose, mannose or dextrins). The carbohydrate may be non-reducing sugars, such as trehalose, sucrose, octasulfate, sorbitol or xylitol. Preferably, the saccharide of the current formulation comprises or consists of trehalose.

[0116] The amount of the saccharide in the pharmaceutical composition can be about 4% to about 15% (w / v), about 6% to about 10% (w / v), about 7% to about 9% (w / v) or about 8% (w / v).

[0117] Preferably, the saccharide is trehalose, and the amount of trehalose comprised in the pharmaceutical composition is about 4% to about 15% (w / v), about 6% to about 10% (w / v), about 7% to about 9% (w / v) or about 8% (w / v). Unless stated otherwise herein, all weight percentages for trehaloses are based on the weight of trehalose dihydrate.

[0118] The surfactant comprised in the pharmaceutical composition of the disclosure may comprise one or more of the following: pluronics, PEG, a sorbitan ester, a polysorbate, such as polysorbate 20, triton, tromethamine, lecithin, cholesterol, and / or tyloxapal. A surfactant comprised in the pharmaceutical composition of the disclosure may be a detergent, preferably with a molecular weight of >1.2 kDa and / or a polyether, preferably with a molecular weight of >3 kDa. Non-limiting examples for detergents are Tween 20, Tween 40, Tween 60, Tween 80 and Tween 85; non-limiting examples for polyethers are PEG 3000, PEG 3350, PEG 4000 and PEG 5000. Preferably, the surfactant comprises or consists of a polysorbate, such as polysorbate 20.

[0119] The concentration of the surfactant may be about 0.005% to about 0.1% (w / v), 0.006% to about 0.05% (w / v), about 0.008% to about 0.03% (w / v) or about 0.009% to about 0.02 % (w / v), or about 0.01% (w / v). Preferably, the surfactant is polysorbate 20 at a concentration of about 0.005% to about 0.1% (w / v), 0.006% to about 0.05% (w / v), about 0.008% to about 0.03% (w / v) or about 0.009% to about 0.02 % (w / v), or about 0.01% (w / v).

[0120] For example, a pharmaceutical composition (liquid formulation) of the disclosure can comprise:(a) an antibody construct which comprises more than one polypeptide chain and the polypeptide chains bind to each other non-covalently (i.e., without inter-chain covalent bond), wherein the concentration of the antibody construct is 3-30 mg / ml;(b) a buffer which comprises or consists of one or more of glutamic acid, histidine, aspartic acid and the salt thereof at the concentration of 12-17 mM;(c) a saccharide at the concentration of 4-15% (w / v);(d) a surfactant at the concentration of 0.005-0.1% (w / v).

[0121] A pharmaceutical composition (liquid formulation) of the disclosure may comprise:(a) an anti-CD16A and anti-CD30 bi-specific antibody construct at the concentration of 3-30 mg / ml,(b) glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 12-17 mM;(c) trehalose at the concentration of 5-10% (w / v); and(d) polysorbate 20 at the concentration of 0.005-0.02% (w / v), and wherein the pH of the pharmaceutical composition is 3.8 to 4.2, and wherein the antibody construct comprises more than one polypeptide chains and each polypeptide chain of the antibody construct has an amino acid sequence with at least 80% identity to SEQ ID NO: 13. The antibody construct preferably has the arrangement of VH1-L1-VL2-L2-VH2-L3-VL1, and VH1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VIE preferably comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 10-12. For example, the antibody construct can comprise two polypeptide chains and each polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0122] Preferably, a pharmaceutical composition (liquid formulation) of the disclosure comprises:(a) an anti-CD16A and anti-CD30 bi-specific antibody construct at the concentration of 5-25 mg / ml;(b) glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 14-16 mM;(c) trehalose at the concentration of 6-9% (w / v); and(d) polysorbate 20 at the concentration of 0.008-0.015% (w / v); and wherein the pH of the pharmaceutical composition is 3.8 to 4.2, and wherein the antibody construct is a dimeric construct consisting of two identical polypeptide chains and the polypeptide chain has an amino acid sequence with at least 90% identity to SEQ ID NO: 13. The antibody construct preferably has the arrangement of VH1-L1-VL2-L2-VH2-L3-VL1, and VH1preferably comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VH2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 10-12. For example, the antibody construct can comprise two polypeptide chains and each polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0123] Preferably, a pharmaceutical composition (liquid formulation) of the disclosure comprises:(a) an anti-CD16A and anti-CD30 bi-specific antibody construct at the concentration of 5-30 mg / ml;(b) glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 12-17 mM;(c) trehalose at the concentration of 5-10% (w / v); and(d) polysorbate 20 at the concentration of 0.005-0.02% (w / v); and wherein the pH of the pharmaceutical composition is 3.8 to 4.2, and wherein the antibody construct is a dimeric construct consisting of two identical polypeptide chains and the polypeptide chain has an amino acid sequence with at least 95% identity to SEQ ID NO: 13. The antibody construct preferably has the arrangement of VH1-L1-VL2-L2-VH2-L3-VL1, and VH1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VH2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 10-12. For example, the antibody construct can comprise two polypeptide chains and each polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13. The antibody construct is preferably at the concentration of about 7 mg / ml to about 20 mg / ml.

[0124] Preferably, a pharmaceutical composition (liquid formulation) of the disclosure comprises:(a) an anti-CD16A and anti-CD30 bi-specific antibody construct at the concentration of 8-18 mg / ml;(b) a buffer which comprises glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 14-16 mM;(c) a saccharide comprising trehalose at the concentration of 6-9% (w / v); and(d) a surfactant comprising polysorbate 20 at the concentration of 0.008-0.012% (w / v); and wherein the pH of the pharmaceutical composition is 3.9 to 4.1, and wherein the antibody construct is a dimeric construct consisting of two identical polypeptide chains, and each polypeptide chain has an amino acid sequence with at least 90% identity to SEQ ID NO: 13. The antibody construct preferably has the arrangement of VH1-L1-VL2-L2-VH2-L3-VL1, and VH1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 preferably comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VEH preferably comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 preferably comprises the CDR sequences as shown in SEQ ID NOs: 10-12. For example, the antibody construct can comprise two polypeptide chains and each polypeptide chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13.

[0125] A pharmaceutical composition disclosed herein may further comprise suitable formulations of one or more (pharmaceutically acceptable) carriers, stabilizers, excipients, diluents, solubilizers, emulsifiers, preservatives and / or adjuvants. The pharmaceutical composition may also contain formulation materials for the purpose of modifying, maintaining or preserving, e.g., the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition (see, REMINGTON'S PHARMACEUTICAL SCIENCES, 18" Edition, (A.R. Genrmo, ed ), 1990, Mack Publishing Company). Acceptable constituents of the composition are preferably nontoxic to recipients at the dosages and concentrations employed. For example, suitable formulation materials may include, but are not limited to:• antimicrobials such as antibacterial and antifungal agents• antioxidants such as ascorbic acid, methionine, sodium sulfite or sodium hydrogensulfite;• buffers, buffer systems and buffering agents as disclosed herein;• aqueous carriers including water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media;• biodegradable polymers such as polyesters;• bulking agents such as mannitol or glycine;• chelating agents such as ethylenediamine tetra acetic acid (EDTA);• isotonic and absorption delaying agents;• fillers;• (low molecular weight) proteins, polypeptides or proteinaceous carriers such as human or bovine serum albumin, gelatin or immunoglobulins, preferably of human origin;• sulfur containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha]-monothioglycerol, and sodium thio sulfate;• diluting agents;• emulsifying agents;• hydrophilic polymers such as polyvinylpyrrolidone;• salt-forming counter-ions such as sodium;• preservatives such as antimicrobials, anti-oxidants, chelating agents, inert gases and the like; examples are: benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide);• solvents and co-solvents (such as glycerin, propylene glycol or polyethylene glycol);• saccharides as disclosed herein;• suspending agents;• tonicity enhancing agents such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol;• parenteral delivery vehicles including sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils;• intravenous delivery vehicles including fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose).

[0126] Once a formulation is prepared as described herein, stability can be assessed using methods known in the art. Several methods are exemplified below in the Examples and may include one of more methods of turbidity measurement, quantification ofHWMS%, homodimer content quantification, and / or particle counting. Other methods can comprise any of a variety of functional assays including, for example, measuring the binding activity, other biochemical activity and / or physiological activity at two or more different time points to determine the stability of the biopharmaceutical in the buffered formulation of the invention.

[0127] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the HMWS% in the composition increases by up to 8 %, up to 7 %, up to 6 %, up to 5 %, up to 4.5 %, up to 4 %, up to 3.5 %, up to 2.5 %, up to 2.0 %, up to 1.5 %, up to 1.0 %, or up to 0.5 % relative HMWS% content as determined by HP-SEC after storage at about 2-8°C for 7 days. For example, the HMWS% in the composition increases by up to 8 % relative HMWS% content after storage at about 2-8°C for 7 days. Preferably, the HMWS% in the composition increases by up to 5 % relative HMWS% content after storage at about 2-8°C for 7 days. As usedherein, “HMWS%” or “relative HMWS% content” relates to the percentage of high molecular weight species that can be detected using the SE-HPLC method. As such, the % value preferably refers to relative peak area detected using the SE-HPLC method. The reference value (100%) is the sum of all integrated peak areas detected using the SE-HPLC method. HMWS% can preferably be measured as essentially described in Method 4 of the Examples.

[0128] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the HMWS% in the composition may increase by up to 10 %, up to 9 %, up to 8 %, up to 7 %, up to 6 %, up to 5 %, up to 4.5 %, up to 4 %, up to 3.5 %, up to 2.5 %, up to 2.0 %, up to 1.5 %, up to 1.0 %, or up to 0.5 % relative HMWS% content as determined by HP-SEC after storage at about 2-8°C for 14 days. For example, the HMWS% in the composition increases by up to 10 % relative HMWS% content after storage at about 2-8°C for 14 days. Preferably, the HMWS% in the composition increases by up to 5 % relative HMWS% content after storage at about 2-8°C for 14 days.

[0129] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the HMWS% in the composition may increase by up to 50 %, up to 45 %, up to 40 %, up to 35 %, up to 30 %, up to 25 %, up to 20 %, up to 18 %, up to 16 %, up to 14 %, up to 12 %, up to 10 %, up to 9 %, up to 8 %, or up to 7% relative HMWS% content as determined by HP- SEC after storage at about 25°C for 7 days. For example, the HMWS% in the composition increases by up to 50 % relative HMWS% content after storage at about 25°C for 7 days. Preferably, the HMWS% in the composition increases by up to 20 % relative HMWS% content after storage at about 25°C for 7 days. Preferably, the HMWS% in the composition increases by up to 10 % relative HMWS% content after storage at about 25°C for 7 days.

[0130] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the HMWS% in the composition may increase by up to 50 %, up to 45 %, up to 40 %, up to 35 %, up to 30 %, up to 25 %, up to 20 %, up to 18 %, up to 16 %, up to 14 %, up to 12 %, up to 10 %, up to 9 %, up to 8 %, or up to 7% relative HMWS% content as determined by HP- SEC after storage at about 25°C for 14 days. For example, the HMWS% in the composition increases by up to 50 % relative HMWS% content after storage at about 25°C for 14 days. Preferably, the HMWS% in the composition increases by up to 20 % relative HMWS% content after storage at about 25°C for 14 days. Preferably, the HMWS% in the composition increases by up to 10 % relative HMWS% content after storage at about 25°C for 14 days.

[0131] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the HMWS% in the composition may increase by up to 60 %, up to 55 %, up to 50 %, up to 45 %, up to 40 %, up to 35 %, up to 30 %, or up to 25% relative HMWS% content as determined by HP-SEC after storage at about 40°C for 7 days. For example, the HMWS% in the composition increases by up to 60 % relative HMWS% content after storage at about 40°C for 7 days. Preferably, the HMWS% in the composition increases by up to 50 % relative HMWS% content after storage at about 40°C for 7 days. Preferably, the HMWS% in the composition increases by up to 30 % relative HMWS% content after storage at about 40°C for 7 days.

[0132] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the relative homodimer content may maintain at least 85 %, at least 90 %, or at least 95 % of its original value as determined by HP-SEC after storage at about 2-8°C for 7 days. For example, the relative homodimer content maintains at least 85 % of its original value after storage at about 2-8°C for 7 days. Preferably, the relative homodimer content maintains at least 90 % of its original value after storage at about 2-8°C for 7 days. As used herein, “homodimer content” relates to the percentage of homodimers among the total amount of antibody constructs. The term “homodimer” does not include HMWS. The homodimer content can preferably be measured as essentially described in Method 4 of the Examples.

[0133] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the relative homodimer content may maintain at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, or at least 95 % of its original value as determined by HP-SEC after storage at about 2-8°C for 14 days. For example, the relative homodimer content maintains at least 65 % of its original value after storage at about 2-8°C for 14 days. Preferably, the relative homodimer content maintains at least 85 % of its original value after storage at about 2-8°C for 14 days.

[0134] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the relative homodimer content may maintain at least 45 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, or at least 90% of its original value as determined by HP-SEC after storage at about 25°C for 7 days. For example, the relative homodimer content maintains at least 45 % of its original value after storage at about 25°C for 7 days. Preferably, the relative homodimer content maintains at least 70 % of its original value after storage at about 25°C for 7 days.

[0135] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the relative homodimer content may maintain at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85%, or at least 90% of its original value as determined by HP-SEC after storage at about 25°C for 14 days. For example, the relative homodimer content maintains at least 40 % of its original value after storage at about 25°C for 14 days. Preferably, the relative homodimer content maintains at least 70 % of its original value after storage at about 25°C for 14 days.

[0136] The present disclosure contemplates that in a pharmaceutical composition disclosed herein, the relative homodimer content may maintain at least 45 %, at least 50 %, at least 60 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, or at least 90% of its original value as determined by HP-SEC after storage at about 40°C for 7 days. For example, the relative homodimer content maintains at least 45 % of its original value after storage at about 40°C for 7 days. Preferably, the relative homodimer content maintains at least 70 % of its original value after storage at about 40°C for 7 days.

[0137] The present disclosure contemplates that the pharmaceutical composition disclosed herein comprises antibody constructs, that have preferably already undergone purification and polishing. As such, the pharmaceutical composition disclosed herein and / or the antibody construct comprised in the pharmaceutical composition of the disclosure has preferably undergone one or more steps for depletion of contaminants (polishing). Preferably, the pharmaceutical composition disclosed herein and / or the antibody construct comprised in the pharmaceutical composition of the disclosure has undergone one or more steps (including all steps) of anion exchange chromatography (AEX), preferably run in bind-and-elute mode, hydroxyapatite chromatography (HAC), preferably run in bind-and-elute mode, and / or virus filtration. Compounds such as a saccharide, such as trehalose, or a surfactant, such as polysorbate 20, are generally added to the formulation only after polishing of the antibody construct, because such compounds are considered to interfere with the aforementioned polishing steps. Preferably, the antibody construct comprised in the pharmaceutical composition is in a purity that is suitable for formulation and / or administration to a human patient. The present disclosure contemplates that the pharmaceutical composition of the disclosure is in a buffer that is preferably suitable for formulation.

[0138] After the liquid format of the final formulation is produced, the formulation is usually frozen or lyophilized. The current invention also includes the formulation as described above in a frozen or lyophilized form. Accordingly, the present disclosure also contemplates a frozenpharmaceutical composition obtainable or obtained by freezing of the (liquid) pharmaceutical composition disclosed herein. The present disclosure also contemplates a solid pharmaceutical composition obtainable or obtained by lyophilization of the (liquid) pharmaceutical composition of the disclosure.

[0139] The present disclosure also contemplates a method of producing a frozen pharmaceutical composition, comprising freezing the (liquid) pharmaceutical composition of the disclosure.

[0140] The present disclosure also contemplates a method of producing a lyophilized pharmaceutical composition, comprising lyophilization of the (liquid) pharmaceutical composition of the disclosure.

[0141] Sometimes, the (liquid) pharmaceutical composition disclosed herein is not lyophilized or frozen directly after its production. Accordingly, a method of producing a frozen pharmaceutical composition or a method of producing a lyophilized pharmaceutical composition of the disclosure can comprise holding the (liquid) composition at a temperature of about 0°C to about 25°C for about 1-24 hours, such as about 1-12 hours or about 12-24 hours before starting the lyophilization or freezing step.

[0142] Sometimes, a pharmaceutical composition of the disclosure is frozen first, then thawed and then lyophilized. This can, e.g., be done if lyophlization is not carried out immediately after the production of the (liquid) pharmaceutical composition of the disclosure. Accordingly, a method of producing a lyophilized pharmaceutical composition of the disclosure can comprise the steps of (i) thawing a frozen pharmaceutical composition of the disclosure to obtain a liquid composition; (ii) holding the liquid composition at a temperature of about 0 to about 25 °C for about 1-24 hours, such as about 1-12 hours or about 12-24 hours; and lyophilization of the liquid composition to obtain a lyophilized pharmaceutical composition.

[0143] A lyophilized pharmaceutical composition of the disclosure can be reconstituted before it is administered to a subject. Accordingly, the present disclosure contemplates a method of producing a liquid pharmaceutical composition comprising reconstituting a solid (lyophilized) pharmaceutical composition disclosed herein with a liquid pharmaceutically acceptable carrier.

[0144] The formulation of the current invention is typically provided as sterile preparations. Sterilization can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted either prior to or following lyophilization and reconstitution.

[0145] The pharmaceutical composition of the disclosure can be provided inside a vial. Accordingly, the present disclosure contemplates a vial comprising the pharmaceutical composition of the disclosure. Such pharmaceutical composition can be a liquid pharmaceutical composition of the disclosure, such as a liquid pharmaceutical composition before freezing or lyophilization or without having been frozen or lyophilized. Such pharmaceutical composition can also be a reconstituted liquid pharmaceutical composition of the disclosure. Such pharmaceutical composition can also be solid and / or lyophilized pharmaceutical composition of the disclosure. Such pharmaceutical composition can also be a frozen pharmaceutical composition of the disclosure.4. Kit

[0146] A kit is also provided herein. The kit comprises the pharmaceutical composition of the current disclosure which can be in one or more containers (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material (preferably waterproof, e.g. plastic or glass). A kit may comprise a liquid pharmaceutical composition of the disclosure. A kit may comprise a frozen pharmaceutical composition of the disclosure. A kit may comprise a solid or lyophilized pharmaceutical composition of the disclosure, which can be in one container. The kit can further comprise a second container comprising a suitable pharmaceutically acceptable carrier for reconstituting the solid or lyophilized pharmaceutical composition of the disclosure.

[0147] The kit may additionally contain instructions for use (e.g., in the form of a leaflet or instruction manual), means for administering the pharmaceutical composition of the present invention such as a syringe, pump, infuser or the like, means for reconstituting the pharmaceutical composition of the invention and / or means for diluting the pharmaceutical composition of the invention. The invention also provides kits for a single-dose administration unit. The kit of the invention may also contain a first recipient comprising the formulation in a dried / lyophilized form and a second recipient comprising an aqueous formulation. Kits containing single-chambered and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes) are also provided. The kit of the invention may comprise a container comprising the pharmaceutical composition of the invention.5. Method of producing the antibody and the formulation

[0148] The disclosure also relates to a method for the production of an antibody construct comprised in the pharmaceutical composition of the disclosure. The antibody construct is produced starting from the nucleic acid coding for the antibody construct or any subunit therein.The method can be carried out in vivo, the polypeptide can, for example, be produced in a bacterial or eukaryotic host organism and then isolated from this host organism or its culture. It is also possible to produce an antibody construct of the disclosure in vitro, for example by use of an in vitro translation system.

[0149] When producing the antibody construct in vivo, a nucleic acid encoding such polypeptide is introduced into a suitable bacterial or eukaryotic host organism by means of recombinant DNA technology. For this purpose, the host cell may be transformed with a cloning vector that includes a nucleic acid molecule encoding an antibody construct as described herein using established standard methods. The host cell may then be cultured under conditions, which allow expression of the heterologous DNA and thus the synthesis of the corresponding polypeptide or antibody construct. Subsequently, the polypeptide or antibody construct is recovered either from the cell or from the cultivation medium.

[0150] Suitable host cells can eukaryotic, such as immortalized mammalian cell lines (e.g., HeLa cells or CHO cells) or primary mammalian cells.

[0151] An antibody construct as described herein, however, is not necessarily generated or produced only by use of genetic engineering. Rather, such polypeptide can also be obtained by chemical synthesis such as Merrifield solid phase polypeptide synthesis or by in vitro transcription and translation. Methods for the solid phase and / or solution phase synthesis of proteins are well known in the art (see e.g., Bruckdorfer, T. et al. (2004) Curr. Pharm. Biotechnol. 5, 29-43).

[0152] An antibody construct of the disclosure may be produced by in vitro transcription / translation employing well-established methods known to those skilled in the art.

[0153] After production of the antibody construct, the antibody construct can be subjected to one ore more downstream processing steps, such as one or more of the following steps: anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction, chromatography (HIC), mixed mode chromatography (MMX), hydrophobic charge induction chromatography (HCIC), protein L chromatography, hydroxyapatite chromatography (HAC), diafiltration, and / or virus filtration.6. Method for treating a patient

[0154] The pharmaceutical compositions of the current disclosure may be formulated for intravenous, parenteral or subcutaneous injection. Preferably, the formulation of the invention may be administered to a subject intravenously.

[0155] The subject is a mammalian subject, for whom administration of the formulation is desired. Mammalian subjects include humans, non- human primates such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and the like, with humans being preferred.

[0156] When the target antigen of the antibody construct is CD30, the formulation of the current invention can be used in the prevention, treatment or amelioration of a CD30 positive proliferative disease or a tumorous disease. Preferably, said tumorous disease is a malignant disease, such as cancer. The malignant disease can be selected from the group consisting of Hodgkin lymphoma, Non-Hodgkin lymphoma, leukemia, multiple myeloma and solid tumors.

[0157] The present disclosure also provides a method for the treatment or amelioration of a CD30 positive proliferative disease or a tumorous disease, the method comprising the step of administering to a subject in need thereof a pharmaceutical composition of the current disclosure. The present disclosure also contemplates a method of treating a patient suffering from a CD30 positive cancer, the method comprising administering a pharmaceutical composition of the disclosure to the patient.

[0158] The present disclosure also contemplates a pharmaceutical composition of the disclosure or a pharmaceutical composition obtained by reconstituting a solid pharmaceutical composition of the disclosure or produced by a method of the disclosure for use in a method for treating a patient suffering from a CD30 positive proliferative disease and / or a tumorous species, such as CD30 positive cancer.

[0159] The present disclosure also contemplates the use of a (liquid) pharmaceutical composition of the disclosure, a solid pharmaceutical composition of the disclosure, a frozen pharmaceutical composition of the disclosure, a lyophilized pharmaceutical composition of the disclosure, or a pharmaceutical composition obtained by reconstituting a solid pharmaceutical composition of the disclosure or a pharmaceutical composition produced by a method of the disclosure for the manufacture of a medicament for treating a patient suffering from a CD30 positive proliferative disease and / or a tumorous species, such as CD30 positive cancer.7. Embodiments

[0160] The present invention is further characterized by the following items:1. A pharmaceutical composition comprising:(a) an antibody construct;(b) a buffer agent comprising one or more of the following agents: glutamic acid, histidine, aspartic acid, and one or more salts thereof;(c) a saccharide; and(d) a surfactant; wherein the pH of the pharmaceutical composition is about 3.5 to about 5.2, wherein the antibody construct comprises two polypeptide chains, wherein said antibody construct comprises (i) a first binding domain (A) which is specific for a first target and comprises the variable domains VH1 and VL1, and (ii) a second binding domain (B) which is specific for a second target and comprises the variable domains VH2 and VL2, wherein the variable domains are positioned within each of the two polypeptide chains from the N-terminus to the C-terminus in the order:VH 1 -L 1 - VL2-L2- VH2-L3 - VL 1 , wherein LI is a linker, wherein L2 is a linker, and wherein L3 is a linker. The antibody construct of item 1, wherein said two polypeptide chains associate with each other non-covalently. The antibody construct of item 1 or 2, wherein each of the VL1, VH2, VL2, and VH1 of one of the two polypeptide chains forms an antibody variable region with the VH1, VL2, VH2, and VL1 of the other one of the two polypeptide chains. The pharmaceutical composition of any one of the preceding items, wherein the two polypeptide chains in the antibody construct are identical to each other. The pharmaceutical composition of any one of the preceding items, wherein the first variable region (VH1 and VL1) is specific for CD16A and the second variable region (VH2 and VL2) is specific for a target cell antigen. The pharmaceutical composition of any one of the preceding items, wherein the second target is a target other than CD 16 A. The pharmaceutical composition of any one of the preceding items, wherein(i) the heavy chain variable domain specific for CD16A (VH1) comprises: a) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; b) a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; andc) a heavy chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:3; and(ii) the light chain variable domain specific for CD16A (VL1) comprises: a) a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; b) a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and c) a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6. The pharmaceutical composition of any one of the preceding items, wherein i) VH1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 14, and / or ii) VL1 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, or 100% identity to SEQ ID NO: 15. The pharmaceutical composition of any one of the preceding items, wherein VH1 comprises the amino acid sequence shown in SEQ ID NO: 14 and VL1 comprises the amino acid sequence shown in SEQ ID NO: 15. The pharmaceutical composition of any one of the preceding items, wherein said target cell antigen is selected from CD30, EGFR, CD123, CD19, CD20, and CD38. The pharmaceutical composition of any one of the preceding items, wherein said target cell antigen is CD30. The pharmaceutical composition of the preceding items, wherein(i) VH2 comprises: a) a heavy chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 7; b) a heavy chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 8; and c) a heavy chain CDR3 comprising the amino acid sequence shown in SEQ IDNO: 9; and(ii) VL2 comprises:a) a light chain CDR1 comprising the amino acid sequence shown in SEQ ID NO: 10; b) a light chain CDR2 comprising the amino acid sequence shown in SEQ ID NO: 11 ; and c) a light chain CDR3 comprising the amino acid sequence shown in SEQ ID NO:12. The pharmaceutical composition of any one of the preceding items, wherein a) VH2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 16, and / or b) VL2 comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity to SEQ ID NO: 17. The pharmaceutical composition of any one of the preceding items, wherein VH2 comprises the amino acid sequence shown in SEQ ID NO: 16 and VL2 comprises the amino acid sequence shown in SEQ ID NO: 17. The pharmaceutical composition of any one of the preceding items, wherein LI, L2 and L3, each consist of 12 or less amino acid residues. The pharmaceutical composition of any one of the preceding items, wherein LI is a glycine-serine linker, preferably a linker as shown in SEQ ID NO: 18. The pharmaceutical composition of any one of the preceding items, wherein L2 is a glycine-serine linker, preferably a linker as shown in SEQ ID NO: 18. The pharmaceutical composition of any one of the preceding items, wherein L3 is a glycine-serine linker, preferably a linker as shown in SEQ ID NO: 18. The pharmaceutical composition of any one of any one of the preceding items, wherein each of the two polypeptide chains comprises an amino acid sequence having at least 85%, at least 90%, at least 95% at least 98%, at least 99%, or 100% identity to SEQ ID NO: 13. The pharmaceutical composition of any one of the preceding items, wherein each of the two polypeptide chains comprises the sequence shown in SEQ ID NO: 13. The pharmaceutical composition of any one of the preceding items, wherein the pH of the pharmaceutical composition is from about 3.8 to about 4.4.The pharmaceutical composition of any one of the preceding items, wherein the pH of the pharmaceutical composition is from about 3.9 to about 4.2. The pharmaceutical composition of any one of the preceding items, wherein the pH of the pharmaceutical composition is about 4.0. The pharmaceutical composition of any one of the preceding items, wherein the buffer agent comprises one or more of the following agents: glutamic acid, aspartic acid, and one or more salts thereof. The pharmaceutical composition of any one of the preceding items, wherein the buffer agent comprises glutamic acid and / or one or more salts thereof. The pharmaceutical composition of any one of the preceding items, wherein the buffer agent comprises aspartic acid and / or salt thereof. The pharmaceutical composition of any one of the preceding items, wherein the concentration of the antibody construct is up to about 50 mg / ml, up to about 40 mg / ml, or up to about 30 mg / ml. The pharmaceutical composition of any one of the preceding items, wherein the concentration of the antibody construct is at least about 5 mg / ml, at least about 6 mg / ml, or at least about 7 mg / ml. The pharmaceutical composition of any one of the preceding items, wherein the concentration of the antibody construct is from about 3 mg / ml to about 50 mg / ml, from about 4 mg / ml to about 40 mg / ml, from about 5 mg / ml to about 30 mg / ml, from about 6 mg / ml to about 25 mg / ml, from about 7 mg / ml to about 20 mg / ml, or about 8 mg / ml to about 15 mg / ml. The pharmaceutical composition of any one of the preceding items, wherein the concentration of the buffer is about 3 to about 50 mM, about 5 to about 25 mM, about 10 to about 20 mM, about 12 to about 17 mM, or about 15 mM. The pharmaceutical composition of any one of the preceding items, wherein said saccharide comprises trehalose. The pharmaceutical composition of any one of the preceding items, wherein the amount of saccharide is about 4% to about 15% (w / v), about 6% to about 10% (w / v), or about 8% (w / v).The pharmaceutical composition of any one of the preceding items, wherein the amount of trehalose is about 4% to about 15% (w / v), or about 6% to about 10% (w / v), or about 8% (w / v), based on the weight of trehalose dihydrate. The pharmaceutical composition of any one of the preceding items, wherein the surfactant comprises one or more of the agents: a pluronic, PEG, a sorbitan ester, a polysorbate, triton, tromethamine, lecithin, cholesterol, and / or tyloxapal. The pharmaceutical composition of any one of the preceding items, wherein the surfactant comprises polysorbate 20. The pharmaceutical composition of any one of the preceding items, wherein the amount of surfactant is about 0.005% to about 0.1% (w / v), about 0.006% to about 0.05% (w / v), about 0.008% to about 0.03% (w / v), about 0.009% to about 0.02 % (w / v), or about 0.01%(w / v). The pharmaceutical composition of any one of the preceding items, wherein the amount of polysorbate 20 is about 0.005% to about 0.1% (w / v), 0.006% to about 0.05% (w / v), about 0.008% to about 0.03% (w / v) or about 0.009% to about 0.02 % (w / v), or about 0.01% (w / v). The pharmaceutical composition of any one of the preceding items, wherein:(a) the concentration of the antibody construct is from 5 mg / ml to 30 mg / ml, wherein said two polypeptide chains of the antibody construct are identical and have at least 95% identity to SEQ ID NO: 13, wherein VH1 comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VH2 comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 comprises the CDR sequences as shown in SEQ ID NOs: 10-12;(b) the buffer comprises glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 12-17 mM;(c) the saccharide comprises trehalose, which is in a concentration of 5-10% (w / v) based on the weight of trehalose dihydrate; and(d) the surfactant comprises polysorbate 20 and the concentration of the polysorbate 20 is 0.005-0.02% (w / v); and wherein the pH of the pharmaceutical composition is from about 3.8 to about 4.2. The pharmaceutical composition of item 38, wherein said two polypeptide chains comprise the amino acid sequence shown in SEQ ID NO: 13.The pharmaceutical composition of item 38 or 39, wherein the amino acid sequence of said two polypeptide chains consist of the amino acid sequence shown in SEQ ID NO: 13. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 8 % relative HMWS content as determined by HP- SEC after storage at about 2-8°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to up to 5 % relative HMWS content as determined by HP-SEC after storage at about 2-8°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 10 % relative HMWS content as determined by HP- SEC after storage at about 2-8°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 5 % relative HMWS content as determined by HP- SEC after storage at about 2-8°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 50 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 20 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 10 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 50 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 20 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 10 % relative HMWS content as determined by HP- SEC after storage at about 25°C for 14 days.The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 60 % relative HMWS content as determined by HP- SEC after storage at about 40°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 50 % relative HMWS content as determined by HP- SEC after storage at about 40°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the HMWS% in the composition increases by up to 30 % relative HMWS content as determined by HP- SEC after storage at about 40°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 85 % of its original value as determined by HP-SEC after storage at about 2-8°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 90 % of its original value as determined by HP-SEC after storage at about 2-8°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 65 % of its original value as determined by HP-SEC after storage at about 2-8°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 85 % of its original value as determined by HP-SEC after storage at about 2-8°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 45 % of its original value as determined by HP-SEC after storage at about 25°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 70 % of its original value as determined by HP-SEC after storage at about 25°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 40 % of its original value as determined by HP-SEC after storage at about 25°C for 14 days.The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 70 % of its original value as determined by HP-SEC after storage at about 25°C for 14 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 45 % its original value as determined by HP-SEC after storage at about 40°C for 7 days. The pharmaceutical composition of any one of the preceding items, wherein the relative homodimer content maintains at least 45 % its original value as determined by HP-SEC after storage at about 40°C for 7 days. A solid pharmaceutical composition obtainable or obtained by lyophilisation of the pharmaceutical composition of any one of the preceding items. A frozen pharmaceutical composition obtainable or obtained by freezing of the pharmaceutical composition of any one of items 1-63. A vial comprising the pharmaceutical composition of any one of items 1-63. A method of producing a lyophilized pharmaceutical composition, comprising lyophilization of the pharmaceutical composition of any one of items 1-63. A method of producing a frozen pharmaceutical composition, comprising freezing the pharmaceutical composition of any one of items 1-63. The method of item 68, wherein the method comprises holding the pharmaceutical composition at a temperature of 0-25 °C for 1-24 hours before starting the lyophilization or freezing step. The method of item 67, comprising:(i) thawing the frozen pharmaceutical composition of item 65 to obtain a liquid composition;(ii) holding the liquid composition at a temperature of 0-25 °C for 1-24 hours; and(iii) lyophilization of the liquid composition to obtain a lyophilized pharmaceutical composition. A method of producing a liquid pharmaceutical composition comprising reconstituting the solid pharmaceutical composition of item 64 or a lyophilized pharmaceutical composition obtainable or obtained by the method of any one of items 67 and 69-70 with a liquid pharmaceutically acceptable carrier.2. A method of treating a patient suffering from a CD30 positive cancer, the method comprising administering the pharmaceutical composition of any one of items 1-63 or a pharmaceutical composition obtained by reconstituting the solid pharmaceutical composition of item 64 or produced by the method of item 71. 3. A pharmaceutical composition of any one of items 1-63 or a pharmaceutical composition obtained by reconstituting the solid pharmaceutical composition of item 64 or produced by the method of item 71 for use in a method for treating a patient suffering from a CD30 positive cancer. 4. Use of a pharmaceutical composition of any one of items 1-63, a solid pharmaceutical composition of item 64, a frozen pharmaceutical composition of item 65, a lyophilized pharmaceutical composition or frozen pharmaceutical composition produced by the method of any one of items 67-70, or a liquid pharmaceutical composition produced by the method of item 71 for the manufacture of a medicament for treating a CD30 positive cancer. 5. A kit comprising a pharmaceutical composition of any one of items 1-63, a solid pharmaceutical composition of item 64, a frozen pharmaceutical composition of item 65, a lyophilized pharmaceutical composition or frozen pharmaceutical composition produced by the method of any one of items 67-70, or a liquid pharmaceutical composition produced by the method of item 71. . Sequences

[0161] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0162] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0163] A better understanding of the present invention and of its advantages will be obtained from the following examples, offered for illustrative purposes only. The examples are not intended to limit the scope of the present invention in any way.

[0164] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.EXAMPLES

[0165] The examples below describe the experiments carried out using the CD30xCD16A bispecific tandem diabody, which consists of two identical polypeptide chains each having the amino acid sequence as depicted in SEQ ID NO: 13.

[0166] In the following examples, the details of the measurement methods used at the time for the experiments are the following. The herein described methods can be applied in a general way, not limited to the samples tested in the examples. Moreover, it is understood that variations of these methods can be applied using the common general knowledge.Measurement methods:Method 1 : pH measurement:

[0167] pH was measured with a calibrated pH meter (SevenEasy®, Mettler Toledo AG, Schwerzenbach, Switzerland) by using a high / normal ionic strength electrode (InLab® Micro).Method 2: UV spectroscopy for turbidity measurement

[0168] UV-spectroscopy was performed in 96-well plates (Corning Incorporation, NY, USA) using a Tecan Safire plate reader (Tecan Austria GmbH, Grbdig, Austria). The samples were gravimetrically diluted to 0.5 mg / ml with dilution buffer prior to each measurement. For each data point 3 wells (n=3), each with 200 pl solution were measured to minimize measurement errors. After the measurement, the data was subtracted with corresponding blank spectrum (i.e., corresponding placebo buffer) and corrected for the path length.

[0169] The turbidity at Assonm was measured. The aggregation index determined at 350 nm was used to assess the turbidity and the extent of light scattering in the sample, respectively, which is expressed as the aggregation index (A. I.):A.I.=(A (280 nm)) / (A (280 nm)-A (350 nm))*100Method 3 : Micro-Flow Imaging (MFI)

[0170] The Micro-Flow Imaging measurements were conducted at a MFI-5200 particle analyzer system (ProteinSimple, Santa Clara, CA, USA) equipped with a silane-coated high-resolution 100-pm flow cell. The samples were analyzed undiluted. Prior to measurement, the system wasflushed with 0.25 ml formulation buffer using a dispense volume of 0.2 ml and a flow rate of 1 ml / min. Afterwards, 0.23 ml of the sample were loaded with a dispense volume of 0.2 ml and a flow rate of 0.2 ml / min. Then, 0.47 ml of sample were loaded and used to optimize the background illumination. Sample measurement was started with the 0.47 ml afterwards. Approximately 1100 images were taken per sample. Between the measurements, the flow cell was cleaned with ultra-pure water. MFI View System Software (MVSS) version 2-R2- 6.1.20.1915 was used to perform the measurements and MFI View Analysis Suite (MVAS) software version 1.3.0.1007 was used to analyze the samples.Method 4: High performance size exclusion chromatography (HP-SEC)

[0171] High molecule weight species (HMWS) refers to those molecules which are oligomers from the active TandAb homodimer, i.e., higher than 104 kDa. The percentage of HMWS is measured in the examples by the HP-SEC method.

[0172] HP-SEC separates molecules according to their apparent molecular weight. Compounds with higher molecular weight elute earlier than those of lower molecular weight. Thus, the AFM13 homodimer can be separated from its high molecular weight species and fragments.

[0173] HP-SEC was performed according to AIM-QC-003; Version 4. A summary of the isocratic HP-SEC method is provided in Table 3. Prior to SEC analysis, the samples were diluted to 1 mg / ml with mobile phase and afterwards centrifuged for 3 min at 10,000 rpm. 250 pl of the supernatant were then transferred into HPLC vials.

[0174] The sample is separated isocratically using 0.04 M NaH2PO4 x H2O; pH 7 as mobile phase. A chromatogram is recorded at a wavelength of 214 nm. The obtained peaks are assigned to homodimer and HMWS by their retention time. Quantification of high molecular weight species is achieved by dividing the area in milli absorption units [mAUs] of the HMWS by the area [mAUs] of all AFM13 peaks.Table 3: Details of the HP-SEC method.Stability test under various storage conditions:

[0175] The formulations were subjected to various storage conditions of temperature 2-8°C, 25°C or 40°C for 2 weeks. Stability tests were carried out at various time points during the storage period.Example 1: Comparison between various buffer systems

[0176] In the current example, the formulations to be prepared and tested are summarized below in Table 4Table 4 Formulations tested in Example 1

[0177] The corresponding placebo formulations without AFM13 were also prepared as reference samples.

[0178] Immediately after the preparation of the formulation, a first round of stability measurement was performed and noted under the time point of TO. Then the formulations were subjected to the tests under various stress conditions:

[0179] Test results: a) Evaluation at TO

[0180] The turbidity, particle count and HMWS% at TO were measured and the data results are summarized in Table 5 below. Generally, acceptance criteria for particles are set based on Ph. Eur. 2.9.19 or USP <788>. In general, for particles > 10 pm: < 6000 particles / vial are accepted, and for particles > 25 pm: < 600 particles / vial are accepted. For AFM13, a vial commonly contains 5.5 mL of the liquid pharmaceutical composition.Table 5 Turbidity at A350, particle count by MFI and HMWS% at TO* precipitation was detected.** assuming a volume of 5.5 mL

[0181] After the sample preparation and before the storage, the formation of HMW species of AFM13 was already apparent with certain samples. The formulation F2 (phosphate pH 6.0), F3 (histidine buffer pH 6.0), F10 (citrate pH 5.0) and Fl l (citrate pH 4.5) showed precipitation and particles could be detected due to possible colloidal instability.

[0182] In contrast, the results showed that the placebo formulations, F5 (histidine pH5.2), F6 (histidine pH5.0), F7 (glutamic acid pH5.2), F8 (glutamic acid pH5.0), and F9 (glutamic acid pH4.5) were essentially free of visible particles.

[0183] In the case of histidine buffer, as can be seen by comparing F3 (histidine pH 6.0) and F4 (histidine pH 5.5), F5 (histidine pH5.2), F6 (histidine pH5.0), the formulations at a pH <5.2 achieved lower turbidity, lower particle counts and lower HMWS% than those with a higher pH.

[0184] In the case of glutamic acid buffer, significant improvement was also seen by lowering pH: when lowering the pH from 5.2, to 5.0 and then to 4.5, the HMWS% decreased significantly. Moreover, the formulations with glutamic acid buffer at pH<5.2 also achieved low turbidity and low particle counts.

[0185] In summary, the above shows that the formulation with histidine buffer at pH<5.2 and the formulation with glutamate buffer at pH<5.2 achieved low turbidity, particle counts and HMWS% which means a better stability. Hence, a low pH at pH<5.2 combined with histidine and / or glutamate buffers can significantly improve the stability of the antibody construct already at TO. However, a similar low pH did not stabilize the formulation when citrate buffer was used. A significantly higher particle count and turbidity with a low HMWS% means that a colloidal instability of the matrix could be detected. b) Results for stress testing (storage stability)

[0186] In order to test the stability of the formulation, the formulations were stored under 2-8°C, 25°C or 40°C degrees for 2 weeks. pH shifts were minor throughout the study at all temperatures (within max. ±0.2).Turbidity results:

[0187] The results of turbidity changes are presented in Table 6, Table 7 and Table 8 below and Fehler! Verweisquelle konnte nicht gefunden werden.:Table 6 turbidity (A350) after storage at 2-8°CTable 7 turbidity (A350) after storage at 25°CTable 8 turbidity after storage at 40°C

[0188] As can be seen in Table 6, Table 7 and Table 8 and Fehler! Verweisquelle konnte nicht gefunden werden., concerning the formulations with histidine buffer at pH<5.2 (i.e. F5, F6) and glutamate buffer at pH<5.2 (i.e. F7, F8, F9), the turbidity were maintained at a stable and low level without significant fluctuations. This is important for the quality of the pharmaceutical formulations, as changes in the drug properties throughout the shelf life should be reduced to the minimum. Unpredictable changes are especially undesirable.

[0189] In contrast, the formulations with histidine at a higher pH (i.e., F3 and F4) and especially with phosphate buffer at pH 6.0 showed not only a generally higher turbidity level, but also a larger fluctuation in the turbidity. Moreover, a low pH at 5.0 or 4.5 in combination with citrate buffer did not stabilize the formulation, but rather resulted in the largest fluctuations in some tests.Particle counts by MFI (data not shown):

[0190] When stored under various temperature, the formulations of F2 (phosphate, pH6.0), F3 (histidine, pH6.0), F10 (citrate pH5.0) and Fl l (citrate pH4.5) developed significantly higher amounts of visible particles. As for sub-visible particles, F5 (histidine pH5.2), F6 (histidine pH5.0), F7 (glutamic acid pH5.2), F8 (glutamic acid pH5.0), and F9 (glutamic acid pH4.5) showed comparatively small changes, whereas the formulations F2 (phosphate, pH6.0), F3 (histidine, pH6.0), F4 (histidine pH5.5), F10 (citrate pH5.0) and Fl l (citrate pH4.5) showed significant changes.Example 2: HMWS% results

[0191] Further, storage stability was also analyzied in term of HMWS%. HMWS% is considered an important quality attribute for AFM13 preparations taking in consideration potency (biological activity or efficacy), PK / PD, immunocenicity and safety. The test data are presented in Table 9 below and summarized in Fehler! Verweisquelle konnte nicht gefunden werden.:Table 9 Relative peak area HMWS%

[0192] Several conclusions can be drawn from the data in Table 9 and Fehler! Verweisquelle konnte nicht gefunden werden..

[0193] Firstly, for the formulations with glutamate, regardless of the storage temperature, the lower the pH was, the lower HMWS% level can be obtained at any specific time point as compared to histidine. Moreover, from the fluctuation point of view, the lower the pH was, the less HWMS% fluctuation was observed, which means a more stable quality. These two aspects mean that lowering pH combined with glutamate buffer not only reduced the absolute value of HMWS% at all time points and storage temperatures, but also reduced the delta in quality changes during storage.

[0194] Secondly, for the formulations with histidine, regardless of the storage temperature, lower pH also resulted in decreased HMWS% level at any specific time point. Moreover, a clear trend of decreased delta in HMWS% changes was observed when lowering the pH.

[0195] In summary, the tests in Example 1 and 2 showed a ranking concerning the overall stability as the following: glutamic acid > Histidine > Phosphate » citrate. It is advantageous to formulate an antibody without covalent inter-chain bonds, such as AFM13, with glutamic acid buffer at pH<5.2 or histidine buffer at pH<5.2. These formulations not only had higher quality, but also had minimal changes in their properties with time.Example 3: Comparison of various pH in the amino acid buffer systems

[0196] This example shows that for formulations with glutamic acid buffer, lowering the pH from 4.8 to 4.2 surprisingly improves the stability of the AFM13 formulation even further.

[0197] In the current example, the following formulations were tested as presented in Table 10:Table 10 Formulation tested in Example 2

[0198] All formulations revealed consistent pH values throughout the stability study with small variations of ± 0.4 units. No time dependent differences of pH were determined in any of the formulations.

[0199] In addition to the storage test, an additional stability test was carried out by subjecting the formulations F12 and F21 to mechanical stress. F12 differed from F9 in that a different starting material was used that already had higher HMWS% content at the beginning of the test as compared to F9. Mechanical stress was performed using an IKA® KS4000ic orbital shaker at room temperature. Shaking was performed with 400 rpm for 2 hours. Samples were covered with aluminum foil in order to avoid additional stress by light sources.HP- SEC results

[0200] The HP-SEC results are summarized in Table 11 and Table 12. The same data are also reflected in Fehler! Verweisquelle konnte nicht gefunden werden..Table 11 : Results of relative homodimer content determined by HP-SEC.Table 12: Results of relative HMWS content determined by HP-SEC.

[0201] Relative homodimer content at TO already showed distinguishable differences between the formulations. Homodimer content of F12 (pH 4.5), F14 (pH 4.2), and F21 (aspartic acid, pH 4.5) ranged from 78 - 83%. For F13 (pH 4.8), homodimer content was between 72 - 73%. Moreover, among all the formulations at the time point of TO, glutamic acid with pH 4.2 achieved the best results.

[0202] After storage for 14 days at 2 - 8 °C, HMWS content slightly increased by 2 - 4% for F12 (pH 4.5), F14 (pH 4.2) and F21 (aspartic acid, pH 4.5) A more pronounced increase in HMWS content was observed for F13 (pH 4.8) with approx. 9 - 12% more HMWS. The total peak area of the remaining formulations was in a comparable range to TO.

[0203] After storage for 14 days at 25 °C, HMWS content further increased for all formulations. HMWS content of F12 (pH 4.5) and F21 (aspartic acid, pH 4.5) increased by 10 - 13%. A more pronounced increase in HMWS content was observed for F13 (pH 4.8) with 17 - 21%. The total peak areas of the remaining formulations remained in the same range as at TO.

[0204] HMWS content further increased after storage of the formulations for 7 days at 40 °C. HMWS content of F12 (pH 4.5) and F21 (aspartic acid, pH 4.5) increased by 9 - 10%, whereas the HMWS content of F13 (pH 4.8) increased by 12 - 17%. Total peak areas of the remaining formulations were in a comparable range to TO.

[0205] In summary, F14 (pH 4.2) was the most stable formulation in terms of HP-SEC analysis with > 75% relative homodimer content detected at all time points. No matter at which time point, F14 (pH 4.2) always achieved the best quality (i.e., highest homodimer amount and lowest HMWS amount). Moreover, from the stability point of view, F14 (pH 4.2) also had the smallest changes in the properties over time.Example 4: Comparison of various pH in the glutamic acid buffer systemThe following samples were tested as shown in Table 13:Table 13 formulations tested in Example 3Particle count results:

[0206] The cumulative particle concentrations were measured starting from TO (i.e. immediately after the preparation of the formulation) by the MFI method for several days.

[0207] As shown in Table 14 below, the cumulative particle concentrations obtained for the AFM13 samples at pH 4.0 and 4.5 both remained low (< ca. 6,600 / ml for particle sizes > 1 pm, and < ca. 2,700 / ml for particle sizes > 2 pm) throughout the time points between TOd and T7d. For particles > 10 pm, the samples at pH 4.5 continuously yielded substantially higher cumulative particle concentrations.Table 14: Cumulative particle concentrations obtained from MFI of AFM13 samples at pH 4.0 and 4.5.Turbidity measurementMeasurement method:

[0208] The sample turbidity measurements were performed by using a NEPHLA turbidimeter (Dr. Lange, Germany) at 860 nm and 90° detection angle. Prior to measurements of 2 ml undiluted samples, the system was calibrated by using a 6 NTU (nephelometric turbidity unit) formazin standard. The obtained results in units of NTU were converted into formazin nephelometric units (FNU).Turbidity results:

[0209] The nephelometry measurements of the AFM13 samples at pH 4.0 and 4.5 yielded constant turbidity values throughout the time points between TOd and T7d. However, the obtained turbidity values of ca. 1.4-1.6 FNU for the pH 4.5 samples were higher than the turbidity values of ca. 1.0-1.1 FNU obtained for the pH 4.0 samples (see Table 15).Table 15: Results obtained from turbidity measurements (n=2) of AFM13 samples at pH 4,0 and 4,5,Results of HP-SEC:

[0210] Throughout the time points between TOd and T7d, both AFM13 samples yielded increasing high- molecular- weight species (HMWS) and decreasing homodimer contents (see Table 16 and Fehler! Verweisquelle konnte nicht gefunden werden.). However, the samples at pH 4.0 exhibited substantially lower HMWS contents, starting from 2.1% at time point TOd up to 7.1% at time point T7d, than the samples at pH 4.5, which yielded starting HMWS contents of 4.1%, and 16.7% at time point T7d. The total peak areas remained constant throughout time points TOd to T7d.

[0211] Moreover, the samples with pH4.0 showed smaller changes from TOd to T7d than the samples with pH4.5, showing that the lower pH of 4.0 had a higher stabilizing effect than the higher pH of 4.5.Table 16: Results obtained from HP-SEC analyses of AFM13 samples at pH 4.0 and 4.5.

[0212] In summary, this example showed that lowering the pH beyond 4.5 even further improves the quality and stability of the AFM13 formulation. With the other conditions being the same, AFM13 formulation with pH4.0 achieved lower turbidity, lower accumulated particles and lower HMW% than AFM13 formulation with pH4.5. Moreover, the lower pH of 4.0 stabilized the formulation better as the changes in the properties were smaller than the higher pH of 4.5.* * *

[0213] Embodiments illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present embodiments have been specifically disclosed by preferred embodiments and optional features, modification and variations thereof may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. In addition, where features are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0214] Equivalents: Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0215] It should be understood that this invention is not limited to the particular methodology, protocols, material, reagents, and substances, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0216] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer’s specifications, instructions, etc.) are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.

[0217] Further embodiments will become apparent from the following claims.

Claims

CLAIMS1. A pharmaceutical composition comprising:(a) an antibody construct;(b) a buffer agent comprising one or more of the following agents: glutamic acid, histidine, aspartic acid, and one or more salts thereof;(c) a saccharide; and(d) a surfactant; wherein the pH of the pharmaceutical composition is about 3.5 to about 5.2, wherein the antibody construct comprises two polypeptide chains, wherein said antibody construct comprises (i) a first binding domain (A) which is specific for a first target and comprises the variable domains VH1 and VL1, and (ii) a second binding domain (B) which is specific for a second target and comprises the variable domains VH2 and VL2, wherein the variable domains are positioned within each of the two polypeptide chains from the N-terminus to the C-terminus in the order:VH 1 -L 1 - VL2-L2- VH2-L3 - VL 1 , wherein LI is a linker, wherein L2 is a linker, and wherein L3 is a linker.

2. The antibody construct of claim 1, wherein said two polypeptide chains associate with each other non-covalently.

3. The pharmaceutical composition of claim 1 or 2, wherein VH1 comprises the amino acid sequence shown in SEQ ID NO: 14 and VL1 comprises the amino acid sequence shown in SEQ ID NO: 15, and / or wherein VH2 comprises the amino acid sequence shown in SEQ ID NO: 16 and VL2 comprises the amino acid sequence shown in SEQ ID NO: 17.

4. The pharmaceutical composition of any one of the preceding claims, wherein each of the two polypeptide chains comprises the sequence shown in SEQ ID NO: 13.

5. The pharmaceutical composition of any one of the preceding claims, wherein the pH of the pharmaceutical composition is from about 3.8 to about 4.4.

6. The pharmaceutical composition of any one of the preceding claims, wherein the buffer agent comprises glutamic acid and / or one or more salts thereof.

7. The pharmaceutical composition of any one of the preceding claims, wherein the concentration of the antibody construct is from about 3 mg / ml to about 50 mg / ml, from about 4 mg / ml to about 40 mg / ml, from about 5 mg / ml to about 30 mg / ml, from about 6 mg / ml to about 25 mg / ml, from about 7 mg / ml to about 20 mg / ml, or about 8 mg / ml to about 15 mg / ml.

8. The pharmaceutical composition of any one of the preceding claims, wherein:(a) the concentration of the antibody construct is from 5 mg / ml to 30 mg / ml, wherein said two polypeptide chains of the antibody construct are identical and have at least 95% identity to SEQ ID NO: 13, wherein VH1 comprises the CDR sequences as shown in SEQ ID NOs: 1-3, VL1 comprises the CDR sequences as shown in SEQ ID NOs: 4-6, VH2 comprises the CDR sequences as shown in SEQ ID NOs: 7-9, and VL2 comprises the CDR sequences as shown in SEQ ID NOs: 10-12;(b) the buffer comprises glutamic acid and / or the salt of glutamic acid, wherein the total concentration of glutamic acid and the salt thereof is 12-17 mM;(c) the saccharide comprises trehalose, which is in a concentration of 5-10% (w / v) based on the weight of trehalose dihydrate; and(d) the surfactant comprises polysorbate 20 and the concentration of the polysorbate 20 is 0.005-0.02% (w / v); and wherein the pH of the pharmaceutical composition is from about 3.8 to about 4.2, wherein said two polypeptide chains preferably comprise the amino acid sequence shown in SEQ ID NO: 13.

9. A solid pharmaceutical composition obtainable or obtained by lyophilisation of the pharmaceutical composition of any one of the preceding claims.

10. A frozen pharmaceutical composition obtainable or obtained by freezing of the pharmaceutical composition of any one of claims 1-8.

11. A vial comprising the pharmaceutical composition of any one of claims 1-8.

12. A method of producing a lyophilized pharmaceutical composition, comprising lyophilization of the pharmaceutical composition of any one of claims 1-8.

13. A method of producing a frozen pharmaceutical composition, comprising freezing the pharmaceutical composition of any one of claims 1-8.

14. A method of producing a liquid pharmaceutical composition comprising reconstituting the solid pharmaceutical composition of claim 9 or a lyophilized pharmaceutical composition obtainable or obtained by the method of claim 12 with a liquid pharmaceutically acceptable carrier.

15. A pharmaceutical composition of any one of claims 1-8 or a pharmaceutical composition obtained by reconstituting the solid pharmaceutical composition of claim 9 or produced by the method of claim 14 for use in a method for treating a patient suffering from a CD30 positive cancer.

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