Formulations of Anti-gremlin-1 antibodies

Abstract

The present invention relates to a pharmaceutical composition comprising an anti-Gremlin-1-antibody, or an antigen-binding fragment thereof, L-histidine, its hydrochloride salt, or the combination thereof, and L-proline. Furthermore, the present invention relates to the use of the pharmaceutical composition for preparing a pharmaceutical solution suitable for infusion to a human patient as well as to a pharmaceutical solution comprising the pharmaceutical composition. Furthermore, the present invention relates to the pharmaceutical composition or the pharmaceutical solution for use as a medicament, e.g., in the treatment of cancer.

Description

[0001] FORMULATIONS OF ANTI-GREMLIN-1 ANTIBODIES

[0002] Co-filed SEQUENCE LISTING

[0003] The present specification makes reference to a sequence listing (submitted electronically on the same date as the present application). The entire contents of the Sequence Listing are incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a pharmaceutical composition comprising an anti-Gremlin-1 -antibody, or an antigen-binding fragment thereof, L-histidine, its hydrochloride salt, or the combination thereof, and L-proline. Furthermore, the present invention relates to the use of the pharmaceutical composition for preparing a pharmaceutical solution suitable for infusion to a human patient as well as to a pharmaceutical solution comprising the pharmaceutical composition. Furthermore, the present invention relates to the pharmaceutical composition or the pharmaceutical solution for use as a medicament, e.g., in the treatment of cancer.

[0006] BACKGROUND OF THE INVENTION

[0007] Gremlin-1 (also known as Drm and CKTSF1 B1) is a 184 amino acid glycoprotein which forms part of the DAN (differential screening-selected gene aberrative in neuroblastoma) family of cys- teine-knot secreted proteins (along with Cerberus and Dan amongst others). Gremlin binds and inhibits the ability of bone morphogenetic proteins (BMPs)-2, 4, and 7 to signal along with a documented pro-angiogenic role possibly through agonism of VEGFR2. The main role of Gremlin-1 is during development, in which it is vital during kidney formation and during limb bud formation. These vital roles make gremlin homozygous knock-outs lethal in embryonic mice.

[0008] In adulthood, increased levels of gremlin have been associated with idiopathic pulmonary fibrosis and pulmonary arterial hypertension in which BMP-2, 4 and 7 signalling is reduced with an associated rise in TGF-p levels. In both diabetic and chronic allograft nephropathy, Gremlin-1 expression has been correlated with fibrosis score.

[0009] Increased levels of gremlin are also linked to scleroderma, diabetic nephropathy and colorectal cancer. Gremlin-1 has been shown to activate cancer cell invasion and proliferation and is thought to play a role in uterine cervix, lung, ovary, kidney, breast, colon, pancreatic and sarcoma carcinomas.

[0010] In view of the above, Gremlin-1 has been a prospective target of interest in oncology and other diseases for several years. Against this background, inhibitors of Gremlin-1 activity have been developed. Preferred inhibitors include anti-Gremlin-1 antibodies and antigen-binding fragments thereof. WO2019 / 243801 relates to an anti-Gremlin-1 antagonist for use in a method of treatment of cancer. WO 2019 / 158658 A1 relates to an anti-Gremlin-1 antibody for use in the treatment of a bone fracture or bone defect. WO 2018 / 115017 A2 inter alia provides antibodies which bind an inhibitory site on Gremlin-1. Ginisortamab is a first-in-class, fully human immunoglobulin G4P (lgG4P) monoclonal antibody (mAb) that demonstrates the ability to restore Gremlin-1-sup- pressed BMP-signaling (MABS 2023, Vol. 15, No. 1 , 2289681).

[0011] One of the long-standing problems with pharmaceutical compositions and solutions comprising antibodies or fragments thereof is physical and chemical stability. As a result, particle formation may, e.g., be observed.

[0012] Against this background, there is a need for a pharmaceutical composition and a pharmaceutical solution comprising an anti-Gremlin-1 -antibody, or an antigen-binding fragment thereof, which have advantageous stability properties.

[0013] OBJECTS AND SUMMARY OF THE INVENTION

[0014] It is an object of the invention to provide a pharmaceutical composition comprising an anti-Grem- lin-1-antibody, or an antigen-binding fragment thereof, which has advantageous properties regarding the stability. In particular, it is an object of the invention to provide a pharmaceutical composition comprising an anti-Gremlin-1-antibody, or an antigen-binding fragment thereof, for which the problem of particle formation upon dilution is reduced. Thus, it is another object of the invention to provide a pharmaceutical composition comprising an anti-Gremlin-1 -antibody, or an anti- gen-binding fragment thereof, which can advantageously be used for preparing a pharmaceutical solution suitable for infusion to a human patient, such that, e.g., no particles have to be filtered off prior to administration or such that no clogging of the filter occurs during administration. It is yet another object of the invention to provide a pharmaceutical solution comprising the pharmaceutical composition and a 0.9% NaCI solution in water, or a 5% glucose solution in water, which has advantageous properties regarding the stability, and preferably has a reduced tendency to form particles.

[0015] These objects are achieved by the pharmaceutical compositions and the pharmaceutical solutions as defined herein. Without being bound to any theory, it has surprisingly been found by the inventors of the present invention that the use of L-proline in combination with a pH of from 6.4 to 7.2 results in advantageous properties regarding the stability of the pharmaceutical compositions of the invention and regarding the stability of the pharmaceutical solutions of the invention comprising such a pharmaceutical composition.

[0016] In a first aspect, the present invention relates to a pharmaceutical composition a. an anti-Gremlin-1 antibody, or an antigen-binding fragment thereof; b. L-histidine, its hydrochloride salt, or the combination thereof; and c. L-proline; wherein the pharmaceutical composition has a pH of from 6.4 to 7.2.

[0017] In one embodiment, the pharmaceutical composition further comprises d. sucrose.

[0018] In a preferred embodiment, the sucrose is present in a molar concentration of from 150 mM to 350mM.

[0019] In one embodiment, the pharmaceutical composition further comprises e. L-methionine. In a preferred embodiment, the L-methionine is present in a molar concentration of from 5 mM to 25 mM.

[0020] In one embodiment, the pharmaceutical composition further comprises f. a polysorbate.

[0021] In a preferred embodiment, the polysorbate is present in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition.

[0022] In another preferred embodiment, the polysorbate is polysorbate 80.

[0023] In one embodiment, the anti-gremlin-1 antibody or the antigen-binding fragment thereof is present in a concentration of from 20 mg / mL to 300 mg / mL.

[0024] In one embodiment,

[0025] (i) the HCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 4;

[0026] (ii) the HCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 5;

[0027] (iii) the HCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 6;

[0028] (iv) the LCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 7;

[0029] (v) the LCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 8; and

[0030] (vi) the LCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 9.

[0031] In one embodiment, the anti-gremlin-1 antibody comprises the heavy chain / light chain pair of SEQ ID NOs: 22 / 23.

[0032] In one embodiment, the L-histidine, its hydrochloride salt, orthe combination thereof is present in a molar concentration of from 20 mM to 80 mM.

[0033] In one embodiment, the L-proline is present in a molar concentration of from 150 mM to 350mM.

[0034] In one embodiment, the pharmaceutical composition comprises: a. an anti-Gremlin1 antibody comprising a heavy chain / light chain pair of SEQ ID Nos: 22 / 23 in a concentration of from 50 mg / mL to 150 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 40 mM to 60 mM; c. L-proline in a molar concentration of from 200 mM to 300mM; d. sucrose in a molar concentration of from 200 mM to 300mM; e. L-methionine in a molar concentration of from 10 mM to 16 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0035] In another aspect, the present invention relates to the use of a pharmaceutical composition as defined above, for preparing a pharmaceutical solution suitable for infusion to a human patient.

[0036] In yet another aspect, the present invention relates to a pharmaceutical solution comprising

[0037] (i) the pharmaceutical composition as defined above; and

[0038] (ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water; wherein the anti-gremlin-1 antibody or an antigen-binding fragment thereof comprised in the pharmaceutical composition is present in the pharmaceutical solution in a concentration of from 1 mg / mL to 30 mg / mL.

[0039] In another aspect, the present invention relates to the pharmaceutical composition or the pharmaceutical solution as defined herein for use as a medicament.

[0040] BRIEF DESCRIPTION OF THE FIGURES

[0041] Figure 1 shows the results regarding the stability of pharmaceutical compositions as described herein based on size exclusion chromatography regarding the rate of formation of HMWS, i.e., aggregates, at different temperatures, expressed as % per month.

[0042] Figure 2 shows the results regarding the stability of pharmaceutical compositions as described herein based on size exclusion chromatography regarding the change in relative monomer content after storage at different temperatures, expressed as % per month.

[0043] Figure 3 shows the results regarding the stability of pharmaceutical compositions as described herein based on turbidity measurements (absorbance at 350 nm) after storage at 40°C at different timepoints.

[0044] Figure 4 shows the results regarding the stability of pharmaceutical compositions as described herein based on turbidity measurements (absorbance at 600 nm) after storage at 40°C at different timepoints.

[0045] Figure 5 shows clinical ancillaries used for the preparation of dose for infusion based on the pharmaceutical compositions as described herein.

[0046] Figure 6 shows different levels of visible particles of protein nature (i.e. insoluble visible ginisortamab molecule aggregates) for P1 and P1a (formulation F03) material at c.a. 9mg / ml_ in 0.9% NaCI solution in water. Sample A1 (left) = ginisortamab P1 formulation at c.a. 9mg / ml_ in 0.9% NaCI solution in water. Sample A4 (right) = ginisortamab P1 a formulation at c.a. 9mg / ml_ in 0.9% NaCI solution in water. DETAILED DESCRIPTION

[0047] The present invention provides pharmaceutical compositions and pharmaceutical solutions comprising an anti-Gremlin-1 antibody, or an antigen-binding fragment thereof.

[0048] Gremlin-1

[0049] The term Gremlin-1 or GREMI as used herein typically has the sequence as set out in the UniProt entry 060565 (SEQ ID NO:1). The term Gremlin-1 may also refer to a Gremlin-1 polypeptide which:

[0050] (a) comprises or consists of the amino acid sequence of SEQ ID NO: 1 with or without the N- terminal signal peptide, i.e. may comprise or consist of the mature peptide sequence as shown in SEQ ID NO: 21 ; or

[0051] (b) is a derivative having one or more amino acid substitutions, modifications, deletions or insertions relative to the amino acid sequence of SEQ ID NO: 1 with or without the N-terminal signal peptide (as shown in SEQ ID NO: 21), which retains the activity of Gremlin-1 , such as the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 20.

[0052] (c) a variant thereof, such variants typically retain at least about 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94% or 95% identity to SEQ ID NO: 1 (or SEQ ID NO: 20 or 21) (or even about 96%, 97%, 98% or 99% identity). In other words, such variants may retain about 60% - about 99% identity to SEQ ID NO: 1 , suitably about 80% - about 99% identity to SEQ ID NO: 1 , more suitably about 90% - about 99% identity to SEQ ID NO: 1 and most suitably about 95% - about 99% identity to SEQ ID NO: 1. Variants are described further below.

[0053] As discussed further below, residue numbers are typically quoted based on the sequence of SEQ ID NO: 1. However, residue numbering could readily be extrapolated by the skilled person to a derivative or variant sequence as discussed above. Where residue numbers are quoted, the invention also encompasses these residues on a variant or derivative sequence.

[0054] Human Gremlin-1 has been crystallized alone, and in complex with an antibody termed Ab 7326 (Fab fragments). Crystallization of Gremlin-1 has allowed putative residues in the BMP binding site to be determined. Furthermore, crystallization with Ab 7326, which is an inhibitory antibody, has allowed residues in the antibody epitope to be determined (WO 2018 / 115017 A2).

[0055] Inhibitors of Gremlin-1 activity

[0056] An inhibitor of Gremlin-1 activity according to the present invention is an agent that reduces or blocks the activity of Gremlin-1 . Inhibitors according to the present invention may partially or completely inhibit Gremlin-1 activity. Inhibitors of use in the present invention include without limitation, inhibitors that are capable of binding to Gremlin-1 or to a nucleic acid molecule encoding gremlin-1 , or are capable of inhibiting the expression of Gremlin-1 .

[0057] In one embodiment, the inhibitor of Gremlin-1 activity is an anti-Gremlin-1 antibody or a functionally active fragment, variant or derivative thereof, e.g., an antigen-binding fragment thereof.

[0058] The term “antibody” as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., “antigen-binding portion”) or single chains thereof. An antibody or immunoglobulin typically refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).

[0059] The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0060] An antibody for use in the present invention may be a monoclonal antibody or a polyclonal antibody, and will typically be a monoclonal antibody. An antibody for use in the invention may be a chimeric antibody, a CDR-grafted antibody, a nanobody, a human or humanized antibody or an antigen-binding portion of any thereof.

[0061] Polyclonal antibodies may be produced by routine methods, such as immunization of a suitable animal with the antigen of interest. Blood may be subsequently removed from the animal and the immunoglobulin fraction purified.

[0062] Antibodies against Gremlin-1 may be obtained, where immunization of an animal is necessary, by administering the polypeptides to an animal, e.g. a non-human animal, using well-known and routine protocols, see for example Handbook of Experimental Immunology, D. M. Weir (ed.), Vol 4, Blackwell Scientific Publishers, Oxford, England, 1986). Many warm-blooded animals, such as rabbits, mice, rats, sheep, goats, cows, camels, lamas or pigs may be immunized. However, rabbits, mice, and rats are generally most suitable.

[0063] Monoclonal antibodies may be prepared by any method known in the art such as the hybridoma technique (Kohler & Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp77-96, Alan R Liss, Inc., 1985).

[0064] Antibodies for use in the invention may also be generated using single lymphocyte antibody methods by cloning and expressing immunoglobulin variable region cDNAs generated from single lymphocytes selected for the production of specific antibodies by for example the methods described by Babcook, J. et al., 1996, Proc. Natl. Acad. Sci. USA 93(15): 7843-7848I; WO 92 / 02551 ; WO 2004 / 051268 and WO 2004 / 106377.

[0065] The antibodies for use in the present invention can also be generated using various phage display methods known in the art and include those disclosed by Brinkman et al. (in J. Immunol. Methods, 1995, 182: 41-50), Ames et al. (J. Immunol. Methods, 1995, 184:177-186), Kettleborough et al. (Eur. J. Immunol. 1994, 24:952-958), Persic et al. (Gene, 1997, 1879-18), Burton et al. (Advances in Immunology, 1994, 57:191-280) and WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401 ; and US 5,698,426; US 5,223,409; US 5,403,484; US 5,580,717; US 5,427,908; US 5,750,753; US 5,821 ,047; US 5,571 ,698; US 5,427,908; US 5,516,637; US 5,780,225; US 5,658,727; US 5,733,743 and US 5,969,108. Fully human antibodies are those antibodies in which the variable regions and the constant regions (where present) of both the heavy and the light chains are all of human origin, or substantially identical to sequences of human origin, but not necessarily from the same antibody. Examples of fully human antibodies may include antibodies produced, for example by the phage display methods described above and antibodies produced by mice in which the murine immunoglobulin variable and optionally the constant region genes have been replaced by their human counterparts e.g. as described in general terms in EP 0546073, US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,661 ,016, US 5,770,429, EP 0438474 and EP 0463151.

[0066] Alternatively, an antibody according to the invention may be produced by a method comprising immunizing a non-human mammal with a Gremlin-1 immunogen; obtaining an antibody preparation from said mammal; deriving therefrom monoclonal antibodies that recognize Gremlin-1.

[0067] The antibody molecules for use in the present invention may comprise a complete antibody molecule having full length heavy and light chains or a fragment or antigen-binding portion thereof. The term “antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to selectively bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. The antibodies and fragments and antigen binding portions thereof may be, but are not limited to Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, single domain antibodies (eg. VH or VL or VHH), scFv, bi, tri ortetra-valent antibodies, Bis-scFv, diabodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). Other antibody fragments for use in the present invention include the Fab and Fab’ fragments described in International patent applications WO 2005 / 003169, WO 2005 / 003170 and WO 2005 / 003171 and Fab- dAb fragments described in International patent application WO 2009 / 040562. Multi-valent antibodies may comprise multiple specificities or may be monospecific (see for example WO 92 / 22853 and WO 2005 / 113605). These antibody fragments may be obtained using conventional techniques known to those of Skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

[0068] In a preferred embodiment, the anti-Gremlin-1 antibody or the antigen-binding fragment thereof is capable of at least partially neutralizing the activity of Gremlin-1 . The neutralizing activity of an anti-Gremlin-1 antibody or antigen-binding fragment may be determined by its at least partially blocking activity of recombinant human Gremlin-1 , and / or by its at least partial activity to restore BMP signaling pathways in human colorectal cancer (CRC) cell lines. For example, the CRC lines may be HCT 116, DLD-1 or LS174T. A suitable assay may be the pSMAD1 / 8 phospho-flow assay on CRC cell lines as described in Davies et al., MABS 15 (2023), 2289681. The disclosure of this document is incorporated herein by reference.

[0069] The assay may be carried out as follows: Cells are plated at 1.25 x 105per well in a 24-well plate (Corning 353047) and incubated for 24 h in complete DMEM (10% FBS / Glutamax) before addition of 15 nM (300 ng / mL) recombinant human gremlin-1 (R&D Systems, 5190-GR) or media-only control for 16 h. Next, 150 nM (22.5 pg / mL, or a titration, as indicated) ginisortamab or a matched human lgG4 isotype control (Biolegend 403702) are either pre-incubated together with gremlin-1 for 30 min before addition to cells or added for the last 30 to 120 min of the 16-h gremlin-1 incubation (as indicated in Figure 4). At the end of the assay, cells are fixed, permeabilized, and stained for pSMAD1 / 8 according to the BD Phosflow protocol. Briefly, cells are detached with TrypLE Express and transferred to a 96-well plate (Corning, 3799). Cells are fixed with Lyse / Fix (BD, Phosflow 558049) for 15 min at 37°C, washed with PBS (Gibco) then permeabilized with cold Perm III (BD, Phosflow 558050) for 30 min on ice. Cells are washed with stain buffer (PBS, 2% FBS, 0.09% NaN3) and incubated for 1 h at room temperature with a 1 :20 dilution of PE- labeled anti-SMAD1 (pS436 / pS465) / SMAD8 (pS465 / pS467) (BD 562509) before washing again with stain buffer and analyzed on a BD FACS CANTO II flow cytometer. Data analysis is performed using FlowJo v10.6.0 and GraphPad Prism v8.1.1 software. Normalized percentage recovery of pSMAD1 / 8 signal is calculated using % recovery = ([Value - Background] / [Max signal - Background]) x 100%, where Background = pSMAD1 / 8 signal in presence of gremlin-1 + hlgG4 and Max signal = media-only control.

[0070] In one example, the functionally active antibody fragment for use in the present invention is a Fab, Fab', F(ab')2, Fv or scFv.

[0071] The constant region domains of the antibody molecule for use in the present invention, if present, may be selected having regard to the effector functions which may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG or IgM domains. In particular, human IgG constant region domains may be used, especially of the lgG1 and lgG3 isotypes when antibody effector functions are required. Alternatively, lgG2 and lgG4 isotypes may be used when antibody effector functions are not required. In one example, the isotype is lgG4P, as described by Angal S. et al, Mol Immunol, Vol 30(1), p105-108, 1993.

[0072] An antibody for use in the invention may be prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for the immunoglobulin genes of interest or a hybridoma prepared therefrom, (b) antibodies isolated from a host cell transformed to express the antibody of interest, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of immunoglobulin gene sequences to other DNA sequences.

[0073] An antibody for use in the invention may be a human antibody or a humanized antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies for use in the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0074] Such a human antibody may be a human monoclonal antibody. Such a human monoclonal antibody may be produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0075] Human antibodies may be prepared by in vitro immunization of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. The term “derivative" refers to any modified form of the antibody, for example a conjugate of the antibody and another agent or effector molecule.

[0076] An effector molecule may comprise a single effector molecule or two or more such molecules so linked as to form a single moiety that can be attached to the antibodies for use in the present invention. Where it is desired to obtain an antibody fragment linked to an effector molecule, this may be prepared by standard chemical or recombinant DNA procedures in which the antibody fragment is linked either directly or via a coupling agent to the effector molecule. Techniques for conjugating such effector molecules to antibodies are well known in the art (see, Hellstrom et al., Controlled Drug Delivery 2nd Ed., Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67- 123). Particular chemical procedures include, for example, those described in WO 93 / 06231 , WO 92 / 22583, WO 89 / 00195, WO 89 / 01476 and WO 2003 / 031581 . Alternatively, where the effector molecule is a protein or polypeptide the linkage may be achieved using recombinant DNA procedures, for example as described in WO 86 / 01533 and EP 0392745.

[0077] The effector molecule may increase the half-life of the antibody in vivo, and / or reduce immunogenicity of the antibody and / or enhance the delivery of an antibody across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin binding proteins or albumin binding compounds such as those described in WO 2005 / 117984.

[0078] The term “humanized antibody” is intended to refer to CDR-grafted antibody molecules in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0079] As used herein, the term “CDR-grafted antibody molecule” refers to an antibody molecule wherein the heavy and / or light chain contains one or more CDRs (including, if desired, one or more modified CDRs) from a donor antibody (e.g. a murine or rat monoclonal antibody) grafted into a heavy and / or light chain variable region framework of an acceptor antibody (e.g. a human antibody). For a review, see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998. In one embodiment rather than the entire CDR being transferred, only one or more of the specificity determining residues from any one of the CDRs described herein above are transferred to the human antibody framework (see for example, Kashmiri et al., 2005, Methods, 36, 25-34). In one embodiment only the specificity determining residues from one or more of the CDRs described herein above are transferred to the human antibody framework. In another embodiment only the specificity determining residues from each of the CDRs described herein above are transferred to the human antibody framework.

[0080] When the CDRs or specificity determining residues are grafted, any appropriate acceptor variable region framework sequence may be used having regard to the class / type of the donor antibody from which the CDRs are derived, including mouse, primate and human framework regions. Suitably, the CDR-grafted antibody for use in the present invention has a variable domain comprising human acceptor framework regions as well as one or more of the CDRs or specificity determining residues described above. Thus, provided in one embodiment is a neutralizing CDR-grafted antibody wherein the variable domain comprises human acceptor framework regions and non-hu- man donor CDRs. Examples of human frameworks which can be used in the present invention are KOL, NEWM, REI, EU, TUR, TEI, LAY and POM (Kabat et al., supra). For example, KOL and NEWM can be used for the heavy chain, REI can be used for the light chain and EU, LAY and POM can be used for both the heavy chain and the light chain. Alternatively, human germline sequences may be used; these are available for example at: http: / / www.vbase2.org / (see Retter et al, Nucl. Acids Res. (2005) 33 (supplement 1), D671-D674).

[0081] In a CDR-grafted antibody for use in the present invention, the acceptor heavy and light chains do not necessarily need to be derived from the same antibody and may, if desired, comprise composite chains having framework regions derived from different chains.

[0082] Also, in a CDR-grafted antibody for use in the present invention, the framework regions need not have exactly the same sequence as those of the acceptor antibody. For instance, unusual residues may be changed to more frequently occurring residues for that acceptor chain class or type. Alternatively, selected residues in the acceptor framework regions may be changed so that they correspond to the residue found at the same position in the donor antibody (see Reichmann et al., 1998, Nature, 332, 323-324). Such changes should be kept to the minimum necessary to recover the affinity of the donor antibody. A protocol for selecting residues in the acceptor framework regions which may need to be changed is set forth in WO 91 / 09967.

[0083] It will also be understood by one skilled in the art that antibodies may undergo a variety of post- translational modifications. The type and extent of these modifications often depends on the host cell line used to express the antibody as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of a carboxy-ter- minal basic residue (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, RJ. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine of the antibody heavy chain of the invention may be absent.

[0084] In one embodiment the antibody heavy chain comprises a CH1 domain and the antibody light chain comprises a CL domain, either kappa or lambda.

[0085] Biological molecules, such as antibodies or fragments, contain acidic and / or basic functional groups, thereby giving the molecule a net positive or negative charge. The amount of overall “observed” charge will depend on the absolute amino acid sequence of the entity, the local environment of the charged groups in the 3D structure and the environmental conditions of the molecule. The isoelectric point (pl) is the pH at which a particular molecule or surface carries no net electrical charge. In one embodiment the antibody or fragment according to the present disclosure has an isoelectric point (pl) of at least 7. In one embodiment the antibody or fragment has an isoelectric point of at least 8, such as 8.5, 8.6, 8.7, 8.8 or 9. In one embodiment the pl of the antibody is 8. Programs such as ** EXPASY http: / / www.expasy.ch / tools / pi_tool.html (see Walker, The Proteomics Protocols Handbook, Humana Press (2005), 571-607) may be used to predict the isoelectric point of the antibody or fragment.

[0086] Antibodies for use in the invention may comprise at least one, at least two or all three heavy chain CDR sequences of SEQ ID NOS: 4 to 6 (HCDR1 / HCDR2 / HCDR3 respectively). These are the HCDR1 / HCDR2 / HCDR3 sequences of the antibody Ab7326 mouse immunoglobulin G1 (mlgG1) as determined using Kabat methodology (see WO 2019 / 158658A1). The Kabat and Chothia methods for determining CDR sequences are well known in the art (as well as other techniques). CDR sequences may be determined using any appropriate method and in the present invention, whilst Kabat is typically employed, other techniques could be used as well. In the present instance, SEQ ID NO: 3 presents the Ab7326 HCDR1 sequence as determined using a combined Chothia & Kabat definition.

[0087] Antibodies for use in the invention may comprise at least one, at least two or all three light chain CDR sequences of SEQ ID NOS: 7 to 9 (LCDR1 / LCDR2 / LCDR3 respectively). These are the LCDR1 / LCDR2 / LCDR3 sequences of Ab7326 using Kabat methodology.

[0088] In one embodiment, the antibody comprises at least a HCDR3 sequence of SEQ ID NO: 6.

[0089] Typically, the antibody comprises at least one heavy chain CDR sequence selected from SEQ ID NOS: 4 to 6 and at least one light chain CDR sequence selected from SEQ ID NOS 7 to 9. The antibody may comprise at least two heavy chain CDR sequences selected from SEQ ID NOS: 4 to 6 and at least two light chain CDR sequences selected from SEQ ID NOS: 7 to 9. The antibody typically comprises all three heavy chain CDR sequences of SEQ ID NOS: 4 to 6 (HCDR1 / HCDR2 / HCDR3 respectively) and all three light chain CDR sequences SEQ ID NOS: 7 to 9 (LCDR1 / LCDR2 / LCDR3 respectively). The antibodies may be chimeric, human or humanized antibodies.

[0090] The antibody may comprise a heavy chain variable region (HCVR) sequence of SEQ ID

[0091] NO: 10 or 12 (the HCVR of Ab7326 variants 1 and 2). The antibody may comprise a light chain variable region (LCVR) sequence of SEQ ID NO: 11 or 13 (the LCVR of Ab7326 variants 1 and 2). The antibody preferably comprises the heavy chain variable region sequence of SEQ ID NO: 10 or 12 and the light chain variable region sequence of SEQ ID NO: 11 or 13 (especially HCVR / LVCR pairs of SEQ ID NOs: 10 / 11 or12 / 13).

[0092] Ab7326 variants 1 and 2 differ by a single amino acid in the heavy chain variable region, and by a single amino acid in the light chain variable region, as follows:

[0093] Heavy chain variable region variant 1 has glutamic acid (E) at position 6. (SEQ ID NO: 10) Heavy chain variable region variant 2 has glutamine (Q) at position 6. (SEQ ID NO: 12) Light chain variable region variant 1 has serine (S) at position 7. (SEQ ID NO:11) Light chain variable region variant 2 has threonine (T) at position 7. (SEQ ID NO: 13)

[0094] Thus, in one embodiment, the antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO: 10, wherein the glutamic acid residue at position 6 is substituted with a glutamine residue (E6Q); wherein the residue numbering is according to SEQ ID NO: 10.

[0095] In one embodiment, the antibody comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO: 12, wherein the glutamine residue at position 6 is substituted with a glutamic acid residue (Q6E); wherein the residue numbering is according to SEQ ID NO: 12.

[0096] In one embodiment, the antibody comprises a light chain variable region (LCVR) sequence of SEQ ID NO: 11 , wherein the serine residue at position 7 is substituted with a threonine residue (S7T); wherein the residue numbering is according to SEQ ID NO: 11 . In one embodiment, the antibody comprises a light chain variable region (LCVR) sequence of SEQ ID NO: 13, wherein the threonine residue at position 7 is substituted with a serine residue (T7S); wherein the residue numbering is according to SEQ ID NO: 13.

[0097] In one embodiment, the antibody comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99% identity), to the sequence of SEQ ID NO: 10 and the light chain variable region comprises a sequence having at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99% identity), to the sequence of SEQ ID NO: 11 .

[0098] In one embodiment, the antibody comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99% identity), to the sequence of SEQ ID NO: 12 and the light chain variable region comprises a sequence having at least 95% identity, (e.g. at least 95%, 96%, 97%, 98% or 99% identity) to the sequence of SEQ ID NO: 13.

[0099] The antibody may comprise a heavy chain (H-chain) sequence of

[0100] SEQ ID NO: 14 mouse full length IgG 1 heavy chain variant 1 , or

[0101] SEQ ID NO: 28 mouse full length IgG 1 heavy chain variant 2, or

[0102] SEQ ID NO: 30 human full length lgG1 heavy chain variant 1 , or

[0103] SEQ ID NO: 16 human full length IgG 1 heavy chain variant 2, or

[0104] SEQ ID NO: 22 human full length lgG4P heavy chain variant 1 , or SEQ ID NO: 34 human full-length lgG4P heavy chain variant 2, or SEQ ID NO: 18 Fab heavy chain variant 1 , or SEQ ID NO: 32 Fab heavy chain variant 2.

[0105] The antibody may comprise a light chain (L-chain) sequence of

[0106] SEQ ID NO: 15 mouse full length IgG 1 light chain variant 1 , or

[0107] SEQ ID NO: 29 mouse full length IgG 1 light chain variant 2, or

[0108] SEQ ID NO: 31 human full length lgG1 light chain variant 1 , or

[0109] SEQ ID NO: 17 human full length IgG 1 light chain variant 2, or

[0110] SEQ ID NO: 23 human full length lgG4P light chain variant 1 , or SEQ ID NO: 35 human full-length lgG4P light chain variant 2, or SEQ ID NO: 19 Fab light chain variant 1 , or SEQ ID NO: 33 Fab light chain variant 2

[0111] In one example, the antibody comprises a heavy chain I light chain sequence pair of

[0112] SEQ ID NOs: 14 / 15 mouse full length lgG1 variant 1 , or

[0113] SEQ ID NOs: 28 / 29 mouse full length IgG 1 variant 2, or

[0114] SEQ ID NOs: 30 / 31 human full length lgG1 variant 1 , or

[0115] SEQ ID NOs: 16 / 17 human full length IgG 1 variant 2, or

[0116] SEQ ID NOS: 22 / 23 human full length lgG4P variant 1 , or

[0117] SEQ ID NOs: 34 / 35 human full-length lgG4P variant 2, or

[0118] SEQ ID NOs: 18 / 19 Fab variant 1 , or

[0119] SEQ ID NOs: 32 / 33 Fab variant 2 The variant forms of corresponding sequences may be interchanged. For example, the antibody may comprise a heavy chain I light chain sequence pair of

[0120] SEQ ID NOs: 14 / 29 mouse full length IgG 1 heavy chain variant 1 / light chain variant 2, or

[0121] SEQ ID NOs: 28 / 15 mouse full length lgG1 heavy chain variant 2 / light chain variant 1 , or

[0122] SEQ ID NOs: 30 / 17 human full length IgG 1 heavy chain variant 1 / light chain variant 2, or

[0123] SEQ ID NOs: 16 / 31 human full length IgG 1 heavy chain variant 2 / light chain variant 1 , or

[0124] SEQ ID NOs: 22 / 35 human full length lgG4P heavy chain variant 1 / light chain variant 2, or

[0125] SEQ ID NOs: 34 / 23 human full-length lgG4P heavy chain variant 2 / light chain variant 1 , or

[0126] SEQ ID NOs: 18 / 33 Fab heavy chain variant 1 / light chain variant 2, or

[0127] SEQ ID NOs: 32 / 19 Fab heavy chain variant 2 / light chain variant 1.

[0128] In a preferred embodiment

[0129] (i) the HCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 4;

[0130] (ii) the HCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 5;

[0131] (iii) the HCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 6;

[0132] (iv) the LCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 7;

[0133] (v) the LCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 8; and

[0134] (vi) the LCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 9.

[0135] In a preferred embodiment the heavy chain variable region of the anti-Gremlin-1 antibody or the antigen-binding fragment thereof has at least 95% identity to the SEQ ID NO: 10 or SEQ ID NO: 12 and / or the light chain variable region of the anti-Gremlin-1 antibody or the antigen-binding fragment thereof has at least 95% identity to the SEQ ID NO: 11 or SEQ ID NO: 13.

[0136] In a further preferred embodiment the heavy chain of the anti-Gremlin-1 antibody or the antigenbinding fragment thereof has the amino acid sequence set forth in SEQ ID NOs: 30, 16, 22, 34, 18 or 32; and the light chain of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof has the amino acid sequence set forth in SEQ ID NOs: 31 , 17, 23, 35, 19 or 33.

[0137] In a further preferred embodiment the anti-Gremlin-1 antibody comprises a heavy chain / light chain pair selected from SEQ ID NOs: 30 / 31 , SEQ ID NOs: 16 / 17, SEQ ID NOs: 22 / 23, SEQ ID NOs: 34 / 35, SEQ ID NOs: 18 / 19, SEQ ID NOs: 32 / 33, SEQ ID NOs: 30 / 17, SEQ ID NOs: 16 / 31 , SEQ ID NOs: 22 / 35, SEQ ID NOs: 34 / 23, SEQ ID NOs: 18 / 33 and SEQ ID NOs: 32 / 19.

[0138] In a further preferred embodiment the anti-Gremlin-1 antibody is an lgG1 subtype or an lgG4 subtype, preferably a human lgG4 subtype. In a particularly preferred embodiment, the anti- Gremlin-1 antibody is an lgG4P.

[0139] In a particular preferred embodiment the anti-Gremlin-1 antibody comprises the heavy chain / light chain pair of SEQ ID NOs: 22 / 23.

[0140] The antibodies may be chimeric, human or humanized antibodies. The antibody may alternatively be or may comprise a variant of one of the specific sequences recited above. For example, a variant may be a substitution, deletion or addition variant of any of the above amino acid sequences.

[0141] A variant antibody may comprise 1 , 2, 3, 4, 5, up to 10, up to 20 or more (typically up to a maximum of 50) amino acid substitutions and / or deletions from the specific sequences discussed above. “Deletion” variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features. "Substitution" variants typically involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

[0142] Table 1 : Amino acid properties.

[0143] “Derivatives" or "variants" generally include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.

[0144] Derivatives and variants as described above may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation of nucleic acids.

[0145] Variant antibodies may have an amino acid sequence which has more than about 60%, or more than about 70%, e.g. 75 or 80%, typically more than about 85%, e.g. more than about 90 or 95% amino acid identity to the amino acid sequences disclosed herein (particularly the HCVR / LCVR sequences and the H- and L-chain sequences). Furthermore, the antibody may be a variant which has more than about 60%, or more than about 70%, e.g. 75 or 80%, typically more than about 85%, e.g. more than about 90 or 95% amino acid identity to the HCVR / LCVR sequences and the H- and L-chain sequences disclosed herein, whilst retaining the exact CDRs disclosed for these sequences. Variants may retain at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the HCVR / LCVR sequences and to the H- and L-chain sequences disclosed herein (in some circumstances whilst retaining the exact CDRs).

[0146] Variants typically retain about 60% - about 99% identity, about 80% - about 99% identity, about 90% - about 99% identity or about 95% - about 99% identity. This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across about 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

[0147] In connection with amino acid sequences, ’’sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters:

[0148] Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

[0149] Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.

[0150] Antibodies having specific sequences and derivatives and variants which maintain the function or activity of these chains are therefore provided for use in the present invention.

[0151] “Derivatives” as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides. The reactive group may be linked directly or through a linker segment to a polymer. It will be appreciated that the residue of such a group will in some instances form part of the product as the linking group between the antibody fragment and the polymer.

[0152] The polymer may be a synthetic or a naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, e.g. a homo- or hetero- polysaccharide. Specific optional substituents which may be present on a synthetic polymer include one or more hydroxy, methyl or methoxy groups. Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethyleneglycol), poly(propyleneglycol) poly(vinylalcohol) or derivatives thereof, especially optionally substituted poly(ethyleneglycol) such as methoxypoly(eth- yleneglycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen or derivatives thereof. The size of the polymer may be varied as desired, but will generally be in an average molecular weight range from 500Da to 50000Da, for example from 5000 to 40000Da such as from 20000 to 40000Da. The polymer size may in particular be selected on the basis of the intended use of the product for example ability to localize to certain tissues or extend circulating half-life (for review see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531-545). Thus, for example, where the product is intended to leave the circulation and penetrate tissue, it may be advantageous to use a small molecular weight polymer, for example with a molecular weight of around 5000Da. For applications where the product remains in the circulation, it may be advantageous to use a higher molecular weight polymer, for example having a molecular weight in the range from 20000Da to 40000Da.

[0153] Suitable polymers include a polyalkylene polymer, such as a poly(ethyleneglycol) or, especially, a methoxypoly(ethyleneglycol) or a derivative thereof, and especially with a molecular weight in the range from about 15000Da to about 40000Da.

[0154] In one example antibodies for use in the present invention are attached to poly(ethyleneglycol) (PEG) moieties. In one particular example the antibody is an antibody fragment and the PEG molecules may be attached through any available amino acid side-chain or terminal amino acid functional group located in the antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using recombinant DNA methods (see for example US 5,219,996; US 5,667,425; W098 / 25971 , W02008 / 038024). In one example the antibody molecule is a modified Fab fragment wherein the modification is the addition to the C-terminal end of its heavy chain one or more amino acids to allow the attachment of an effector molecule. Suitably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule may be attached. Multiple sites can be used to attach two or more PEG molecules.

[0155] Antibodies may compete for binding to Gremlin-1 with, or bind to the same epitope as, those defined above in terms of H-chain / L-chain, HCVR / LCVR or CDR sequences. In particular, an antibody may compete for binding to Gremlin-1 with, or bind to the same epitope as, an antibody which comprises a HCDR1 IHCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 sequence combination of SEQ ID NOs: 4 / 5 / 6 / 7 / 8Z9. An antibody may compete for binding to Gremlin-1 with, or bind to the same epitope as, an antibody which comprises a HCVR and LCVR sequence pair of SEQ ID NOs: 10 / 11 or 12 / 13 or full length chains of SEQ ID NOs: 22 / 23 or 34 / 35.

[0156] An "epitope” is a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of non-linear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, in certain embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics.

[0157] One can easily determine whether an antibody binds to the same epitope as, or competes for binding with, a reference antibody by using routine methods known in the art. For example, to determine if a test antibody binds to the same epitope as a reference antibody for use in the invention, the reference antibody is allowed to bind to a protein or peptide under saturating conditions. Next, the ability of a test antibody to bind to the protein or peptide is assessed. If the test antibody is able to bind to the protein or peptide following saturation binding with the reference antibody, it can be concluded that the test antibody binds to a different epitope than the reference antibody. On the other hand, if the test antibody is not able to bind to protein or peptide following saturation binding with the reference antibody, then the test antibody may bind to the same epitope as the epitope bound by the reference antibody of the invention.

[0158] To determine if an antibody competes for binding with a reference antibody, the above-described binding methodology is performed in two orientations. In a first orientation, the reference antibody is allowed to bind to a protein / peptide under saturating conditions followed by assessment of binding of the test antibody to the protein / peptide molecule. In a second orientation, the test antibody is allowed to bind to the protein / peptide under saturating conditions followed by assessment of binding of the reference antibody to the protein / peptide. If, in both orientations, only the first (saturating) antibody is capable of binding to the protein / peptide, then it is concluded that the test antibody and the reference antibody compete for binding to the protein / peptide. As will be appreciated by the skilled person, an antibody that competes for binding with a reference antibody may not necessarily bind to the identical epitope as the reference antibody, but may sterically block binding of the reference antibody by binding an overlapping or adjacent epitope.

[0159] Two antibodies bind to the same or overlapping epitope if each competitively inhibits (blocks) binding of the other to the antigen. That is, a 1-, 5-, 10-, 20- or 100-fold excess of one antibody inhibits binding of the other by at least 50%, 75%, 90% or even 99% as measured in a competitive binding assay (see, e.g., Junghans et al., Cancer Res, 1990:50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate binding of one antibody reduce or eliminate binding of the other.

[0160] Additional routine experimentation (e.g., peptide mutation and binding analyses) can then be carried out to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody or if steric blocking (or another phenomenon) is responsible for the lack of observed binding. Experiments of this sort can be performed using ELISA, RIA, surface plasmon resonance, flow cytometry or any other quantitative or qualitative antibody-binding assay available in the art.

[0161] The anti-gremlin-1 antibody Ab7326, has been found to bind the following residues of Gremlin-1 : Ile131 , Lys147, Lys148, Phe149, Thr150, Thr151 , Arg169, Lys174 and Gln175; where Lys147, Lys148, Phe149, Thr150, Thr151 , Arg169, Lys174 and Gln175 are present on one Gremlin-1 monomer and Ile131 is present on the second Gremlin-1 monomer. The numbering is based on the UniProt entry 060565 of SEQ ID NO: 1. As discussed in WO 2019 / 158658 A1 these epitope residues were identified using NCONT analysis at 4 A from the Gremlin-1-Ab7326 Fab complex.

[0162] Antibodies for use in the invention may therefore bind to an epitope which comprises at least one residue selected from Ile131 , Lys147, Lys148, Phe149, Thr150, Thr151 , Arg169, Lys174 and Gln175 (With residue numbering based on SEQ ID NO: 1). Antibodies for use in the invention may bind an epitope which comprises 2, 3, 4, 5, 6, 7, 8 or all 9 of these residues (preferably at least 5 residues). Antibodies for use in the invention may also recognize an epitope where Ile131 is present on a different Gremlin-1 monomer to the other residues.

[0163] Although these residues are provided for a particular sequence of human Gremlin-1 , the skilled person could extrapolate the positions of these residues to other corresponding Gremlin sequences using routine techniques. Antibodies binding to epitopes comprising the corresponding residues within these other Gremlin sequences are therefore also provided for use in the invention.

[0164] To screen for antibodies that bind to a particular epitope, a routine cross-blocking assay such as that described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY) can be performed. Other methods include alanine scanning mutants, peptide blots (Reineke (2004) Methods Mol Biol 248:443-63), or peptide cleavage analysis. In addition, methods such as epitope excision, epitope extraction and chemical modification of antigens can be employed (Tomer (2000) Protein Science 9: 487-496). Such methods are well known in the art.

[0165] Antibody epitopes may also be determined by x-ray crystallography analysis. Antibodies for use in the present invention may therefore be assessed through x-ray crystallography analysis of the antibody bound to Gremlin-1 . Epitopes may, in particular, be identified in this way by determining residues on Gremlin-1 within 4 of an antibody paratope residue.

[0166] Antibodies can be tested for binding to Gremlin-1 by, for example, standard ELISA or Western blotting. An ELISA assay can also be used to screen for hybridomas that show positive reactivity with the target protein. The binding selectivity of an antibody may also be determined by monitoring binding of the antibody to cells expressing the target protein, for example by flow cytometry. Thus, a screening method may comprise the step of identifying an antibody that is capable of binding Gremlin-1 by carrying out an ELISA or Western blot or by flow cytometry.

[0167] Antibodies may selectively (or specifically) recognize Gremlin-1 . An antibody, or other compound, “selectively binds” or “selectively recognizes" a protein when it binds with preferential or high affinity to the protein for which it is selective but does not substantially bind, or binds with low affinity, to other proteins. The selectivity of an antibody may be further studied by determining whether or not the antibody binds to other related proteins as discussed above or whether it discriminates between them. Antibodies for use in the invention typically recognize human Gremlin- 1.

[0168] Antibodies may also have cross-reactivity for related proteins, or for human Gremlin-1 and for Gremlin-1 from other species.

[0169] By specific (or selective), it will be understood that the antibody binds to the protein of interest with no significant cross- reactivity to any other molecule. Cross-reactivity may be assessed by any suitable method described herein. Cross-reactivity of an antibody may be considered significant if the antibody binds to the other molecule at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% as strongly as it binds to the protein of interest. An antibody that is specific (or selective) may bind to another molecule at less than about 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25% or 20% the strength that it binds to the protein of interest. The antibody may bind to the other molecule at less than about 20%, less than about 15%, less than about 10% or less than about 5%, less than about 2% or less than about 1% the strength that it binds to the protein of interest.

[0170] Thus, antibodies suitable for use in the present invention may have a high affinity binding for (human) Gremlin-1 . The antibody may have a dissociation constant (KD) of less than <1 nM, and preferably <500 pM. In one example, the antibody has a dissociation constant (KD) of less than 200pM. In one example, the antibody has a dissociation constant (KD) of less than 100pM. A variety of methods can be used to determine the binding affinity of an antibody for its target antigen such as surface plasmon resonance assays, saturation assays, or immunoassays such as ELISA or RIA, as are well known to persons of skill in the art. An exemplary method for determining binding affinity is by surface plasmon resonance analysis on a BIAcore™ 2000 instrument (Biacore AB, Freiburg, Germany) using CM5 sensor chips, as described by Krinner et al., (2007) Mol. Immunol. February; 44 (5):916-25. (Epub 2006 May 11)).

[0171] The anti-Gremlin-1 antibody Ab7326, is an inhibitor of Gremlin-1 activity which binds to an epitope distal from the BMP binding site. (WO 2018 / 115017 A2) Ab7326 binds to Gremlin-1 with exceptionally high affinity with a Kd value <100pM, and is expected to be particularly useful in the context of the present invention.

[0172] An inhibitor of gremlin-1 activity may have an effect on any of the functions of Gremlin-1 , but typically reduces binding of Gremlin-1 to BMP (BMP 2, 4, and / or 7). Gremlin-1 is a negative regulator of BMP and so reduced binding increases signalling through BMP.

[0173] Once a suitable antibody has been identified and selected, the amino acid sequence of the antibody may be identified by methods known in the art. The genes encoding the antibody can be cloned using degenerate primers. The antibody may be recombinantly produced by routine methods.

[0174] Examples of DNA sequences encoding full length heavy chains and light chains of Ab7326 are provided in the sequence listing:

[0175] - SEQ ID NO: 24 (Human IgG 1 heavy chain DNA variant 1)

[0176] - SEQ ID NO: 25 (Human IgG 1 light chain DNA variant 1)

[0177] - SEQ ID NO: 26 (Human lgG4P heavy chain DNA variant 1)

[0178] - SEQ ID NO: 27 (Human lgG4P light chain DNA variant 1)

[0179] In one embodiment, the anti-gremlin-1 antibody is ginisortamab comprising the heavy chain / light chain pair of SEQ ID NOs: 22 / 23. Ginisortamab is a fully human immunoglobulin G4P (lgG4P) monoclonal antibody (mAb) optimized for neutralizing activity against human Gremlin-1 protein. The nomenclature lgG4P signifies that the lgG4 heavy chain sequence has been modified in its hinge region by mutating serine at position 241 to proline in order to reduce the occurrence of chain exchange (separation of the 2 heavy chain-light chain pairs of the IgG followed by re-asso- ciation with equivalent heavy-light chain pairs of lgG4 of different specificity). The light chain is kappa Km3 allotype and the heavy chain is gamma-4. The total molecular weight of the active pharmaceutical ingredient is approximately 150 kDa. A report on ginisortamab is provided in MABS 2023, Vol. 15, No. 1 , 2289681 .

[0180] In a preferred embodiment, the anti-gremlin-1 antibody, in particular ginisortamab, is present in the pharmaceutical composition in a concentration of from 20 mg / mL to 300 mg / mL. In a more preferred embodiment, the anti-gremlin-1 antibody, in particular ginisortamab, is present in the pharmaceutical composition in a concentration of form 30 mg / mL to 250 mg / mL. In an even more preferred embodiment, the anti-gremlin-1 antibody, in particular ginisortamab, is present in the pharmaceutical composition in a concentration of form 50 mg / mL to 150 mg / mL. In a yet even more preferred embodiment, the anti-gremlin-1 antibody, in particular ginisortamab, is present in the pharmaceutical composition in a concentration of form 80 mg / mL to 120 mg / mL. In an even more preferred embodiment, the anti-gremlin antibody, in particular ginisortamab, is present in the pharmaceutical composition in a concentration of from 90 mg / mL to 110 mg / mL.

[0181] Pharmaceutical composition comprising an anti-Gremlin-1 antibody

[0182] As indicated above, the pharmaceutical composition of the invention comprises not only the anti- gremlin-1 antibody, but also other ingredients. In particular, the pharmaceutical composition of the invention comprises a. an anti-Gremlin-1 antibody, or an antigen-binding fragment thereof; b. L-histidine, its hydrochloride salt, or the combination thereof; and c. L-proline; wherein the pharmaceutical composition has a pH of from 6.4 to 7.2.

[0183] The pharmaceutical composition can be formulated as a solution. As used herein a pharmaceutical composition of the invention may also be referred to as formulation of the invention. The pharmaceutical composition is typically sterile. It will preferably be diluted prior to administration as explained in further detail below.

[0184] As indicated above, the pharmaceutical composition exhibits advantageous stability properties. The term “stability” as used herein, refers to the physical and chemical stability of the pharmaceutical composition. Stability is typically assessed at a selected temperature (for instance -60 °C, 5 °C, 25 °C, 40 °C or more) for a selected time-period (e.g. 4 weeks, 8 weeks, 13 weeks, 26 weeks, 39 weeks, 52 weeks, 36 months). Various values can be used to characterize stability over a given time-period (in comparison with the initial data), such as (and not limited to):

[0185] Measurement of the rate of formation of high molecular weight species (HMWS), i.e. aggregates, after storage at different temperatures based on size exclusion chromatography (SEC);

[0186] Measurement of the change in relative monomer content after storage at different temperatures based on size exclusion chromatography (SEC);

[0187] Measurement of the turbidity (measuring the absorbance, e.g., at 350 nm or 600 nm) after storage at a certain temperature at different timepoints;

[0188] - Visual assessment to detect visible particles and assess opalescence / turbidity by observing the samples under controlled light intensity with both a black background and a white background following European Pharmacopeia § 2.2.1. as well as with the sample between the observer and the light source which allows observer to detect particles more easily.

[0189] Without being bound to any theory, the excipients, e.g., the presence of L-proline, in combination with the pH of from 6.4 to 7.2 of the pharmaceutical composition were shown to result in advantageous stability properties of the pharmaceutical composition. In one embodiment, the pharmaceutical composition comprises L-proline in a molar concentration of from 150 mM to 350 mM. In a preferred embodiment, the L-proline is present in a molar concentration of from 200 mM to 300 mM. In an even more preferred embodiment, the L-proline is present in a molar concentration of from 225 to 250 mM, e.g. from 240 mM to 260 mM, from 245 mM to 255 mM or from 249 mM to 251 mM.

[0190] In one embodiment, the pharmaceutical composition has a pH of from 6.6 to 7.0. In a preferred embodiment, the pharmaceutical composition has a pH of from 6.7 to 6.9. In a more preferred embodiment, the pharmaceutical composition has a pH of 6.8.

[0191] In one embodiment, the pharmaceutical composition comprises L-arginine, its hydrochloride salt, or a combination thereof in a molar concentration of less than 40 mM. In a preferred embodiment, the pharmaceutical composition comprises L-arginine, its hydrochloride salt, or a combination thereof in a molar concentration of less than 20 mM, preferably less than 10 mM. In an even more preferred embodiment, the pharmaceutical composition comprises L-arginine or L-arginine hydrochloride in a molar concentration of less than 1 mM. In a particularly preferred embodiment, the pharmaceutical composition does not comprise L-arginine or L-arginine hydrochloride.

[0192] In another embodiment, the pharmaceutical composition is substantially free of L-arginine or L- arginine hydrochloride. As used herein “substantially free” means a concentration of less than 1 % (w / v), preferably less than 0.5 % (w / v) based on the total volume of the pharmaceutical composition, more preferably entirely free of L-arginine or L-arginine hydrochloride.

[0193] The term “w / v” as used herein expresses the amount of a substance in the pharmaceutical composition in terms of the substance’s weight per 100mL of the composition.

[0194] In one embodiment, the pharmaceutical composition comprises L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 20 mM to 80 mM. In a preferred embodiment, the L-histidine, its hydrochloride salt, or the combination thereof is present in a molar concentration of from 30 mM to 70 mM. In a more preferred embodiment, L-histidine, its hydrochloride salt, or the combination thereof is present in a molar concentration of from 40 mM to 60 mM. In an even more preferred embodiment, L-histidine, its hydrochloride salt, orthe combination thereof is present in a molar concentration of from 45 mM to 55 mM, e.g. from 49 mM to 51 mM.

[0195] In one embodiment, the pharmaceutical composition further comprises sucrose. Without being bound to any theory, the sucrose was shown to further improve the stability of the pharmaceutical composition. In a preferred embodiment, the sucrose is present in a molar concentration of from 150 mM to 350 mM. In a more preferred embodiment, the sucrose is present in a molar concentration of from 200 mM to 300 mM. In an even more preferred embodiment, the sucrose is present in a molar concentration of from 225 to 275mM, e.g. from 240 mM to 260 mM, from 245 mM to 255 mM or from 249 mM to 251 mM.

[0196] In one embodiment, the pharmaceutical composition further comprises L-methionine. Without being bound to any theory, the presence of L-methionine was shown to further improve the stability of the pharmaceutical composition. In a preferred embodiment, the L-methionine is present in a molar concentration of from 5 mM to 25 mM. In a more preferred embodiment, the L-methionine is present in a molar concentration of from 8 mM to 18 mM. In an even more preferred embodiment, the L-methionine is present in a molar concentration of from 10 mM to 16 mM. In an even more preferred embodiment, the L-methionine is present in a molar concentration of from 11 mM to 15 mM.

[0197] In one embodiment, the pharmaceutical composition further comprises a polysorbate. In a preferred embodiment, the polysorbate is present in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition. In a preferred embodiment, the polysorbate is present in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition. In a more preferred embodiment, the polysorbate is present in an amount of from 0.025% (w / v) to 0.035%(w / v), based on the total volume of the pharmaceutical composition. In another preferred embodiment, the polysorbate is polysorbate 80 or polysorbate 20. In a more preferred embodiment, the polysorbate is polysorbate 80.

[0198] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises at least one HCDR sequence selected from SEQ ID Nos: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from SEQ ID Nos: 7, 8 and 9, in a concentration of from 30 mg / mL to 250 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 30 mM to 70 mM; c. L-proline in a molar concentration of from 150 mM to 350mM; d. sucrose in a molar concentration of from 150 mM to 350mM; e. L-methionine in a molar concentration of from 8 mM to 18 mM; and f. polysorbate 80 in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0199] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 10 and the light chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 11 , in a concentration of from 30 mg / mL to 250 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 30 mM to 70 mM; c. L-proline in a molar concentration of from 150 mM to 350mM; d. sucrose in a molar concentration of from 150 mM to 350mM; e. L-methionine in a molar concentration of from 8 mM to 18 mM; and f. polysorbate 80 in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0200] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. ginisortamab in a concentration of from 30 mg / mL to 250 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 30 mM to 70 mM; c. L-proline in a molar concentration of from 150 mM to 350mM; d. sucrose in a molar concentration of from 150 mM to 350mM; e. L-methionine in a molar concentration of from 8 mM to 18 mM; and f. polysorbate 80 in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0201] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises at least one HCDR sequence selected from SEQ ID Nos: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from SEQ ID Nos: 7, 8 and 9, in a concentration of from 50 mg / mL to 150 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 40 mM to 60 mM; c. L-proline in a molar concentration of from 200 mM to 300mM; d. sucrose in a molar concentration of from 200 mM to 300mM; e. L-methionine in a molar concentration of from 10 mM to 16 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0202] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 10 and the light chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 11 , in a concentration of from 50 mg / mL to 150 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 40 mM to 60 mM; c. L-proline in a molar concentration of from 200 mM to 300mM; d. sucrose in a molar concentration of from 200 mM to 300mM; e. L-methionine in a molar concentration of from 10 mM to 16 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0203] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. ginisortamab in a concentration of from 50 mg / mL to 150 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 40 mM to 60 mM; c. L-proline in a molar concentration of from 200 mM to 300mM; d. sucrose in a molar concentration of from 200 mM to 300mM; e. L-methionine in a molar concentration of from 10 mM to 16 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0204] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises at least one HCDR sequence selected from SEQ ID Nos: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from SEQ ID Nos: 7, 8 and 9, in a concentration of from 90 mg / mL to 110 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 45 mM to 55 mM; c. L-proline in a molar concentration of from 225 mM to 275 mM; d. sucrose in a molar concentration of from 225 mM to 275 mM; e. L-methionine in a molar concentration of from 11 mM to 15 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0205] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 10 and the light chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 11 , in a concentration of from 90 mg / mL to 110 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 45 mM to 55 mM; c. L-proline in a molar concentration of from 225 mM to 275 mM; d. sucrose in a molar concentration of from 225 mM to 275 mM; e. L-methionine in a molar concentration of from 11 mM to 15 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0206] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. ginisortamab in a concentration of from 90 mg / mL to 110 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 45 mM to 55 mM; c. L-proline in a molar concentration of from 225 mM to 275 mM; d. sucrose in a molar concentration of from 225 mM to 275 mM; e. L-methionine in a molar concentration of from 11 mM to 15 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

[0207] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises at least one HCDR sequence selected from SEQ ID Nos: 3, 4, 5 and 6 and / or at least one LCDR sequence selected from SEQ ID Nos: 7, 8 and 9, in a concentration of 100 mg / ml; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of 50 mM; c. L-proline in a molar concentration of 250 mM; d. sucrose in a molar concentration of 250 mM; e. L-methionine in a molar concentration of 13 mM; and f. polysorbate 80 in an amount of 0.03%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of 6.8.

[0208] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, which comprises the sequence of SEQ ID NO: 3 or 4 for HCDR1 , the sequence of SEQ ID NO: 5 for HCDR2, the sequence of SEQ ID NO: 6 for HCDR3, the sequence of SEQ ID NO: 7 for LCDR1 , the sequence of SEQ ID NO: 8 for LCDR2 and the sequence of SEQ ID NO: 9 for LCDR3; and wherein the heavy chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 10 and the light chain variable region comprises a sequence having at least 95% identity to the sequence of SEQ ID NO: 11 , in a concentration of 100 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of 50 mM; c. L-proline in a molar concentration of 250 mM; d. sucrose in a molar concentration of 250 mM; e. L-methionine in a molar concentration of 13 mM; and f. polysorbate 80 in an amount of 0.03%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of 6.8.

[0209] In one embodiment, the present invention relates to a pharmaceutical composition comprising a. ginisortamab in a concentration of 100 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of 50 mM; c. L-proline in a molar concentration of 250 mM; d. sucrose in a molar concentration of 250 mM; e. L-methionine in a molar concentration of 13 mM; and f. polysorbate 80 in an amount of 0.03%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of 6.8.

[0210] In one embodiment, the present invention relates to a pharmaceutical composition comprising ginisortamab in a concentration of 100 mg / mL, L-histidine in a concentration of 6.7 mg / mL, L- histidine hydrochloride monohydrate in a concentration of 1 .5 mg / mL, L-proline in a concentration of 28.8 mg / mL, sucrose in a concentration of 85.6 mg / mL, L-methionine in a concentration of 1.9 mg / mL, and polysorbate 80 in a concentration of 0.3 mg / mL.

[0211] In connection with the above embodiments, it is particularly preferred that the pharmaceutical composition is substantially free of L-arginine or L-arginine hydrochloride. In a preferred embodiment, the composition does not comprise L-arginine or L-arginine hydrochloride.

[0212] The pharmaceutical composition according to the invention may be administered in a therapeutically effective amount. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent (i.e. an antibody) needed to treat, ameliorate or prevent a targeted disease, disorder or condition, or to exhibit a detectable therapeutic, pharmacological or preventative effect. The precise therapeutically effective amount for a human subject will depend upon the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities and tolerance / response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, a therapeutically effective amount of antibody will be from 0.01 mg / kg to 500 mg / kg, for example 0.1 mg / kg to 200 mg / kg or 1 to 100 mg / kg.

[0213] In a preferred embodiment, the pharmaceutical composition according to the invention is administered by intravenous route. For this purpose, the pharmaceutical composition needs to be diluted. It has been found that the pharmaceutical composition of the invention is also stable upon dilution.

[0214] The pharmaceutical composition according to the invention may thus advantageously be used for preparing a pharmaceutical solution suitable for infusion to a human patient. Therefore, the present invention also relates to the use of a pharmaceutical composition as defined herein for preparing a pharmaceutical solution suitable for infusion to a human patient.

[0215] In another embodiment, the present invention relates to a kit comprising

[0216] (i) the pharmaceutical composition as defined herein; and

[0217] (ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water.

[0218] The kit is useful for preparing a pharmaceutical solution suitable for infusion to a human patient.

[0219] In another embodiment, the present invention relates to a process of preparing a pharmaceutical solution comprising combining

[0220] (i) the pharmaceutical composition as defined herein; and

[0221] (ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water.

[0222] In a preferred embodiment, the desired amount of the pharmaceutical composition as defined herein (the dose) is injected in a bag of the NaCI or the glucose solution as defined above.

[0223] In another embodiment, the present invention relates to a pharmaceutical solution comprising

[0224] (i) the pharmaceutical composition as defined herein; and

[0225] (ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water; wherein the anti-gremlin-1 antibody or an antigen-binding fragment thereof comprised in the pharmaceutical composition is present in the pharmaceutical solution in a concentration of from 1 mg / mL to 30 mg / mL.

[0226] In one embodiment of the pharmaceutical solution, the anti-gremlin antibody or an antigen-binding fragment thereof comprised in the pharmaceutical composition is present in the pharmaceutical solution in a concentration of from 5 mg / mL to 25 mg / mL. In another embodiment of the pharmaceutical solution, the anti-gremlin antibody or an antigen-binding fragment thereof comprised in the pharmaceutical composition is present in the pharmaceutical solution in a concentration of from 8 mg / mL to 25 mg / mL.

[0227] The pharmaceutical solution is preferably infused into the patient, no later than 4 hours after preparation, through a canula. Preferably, the canula comprises two inline filters (e.g., 15 pm and 0.22 pm). A pump may be used to control the flow.

[0228] The liquid pharmaceutical formulation of the invention is suitably administered to the patient at one time or over a series of treatments and may be administered to the patient at any time from diagnosis onwards; it may be administered as the sole treatment or in conjunction with other drugs or therapies useful in treating the conditions as described herein.

[0229] The anti-Gremlin-1 antibody or antigen-binding fragment thereof may be the sole active ingredient in the pharmaceutical composition or pharmaceutical solution. Alternatively, the antibody or anti- gen-binding fragment thereof may be administered in combination, e.g. simultaneously, sequentially or separately, with one or more other therapeutically active ingredients. Active ingredient as employed herein refers to an ingredient with a pharmacological effect, such as a therapeutic effect, at a relevant dose.

[0230] Other therapeutically active ingredients which may be administered in combination with the anti- Gremlin-1 antibody or antigen-binding fragment include, without being limited, chemotherapeutic agents, as for example described in WO 2019 / 243801 ; proliferation-dependent cytotoxic agents, as for example described in WO 2023 / 194584; or inhibitors of Ras-Raf- MEK-ERK signalling, as for example described in WO 2023 / 194583.

[0231] Inhibitors of the Ras-Raf-MEK-ERK signalling for administration with the anti-Gremlin1-antibody or antigen-binding fragment thereof include MEK inhibitors, ERK inhibitors, RAS inhibitors or Raf- inhibitors. In a preferred embodiment, the inhibitor of the Ras-Raf-MEK-ERK signalling is a MEK inhibitor. Preferred MEK inhibitors for administration in combination with the anti-Gremlin-1 antibody or antigen-binding fragment thereof are cobimetinib (GDC- 0973; (S)-[3,4-Difluoro-2-(2- fluoro-4-iodophenylamino)phenyl] [3-hydroxy-3-(piperidin-2-yl)azetidin-1-yl] methanone), selu- metinib (AZD6244; ARRY- 142886; 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3- methylbenzimidazole-5-carboxamide) or WX-554 (UCB-554; [3-(Aminomethyl)azetidin-1-yl]-{2- [(2-fluoro-4-iodophenyl)amino]thieno[2,3-b]pyridin-3-yl}-methanone).

[0232] Proliferation-dependent cytotoxic agents for administration in combination with the anti-Gremlin1- antibody or antigen-binding fragment thereof include cytidine analogues, deoxycytidine analogues or mitotic inhibitors. A preferred cytidine analogue for administration in combination with the anti-Gremlin1-antibody or antigen-binding fragment thereof is gemcitabine (2'-deoxy-2',2'- difluorocytidine). Preferred mitotic inhibitors for administration in combination with the anti-Grem- lin 1 -antibody or antigen-binding fragment thereof are paclitaxel or abraxane (NAB-paclitaxel).

[0233] Preferred chemotherapeutic agents for administration in combination with the anti-Gremlin1-anti- body or antigen-binding fragment thereof include 5-fluorouracil, oxaliplatin, leucovorin (folinic acid), FOLFOX (5-fluorouracil, oxaliplatin, and leucovorin), trifluridine and tipiracil.

[0234] In some embodiments the anti-Gremlin-1 antibody or antigen-binding fragment thereof in the pharmaceutical composition may be accompanied by other active ingredients including other antibodies or non-antibody ingredients, administered by the same or by a different route of administration.

[0235] The other active ingredients for administration in combination with the anti-Gremlin-1 antibody or antigen-binding fragment thereof may be provided separately from the pharmaceutical composition. Alternatively, the other active agent may be provided together with the pharmaceutical composition in a kit.

[0236] In one embodiment, the present invention relates to a kit which comprises (i) the pharmaceutical composition as defined herein; and

[0237] (ii) cobimetinib, selumetinib, or WX-554; gemcitabine; gemcitabine and abraxane;

[0238] FOLFOX; or trifluridine and tipiracil.

[0239] In another embodiment, the present invention relates to a kit which comprises

[0240] (i) the pharmaceutical composition as defined herein;

[0241] (ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water; and

[0242] (iii) cobimetinib, selumetinib, or WX-554; gemcitabine; gemcitabine and abraxane;

[0243] FOLFOX; or trifluridine and tipiracil.

[0244] The present invention also relates to the pharmaceutical composition or the pharmaceutical solution as defined herein for use as a medicament. In yet another embodiment, the present invention relates to the pharmaceutical composition of the pharmaceutical solution as defined herein for use in the treatment or prevention of a cancer, in particular a solid cancer. In yet another embodiment, the present invention relates to the pharmaceutical composition of the pharmaceutical solution as defined herein for use in the treatment or prevention of a cancer, which is selected from colorectal cancer, multiple myeloma, pancreatic cancer, bladder cancer, breast cancer, lung cancer, stomach cancer, duodenal cancer, oesophageal cancer, head and neck cancer, prostate cancer, glioma, endometrial cancer, liver cancer, spleen cancer, bone-resident cancer, and osteosarcoma. In yet another embodiment, the present invention relates to the pharmaceutical composition of the pharmaceutical solution as defined herein for use in the treatment of renal fibrosis such as diabetic nephropathy, idiopathic pulmonary fibrosis, pulmonary arterial hypertension, and angiogenesis. In yet another embodiment, the present invention relates to the pharmaceutical composition of the pharmaceutical solution as defined herein for use in the treatment of bone fracture or bone defect.

[0245] The term “treating” or “treatment” of a disease state includes: (i) inhibiting the disease state, i.e. arresting the development of the disease state or its clinical symptoms, or (ii) relieving the disease state, i.e. causing temporary or permanent regression of the disease state or its clinical symptoms.

[0246] The term “preventing” or “prevention” of a disease state includes causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

[0247] The present invention will be further illustrated by the following examples.

[0248] EXAMPLES

[0249] Abbreviations and definitions ELISA (enzyme-linked immunosorbent assay); Cone, (concentration); SEC (Size-exclusion Chromatography); SE-HPLC (Size-exclusion high-performance liquid chromatography); CGE-NR (Capillary Gel Electrophoresis, Non-Reduced method); iCE (Imaged Capillary Electrophoresis); DLS (Dynamic light scattering); P1 formulation (process 1 formulation); P1 a formulation (process 1a formulation); UV / Vis (Ultraviolet and Visible light); Suer (Sucrose); PS80 (Polysorbate 80); L- Arg.HCI (L-arginine hydrochloride); L-Pro (L-Proline); L-His I L-His.HCI (L-histidine I L-histidine hydrochloride); L-Met (L-methionine); w / v (weight per volume); P1 formulation (ginisortamab at 100mg / mL in 50mM L-His / L-His.HCI, 150mM L-Arg.HCI, 0.02%(w / v) PS80, pH6.0); P1a formulation (ginisortamab at 10Omg / mL in 50mM L-His I L-His.HCI, 250mM L-Pro, 250mM Suer, 13mM L-Met, 0.03%(w / v) polysorbate 80, pH6.8); HMWS (high molecular weight species / insoluble aggregates); LMWS (low molecular weight species); UPLC (Ultra Performance Liquid Chromatography); PDA (Photo-Diode Array); timepoint to (initial timepoint just after sample preparation); t4w (4 weeks timepoint); t8w (8 weeks timepoint); t13w (13 weeks timepoint); t26w (26 weeks timepoint); t39w (39 weeks timepoint); t52w (52 weeks timepoint); Cat. No. (catalog number); q.s. (quantum satis); c.a. circa, latin for “about, around”, typically ±10%); i.e. (jd est, latin for “that is").

[0250] Material

[0251] The active pharmaceutical ingredient ginisortamab is a fully human immunoglobulin G4P (lgG4P) monoclonal antibody (mAb) optimized for neutralizing activity against human gremlin-1 protein. Ginisortamab has the heavy chain / light chain pair of SEQ ID NOs: 22 / 23. Gremlin-1 is a high affinity antagonist regulating the activity of the bone morphogenetic proteins (BMPs) 2, 4, and 7. In tumor biology, BMPs inhibit expansion and self-renewal of tumor cells, which gremlin-1 secreted by the stroma may reverse, allowing tumor cell expansion. Inhibition of gremlin-1 activity by ginisortamab is expected to restore BMP function, thereby inhibiting tumor growth. The antibody is manufactured by mammalian expression using a Chinese Hamster Ovary CHO DG44 cell line and purified with a 3-step chromatography process.

[0252] The nomenclature lgG4P signifies that the lgG4 heavy chain sequence has been modified in its hinge region by mutating serine at position 241 to proline in order to reduce the occurrence of chain exchange (separation of the 2 heavy chain-light chain pairs of the IgG followed by re-asso- ciation with equivalent heavy-light chain pairs of lgG4 of different specificity). The light chain is kappa Km3 allotype and the heavy chain is gamma-4. The total molecular weight of the active pharmaceutical ingredient is approximately 150 kDa.

[0253] Ginisortamab was discovered using a human derived phage display library panned against human gremlin-1 protein. It is a fully human lgG4P antibody with a specificity for the dysfunctional human Gremlin protein, produced from mammalian cell culture.

[0254] Stability indicating analytical methods

[0255] Visual assessment

[0256] Visual assessment was performed to detect visible particles and assess opalescence / turbidity by observing the samples under controlled light intensity with both a black background and a white background following European Pharmacopeia § 2.2.1. as well as with the sample between the observer and the light source which allows observer to detect particle more easily. The reference suspensions described the European Pharmacopeia § 2.2.1 can be purchased (formazin turbidity standards 3, 6, 18, 30 NTU, e.g. Hach, Cat. No. 2896742, 2896842, 2896942, and cat. 2897042). These ready to use solutions correspond to reference suspensions I, II, II and IV, respectively. These reference suspensions can also be prepared from a stock reference suspension as described below. The stock suspension is prepared by diluting 1.5 mL of the 4000 NTU formazin turbidity standard (formazin turbidity standard, 4000 NTU, Hach, Cat. No. 246149) with ultrapure water (q.s. 100ml_).

[0257] Table 1- Preparation of Reference Suspensions for Opalescence testing

[0258] Turbidity- absorbance at 350 and 600nm

[0259] Turbidity was determined using a calibrated UV / Vis spectrophotometer by measuring the absorbance of the samples at 350 and 600nm.

[0260] Protein concentration

[0261] Protein concentration was determined by measuring the absorbance at 280nm using a calibrated UV / Vis spectrophotometer after dilution to approximately 0.5mg / ml_ and analyzed, according to standard methods. In the case where particles are visible, samples may need to be centrifuged briefly and the concentration determined on the supernatant. The sample concentration is then calculated with the following formula using the established extinction coefficient (with c= protein concentration (mg / mL); A2ao = absorbance at 280nm; dil = dilution factor; Em= extinction coefficient (1.47 mL / mg.cm) ; b=path length (cm, determined by the spectrophotometer):

[0262] Size variants distribution - Aggregates (High molecular weight species) and Monomer relative content

[0263] SEC was used (UPLC system equipped with a 30cm Waters Acquity BEH200 SEC 1 ,7pm, 4.6 x 300mm column and a UV detector measuring the absorbance at 280nm, using phosphate sodium chloride buffer pH7.0 as eluent running) to resolve and quantitate product-related species (HMWS, i.e. aggregates, LMWS, and monomer relative content) in the formulations tested.

[0264] Purity - intact monomer relative content

[0265] CGE-NR was used (CGE system equipped with a 50pm x 30.2cm bare-fused silica capillary and a UV PDA detector) to resolve and quantitate intact monomer relative content in the formulations tested.

[0266] Charge variants distribution - Main peak, acid and basic peak groups relative content iCE was used (iCE3 system equipped with a 100pm x 50mm fluorocarbon coated clEF cartridge and a UV detector measuring the absorbance at 280nm) to resolve and quantitate product-related species (e.g., Main peak, acid and basic peak groups relative content) in the formulations tested.

[0267] Dynamic Light Scattering - Polydispersity, hydrodynamic radius, and purity

[0268] DLS was used to determine the polydispersity, hydrodynamic radius, and purity of the protein in the formulations tested. pH

[0269] The pH of the samples was determined using a calibrated pH meter equipped with appropriate pH probe.

[0270] Peptide mapping

[0271] Peptide mapping was performed with liquid chromatography and mass spectrometry for the confirmation of protein primary structure and detection of post-translational modifications such as oxidation, deamidation, glycosylation and C-terminal clipping. Thereto, the sample was enzymatically digested into fragment peptides, which were subsequently separated using liquid chromatography prior to online analysis using electrospray ionization with mass spectrometry. The mass- to-charge ratio of the peptides was measured by mass spectrometry, from which their mass was inferred.

[0272] Binding ELISA

[0273] A binding ELISA was used to quantitatively measure the specific binding activity of ginisortamab to human Gremlin-1 . ELISA plates were coated with a solution of Gremlin-1 (Sigma-Aldrich; Cat No. SRP3285 or R&D System; Cat No. 5190GR) followed by a blocking step with Fish Serum Blocking Buffer in dPBS. The plates were washed and incubated with serial dilutions of ginisortamab reference standard (aliquots of ginisortamab GMP batch material suitably characterized with documented identity, purity, and characteristics, following ICH Q2(R2) guideline) and samples. After a washing step, the plates were incubated with an anti-kappa light chain peroxi- dase-conjugated antibody (MP Biomedical; Cat. No. 55233 or Sigma Aldrich; Cat. no.P502P) that binds the Fab fragment of ginisortamab. The plates were washed and incubated with the substrate solution (TMB). After having stopped the reaction by adding sulfuric acid, the readout was performed at 450nm / 620nm. The response function curves were generated for the ginisortamab reference standard and the samples and the relative potency of the sample was then estimated by comparison to the standard curve. The binding activity of the test sample was calculated as a percentage relative activity to the reference standard.

[0274] Stability of the pharmaceutical composition of the invention

[0275] Ginisortamab was originally presented as a liquid formulation at 100mg / mL in 50mM L-His I L- His.HCI, 150mM L-Arg.HCI, 0.02%(w / v) PS80, pH6.0 (herein named Process 1 (P1) formulation). The P1 formulation presented suboptimal physical and chemical stability profiles. More particularly upon dilution in a 0.9% NaCI solution in water, particle formation was observed. A study was undertaken to develop a formulation with improved physical and chemical stability to address this issue. The impact of pH (6.0, 6.4, and 6.8) and alternative stabilizers (L-arginine hydrochloride, L-me- thionine and sucrose) on the physical and chemical stability of ginisortamab was evaluated. A total of 17 formulations, including a formulation very similar to and representative for the P1 formulation (i.e. formulation F17), were prepared by buffer exchange and concentrated to the required concentration of 100mg / ml_. Polysorbate 80 (PS80) was added from stock solution to reach the required level of 0.02 (P1 formulation) or 0.03%(w / v) (P1a and other formulations). A longterm (up to 52 weeks) stability study was performed under various storage conditions, i.e., <- 60°C, 5°C, 25°C, and 40°C to evaluate the stability of ginisortamab in the different formulations. Samples were analyzed at various timepoints (7 timepoints at <-60°C and 5°C, i.e. to, t4w, t8w, t13w, t26w, t39w, and t52w, 5 timepoints at 25°C , i.e. t4w, t8w, t13w, and t26w, and 4 timepoints at 40°C, i.e. t4w, t8w, and t13w) using stability indicating analytical techniques, i.e. SEC, iCE, CGE-NR, DLS, visual assessment, pH, turbidity by absorbance at 350nm and 600nm, and peptide mapping.

[0276] Results from a subset of 6 formulations, as described in Table 2, are described below.

[0277] Table 2- Description of formulations

[0278] Table 3- Size Exclusion Chromatography

[0279] Slope HMWS or Monomer are respectively the rates of change in HMWS or Monomer relative content. Values are calculated using data from all timepoints available and expressed as the percentage of change per month. A positive or negative value indicates an increase, or respective decrease of the measured species.

[0280] Table 4- Visual assessment - turbidity

[0281] Cl = Clear: clarity is the same as that of the formulation buffer; T+: turbid; T++: very turbid

[0282] Table 5- Turbidity (absorbance at 350nm) Table 6- Turbidity (absorbance at 600nm)

[0283] No significant differences between the tested formulations could be observed in iCE, CGE-NR, DLS, pH, ELISA, and peptide mapping. The results based on size exclusion chromatography regarding the formation of HMWS, i.e., aggregates, and the relative monomer content after storage at different temperatures per month are further illustrated in Figure 1 and Figure 2, respectively. The results regarding the turbidity (absorbance at 350 nm) after storage at 40°C provided in Table 5 are further illustrated in Figure 3. The results regarding the turbidity (absorbance at 600 nm) after storage at 40°C provided in Table 6 are further illustrated in Figure 4.

[0284] The results of this study inter alia lead to ginisortamab being presented as a liquid formulation at 100mg / mL in 50mM L-His / L-His.HCI, 250mM L-Pro, 250mM Suer, 13mM L-Met, 0.03%(w / v) polysorbate 80, pH6.8 (herein named P1a or formulation F03). Indeed, overall, the P1a formulation (formulation F03) was the most stable of all the formulations tested in this study.

[0285] The SEC results showed that the P1a formulation (formulation F03) is significantly more stable over time at 25°C than the P1 formulation (as represented by formulation F17) (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in the P1 a formulation than in the F17 formulation. The turbidity data (from visual assessment of turbidity) also showed that the P1a formulation is significantly more stable than the F17 formulation. The F17 formulation remained very turbid from the day of preparation till the end of the study whilst the P1 a formulation, which was clear from the day of preparation, turned turbid only after 26 weeks on storage at 5°C, yet less turbid than the F17 formulation (see Table 4).

[0286] Finally, a long-term stability study of 36 months was performed to evaluate the stability of ginisortamab in the P1a formulation at 5°C (i.e. the foreseen storage temperature of the P1a formulation). A Drug product batch was placed at 5°C for 36 months in inverted position and the following characteristics which were monitored did not show any statistical significant changes after the storage period of 36 months: %Relative Potency as determined by Binding ELISA, Charge variant distribution (%Acidic Peak Group, %Main Peak %Basic Peak Group) as determined by iCE,%lntact IgG Monomer as determined by CGE in Non-reducing conditions, %lgG Monomer as sum of Heavy and Light chains as determined by CGE in Reducing conditions, %lgG Monomer, %HMWS, and %LMWS as determined by SEC, Appearance of the solution, Degree of clarity and opalescence, Degree of coloration, pH (23-25°C), Osmolality, Identity by tryptic peptide mapping, Protein concentration, Visible particles, and Sub-visible particles (> 25pm and > 10pm). This demonstrates that the P1a formulation is stable upon storage at 5°C over a long period of time.

[0287] The effect could be attributed to the (combination of) excipients that were added or changed in the formulation and by the change in pH.

[0288] An increase in pH from pH6.0 to 6.8, was shown to have a stabilizing effect on ginisortamab. Formulation F02 and formulation F09 have the same composition but for the pH. The SEC results showed that the formulation F02, with a pH of 6.8 was significantly more stable at 25°C over time than the formulation F09, with a pH of 6.0 (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in formulation F09 than in the formulation F02. The impact of pH 6.8 compared to pH 6.0 can also be seen by comparing turbidity data (from visual assessment and absorbances at 350 and 600nm) of the formulations F02 and F09. The formulation F09 was turbid from the day of preparation till the end of the study whilst the formulation F02, which was clear from the day of preparation, turned turbid only after 26 weeks on storage at 5°C, yet less turbid than formulation F09 (see Table 4, Table 5, and Table 6, as well as Figure 3 and Figure 4).

[0289] Omission of L-Arg.HCI was shown to result in better stability for ginisortamab. The SEC results showed that the formulation F11 , which does not contain L-Arg.HCI, was significantly more stable at 5°C over time than the formulation F14, containing L-Arg.HCI (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in the formulation F11 than in the formulation F14. The turbidity data (from visual assessment of turbidity) also showed that the formulation F11 was significantly more stable than the formulation F14. The formulation F14, which was clear on the day of preparation, turned and remained turbid until the end of the study under all storage conditions, whilst the formulation F 11 remained clear from the day of preparation until the end of the study under all storage conditions (see Table 4).

[0290] Sucrose was shown to have a stabilizing effect on ginisortamab. Formulation F03 (the P1a formulation) and formulation F02 have the same composition but for the sucrose. The SEC results showed that the P1 a formulation was significantly more stable at 25°C over time than the formulation F02 (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in the P1a formulation than in the formulation F02, which can be attributed to the presence of sucrose.

[0291] The combination of an increase in pH from pH 6.0 to pH 6.8 and sucrose was shown to have a stabilizing effect on ginisortamab. The SEC results showed that the P1a formulation which has a pH of 6.8 and contains sucrose was significantly more stable at 25°C over time than formulation F09 which has a pH of 6.0 and contains no sucrose and is otherwise identical to formulation P1 a (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in P1 a formulation than in formulation F09. The turbidity data (from visual assessment and absorbances at 350 and 600nm) also showed that the P1a formulation is significantly more stable than formulation F09. Formulation F09 remained turbid from the day of preparation till the end of the study whilst the formulation F03, which was clear from the day of preparation, turned turbid only after 26 weeks on storage at 5°C, yet less turbid than the formulation F09 (see Table 4, Table 5, and Table 6, as well as Figure 3 and Figure 4).

[0292] L-Met was shown to have a stabilizing effect on ginisortamab. The SEC results showed that the P1a formulation which contains L-Met is significantly more stable at 25°C over time than the formulation F11 which does not contain L-Met but is otherwise identical to the P1a formulation (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in the P1 a formulation) than in formulation F11 .

[0293] The combination of an increase in pH from pH 6.0 to pH 6.8, replacement of L-Arg.HCI by L-Pro and the addition of L-Met was shown to have a stabilizing effect on ginisortamab. The SEC results showed that formulation F02 is significantly more stable at 25°C over time than the P1 formulation (as represented by formulation F17) (see Table 3, as well as Figure 1 and Figure 2). The HMWS (aggregates) relative content increased at a significantly lower rate in formulation F02 which has a pH of 6.8 and contains L-Pro and L-Met, but no L-Arg.HCI, than in the F17 formulation. The turbidity data (from visual assessment of turbidity) also showed that formulation F02 was significantly more stable than the F17 formulation. The F17 formulation remained turbid from the day of preparation until the end of the study under all storage conditions, whilst formulation F02, which was clear from the day of preparation, turned turbid only after 26 weeks on storage at 5°C, yet less turbid than formulation F17 (see Table 4).

[0294] Stability of the pharmaceutical solution of the invention

[0295] For intravenous administration, the ginisortamab formulation needs to be diluted. Thereto, a desired amount of the ginisortamab formulation (the dose) is injected in a bag of 0.9% NaCI solution in water for infusion into the patient, no later than 4 hours after preparation, through a canula comprising two inline filters (15pm and 0.22pm) (see Figure 1) and using a pump to control the flow.

[0296] The propensity of antibodies to form particles is a known phenomenon. When they are diluted, monoclonal antibodies tend to aggregate or precipitate in a reversible way and this cannot be totally overcome (Ref 1, 2, and 3).

[0297] Consequently, investigations were undertaken to assess the impact of particle formation during the preparation of the ginisortamab solutions for infusion. These investigations were especially concerned with the impact of particles on the visual aspect (whether the number of particles could potentially alarm clinicians so much they would have doubt on the safety for the patient) and filterability (whether the particles trapped in the inline filters, would potentially slow down excessively or even stop the infusion which typically can take from 30 minutes up to 2 hours).

[0298] In one investigation focusing on particle formation, the P1 and P1a formulation were diluted and the propensity for particle formation was compared. The P1 formulation was diluted to c.a. 9 mg / mL or c.a. 14 mg / mL in 0.9% NaCI solution in water and 5% glucose solution in water, and the P1a formulation was diluted to c.a. 9mg / ml_ in 0.9% NaCI solution in water and 5% glucose solution in water, as well as c.a. 14 and c.a. 24 mg / mL in 0.9% NaCI solution in water. 5% Glucose solution in water is a potential alternative diluent to 0.9% NaCI solution in water, although more seldomly used as it precludes diabetic patient from participating to a clinical study. All dilutions were prepared in transparent glass type 1 (Fiolax I VCDIN20R) vials and tested after standing 4 hours at room temperature for the presence of particles, changes in size and charge species distributions, changes in concentration and pH. No clinical ancillaries were used in this study as it had already been demonstrated that ginisortamab was compatible with the clinical ancillaries. All diluted solutions were analyzed using stability indicating analytical techniques, i.e. SEC, iCE, CGE-NR, visual assessment, concentration determination, as well as activity assessment (binding ELISA).

[0299] An impact was observed on the visual aspect of all the ginisortamab diluted solutions. However, different levels of visible particles of protein nature (i.e. insoluble visible ginisortamab molecule aggregates) were observed, see Figure 6. This impact was especially evident when observing the samples with a light behind and was more accentuated when using the P1 formulation (at c.a. 9mg / mL in 0.9% NaCI solution in water or in 5% glucose solution in water) compared to the P1a formulation (at ca. 9mg / mL in 0.9% NaCI solution in water or in 5% glucose solution in water), see Table 7. The P1a formulation could be used at higher concentrations (up to c.a. 24mg / mL), without an increased impact of the visual aspect of the ginisortamab diluted solutions. Additionally, the P1a formulation showed compatibility with glucose, which was not the case for the P1 formulation. The P1a formulation presents a clear advantage compared to P1 formulation.

[0300] Table 7 - Visual assessment using light between the samples and the observer

[0301] No significant differences could be observed for the diluted formulations using iCE, CGE-NR, DLS, pH, ELISA, and peptide mapping.

[0302] Another investigation focused on the potential impact of the presence of particles on the filterability through the 15 pm filter from the drip chamber and 0.22 pm in-line filter of the ginisortamab solutions for infusion. During this investigation, the P1 formulation and the P1a formulation were injected though the injection port using syringes equipped with needles into the bags of 0.9% NaCI solution in water to reach a concentration of c.a. 14 mg / mL or c.a. 9 mg / ml. The clinical ancillaries used in this study had already been demonstrated to be compatible with ginisortamab. The bags prepared were left standing for 4 hours at room temperature before a cannula was added to the port designed for cannula inlet, and the content of the bag was dispensed, thereby passing through the 15pm filter from the drip chamber and the 0.22 pm in-line filter of the cannula, into an empty recipient, see Figure 5. The peristatic pump placed between the two filters was set to pump for 1 hour at 287 mL / h. Pressure and flow were monitored during the entire dispensing process. Samples were taken during the infusion from the bags as well as after the cannula, and were analyzed using stability indicating analytical techniques, i.e. pH, SEC, iCE, visual assessment, and concentration.

[0303] No decrease in flow rate and no increase of the back pressure was observed when using the P1 or P1 a formulation diluted at c.a. 9 mg / mL in 0.9% NaCI solution in water. A decrease in flow rate and an increase of the back pressure was observed when using the P1 formulation diluted at c.a. 14 mg / mL in 0.9% NaCI solution in water due to filter clogging, whereas no decrease of flow rate and no increase of the back pressure was observed when using the P1a formulations diluted at c.a. 14 mg / mL in 0.9% NaCI solution in water. The P1a formulation can thus be used at higher concentrations (at least up to c.a. 14 mg / mL) in 0.9% NaCI solution as compared to the P1a formulation, where clogging was already observed at c.a. 14 mg / ml.

[0304] Table 8 - Filterability

[0305] No significant differences could be observed for the diluted formulations using pH, SEC, iCE, and concentration determination. Results from the visual assessment study were consistent with the results from the investigation focusing on particle formation.

[0306] Ref 1 : Mathonet et al., 2016, ‘A biopharmaceutical industry perspective on the control of visible particles in biotechnology-derived injectable drug products’, PDA J Pharm Sci and Tech, 392- 408, 70

[0307] “biologies, including monoclonal antibodies, can have an inherent molecular property to self-associate, or aggregate and form proteinaceous particles despite formulation, manufacturing process, and container closure development to minimize visible protein particles. This propensity is a basic thermodynamic property of the molecule that cannot be totally overcome. Such particles may form over time, often exist in equilibrium, and may or may not be reversible as a result of non-covalent interactions."

[0308] Ref 2: Pardeshi et al, “Micro- And Nanoparticles Delivered in Intravenous 0.9% NaCI solution in water and in an Intravenous Solution of a Therapeutic Antibody Product”, J Pharm Sci. 2017 February; 106(2): 511-520

[0309] “because of the high level of dilution occurring when IV solutions are prepared, protein stability provided by the product formulation is greatly reduced, increasing the likelihood of protein aggregation. For example, Kumru et al found that upon dilution into IV 0.9% NaCI solution in water, an igG4 mAb formed soluble aggregates and subvisible particles, which were measured by microflow imaging (MFI) and nanoparticle tracking analysis (NTA)."

[0310] Ref 3: Mahler et al., “Protein aggregation: Pathways, induction factors and analysis”, J Pharm Sci. 2009 September; 98(9): 2909-2934

Claims

CLAIMS1 . A pharmaceutical composition comprising a. an anti-Gremlin-1 antibody, or an antigen-binding fragment thereof; b. L-histidine, its hydrochloride salt, or the combination thereof; and c. L-proline; wherein the pharmaceutical composition has a pH of from 6.4 to 7.2.

2. The pharmaceutical composition according to claim 1 further comprising d. sucrose.

3. The pharmaceutical composition according to claim 2, wherein the sucrose is present in a molar concentration of from 150 mM to 350mM.

4. The pharmaceutical composition according to any one of claims 1 to 3 further comprising e. L-methionine.

5. The pharmaceutical composition according to claim 4, wherein the L-methionine is present in a molar concentration of from 5 mM to 25 mM.

6. The pharmaceutical composition according to any one of claims 1 to 5 further comprising f. a polysorbate.

7. The pharmaceutical composition according to claim 6, wherein the polysorbate is present in an amount of from 0.01% (w / v) to 0.05%(w / v), based on the total volume of the pharmaceutical composition.

8. The pharmaceutical composition according to claim 6 or 7, wherein the polysorbate is polysorbate 80.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein(i) the HCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 4;(ii) the HCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 5;(iii) the HCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 6;(iv) the LCDR1 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 7;(v) the LCDR2 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 8; and(vi) the LCDR3 sequence of the anti-Gremlin-1 antibody orthe antigen-binding fragment thereof is SEQ ID NO: 9.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the anti- gremlin-1 antibody comprises the heavy chain / light chain pair of SEQ ID NOs: 22 / 23.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the anti- gremlin-1 antibody or the antigen-binding fragment thereof is present in a concentration of from 20 mg / mL to 300 mg / mL.

12. The pharmaceutical composition according to any one of claims 1 to 11 , wherein the L- histidine, its hydrochloride salt, or the combination thereof is present in a molar concentration of from 20 mM to 80 mM.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the L- proline is present in a molar concentration of from 150 mM to 350mM.

14. The pharmaceutical composition according to any one of the preceding claims, wherein the composition comprises: a. an anti-Gremlin-1 antibody comprising a heavy chain / light chain pair of SEQ ID NOs: 22 / 23 in a concentration of from 50 mg / mL to 150 mg / mL; b. L-histidine, its hydrochloride salt, or the combination thereof in a molar concentration of from 40 mM to 60 mM; c. L-proline in a molar concentration of from 200 mM to 300mM; d. sucrose in a molar concentration of from 200 mM to 300mM; e. L-methionine in a molar concentration of from 10 mM to 16 mM; and f. polysorbate 80 in an amount of from 0.02% (w / v) to 0.04%(w / v), based on the total volume of the pharmaceutical composition; wherein the pharmaceutical composition has a pH of from 6.6 to 7.0.

15. Use of a pharmaceutical composition according to any one of claims 1 to 14 for preparing a pharmaceutical solution suitable for infusion to a human patient.

16. A pharmaceutical solution comprising(i) the pharmaceutical composition according to any one of claims 1 to 14; and(ii) a 0.9% NaCI solution in water, or a 5% glucose solution in water; wherein the anti-gremlin-1 antibody or the antigen-binding fragment thereof comprised in the pharmaceutical composition is present in the pharmaceutical solution in a concentration of from 1 mg / mL to 30 mg / mL.

17. The pharmaceutical composition according to any one of claims 1 to 14 or the pharmaceutical solution according to claim 16 for use as a medicament.

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