CFC1 binding molecule

Abstract

The present invention relates to a binding molecule comprising a CFC1 binding domain. In particular, the present invention relates to a CFC1 binding domain that induces a high rate of internalisation of the binding molecule into a cell expressing CFC1. The present invention further relates to antibodies comprising the binding molecule.

Description

[0001] CFC1 BINDING MOLECULE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a binding molecule comprising a CFC1 binding domain. In particular, the present invention relates to a CFC1 binding domain that induces a high rate of internalisation of the binding molecule into a cell expressing CFC1. The present invention further relates to antibodies comprising the binding molecule.

[0004] BACKGROUND TO THE INVENTION

[0005] CFC1 is a member of the EGF-CFC protein family and can be cell-associated / membrane bound or can be extracellular and present in soluble form.

[0006] CFC1 is involved in Akt and MAPK signalling and is known to be a co-receptor that binds Nodal with ALK4 / 7 receptors, which can result in phosphorylation of SMAD 2 / 3 that in turn associates with SMAD4 and translocates to the nucleus.

[0007] It is also known that CFC1 is involved in regulation of gastrulation, mesoderm induction, and axis formation during embryonic development. Moreover, CFC1 functions as a Nodal coreceptor to establish left right asymmetry of developing organs.

[0008] CFC1 is associated with heterotaxy syndrome in humans. Mice with knock-out of CFC1 survive until birth and display severe left-right laterality defects, but do not exhibit the phenotypes associated with pre-gastrulation patterning and differentiation.

[0009] CFC1 is expressed in tumors.

[0010] There is a need for improved therapies targeting CFC1 expressing tumours and cancers, in particular for CFC1 binding molecules with advantageous properties for in vivo administration and for use as medicaments. SUMMARY OF THE INVENTION

[0011] The present invention provides a binding molecule comprising a CFC1 binding domain.

[0012] In some embodiments, the CFC1 binding domain may bind to CFC1 (such as CFC1 on the surface of a cell) and may induce internalisation of the binding molecule into a cell expressing CFC1.

[0013] In some embodiments, the CFC1 binding domain may induce a high rate of internalisation of the binding molecule into a cell expressing CFC1.

[0014] In some embodiments, the binding molecule may have a high rate of internalisation compared to a control.

[0015] In some embodiments, the high rate of internalisation may be determined by an Immunofluorescence Quenching assay.

[0016] In some embodiments, the high rate of internalisation may be at least 60% at 1 hour as determined by an Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, or at least 80% at 1 hour as determined by the Immunofluorescence Quenching assay.

[0017] In some embodiments, the CFC1 binding domain may comprise a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:

[0018] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (SSDMS),

[0019] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG), and

[0020] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (LWNM).

[0021] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations.

[0022] In some embodiments, the CFC1 binding domain may comprise a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the Kabat definition, wherein: LCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA),

[0023] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), and

[0024] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0025] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations.

[0026] In some embodiments, the CFC1 binding domain may comprise a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the IMGT definition, wherein:

[0027] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and

[0028] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM).

[0029] In some embodiments, one or more of the CDRs may comprise one, two or three amino acid mutations.

[0030] In some embodiments, the CFC1 binding domain may comprise a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the IMGT definition, wherein:

[0031] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR),

[0032] LCDR2 comprises the amino acid sequence DAS, and

[0033] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0034] In some embodiments, one or more of the CDRs comprises one, two or three amino acid mutations.

[0035] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto.

[0036] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto. In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity thereto.

[0037] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 11 ; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 13.

[0038] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity thereto.

[0039] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 14.

[0040] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity thereto.

[0041] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 15.

[0042] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity thereto.

[0043] In some embodiments, the VH domain may comprise the amino acid sequence according to SEQ ID NO: 12; and the VL domain may comprise the amino acid sequence according to SEQ ID NO: 16. In some embodiments, the binding molecule may further comprise an Fc domain.

[0044] In some embodiments, the Fc domain may be a modified Fc domain.

[0045] In some embodiments, the modified Fc domain may comprise a modified hinge region.

[0046] In some embodiments, the Fc domain may have reduced, or essentially no, binding to one or more Fc gamma receptors or C1q.

[0047] In some embodiments, the Fc domain may not be capable of binding to immune cells.

[0048] In some embodiments, the Fc domain may comprise LALA, LALA-KA, STR, or LALA-deltaA mutations.

[0049] In some embodiments, the binding molecule may comprise a heavy chain constant domain comprising the amino acid sequence according to SEQ ID NO: 17 or a variant having at least 80% sequence identity thereto.

[0050] In some embodiments, the binding molecule may comprise a light chain constant domain comprising the amino acid sequence according to SEQ ID NO: 18 or a variant having at least 80% sequence identity thereto.

[0051] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 19 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 21 or a variant having at least 80% sequence identity thereto.

[0052] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 22 or a variant having at least 80% sequence identity thereto.

[0053] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 23 or a variant having at least 80% sequence identity thereto.

[0054] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 24 or a variant having at least 80% sequence identity thereto. In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 19 and a light chain sequence of SEQ ID NO: 21.

[0055] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 and a light chain sequence of SEQ ID NO: 22.

[0056] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 and a light chain sequence of SEQ ID NO: 23.

[0057] In some embodiments, the binding molecule may comprise a heavy chain sequence of SEQ ID NO: 20 and a light chain sequence of SEQ ID NO: 24.

[0058] In some embodiments, the CFC1 binding domain may bind to a CFC1 antigen.

[0059] In some embodiments, the CFC1 antigen may comprise or consist of an amino acid sequence according to SEQ ID NO: 25 or a variant having at least 80% sequence identity thereto.

[0060] The present invention provides an antibody or antigen-binding fragment thereof comprising the binding molecule according to the invention.

[0061] In some embodiments, the antibody or fragment thereof may be selected from the list consisting of: an scFv, a Fab, a single domain antibody, a nanobody, a VHH antibody, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, a chimeric antibody or fragment thereof, and a bispecific antibody.

[0062] In some embodiments, the antibody or fragment thereof may be a monoclonal antibody or fragment thereof.

[0063] In some embodiments, the antibody or fragment thereof may be a VHH antibody or fragment thereof.

[0064] The present invention provides a polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0065] In some embodiments, the nucleic acid sequence may be an RNA sequence. The present invention provides a vector which comprises a polynucleotide according to the invention.

[0066] The present invention provides a cell comprising a polynucleotide according to the invention, or a vector according to the invention.

[0067] In some embodiments, the cell may be capable of expressing the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0068] The present invention provides a method for making a cell according to the invention, comprising the step of introducing a polynucleotide according to the invention, or a vector according to the invention into said cell.

[0069] The present invention provides a method for producing the binding molecule according to the invention or the antibody or fragment thereof according to the invention, wherein the method comprises the steps of:

[0070] (i) introducing a polynucleotide according to the invention, or a vector according to the invention into a cell; and

[0071] (ii) expressing the binding molecule or antibody or fragment thereof in the cell.

[0072] In some embodiments, the method further comprises the step of (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or cell culture supernatant of the cell.

[0073] The present invention provides a composition which comprises the binding molecule according to the invention, the antibody or fragment thereof according to the invention; together with a carrier, diluent or excipient.

[0074] The present invention provides a composition comprising:

[0075] (i) a polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention; and

[0076] (ii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture. In some embodiments, the composition may also comprise (iii) a carrier, diluent or excipient.

[0077] The present invention provides a kit comprising:

[0078] (i) the composition according to the invention.

[0079] In some embodiments, the kit may further comprise (ii) instructions for using the kit to target a cell expressing CFC1 in vitro.

[0080] BRIEF DESCRIPTION OF THE FIGURES

[0081] Figure 1 - Binding of the chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240 and MAB-20-0250 to human CFC1 ECD was determined by an ELISA. Antibodies were diluted in a concentration range from 2,000 - 0.06 ng / ml. As control, an anti- CFC1 antibody was added. The data was analyzed by non-linear regression.

[0082] Figure 2 - Binding of the chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240 and MAB-20-0250 to (A) human or (B) cynomolgus CFC1 overexpressing CHO-K1 were investigated by high content imaging. Antibodies were diluted in a concentration range from 5,000 - 0.05 ng / ml and an anti-CFC1 antibody control was added. The data was analyzed by non-linear regression.

[0083] Figure 3 - Binding and internalization of the chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240 and MAB-20-0250 into ectopically expressing CHO-K1 human CFC1 cells by high-content imaging. Percent internalization of 3pg / ml anti-CFC1 antibody was determined after 0, 1 or 4 hrs incubation at 37°C. At timepoint 0, antibody binding to the cells was determined and plotted as mean total cellular Alexa Fluor 488 Intensity.

[0084] Figure 4 - FACS binding analysis of MAB-20-0235 and isotype control (“negative control”) to (A) NCI-H810 and (B) NCI-H727 cells.

[0085] DETAILED DESCRIPTION OF THE INVENTION

[0086] BINDING MOLECULE

[0087] CFC1 , also known as Cryptic protein, is a NODAL co-receptor involved in the correct establishment of the left-right axis. An illustrative amino acid sequence is the human CFC1 sequence designated as UniProt: P0CG37.

[0088] The present invention provides a binding molecule comprising a CFC1 binding domain. In other words, the binding molecule according to the invention may be capable of binding to CFC1.

[0089] It will be understood that a binding molecule is a molecule that interacts with a target (e.g. a target antigen) such that a stable association is formed. An example of a binding molecule is an antigen (or antibody-like molecule) that binds to its cognate antigen. Such binding interaction will be known in the art. The binding interaction may be non-covalent, reversible covalent or irreversible covalent.

[0090] Suitable assays and techniques for measuring / quantifying binding activity of the binding molecule according to the invention may include, but are not limited to, ELISA, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), bioluminescence assays and flow cytometry. Other suitable techniques will be known in the art.

[0091] It will be understood that EC50 is a measure of the concentration of an antibody that induces a specific response that is 50% between the maximum response and the baseline response. As such, EC50 can be used to assess the ability of an antibody to bind to a target.

[0092] In some embodiments, the binding molecule according to the invention may have an EC50 of 5-30 ng / ml, as determined by ELISA In some embodiments, the binding molecule according to the invention may have an EC50 of 10-20 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 10 ng / ml, 11 ng / ml, 12 ng / ml, 13 ng / ml, 14 ng / ml, 15 ng / ml, 16 ng / ml, 17 ng / ml, 18 ng / ml, 19 ng / ml, or 20 ng / ml, as determined by ELISA.

[0093] In some embodiments, the binding molecule according to the invention may have an EC50 of 10 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 11 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 12 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 13 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 14 ng / ml, as determined by ELISA. In some embodiments, the binding molecule according to the invention may have an EC50 of 15 ng / ml, as determined by ELISA.

[0094] Internalisation

[0095] The interaction of a binding molecule with a target molecule (e.g. an antibody binding to its cognate antigen) may result in trafficking of the target molecule (e.g. an antigen) into the cells. Such trafficking may be referred to as endocytosis or internalisation.

[0096] Binding of the binding molecule according to the invention to CFC1 expressed on the surface of a cell may result in internalisation of CFC1 into the cell (e.g. due to conformational changes of the CFC1 protein). As the binding molecule according to the invention is bound to CFC1 , as the CFC1 is internalised into the cell, the binding molecule according to the invention may also become internalised into the cell.

[0097] Accordingly, the binding molecule according to the invention is capable of inducing internalisation into a cell expressing CFC1 on the surface of the cell.

[0098] The binding molecule according to the invention has improved properties over known molecules that bind to CFC1. In particular, the binding molecule according to the invention may have an improved rate of internalisation into a cell. The improved rate of internalisation may be considered as a high rate of internalisation or a fast rate of internalisation.

[0099] In some embodiments, the CFC1 binding domain induces a high rate of internalisation of the binding molecule according to the invention into a cell expressing CFC1.

[0100] In some embodiments, the high rate of internalisation is determined compared to a suitable control. It will be understood that the control may be a binding molecule that binds to CFC1 but which may be considered to only induce a low rate of internalisation, or no, or substantially no, internalisation over a given period of time. A comparison between binding molecules with a high rate of internalisation and binding molecules with a low rate of internalisation are shown in the appended Examples below and in Figure 3.

[0101] Determining the rate of internalisation may be performed using any suitable assay known in the art. For example, internalisation of the binding molecule according to the invention may be assessed by an Immunofluorescence Quenching assay. The Immunofluorescence Quenching assay involves quenching extracellular fluorescence and measuring intracellular fluorescence in order to assess the rate of internalisation of a binding molecule.

[0102] In brief, binding molecules (such as antibodies) are tagged with a detectable marker such as a fluorescent marker (e.g. AF488) and are incubated with a cell or population of cells expressing a cognate antigen on the cell surface, such that the binding molecules bind to the cognate antigen to form a binding molecule-antigen complex. Endocytosis of the antigen may then occur, transporting (i.e. internalising) the binding molecule-antigen complex into the cell. As the binding molecule is tagged with the detectable marker, this detectable marker is also internalised. A quenching step is then performed to some of the samples, such that the signal of the detectable marker on any binding molecules that remain bound to antigens on the surface of the cell is quenched. However, the detectable markers that have been internalised with the binding molecule-antigen complex will be protected from the quenching step and will continue to emit a signal (e.g. a fluorescent signal). In control samples that not have received the quenching step, both the detectable markers on binding molecules bound to cognate antigens on the cell surface and detectable markers on binding molecules bound to cognate antigens that have been internalised will emit a signal. A comparison can be made of samples where the quenching step was performed compared to samples without the quenching step. As the ratio of intracellular signal (e.g. fluorescent signal) to total (surface + intracellular) cellular signal increases over time proportional to the rate of internalisation, the Immunofluorescence Quenching assay may be used to measure the rate of internalisation of a binding molecule and can be used to determine whether a binding molecule has a high rate of internalisation.

[0103] The Immunofluorescence Quenching assay is further described in Example 5.

[0104] In some embodiments, the high rate of internalisation may be determined by the Immunofluorescence Quenching assay.

[0105] It will be understood that a given % rate of internalisation at a given timepoint refers to the proportion of antibody that has been internalised at that timepoint during the Immunofluorescence Quenching assay. For example, it will be understood that a given % rate of internalisation at 1 hour refers to the proportion of antibody that has been internalised after 1 hour duration of the Immunofluorescence Quenching assay. For example, it will be understood that a % rate of internalisation of 50% at 1 hour means that half of the proportion of antibody has been internalised after 1 hour duration of the Immunofluorescence Quenching assay.

[0106] In some embodiments, the high rate of internalisation may be at least 50% at 1 hour as determined by the Immunofluorescence Quenching assay. In other words, a rate of at least 50% at 1 hour as determined by the Immunofluorescence Quenching assay may be considered as a measure for identifying a high rate of internalisation.

[0107] In some embodiments, the high rate of internalisation may be 50%-100% at 1 hour as determined by the Immunofluorescence Quenching assay.

[0108] In some embodiments, the high rate of internalisation may be at least 55% at 1 hour, as determined by the Immunofluorescence Quenching assay.

[0109] In some embodiments, the high rate of internalisation may be at least 60% at 1 hour as determined by the Immunofluorescence Quenching assay.

[0110] In some embodiments, the high rate of internalisation may be at least 65% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 66% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 67% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 68% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 69% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 70% at 1 hour, as determined by the Immunofluorescence Quenching assay.

[0111] In some embodiments, the high rate of internalisation may be at least 75% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 76% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 77% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 78% at 1 hour, as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 79% at 1 hour, as determined by the Immunofluorescence Quenching assay.

[0112] In some embodiments, the high rate of internalisation may be at least 80% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 81 % at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 82% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 83% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 84% at 1 hour as determined by the Immunofluorescence Quenching assay.

[0113] In some embodiments, the high rate of internalisation may be at least 85% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 86% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 87% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 88% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 89% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 90% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be at least 95% at 1 hour as determined by the Immunofluorescence Quenching assay. In some embodiments, the high rate of internalisation may be 100% at 1 hour as determined by the Immunofluorescence Quenching assay.

[0114] Variable domain

[0115] In some embodiments, the binding molecule according to the invention may comprise a heavy chain variable (VH) domain.

[0116] In some embodiments, the binding molecule according to the invention may comprise a light chain variable (VL) domain.

[0117] In some embodiments, the binding molecule according to the invention may comprise a VH domain and a VL domain. In some embodiments, the VH domain may comprise one or more complementarity determining regions (CDRs). In some embodiments, the VH domain may comprise one, two or three CDRs. In some embodiments, the VH domain may comprise three CDRs. It will be understood that CDRs of the VH domain may be termed HCDRs. It will also be understood that each of the three CDRs of the VH domain may be termed HCDR1 , HCDR2 and HCDR3 respectively.

[0118] In some embodiments, the VL domain may comprise one or more CDRs. In some embodiments, the VL domain may comprise one, two or three CDRs. In some embodiments, the VL domain may comprise three CDRs. It will be understood that CDRs of the VL domain may be termed LCDRs. It will also be understood that each of the three CDRs of the VL domain may be termed LCDR1 , LCDR2 and LCDR3 respectively.

[0119] The terms “heavy chain variable region” or “VH” may refer to the portion of the heavy chain of an antigen-binding molecule, or antibody, that typically contains three CDRs, where each CDR is positioned between stretches of amino acids known as framework regions that form a scaffold to support the CDRs.

[0120] The terms “light chain variable region” or “VL” may refer to the fragment of the light chain of an antigen-binding domain or antibody that contains typically three CDRs, where each CDR is positioned between stretches of amino acids known as framework regions that form a scaffold to support the CDRs.

[0121] The terms “complementarity determining region” or “CDR” refer to a highly variable loop in the variable region which interact with a cognate antigen and is largely responsible for determining the ability of a binding molecule (e.g. an antibody) to bind to an antigen and for determining binding affinity. The CDRs within a variable region are typically numbered from the amino to the carboxy terminus, with CDR1 being closest to the amino terminus of the variable region and CDR3 being closest to the carboxy terminus of the variable region.

[0122] The CDRs may be determined according to the Kabat definition and / or the IMGT definition, both of which are well-known in the art.

[0123] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising HCDRs 1-3 as defined by the Kabat definition, wherein:

[0124] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (SSDMS), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG), and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (LWNM).

[0125] In some embodiments, the binding molecule according to the invention may comprise a VL domain comprising LCDRs 1-3 as defined by the Kabat definition, wherein:

[0126] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA),

[0127] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), and

[0128] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0129] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising HCDRs 1-3 as defined by the Kabat definition and may comprise a VL domain comprising LCDRs 1-3 as defined by the Kabat definition, wherein:

[0130] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (SSDMS),

[0131] HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG),

[0132] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (LWNM), LCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA),

[0133] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), and

[0134] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0135] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising HCDRs 1-3 as defined by the IMGT definition, wherein:

[0136] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and

[0137] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM).

[0138] In some embodiments, the binding molecule according to the invention may comprise a VL domain comprising LCDRs 1-3 as defined by the IMGT definition, wherein:

[0139] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR), LCDR2 comprises the amino acid sequence DAS, and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0140] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising HCDRs 1-3 as defined by the IMGT definition and may comprise a VL domain comprising LCDRs 1-3 as defined by the IMGT definition, wherein:

[0141] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), HCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM), LCDR1 comprises the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR), LCDR2 comprises the amino acid sequence DAS, and

[0142] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

[0143] In some embodiments, one or more of the HCDRs may comprise one, two or three amino acid mutations.

[0144] In some embodiments, HCDR 1 may comprise one, two or three amino acid mutations.

[0145] In some embodiments, HCDR 2 may comprise one, two or three amino acid mutations.

[0146] In some embodiments, HCDR 3 may comprise one, two or three amino acid mutations.

[0147] In some embodiments, one or more of the LCDRs may comprise one, two or three amino acid mutations.

[0148] In some embodiments, LCDR 1 may comprise one, two or three amino acid mutations.

[0149] In some embodiments, LCDR 2 may comprise one, two or three amino acid mutations.

[0150] In some embodiments, LCDR 3 may comprise one, two or three amino acid mutations.

[0151] It will be understood that a mutation in any of the CDRs described herein may encompass a deletion of an amino acid, an insertion of an amino acid, or a substitution of an amino acid. It will also be understood that such a mutation may not prevent the binding molecule according to the invention from binding to CFC1. In other words, a binding molecule according to the invention comprising a mutation in one or more CDRs described herein may suitably maintain the capacity to bind to CFC1.

[0152] In some embodiments, the VH domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the VH domain may consist of the amino acid sequence according to SEQ ID NO: 11.

[0153] SEQ ID NO: 11 :

[0154] QSLEESGGRLVTPGTPLTLTCTVSGFSLSSSDMSWVRQAPGKGLEWIGIIYASDNAYYASWAKGRFTI SKTSTTVDLKITSPTTEDTATYFCVRLWNMWGPGTLVTVSL

[0155] In some embodiments, the VH domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the VH domain may consist of the amino acid sequence according to SEQ ID NO: 12.

[0156] SEQ ID NO: 12:

[0157] QVQLVESGGRLVQPGTPLRLSCAVSGFSLSSSDMSWVRQAPGKGLEWIGI IYASDNAYYASWAKGRFT ISRDTSKTTLYLQMNSLRAEDTATYFCVRLWNMWGPGTLVTVSL

[0158] In some embodiments, the VL domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments, the VL domain may consist of the amino acid sequence according to SEQ ID NO: 13.

[0159] SEQ ID NO: 13:

[0160] AQVLTQTASPVSAAVGGTVTISCQSSQSVYDNRLAWYQQKLGQPPKLLIYDASKLESGVPSRFKGSGS GTQFTLTISDLESDDAATYYCAARYSGNIGGFGGGTEVVVK In some embodiments, the VL domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the VL domain may consist of the amino acid sequence according to SEQ ID NO: 14.

[0161] SEQ ID NO: 14:

[0162] DILLTQTPSVVSASVGDRVTITCQSSQSVYDNRLAWYQQKPGQAPRLLIYDASKLESGVPSRFRGSGS GTDFTLTITSLQPEDFATYYCAARYSGNIGGFGGGTRLEIK

[0163] In some embodiments, the VL domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 15. In some embodiments, the VL domain may consist of the amino acid sequence according to SEQ ID NO: 15.

[0164] SEQ ID NO: 15:

[0165] DIQMTQSPSTLSASVGDRVTITCQSSQSVYDNRLAWYQQKLGKAPRLLIYDASKLESGVPSRFSGSGS GTEFTLTISSLQPDDFVTYYCAARYSGNIGGFGGGTKVEIK

[0166] In some embodiments, the VL domain of the binding molecule according to the invention may comprise the amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 16. In some embodiments, the VL domain may consist of the amino acid sequence according to SEQ ID NO: 16.

[0167] SEQ ID NO: 16:

[0168] AIQMTQSPSTLSASVGGTVTITCQSSQSVYDNRLAWYQQKLGQPPKLLIYDASKLESGVPSRFKGSGS GTEFTLTISDLESDDFATYYCAARYSGNIGGFGGGTEVVVK

[0169] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity thereto. In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 11 ; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 13.

[0170] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity thereto.

[0171] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 14.

[0172] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity thereto.

[0173] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 15.

[0174] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity thereto.

[0175] In some embodiments, the binding molecule according to the invention may comprise a VH domain comprising the amino acid sequence according to SEQ ID NO: 12; and a VL domain comprising the amino acid sequence according to SEQ ID NO: 16.

[0176] It will be understood that a variant of a VH domain as described herein may have an equivalent function to a full length VH domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CFC1 . It will be understood that a variant of a VL domain as described herein may have an equivalent function to a full length VL domain amino acid sequence as described herein and may suitably maintain the capacity to bind to CFC1 .

[0177] Fc domain

[0178] In some embodiments, the binding molecule may comprise an immunoglobulin constant region.

[0179] In some embodiments, the binding molecule according to the invention may comprise an immunoglobulin constant region in combination with the VH or VL domain described above.

[0180] In some embodiments, the binding molecule according to the invention may comprise an immunoglobulin constant region in combination with the VH domain described above and the VL domain described above.

[0181] It will be understood that a VH domain and a VL domain may together form a variable region.

[0182] In some embodiments, the immunoglobulin constant region may comprise one or more immunoglobulin constant domain(s).

[0183] In some embodiments, the immunoglobulin constant domain may be selected from the list consisting of: a constant heavy 1 (CH1) domain, a constant heavy 2 (CH2) domain, and a constant heavy 3 (CH3) domain.

[0184] In some embodiments, the immunoglobulin constant region may comprise one or more constant heavy 2 (CH2) domain(s) or one or more constant heavy 3 (CH3) domain(s). In some embodiments, the immunoglobulin constant region may comprise one or more constant heavy 2 (CH2) domain(s) and one or more constant heavy 3 (CH3) domain(s).

[0185] It will be understood that an immunoglobulin constant region may comprise an Fc domain, such as an Fc domain as described herein.

[0186] In some embodiments, the binding molecule according to the invention may comprise an Fc domain. In some embodiments, the binding molecule according to the invention may comprise an Fc domain in combination with the VH or VL domain described above. In some embodiments, the binding molecule according to the invention may comprise an Fc domain in combination with the VH and VL domain described above. It will be understood that the Fc domain may interact with Fc receptors presented on the surface of a cell and / or may interact with proteins of the complement system. The Fc receptors may be Fc gamma receptors. The proteins of the complement system may include C1q.

[0187] In some embodiments, the Fc domain of the binding molecule according to the invention may be a modified Fc domain.

[0188] In some embodiments, the Fc domain of the binding molecule according to the invention may comprise a hinge region.

[0189] In some embodiments, the Fc domain of the binding molecule according to the invention may comprise a modified hinge region.

[0190] In some embodiments, the modified Fc domain may comprise a modified hinge region.

[0191] In some embodiments, the binding molecule according to the invention may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cysteine residues. In some embodiments, the binding molecule according to the invention may be capable of forming 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 disulphide bonds.

[0192] In some embodiments, the Fc domain may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cysteine residues. In some embodiments, the Fc domain may be capable of forming 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 disulphide bonds.

[0193] In some embodiments, the modified hinge region may comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 cysteine residues. In some embodiments, the modified hinge region may be capable of forming 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 disulphide bonds.

[0194] In some embodiments, the Fc domain of the binding molecule according to the invention may comprise one or more mutation(s) such that Fc domain is not capable of interacting with Fc receptors, complement proteins and / or be unable to recruit immune cells.

[0195] In some embodiments, the Fc domain of the binding molecule according to the invention may have reduced, or essentially no, binding to one or more Fc receptors. In some embodiments, the Fc domain of the binding molecule according to the invention may have reduced, or essentially no, binding to one or more Fc gamma receptors. In some embodiments, the Fc domain of the binding molecule according to the invention may have reduced, or essentially no, binding to one or more complement protein(s). In some embodiments, the Fc domain of the binding molecule according to the invention may have reduced, or essentially no, binding to C1q.

[0196] In some embodiments, the Fc domain of the binding molecule according to the invention may not be capable of binding to immune cells and / or recruiting immune cells.

[0197] In some embodiments, the Fc domain of the binding molecule according to the invention may comprises “LALA”, “LALA-KA”, “STR”, or “LALA-deltaA” mutations. It will be understood that such mutations may prevent or reduce the ability of the binding molecule according to the invention to bind to one or more Fc receptor(s) and / or one or more complement protein(s).

[0198] In some embodiments, the Fc domain of the binding molecule according to the invention may comprises “LALA” mutations.

[0199] In some embodiments, the Fc domain of the binding molecule according to the invention may comprises “LALA-KA” mutations.

[0200] In some embodiments, the Fc domain of the binding molecule according to the invention may comprises “STR” mutations.

[0201] In some embodiments, the Fc domain of the binding molecule according to the invention may comprises “LALA-deltaA” mutations.

[0202] In some embodiments, the LALA mutations may be L234A and L235A.

[0203] In some embodiments, the LALA-KA mutations may be L234A, L235A and K322A.

[0204] In some embodiments, the LALA-deltaA mutations (i.e. “Fc[LALA-Aa]”) may be L234A, L235A, A327G, A330S and P331S.

[0205] In some embodiments, the STR mutations may be L234S, L235T and G236R.

[0206] In some embodiments, the amino acid numbering and residues for L234A, L235A, K322A, A327G, A330S and P331S and for L234S, L235T and G236R may be relative to the lgG1 positions and residues described in Armour et al., (Eur. J. Immunol. 1999. 29: 2613-2624; incorporated herein by reference), or may be relative to the IgG constant region residues and positions according to the Ell numbering system (found in Kabat, E. A., Wu, T. T., Perry, H. M., Gottesman, K. S. and Foeller, C., Sequences of proteins of immunological interest. US Department of Health and Human services, NIH, Bethesda 1991).

[0207] In some embodiments, the binding molecule according to the invention may comprise a heavy chain constant domain. In some embodiments, the heavy chain constant domain may be a heavy chain IgG constant domain. In some embodiments, the heavy chain IgG constant domain may be a heavy chain IgG 1 constant domain. In some embodiments, the heavy chain IgG constant domain may be a heavy chain lgG2 constant domain. In some embodiments, the heavy chain IgG constant domain may be a heavy chain lgG3 constant domain. In some embodiments, the heavy chain IgG constant domain may be a heavy chain lgG4 constant domain.

[0208] In some embodiments, heavy chain constant domain may comprise a constant heavy 2 (CH2) domain or a constant heavy 3 (CH3) domain. In some embodiments, heavy chain constant domain may comprise a constant heavy 2 (CH2) domain and a constant heavy 3 (CH3) domain.

[0209] In some embodiments, the binding molecule according to the invention may comprise a heavy chain constant domain comprising the amino acid sequence according to SEQ ID NO: 17 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 17. In some embodiments, the heavy chain constant domain may consist of the amino acid sequence according to SEQ ID NO: 17.

[0210] SEQ ID NO: 17 (hulgG1):

[0211] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0212] In some embodiments, the binding molecule according to the invention may comprise a light chain constant domain. In some embodiments, the light chain constant domain may be a light chain kappa constant domain. In some embodiments, the light chain constant domain may be a light chain lambda constant domain. In some embodiments, the binding molecule according to the invention may comprise a light chain constant domain comprising the amino acid sequence according to SEQ ID NO: 18 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 18. In some embodiments, the light chain constant domain may consist of the amino acid sequence according to SEQ ID NO: 18.

[0213] SEQ ID NO: 18 (huIgK):

[0214] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0215] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 19 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 21 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 21.

[0216] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 19; and a light chain sequence of SEQ ID NO: 21.

[0217] SEQ ID NO: 19:

[0218] QSLEESGGRLVTPGTPLTLTCTVSGFSLSSSDMSWVRQAPGKGLEWIGIIYASDNAYYASWAKGRFTI SKTSTTVDLKITSPTTEDTATYFCVRLWNMWGPGTLVTVSLASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0219] SEQ ID NO: 21 :

[0220] AQVLTQTASPVSAAVGGTVTISCQSSQSVYDNRLAWYQQKLGQPPKLLIYDASKLESGVPSRFKGSGS GTQFTLT I SDLE SDDAATY YCAARY S GN I GGFGGGTEVWKRTVAAPSVFI FPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS

[0221] SPVTKSFNRGEC In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 22 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 22.

[0222] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20; and a light chain sequence of SEQ ID NO: 22.

[0223] SEQ ID NO: 20:

[0224] QVQLVESGGRLVQPGTPLRLSCAVSGFSLSSSDMSWVRQAPGKGLEWIGIIYASDNAYYASWAKGRFT ISRDTSKTTLYLQMNSLRAEDTATYFCVRLWNMWGPGTLVTVSLASTKGPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0225] SEQ ID NO: 22:

[0226] DILLTQTPSWSASVGDRVTITCQSSQSVYDNRLAWYQQKPGQAPRLLIYDASKLESGVPSRFRGSGS GTDFTLT I TSLQPEDFATY YCAARY S GN I GGFGGGTRLE I KRTVAAPSVFI FPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0227] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 23 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 23.

[0228] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20; and a light chain sequence of SEQ ID NO: 23. SEQ ID NO: 23:

[0229] DIQMTQSPSTLSASVGDRVTITCQSSQSVYDNRLAWYQQKLGKAPRLLIYDASKLESGVPSRFSGSGS GTE FTLT I S SLQPDDFVTY YCAARY S GN I GGFGGGTKVE I KRTVAAPSVFI FPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0230] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 24 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 24.

[0231] In some embodiments, the binding molecule according to the invention may comprise a heavy chain sequence of SEQ ID NO: 20; and a light chain sequence of SEQ ID NO: 24.

[0232] SEQ ID NO: 24:

[0233] AIQMTQSPSTLSASVGGTVTITCQSSQSVYDNRLAWYQQKLGQPPKLLIYDASKLESGVPSRFKGSGS GTE FTLT I SDLE SDDFATY YCAARY S GN I GGFGGGTEVWKRTVAAPSVFI FPPSDEQLKSGTASVVC LLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

[0234] CFC1 antigen

[0235] The present invention provides a binding molecule that binds to a CFC1 antigen.

[0236] In some embodiments, the binding molecule according to the invention may bind to a CFC1 antigen.

[0237] In some embodiments, the CFC1 antigen may be defined according to the UniProt entry: P0CG37.

[0238] In some embodiments, the CFC1 antigen may comprise an amino acid sequence according to SEQ ID NO: 25 or a variant having at least 80% sequence identity thereto. In some embodiments, the variant may have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 25. In some 1 embodiments, the CFC1 antigen may consist of an amino acid sequence according to SEQ ID NO: 25.

[0239] SEQ ID NO: 25:

[0240] YQREKHNGGREEVTKVATQKHRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSRAFGEGASARPRCCRN GGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRACHLCRCIFGALHCLPLQTPDRCDPKD FLASHAHGPSAGGAPSLLLLLPCALLHRLLRPDAPAHPRSLVPSVLQRERRPCGRPGLGHRL

[0241] ANTIBODY

[0242] The present invention provides an antibody, or antigen-binding fragment thereof, comprising the binding molecule according to the invention.

[0243] As used herein, the term “antibody” is well-known in the art and means a protein or polypeptide having an antigen binding site or antigen-binding domain comprising at least one CDR.

[0244] In some embodiments, the antibody may comprise 3 CDRs. It will be understood that such an antibody may be a single domain antibody (sdAb).

[0245] In some embodiments, the antibody may comprise 6 CDRs. It will be understood that such an antibody may be a classical antibody molecule.

[0246] In some embodiments, the antibody or fragment thereof may be selected from the list consisting of: a single-chain variable fragment (scFv), a Fab, a F(ab)’2, a Fv, a single domain antibody, a nanobody, a VHH antibody, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, a chimeric antibody or fragment thereof, a bifunctional antibody, and a bispecific antibody.

[0247] In some embodiments, the antibody may be a non-human antibody, a chimeric antibody, a humanised antibody or a fully human antibody.

[0248] In some embodiments, the antibody may be a humanised antibody.

[0249] In some embodiments, the antibody may be a full-length, classical antibody. In some embodiments, the antibody may be an IgG, IgM, IgD, IgE or IgA molecule.

[0250] In some embodiments, the antibody may be an IgG molecule. In some embodiments, the antibody may be an lgG1 molecule. In some embodiments, the antibody may be an lgG2 molecule. In some embodiments, the antibody may be an lgG3 molecule. In some embodiments, the antibody may be an lgG4 molecule.

[0251] In some embodiments, a composition of antibodies may comprise monoclonal antibodies or polyclonal antibodies.

[0252] In some embodiments, the antibody may be a monoclonal antibody or fragment thereof. In some embodiments, the antibody may be a humanised monoclonal antibody or fragment thereof.

[0253] In some embodiments, the antibody may be a VHH antibody or fragment thereof.

[0254] In some embodiments, the antibody may be a humanised VHH antibody or fragment thereof.

[0255] It will be understood that a VHH antibody may comprise a VH domain and an Fc domain. It will also be understood that a VHH antibody may comprise a VH domain, a CH2 domain and a CH3 domain.

[0256] Techniques for obtaining antibodies are well-known in the art. For example, antibodies may be obtained by techniques comprising immunizing an animal with a target antigen and isolating the antibody from serum.

[0257] It will be understood that an antigen-binding fragment comprises an amino acid sequence that is shorter than the full-length sequence of an antibody, but retains the biological activity (e.g. binding specificity and / or affinity) of the full-length antibody.

[0258] The term "humanised antibody" typically refers to a genetically engineered antibody generated in a non-human animal that has been engineered to reduce immunogenicity when used in a human whilst retaining antigen specificity. The humanised antibody typically contains human antibody constant domains and non-human variable domains that are modified to contain a high level of sequence homology to human variable domains.

[0259] POLYNUCLEOTIDE

[0260] The present invention provides a polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention. The present invention provides one or more polynucleotide(s) comprising a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0261] As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other. The nucleic acid sequence may be an RNA or a DNA sequence. The nucleic acid sequence may be single-stranded or may be double-stranded. The nucleic acid sequence may be, for example, genomic, recombinant, mRNA or cDNA. The nucleic acid sequence may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance the in vivo activity and / or stability of the polynucleotide.

[0262] In some embodiments, the nucleic acid sequence may be a DNA sequence. In some embodiments, the nucleic acid sequence may be a cDNA sequence. In some embodiments, the nucleic acid sequence may be an RNA sequence. In some embodiments, the nucleic acid sequence may be an mRNA sequence.

[0263] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These sequences are encompassed by the present invention. Therefore multiple polynucleotides are envisaged, each with a different nucleic acid sequence but which encodes a polypeptide according to the invention or a further polypeptide as described herein. It is known in the art how to design and produce such nucleic acid sequences.

[0264] In some embodiments, the nucleic acid sequence of the polynucleotide may be codon optimised for production in the host cell of choice.

[0265] In some embodiments, the nucleic acid sequence of the polynucleotide may be operably linked to a sequence such as a control sequence, e.g. a promoter sequence, an enhancer sequence or regulatory sequence, which controls transcription and / or translation. The polynucleotide may be in the form of an expression cassette.

[0266] The polynucleotide may be suitable for expression in prokaryotic cells or in eukaryotic cells, such as mammalian cells. Any promoter may be used in the polynucleotide, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter

[0267] VECTOR

[0268] The present invention provides a vector comprising the polynucleotide according to the invention.

[0269] Accordingly, the vector may comprise a polynucleotide comprising a nucleic acid sequence which encodes the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0270] The vector may be used to introduce polynucleotide according to the invention into a cell so that the cell expresses and / or produces the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0271] As used herein, the term “vector” may be considered interchangeable with the term “expression vector” and “expression construct”. The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[0272] The vector according to the invention may be any agent capable of delivering a polynucleotide according to the invention to a cell and / or expressing a nucleic acid sequence of a polynucleotide according to the invention in a cell. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0273] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retroviral vector or a lentiviral vector.

[0274] The vector may be capable of transfecting or transducing a cell.

[0275] CELL AND RELATED METHODS

[0276] The present invention provides a cell comprising a polynucleotide according to the invention, or a vector according to the invention. The polynucleotide or vector may, for example, be introduced into a cell by transduction or transfection in vitro or ex vivo.

[0277] As such, the present invention also provides a method for making a cell according to the invention comprising the step of introducing a polynucleotide according to the invention, or a vector according to the invention into said cell. In some embodiments, the polynucleotide may be introduced as described herein.

[0278] In some embodiments, the cell may be capable of expressing the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0279] In some embodiments, the cell may be capable of producing the binding molecule according to the invention or the antibody or fragment thereof according to the invention.

[0280] In some embodiments, the cell may be capable of expressing and / or producing the binding molecule according to the invention or the antibody or fragment thereof according to the invention when the cell is cultured under suitable conditions.

[0281] As such, the present invention also provides a method for producing the binding molecule according to the invention or the antibody or fragment thereof according to the invention, wherein the method comprises the steps of:

[0282] (i) introducing a polynucleotide according to the invention, or a vector according to the invention into a cell; and

[0283] (ii) expressing the binding molecule or antibody or fragment thereof in the cell.

[0284] In some embodiments of the methods according to the invention, the polynucleotide or vector may be introduced into the cell by transduction or transfection in vitro or ex vivo.

[0285] In some embodiments of the methods according to the invention, culturing the cell under suitable conditions may result in the cell expressing and / or producing the binding molecule or antibody or fragment thereof.

[0286] In some embodiments, the method for producing the binding molecule or the antibody or fragment thereof may further comprise step (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or cell culture supernatant of the cell. It will be understood that the binding molecule according to the invention or the antibody or fragment thereof according to the invention may be harvested from the cell. It will also be understood that the binding molecule according to the invention or the antibody or fragment thereof according to the invention may be harvested from supernatant of the cell, for example when the binding molecule, antibody or fragment thereof is released out of the cell into the cell culture medium that the cell is cultured in.

[0287] In some embodiments, the cell may be a prokaryotic cell or a eukaryotic cell.

[0288] In some embodiments, the cell may be a bacterial cell, a fungal cell, a yeast cell, a plant cell or an animal cell.

[0289] In some embodiments, the cell may be a mammalian cell or an insect cell. In some embodiments, the cell may be a human cell.

[0290] COMPOSITION

[0291] The present invention provides a composition which comprises the binding molecule according to the invention, the antibody or fragment thereof according to the invention; together with a carrier, diluent or excipient.

[0292] In some embodiments, the composition may comprise the binding molecule according to the invention, together with a carrier, diluent or excipient.

[0293] In some embodiments, the composition may comprise the antibody or fragment thereof according to the invention, together with a carrier, diluent or excipient.

[0294] The present invention also provides a composition which comprises the polynucleotide according to the invention, together with a carrier, diluent or excipient.

[0295] The present invention also provides a composition which comprises the vector according to the invention, together with a carrier, diluent or excipient.

[0296] In some embodiments, the compositions described herein may further comprise one or more selected from this list consisting of: a adjuvant, salt, active polypeptide, compound, component and active agent. Compositions typically should be sterile and stable under the conditions of manufacture and storage. The composition according to the invention may be produced using current good manufacturing practices (CGMP).

[0297] The term “carrier”, as used herein, may refer to a diluent, adjuvant, excipient, or vehicle. Such carriers can be sterile liquids, such as saline solutions in water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil. A sterile saline solution is a preferred carrier.

[0298] Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0299] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition of the invention can be formulated as neutral or salt forms. Salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[0300] In some embodiments, the salt may comprise a metal cation, such as a sodium salt or a potassium salt.

[0301] In some embodiments, the composition may comprise an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be a phosphate buffered saline solution, such as a sterile phosphate buffered saline solution.

[0302] In some embodiments, the composition may comprise one or more vesicles, nanoparticles, lipid nanoparticle (LNPs), liposomes or polymeric mixtures.

[0303] The composition may enable delivery of a polynucleotide according to the invention and / or a vector according to the invention to a cell.

[0304] The present invention also provides a composition comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention; and

[0305] (ii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture.

[0306] Without wishing to be bound by theory, the composition may be used to deliver a nucleic acid sequence encoding the binding molecule according to the invention or the antibody or fragment thereof according to the invention to one or more cells, such that the cell(s) can express and produce the binding molecule or the antibody or fragment thereof.

[0307] In some embodiments, the polynucleotide in (i) may be a polynucleotide according to the invention.

[0308] In some embodiments, in addition to (i) and (ii), the composition may further comprise (iii) a carrier, diluent or excipient.

[0309] In some embodiments, the nucleic acid sequence of the polynucleotide in (i) may be a DNA sequence. In some embodiments, the nucleic acid sequence of the polynucleotide in (i) may be a cDNA sequence. In some embodiments, the nucleic acid sequence of the polynucleotide in (i) may be an RNA sequence. In some embodiments, the nucleic acid sequence of the polynucleotide in (i) may be an mRNA sequence.

[0310] KIT

[0311] The present invention also provides a kit comprising:

[0312] (i) the composition according to the invention.

[0313] In some embodiments, the kit may optionally comprise (ii) instructions for using the kit to target a cell expressing CFC1 in vitro.

[0314] IDENTITY

[0315] The terms “identity” and “% sequence identity” as used herein, may refer to the proportion of amino acids (expressed in percent) of an amino acid sequence in a peptide or protein, which across the amino acid sequence, are identical to a reference sequence. The percentage of identity is thus calculated by counting the number of aligned amino acids that are identical (a Match) between two sequences (in the amino acids sequence of the peptide or protein of the invention and in the reference sequence), dividing that number by the total number of amino acids in the aligned region and multiplying by 100.

[0316] Therefore, Percentage of Identity = (Matches divided by Length of the aligned region) multiplied by 100.

[0317] Insertions and deletions are not allowed in the calculation the percentage of identity of an amino acid sequence.

[0318] Any method of calculating percentage identity is permitted within the scope of the invention.

[0319] GENERAL TERMS AND DEFINITIONS

[0320] This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.

[0321] The term “polypeptide” is used in the conventional sense to mean a series of amino acids, typically L-amino acids, connected one to the other, typically by peptide bonds between the a- amino and carboxyl groups of adjacent amino acids.

[0322] The term “polypeptide” is used interchangeably with the terms “amino acid sequence”, “peptide” and / or “protein”.

[0323] The term “residues” is used to refer to amino acids in an amino acid sequence.

[0324] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0325] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

[0326] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0327] The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of' also include the term "consisting of.

[0328] As used herein, the term “variant” is synonymous with the term “mutant” and refers to an amino acid sequence or a nucleic acid sequence that differs in comparison to the corresponding wildtype sequence. The term “wild-type” is used to mean a protein comprising an amino acid sequence or a polynucleotide comprising an amino acid sequence, which is identical to the native protein or native polynucleotide (e.g. gene) respectively. The variant may have an equivalent function to the amino acid sequences or nucleic acid sequences described herein, but may include one or more amino acid or nucleic acid (respectively) substitutions, insertions or deletions. Amino acid substitutions, insertions and / or deletions may be considered as mutations. Nucleic acid substitutions, insertions and / or deletions may be considered as mutations.

[0329] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0330] SEQUENCES

[0331] The antibody clones used herein are: MAB-18-0129, MAB-20-0235, MAB-20-0240, and MAB-20-0250.

[0332] NUMBERED PARAGRAPHS

[0333] The present invention may be described by way of the following numbered paragraphs:

[0334] 1. A binding molecule comprising a CFC1 binding domain.

[0335] 2. The binding molecule according to paragraph 1 , wherein the CFC1 binding domain binds to CFC1 and induces internalisation of the binding molecule into a cell expressing CFC1 .

[0336] 3. The binding molecule according to paragraph 1 or paragraph 2, wherein the CFC1 binding domain induces a high rate of internalisation of the binding molecule into a cell expressing CFC1.

[0337] 4. The binding molecule according to paragraph 3, wherein the binding molecule has a high rate of internalisation compared to a control.

[0338] 5. The binding molecule according to paragraph 4, wherein the high rate of internalisation is determined by an Immunofluorescence Quenching assay.

[0339] 6. The binding molecule according to paragraph 5, wherein the high rate of internalisation is at least 60% at 1 hour as determined by the Immunofluorescence Quenching assay, optionally wherein the high rate of internalisation is at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, or at least 80% at 1 hour.

[0340] 7. The binding molecule according to any one of paragraphs 1-6, wherein the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:

[0341] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1 (SSDMS), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG), and

[0342] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (LWNM); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences. 8. The binding molecule according to any one of paragraphs 1-7, wherein the CFC1 binding domain comprises a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the Kabat definition, wherein:

[0343] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA),

[0344] LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), and

[0345] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0346] 9. The binding molecule according to any one of paragraphs 1-6, wherein the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the IMGT definition, wherein:

[0347] HCDR1 comprises the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 comprises the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and

[0348] HCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0349] 10. The binding molecule according to any one of paragraphs 1-6 or paragraph 9, wherein the CFC1 binding domain comprises a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the IMGT definition, wherein:

[0350] LCDR1 comprises the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR), LCDR2 comprises the amino acid sequence DAS, and

[0351] LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

[0352] 11. The binding molecule according to any one of paragraphs 7-10, wherein the VH domain comprises (i) the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; or

[0353] (ii) the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto.

[0354] 12. The binding molecule according to paragraph 11 , wherein:

[0355] (i) the VH domain comprises the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity thereto; or

[0356] (ii) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity thereto; or

[0357] (iii) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity thereto; or

[0358] (iv) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity thereto.

[0359] 13. The binding molecule according to any one of paragraphs 1-12, further comprising an Fc domain; optionally wherein the Fc domain is a modified Fc domain.

[0360] 14. The binding molecule according to paragraph 13, wherein the modified Fc domain comprises a modified hinge region.

[0361] 15. The binding molecule according to paragraph 13 or paragraph 14, wherein

[0362] (i) the Fc domain has reduced, or essentially no, binding to one or more Fc gamma receptors or C1q; and / or

[0363] (ii) the Fc domain is not capable of binding to immune cells; and / or

[0364] (iii) the Fc domain comprises LALA, LALA-KA, STR, or LALA-deltaA mutations.

[0365] 16. The binding molecule according to any one of paragraphs 1-15, wherein the binding molecule comprises (i) a heavy chain constant domain comprising the amino acid sequence according to SEQ ID NO: 17 or a variant having at least 80% sequence identity thereto; and / or

[0366] (ii) a light chain constant domain comprising the amino acid sequence according to SEQ ID NO: 18 or a variant having at least 80% sequence identity thereto.

[0367] 17. The binding molecule according to any one of paragraphs 1-16, wherein the binding molecule comprises:

[0368] (i) a heavy chain sequence of SEQ ID NO: 19 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 21 or a variant having at least 80% sequence identity thereto;

[0369] (ii) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 22 or a variant having at least 80% sequence identity thereto;

[0370] (iii) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 23 or a variant having at least 80% sequence identity thereto; or

[0371] (iv) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 24 or a variant having at least 80% sequence identity thereto.

[0372] 18. The binding molecule according to any one of paragraphs 1-17, wherein the CFC1 binding domain binds to a CFC1 antigen.

[0373] 19. The binding molecule according to paragraph 18, wherein the CFC1 antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 25 or a variant having at least 80% sequence identity thereto.

[0374] 20. An antibody or antigen-binding fragment thereof comprising the binding molecule according to any preceding paragraph.

[0375] 21 . The antibody or fragment thereof according to paragraph 20, wherein the antibody or fragment thereof is selected from the list consisting of: an scFv, a Fab, a single domain antibody, a nanobody, a VHH antibody, a monoclonal antibody or fragment thereof, a humanized antibody or fragment thereof, a chimeric antibody or fragment thereof, and a bispecific antibody. 22. The antibody or fragment thereof according to paragraph 20 or paragraph 21 , wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof.

[0376] 23. The antibody or fragment thereof according to paragraph 20 or paragraph 21 , wherein the antibody or fragment thereof is a VHH antibody or fragment thereof.

[0377] 24. A polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to any of paragraphs 1-19 or the antibody or fragment thereof according to any one of paragraphs 20-23.

[0378] 25. The polynucleotide according to paragraph 24, wherein the nucleic acid sequence is an RNA sequence.

[0379] 26. A vector which comprises a polynucleotide according to paragraph 24 or paragraph 25.

[0380] 27. A cell comprising a polynucleotide according to paragraph 24 or paragraph 25, or a vector according to paragraph 26.

[0381] 28. The cell according to paragraph 27, wherein the cell is capable of expressing the binding molecule according to any of paragraphs 1-19 or the antibody or fragment thereof according to any one of paragraphs 20-23.

[0382] 29. A method for making a cell according to paragraph 27 or paragraph 28, comprising the step of introducing a polynucleotide according to paragraph 24 or paragraph 25, or a vector according to paragraph 26 into said cell.

[0383] 30. A method for producing the binding molecule according to any of paragraphs 1-19 or the antibody or fragment thereof according to any one of paragraphs 20-23, wherein the method comprises the steps of:

[0384] (i) introducing a polynucleotide according to paragraph 24 or paragraph 25, or a vector according to paragraph 26 into a cell; and

[0385] (ii) expressing the binding molecule or antibody or fragment thereof in the cell; and optionally (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or cell culture supernatant of the cell. 31 . A composition which comprises the binding molecule according to any of paragraphs 1-19, the antibody or fragment thereof according to any one of paragraphs 20-23; together with a carrier, diluent or excipient.

[0386] 32. A composition comprising:

[0387] (i) a polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to any of paragraphs 1-19 or the antibody or fragment thereof according to any one of paragraphs 20-23; and

[0388] (ii) a vesicle, a nanoparticle, a lipid nanoparticle (LNP), a liposome or a polymeric mixture; and optionally

[0389] (iii) a carrier, diluent or excipient; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymeric mixture.

[0390] 33. A kit comprising:

[0391] (i) the composition according to paragraph 31 or paragraph 32; and optionally (ii) instructions for using the kit to target a cell expressing CFC1 in vitro.

[0392] The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

[0393] EXAMPLES

[0394] Example 1 : Generation of CFC1 -binding monoclonal antibodies

[0395] To obtain CFC1 -specific monoclonal antibodies, New Zealand White rabbits were immunized with recombinant human CFC1 extracellular domain containing a C-terminal 10xHIS protein. Single B-cells were isolated by FACS from peripheral blood and cultivated to obtain monoclonal antibodies in the medium supernatant. Selected monoclonal B-cell clones were lysed in RNA extraction RLT buffer for RNA extraction, RT-PCR and Sanger sequencing of the antibody heavy and light chain variable regions. Antibody heavy and light chain V-regions were gene synthesized, cloned into pCEP4 expression plasmids upstream of hlgG1-constant domains and produced in HEK293-FreeStyle™ cells.

[0396] Example 2: Humanization of CFC1 -binding monoclonal antibodies

[0397] VL and VH-region coding sequences of MAB-18-0129 antibodies were subjected to an in silico humanization procedure based on a combination of CDR grafting and antibody structure guided modifications. Five heavy chain and four light chain humanized V-regions were gene synthesized and cloned into pCEP4 expression plasmids upstream of hlgG1-constant domains. Combinations of all possible humanized antibody heavy and light chains were produced in HEK293-FreeStyle™ cells.

[0398] Example 3: Human CFC1 ELISA

[0399] The specificity and binding potencies of the anti-CFC1 chimeric and humanized antibodies of the invention to human CFC1 were determined by an ELISA (see Table 1 and Figure 1).

[0400] Recombinant human CFC1 ECD (R&D Systems) was coated on 384-well Nunc MaxiSorp™ flat bottom plates at a concentration of 0,6 pg / ml in PBS for 60 minutes at room temperature. After 3 washes with PBS 0.1 % Tween (wash buffer), blocking with PBS, 2% BSA, 0.05% Tween for 60 minutes at room temperature and another 3 washes, the chimeric and humanized antibodies of the invention were added in PBS, 0.5% BSA, 0.05% Tween (ELISA buffer) in concentrations ranging from 2,000 to 0.06 ng / ml and the plate was incubated for 60 minutes at room temperature. As reference antibodies, a control anti-CFC1 antibody was added in same concentration range. After 3 washes with wash buffer, horseradish peroxidase coupled anti-human-IgG (Fab’)2 fragment (AbD Serotec) was added in ELISA buffer at a dilution of 1 :5000. The plate was incubated for 60 minutes at room temperature, washed 6 times with wash buffer before TMB solution (Thermo Fisher Scientific) was added. The color reaction was stopped with HCI and the absorbance was recorded at wavelengths 450 / 620 nm using a Tecan Infinite M1000 reader. Data analysis including 4PL curve fitting and EC50 calculation were done with IDBS XLfit.

[0401] Example 4: CHO-K1 human CFC1 and CHO-K1 cynomolgus cell binding

[0402] Binding potencies of anti-CFC1 chimeric or humanized antibodies of the invention to human or cynomolgus CFC1 ectopically expressing CHO-K1 cell line were analyzed by high content imaging (see Table 1 and Figure 2).

[0403] 1 ,000 CHO-K1 cells were seeded per well in black 384-well cell-culture treated plates and incubated for 2 hours at 37°C and 5% CO2. Chimeric or humanized antibodies of the invention were diluted ranging from 5,000 to 0.05 ng / ml. As reference antibodies, a control anti-CFC1 antibody was used at same concentration range. After overnight incubation at 37°C and 5% CO2, plates were washed once with PBS, 0.05% Tween 20 (cell wash buffer) and Alexa-Fluor- 448-conjugated anti-human-IgG F(ab’)2 fragment was added at a concentration of 0.8 pg / ml. Plates were incubated for 4 hours at 37°C and 5% CO2 in the dark, washed once with cell wash buffer and incubated for 10 minutes with medium containing 5pg / ml Hoechst (Invitrogen). Cell-associated immunofluorescent signals were recorded using a Celllnsight CX5 high content imager (Thermo Fisher) given as the mean spot intensity averaged over all cells . Data analysis including 4PL curve fitting and EC50 calculation were done with IDBS XLfit.

[0404] Table 1 Example 5: High content imaging internalization assay

[0405] The chimeric or humanized anti-CFC1 antibodies of the invention were evaluated in their internalization rate and strength in overexpressing CHO-K1 human CFC1 cells via high- content imaging (see Table 2 and Figure 3).

[0406] Cells were seeded in 384 well plates overnight and then stained with Hoechst 33342. Each of the antibodies was added at 20nM for 1 hr at 4°C to the cells, then washed off and cells were stained with 60nM Alexafluor 488 anti-human IgG-Fab fragment (Dianova) for 30 minutes at 4°C. The plates were cultivated at 37°C in humidified CO2 incubator for either 0, 1 or 4 hrs. Subsequently part of the replicate conditions were either treated with cell medium (nonquenched) or 100nM anti-AlexaFluor 488-lgG quenching antibody for 30 minutes at4°C. After a washing step, plates were measured for Hoechst and Alexa Fluor 488 staining in the high- content imager Celllnsight CX5 (Thermo Fisher). The software module SpotDetector was deployed for cell masking and calculation of the mean Alexa Fluor 488 Intensity per well accounting for each single cell, mean Alexa Fluor 488 spot intensity. Percent internalization was calculated from the ratio of mean Alexa Fluor 488 intensity from the quenched condition to the mean intensity of AlexaFluor 488 without quenching. Data was plotted using GraphPad Prism.

[0407] Table 2

[0408] Example 6: Antibody binding to low CFC1 expressing cells

[0409] Cells (Table 3) were plated at 1E5 cells / well and incubated for 1 hour at 4°C with serial dilutions of MAB-20-0235 antibody or isotype control (anti-HIV antibody) (0-400 nM). Secondary antibody (A647 goat anti-human IgG Fey) was added (1 :500 dilution) and incubated for 1 hour at 4°C. Antibody binding was determined by FACS. Table 3

[0410] MAB-20-0235 showed dose dependent binding to NCI-H727 and NCI-H810 cells (see Table 4 and Figure 4).

[0411] Table 4

[0412] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

CLAIMS1. A binding molecule comprising a CFC1 binding domain.

2. The binding molecule according to claim 1 , wherein the CFC1 binding domain binds to CFC1 and induces internalisation of the binding molecule into a cell expressing CFC1 ; optionally wherein the CFC1 binding domain induces a high rate of internalisation of the binding molecule into a cell expressing CFC1.

3. The binding molecule according to claim 2, wherein the high rate of internalisation is at least 60% at 1 hour as determined by an Immunofluorescence Quenching assay, optionally wherein the high rate of internalisation is at least 65% at 1 hour, at least 70% at 1 hour, at least 75% at 1 hour, at least 76% at 1 hour, at least 77% at 1 hour, at least 78% at 1 hour, at least 79% at 1 hour, or at least 80% at 1 hour as determined by the Immunofluorescence Quenching assay.

4. The binding molecule according to any one of claims 1-3, whereinthe CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein theVH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the Kabat definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 1(SSDMS),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 2(IIYASDNAYYASWAKG), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 3(LWNM); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences; and / or(ii) the CFC1 binding domain comprises a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the Kabat definition, wherein:LCDR1 comprises the amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA),LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), andLCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences; or(b)(i) the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1-3 as defined by the IMGT definition, wherein:HCDR1 comprises the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD),HCDR2 comprises the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), andHCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences; and / or(ii) the CFC1 binding domain comprises a light chain variable (VL) domain; wherein the VL domain comprises light chain complementarity determining regions (LCDRs) 1-3 as defined by the IMGT definition, wherein:LCDR1 comprises the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR),LCDR2 comprises the amino acid sequence DAS, andLCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG); optionally wherein one or more of the CDRs comprises one, two or three amino acid mutations relative to the recited sequences.

5. The binding molecule according to claim 4, wherein the VH domain comprises(a)(i) the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; or(ii) the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; optionally wherein(b)(i) the VH domain comprises the amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity thereto; or(ii) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity thereto; or(iii) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity thereto; or(iv) the VH domain comprises the amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity thereto; and the VL domain comprises the amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity thereto.

6. The binding molecule according to any one of claims 1-5, further comprising an Fc domain; optionally wherein the Fc domain is a modified Fc domain.

7. The binding molecule according to any one of claims 1-6, wherein the binding molecule comprises(i) a heavy chain constant domain comprising the amino acid sequence according to SEQ ID NO: 17 or a variant having at least 80% sequence identity thereto; and / or(ii) a light chain constant domain comprising the amino acid sequence according to SEQ ID NO: 18 or a variant having at least 80% sequence identity thereto.

8. The binding molecule according to any one of claims 1-7, wherein the binding molecule comprises:(i) a heavy chain sequence of SEQ ID NO: 19 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 21 or a variant having at least 80% sequence identity thereto;(ii) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 22 or a variant having at least 80% sequence identity thereto;(iii) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 23 or a variant having at least 80% sequence identity thereto; or(iv) a heavy chain sequence of SEQ ID NO: 20 or a variant having at least 80% sequence identity thereto; and a light chain sequence of SEQ ID NO: 24 or a variant having at least 80% sequence identity thereto.

9. The binding molecule according to any one of claims 1-8, wherein the CFC1 binding domain binds to a CFC1 antigen; optionally wherein the CFC1 antigen comprises or consists of an amino acid sequence according to SEQ ID NO: 25 or a variant having at least 80% sequence identity thereto.

10. An antibody or antigen-binding fragment thereof comprising the binding molecule according to any preceding claim.

11. A polynucleotide comprising a nucleic acid sequence encoding the binding molecule according to any of claims 1-9 or the antibody or fragment thereof according to claim 10.

12. A vector which comprises a polynucleotide according to claim 11.

13. A cell comprising a polynucleotide according to claim 11 , or a vector according to claim 12.

14. A method for producing the binding molecule according to any of claims 1-9 or the antibody or fragment thereof according to claim 10, wherein the method comprises the steps of:(i) introducing a polynucleotide according to claim 11 , or a vector according to claim 12 into a cell; and(ii) expressing the binding molecule or antibody or fragment thereof in the cell;and optionally (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or cell culture supernatant of the cell.

15. A composition which comprises the binding molecule according to any of claims 1-9, the antibody or fragment thereof according to claim 10; together with a carrier, diluent or excipient.

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