TW202542189A - Cfc1 binding molecule - Google Patents

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

CFC1 bound molecules

This invention relates to a binding molecule comprising a CFC1 binding domain. Specifically, this invention relates to a CFC1 binding domain that induces the binding molecule to be internalized at a high rate into cells expressing CFC1. This invention further relates to an antibody comprising the binding molecule.

CFC1 is a member of the EGF-CFC protein family and can be cell-bound/membrane-bound, or it can exist extracellularly in a soluble form.

CFC1 is involved in Akt and MAPK communication and is known to be a co-receptor for Nodal and ALK4/7 receptor binding. It can induce SMAD 2/3 phosphorylation, which in turn binds to SMAD4 and translocates to the cell nucleus.

CFC1 is also known to participate in the regulation of gastrulation, mesoderm induction, and axon formation during embryonic development. In addition, CFC1 acts as a Nodal receptor, establishing left-right asymmetry in developing organs.

CFC1 is associated with visceral ectopic syndrome in humans. Mice with the CFC1 gene knocked out survive to birth and exhibit severe lateral defects, but do not exhibit phenotypes associated with pre-gastrulation patterning and differentiation.

CFC1 is expressed in tumors.

There is a need for improved therapies that target CFC1 expression in tumors and cancers, particularly CFC1 binding molecules with advantageous properties for in vivo administration and as pharmaceuticals.

The present invention provides a binding molecule comprising a CFC1 binding domain.

In some embodiments, the CFC1 binding domain can bind to CFC1 (e.g., CFC1 on the cell surface) and can induce the binding molecule to be internalized into cells expressing CFC1.

In some embodiments, the CFC1 binding domain can induce the binding molecule to be internalized at a high rate into cells expressing CFC1.

In some embodiments, the bound molecules may have a higher internalization rate compared to the control.

In some embodiments, high internalization rates can be determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate may be at least 60% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, the high internalization rate 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 analysis.

In some embodiments, the CFC1 binding domain may include a heavy chain variable (VH) domain; wherein the VH domain includes heavy chain complementarity determining regions (HCDRs) 1 to 3 as defined by Kabat, wherein: HCDR1 includes the amino acid sequence according to SEQ ID NO: 1 (SSDMS), HCDR2 includes the amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG), and HCDR3 includes the amino acid sequence according to SEQ ID NO: 3 (LWNM).

In some embodiments, one or more of the CDRs may contain one, two or three amino acid mutations.

In some embodiments, the CFC1 binding domain may include a light chain variable (VL) domain; wherein the VL domain includes light chain complementary determinant regions (LCDRs) 1 to 3 as defined by Kabat, wherein: LCDR1 includes the amino acid sequence (QSSQSVYDNRLA) according to SEQ ID NO: 4, LCDR2 includes the amino acid sequence (DASKLES) according to SEQ ID NO: 5, and LCDR3 includes the amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6.

In some embodiments, one or more of the CDRs may contain one, two or three amino acid mutations.

In some embodiments, the CFC1 binding domain may include a heavy chain variable (VH) domain; wherein the VH domain includes heavy chain complementarity determining regions (HCDRs) 1 to 3 as defined by IMGT, wherein: HCDR1 includes an amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 includes an amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and HCDR3 includes an amino acid sequence according to SEQ ID NO: 9 (VRLWNM).

In some embodiments, one or more of the CDRs may contain one, two or three amino acid mutations.

In some embodiments, the CFC1 binding domain may include a light chain variable (VL) domain; wherein the VL domain includes light chain complementary determination regions (LCDRs) 1 to 3 as defined by IMGT, wherein: LCDR1 includes an amino acid sequence (QSVYDNR) according to SEQ ID NO: 10, LCDR2 includes the amino acid sequence DAS, and LCDR3 includes an amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6.

In some embodiments, one or more of the CDRs contain one, two, or three amino acid mutations.

In some embodiments, the VH domain may contain an amino acid sequence according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity.

In some embodiments, the VH domain may contain an amino acid sequence according to SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity.

In some embodiments, the VH domain may contain an amino acid sequence according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity; and the VL domain may contain an amino acid sequence according to SEQ ID NO: 13 or a variant thereof having at least 80% sequence identity.

In some embodiments, the VH field may contain an amino acid sequence according to SEQ ID NO: 11; and the VL field may contain an amino acid sequence according to SEQ ID NO: 13.

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

In some embodiments, the VH field may contain an amino acid sequence according to SEQ ID NO: 12; and the VL field may contain an amino acid sequence according to SEQ ID NO: 14.

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

In some embodiments, the VH field may contain an amino acid sequence according to SEQ ID NO: 12; and the VL field may contain an amino acid sequence according to SEQ ID NO: 15.

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

In some embodiments, the VH field may contain an amino acid sequence according to SEQ ID NO: 12; and the VL field may contain an amino acid sequence according to SEQ ID NO: 16.

In some embodiments, the binding molecule may further include an Fc domain.

In some implementations, the Fc field can be a modified Fc field.

In some embodiments, the modified Fc field may contain a modified hinge region.

In some embodiments, the binding of the Fc domain to one or more Fcγ receptors or C1q may be reduced or essentially non-binding.

In some implementations, the Fc domain may be unable to bind to immune cells.

In some embodiments, the Fc domain may contain LALA, LALA-KA, STR, or LALA-deltaA mutations.

In some embodiments, the binding molecule may include a heavy chain constant that comprises an amino acid sequence according to SEQ ID NO: 17 or a variant thereof having at least 80% sequence identity.

In some embodiments, the binding molecule may include a light chain constant that comprises an amino acid sequence according to SEQ ID NO: 18 or a variant thereof having at least 80% sequence identity.

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

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

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

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

In some embodiments, the binding molecule may include the heavy chain sequence of SEQ ID NO: 19 and the light chain sequence of SEQ ID NO: 21.

In some embodiments, the binding molecule may include the heavy chain sequence of SEQ ID NO: 20 and the light chain sequence of SEQ ID NO: 22.

In some embodiments, the binding molecule may include the heavy chain sequence of SEQ ID NO: 20 and the light chain sequence of SEQ ID NO: 23.

In some embodiments, the binding molecule may include the heavy chain sequence of SEQ ID NO: 20 and the light chain sequence of SEQ ID NO: 24.

In some embodiments, the CFC1 binding domain can bind to the CFC1 antigen.

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

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

In some embodiments, the antibody or fragment thereof may be selected from the list of the following: scFv, Fab, single-domain antibody, nano antibody, VHH antibody, monoclonal antibody or fragment thereof, humanized antibody or fragment thereof, chimeric antibody or fragment thereof, and bispecific antibody.

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

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

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

In some embodiments, the nucleic acid sequence may be an RNA sequence.

The present invention provides a carrier comprising a polynucleotide according to the present invention.

The present invention provides a cell comprising a polynucleotide according to the present invention or a carrier according to the present invention.

In some embodiments, cells may be able to express the binding molecules or antibodies or fragments thereof according to the invention.

The present invention provides a method for generating cells according to the present invention, comprising the steps of introducing a polynucleotide according to the present invention or a carrier according to the present invention into the cells.

The present invention provides a method for generating a binding molecule or an antibody or fragment thereof according to the present invention, wherein the method comprises the steps of: (i) introducing a polynucleotide or a carrier according to the present invention into a cell; and (ii) expressing the binding molecule or antibody or fragment thereof in the cell.

In some embodiments, the method further includes step (iii): harvesting the binding molecule or antibody or fragment thereof from the cell or cell culture supernatant.

The present invention provides a composition comprising a binding molecule according to the present invention, an antibody according to the present invention or a fragment thereof; and a carrier, diluent or excipient.

The present invention provides a composition comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding a binding molecule or an antibody or fragment thereof according to the present invention; and (ii) a vesicle, nanoparticle, liposome nanoparticle (LNP), liposome or polymer mixture; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymer mixture.

In some embodiments, the composition may also contain (iii) a carrier, a diluent or an excipient.

The present invention provides a kit comprising: (i) a combination according to the present invention.

In some embodiments, the kit may further include (ii) instructions for using the kit to target cells expressing CFC1 in vitro.

The binding molecule CFC1, also known as the cryptic protein, is a NODAL co-receptor involved in the correct establishment of the left and right axes. An exemplary amino acid sequence is the human CFC1 sequence named UniProt:P0CG37.

This invention provides a binding molecule that includes a CFC1 binding domain. In other words, the binding molecule according to this invention may be able to bind to CFC1.

It should be understood that a binding molecule is a molecule that interacts with a target (e.g., a target antigen) to form a stable binding. Examples of binding molecules are antigens (or antibody-like molecules) that bind to their homologous antigens. Such binding interactions are known in this art. Binding interactions can be nonvalent, reversibly covalent, or irreversibly covalent.

Suitable analyses and techniques for measuring/quantifying the binding activity of the binding molecules according to the present invention may include (but are not limited to) ELISA, surface plasma resonance (SPR), quartz crystal microbalance (QCM), bioluminescence analysis, and flow cytometry. Other suitable techniques will be known in this art.

It should be understood that EC50 is a measure of antibody concentration that induces a specific response at 50% between the maximal and baseline responses. Therefore, EC50 can be used to assess the ability of an antibody to bind to a target.

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.

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

The interaction between an internalized binding molecule and a target molecule (e.g., the binding of an antibody to its homologous antigen) allows the target molecule (e.g., an antigen) to be transported into the cell. This type of transport can be referred to as endocrine function or internalization.

The binding of the binding molecule according to the invention to CFC1 expressed on the cell surface allows CFC1 to be internalized into the cell (e.g., due to conformational changes in the CFC1 protein). When the binding molecule according to the invention binds to CFC1, the binding molecule according to the invention is also internalized into the cell as CFC1 is internalized.

Therefore, the binding molecule according to the present invention can be induced to be internalized into cells that express CFC1 on the cell surface.

The binding molecule according to the present invention has improved properties compared to known molecules that bind CFC1. Specifically, the binding molecule according to the present invention can have an increased rate of internalization into the cell. An increased internalization rate can be considered as a high internalization rate or a rapid internalization rate.

In some embodiments, the CFC1 binding domain induces the binding molecule according to the invention to be internalized at a high rate into cells expressing CFC1.

In some embodiments, a high internalization rate is determined by comparison with a suitable control. It should be understood that a control may be a bound molecule bound to CFC1, but may be considered to induce only a low rate of internalization, or no internalization, or substantially no internalization, within a given time period. Comparisons between bound molecules with high internalization rates and bound molecules with low internalization rates are shown in the accompanying examples and Figure 3.

The internalization rate can be determined using any suitable analysis known in this technique.

For example, the internalization of the binding molecules according to the present invention can be evaluated by immunofluorescence quenching analysis. Immunofluorescence quenching analysis involves quenching extracellular fluorescence and measuring intracellular fluorescence to evaluate the internalization rate of the binding molecules.

In short, binding molecules (such as antibodies) are labeled with detectable markers, such as fluorescent markers (e.g., AF488), and co-cultured with cells or cell populations expressing homologous antigens on their cell surfaces, allowing the binding molecules to bind to the homologous antigens and form binding molecule-antigen complexes. Antigen endocytosis may then occur, transporting (i.e., internalizing) the binding molecule-antigen complex into the cells. When the binding molecules are labeled with a detectable marker, that detectable marker is also internalized. A quenching step is then performed on a portion of the sample, quenching the detectable marker signal on any binding molecules that remain bound to the antigens on the cell surface. However, detectable markers that have been internalized by the molecule-antigen complex are protected from the quenching step and will continue to emit signals (e.g., fluorescent signals). In the control sample where the quenching step was not performed, both detectable markers on the binding molecules that bind to homologous antigens on the cell surface and detectable markers on the binding molecules that bind to internalized homologous antigens will emit signals. Samples that have undergone the quenching step can be compared with those that have not. Since the ratio of intracellular signals (e.g., fluorescent signals) to total (surface + intracellular) cellular signals increases proportionally with time and internalization rate, immunofluorescence quenching assays can be used to measure the internalization rate of bound molecules and to determine whether bound molecules have a high internalization rate.

Immunofluorescence quenching analysis is further described in Example 5.

In some implementations, high internalization rates can be determined by immunofluorescence quenching analysis.

It should be understood that the given internalization rate % at a given time point refers to the proportion of antibodies that have been internalized at that time point during the immunofluorescence quenching assay.

For example, it should be understood that the internalization rate % given at 1 hour refers to the proportion of antibodies that have been internalized after 1 hour of continuous immunofluorescence quenching analysis. For example, it should be understood that an internalization rate % of 50% at 1 hour refers to half of the antibodies that have been internalized after 1 hour of continuous immunofluorescence quenching analysis.

In some embodiments, a high internalization rate can be defined as at least 50% at 1 hour, as determined by immunofluorescence quenching analysis. In other words, a rate of at least 50% at 1 hour, as determined by immunofluorescence quenching analysis, can be considered a measure of identifying a high internalization rate.

In some embodiments, the high internalization rate can be 50% to 100% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate can be defined as at least 55% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate can be defined as at least 60% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate may be at least 65% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 66% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 67% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 68% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 69% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 70% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate may be at least 75% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 76% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 77% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 78% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 79% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate may be at least 80% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 81% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 82% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 83% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 84% at 1 hour, as determined by immunofluorescence quenching analysis.

In some embodiments, a high internalization rate may be at least 85% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 86% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 87% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 88% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 89% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate may be at least 90% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate can be at least 95% at 1 hour, as determined by immunofluorescence quenching analysis. In some embodiments, a high internalization rate can be 100% at 1 hour, as determined by immunofluorescence quenching analysis.

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

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

In some embodiments, the binding molecule according to the present invention may include a VH domain and a VL domain.

In some embodiments, the VH field may contain one or more complementary decision regions (CDRs). In some embodiments, the VH field may contain one, two, or three CDRs. In some embodiments, the VH field may contain three CDRs. It should be understood that the CDRs of the VH field may be referred to as HCDRs. It should also be understood that each of the three CDRs of the VH field may be referred to as HCDR1, HCDR2, and HCDR3, respectively.

In some embodiments, a VL field may contain one or more CDRs. In some embodiments, a VL field may contain one, two, or three CDRs. In some embodiments, a VL field may contain three CDRs. It should be understood that the CDR of the VL field may be referred to as LCDR. It should also be understood that each of the three CDRs of the VL field may be referred to as LCDR1, LCDR2, and LCDR3, respectively.

The term "heavy chain variable region" or "VH" can refer to the heavy chain portion of an antigen-binding molecule or antibody, which typically contains three CDRs, each located between amino acid fragments called backbone regions, which form a scaffold supporting the CDRs.

The term "light chain variable region" or "VL" can refer to an antigen-binding domain or a light chain segment of an antibody, which typically contains three CDRs, each located between amino acid segments called backbone regions, which form a scaffold supporting the CDRs.

The term "complementary determining region" or "CDR" refers to a highly variable ring within the variable region that interacts with homologous antigens and is primarily responsible for determining the ability of a binding molecule (e.g., an antibody) to bind to the antigen and for determining binding affinity. CDRs within the variable region are typically numbered from the amino terminus to the carboxyl terminus, with CDR1 being closest to the amino terminus and CDR3 being closest to the carboxyl terminus.

CDR can be determined according to the Kabat definition and/or the IMGT definition, both of which are well known in this art.

In some embodiments, the binding molecule according to the present invention may include a VH domain comprising HCDR 1 to 3 as defined by Kabat, 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), and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3 (LWNM).

In some embodiments, the binding molecule according to the present invention may include a VL domain comprising LCDR 1 to 3 as defined by Kabat, wherein: LCDR1 comprises the amino acid sequence (QSSQSVYDNRLA) according to SEQ ID NO: 4, LCDR2 comprises the amino acid sequence (DASKLES) according to SEQ ID NO: 5, and LCDR3 comprises the amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6.

In some embodiments, the binding molecule according to the present invention may include a VH domain comprising HCDR 1 to 3 as defined by Kabat; and may include a VL domain comprising LCDR 1 to 3 as defined by Kabat, 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), 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), LCDR2 comprises the amino acid sequence according to SEQ ID NO: 5 (DASKLES), and LCDR3 comprises the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

In some embodiments, the binding molecule according to the present invention may include a VH domain comprising HCDR 1 to 3 as defined by IMGT, 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), and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 9 (VRLWNM).

In some embodiments, the binding molecule according to the present invention may include a VL domain comprising LCDR 1 to 3 as defined by IMGT, wherein: LCDR1 comprises the amino acid sequence (QSVYDNR) according to SEQ ID NO: 10, LCDR2 comprises the amino acid sequence DAS, and LCDR3 comprises the amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6.

In some embodiments, the binding molecule according to the present invention may include a VH domain containing HCDR 1 to 3 as defined by IMGT; and may include a VL domain containing LCDR 1 to 3 as defined by IMGT, wherein: HCDR1 contains the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 contains the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), HCDR3 contains the amino acid sequence according to SEQ ID NO: 9 (VRLWNM), LCDR1 contains the amino acid sequence according to SEQ ID NO: 10 (QSVYDNR), LCDR2 contains the amino acid sequence DAS, and LCDR3 contains the amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG).

In some embodiments, one or more HCDRs may contain one, two or three amino acid mutations.

In some embodiments, HCDR 1 may contain one, two, or three amino acid mutations.

In some embodiments, HCDR 2 may contain one, two, or three amino acid mutations.

In some embodiments, HCDR 3 may contain one, two, or three amino acid mutations.

In some embodiments, one or more of the LCDRs may contain one, two or three amino acid mutations.

In some embodiments, LCDR 1 may contain one, two, or three amino acid mutations.

In some embodiments, LCDR 2 may contain one, two, or three amino acid mutations.

In some embodiments, LCDR 3 may contain one, two, or three amino acid mutations.

It should be understood that any mutation in a CDR described herein may include the deletion, insertion, or substitution of an amino acid. It should also be understood that such mutations may not prevent the binding of the binding molecule according to the invention to CFC1. In other words, the binding molecule according to the invention, containing mutations in one or more of the CDRs described herein, may suitably maintain its ability to bind to CFC1.

In some embodiments, the VH domain of the binding molecule according to the invention may comprise the amino acid sequence of SEQ ID NO: 11 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 11. In some embodiments, the VH domain may consist of the amino acid sequence of SEQ ID NO: 11. SEQ ID NO: 11:

In some embodiments, the VH domain of the binding molecule according to the invention may comprise an amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 12. In some embodiments, the VH domain may consist of an amino acid sequence according to SEQ ID NO: 12. SEQ ID NO: 12:

In some embodiments, the VL domain of the binding molecule according to the invention may comprise an amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 13. In some embodiments, the VL domain may consist of an amino acid sequence according to SEQ ID NO: 13. SEQ ID NO: 13:

In some embodiments, the VL domain of the binding molecule according to the invention may comprise an amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 14. In some embodiments, the VL domain may consist of an amino acid sequence according to SEQ ID NO: 14. SEQ ID NO: 14:

In some embodiments, the VL domain of the binding molecule according to the invention may comprise an amino acid sequence according to SEQ ID NO: 15 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 15. In some embodiments, the VL domain may consist of an amino acid sequence according to SEQ ID NO: 15. SEQ ID NO: 15:

In some embodiments, the VL domain of the binding molecule according to the invention may comprise an amino acid sequence according to SEQ ID NO: 16 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 16. In some embodiments, the VL domain may consist of an amino acid sequence according to SEQ ID NO: 16. SEQ ID NO: 16:

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

In some embodiments, the binding molecule according to the present invention may include a VH domain containing an amino acid sequence according to SEQ ID NO: 11; and a VL domain containing an amino acid sequence according to SEQ ID NO: 13.

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

In some embodiments, the binding molecule according to the present invention may include a VH domain containing an amino acid sequence according to SEQ ID NO: 12; and a VL domain containing an amino acid sequence according to SEQ ID NO: 14.

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

In some embodiments, the binding molecule according to the present invention may include a VH domain containing an amino acid sequence according to SEQ ID NO: 12; and a VL domain containing an amino acid sequence according to SEQ ID NO: 15.

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

In some embodiments, the binding molecule according to the present invention may include a VH domain containing an amino acid sequence according to SEQ ID NO: 12; and a VL domain containing an amino acid sequence according to SEQ ID NO: 16.

It should be understood that the variants of the VH domain described herein may have functions equivalent to the full-length VH domain amino acid sequence described herein and may appropriately maintain the ability to bind to CFC1.

It should be understood that the variants of the VL domain described herein may have functions equivalent to the full-length VL domain amino acid sequence described herein and may appropriately maintain the ability to bind to CFC1.

In some embodiments, the Fc domain of the binding molecule may include an immunoglobulin stationary region.

In some embodiments, the binding molecule according to the present invention may comprise a combination of an immunoglobulin constant region and the VH or VL domain described above.

In some embodiments, the binding molecule according to the present invention may comprise a combination of an immunoglobulin constant region and the VH domain and VL domain described above.

It should be understood that the VH domain and the VL domain can together form a variable region.

In some embodiments, the immunoglobulin constant region may include one or more immunoglobulin constant domains.

In some embodiments, the immunoglobulin constant domain may be selected from the list of the following: constant heavy chain 1 (CH1) domain, constant heavy chain 2 (CH2) domain and constant heavy chain 3 (CH3) domain.

In some embodiments, the immunoglobulin constant region may include one or more constant heavy chain 2 (CH2) domains or one or more constant heavy chain 3 (CH3) domains. In some embodiments, the immunoglobulin constant region may include one or more constant heavy chain 2 (CH2) domains and one or more constant heavy chain 3 (CH3) domains.

It should be understood that the immunoglobulin constant region may contain an Fc domain, such as the Fc domain described herein.

In some embodiments, the binding molecule according to the invention may include an Fc domain. In some embodiments, the binding molecule according to the invention may include a combination of an Fc domain and the VH or VL domains described above. In some embodiments, the binding molecule according to the invention may include a combination of an Fc domain and the VH and VL domains described above.

It should be understood that the Fc domain can interact with Fc receptors presented on the cell surface and/or with proteins of the complement system. The Fc receptor may be an Fcγ receptor. Proteins of the complement system may include C1q.

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

In some embodiments, the Fc domain of the binding molecule according to the invention may contain an erbium chain region.

In some embodiments, the Fc domain of the binding molecule according to the invention may include a modified hindchain region.

In some embodiments, the modified Fc field may contain a modified hinge region.

In some embodiments, the combined molecule according to the invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine residues. In some embodiments, the combined molecule according to the invention may be able to form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds.

In some embodiments, the Fc domain may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine residues. In some embodiments, the Fc domain may be able to form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds.

In some embodiments, the modified hinge region may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine residues. In some embodiments, the modified hinge region may be able to form 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds.

In some embodiments, the Fc domain of the binding molecule according to the invention may contain one or more mutations that prevent the Fc domain from interacting with Fc receptors, complement proteins and/or from recruiting immune cells.

In some embodiments, the binding of the Fc domain of the binding molecule according to the invention to one or more Fc receptors may be reduced or substantially non-binding. In some embodiments, the binding of the Fc domain of the binding molecule according to the invention to one or more Fcγ receptors may be reduced or substantially non-binding.

In some embodiments, the binding of the Fc domain of the binding molecule according to the invention to one or more complement proteins may be reduced or substantially non-binding. In some embodiments, the binding of the Fc domain of the binding molecule according to the invention to C1q may be reduced or substantially non-binding.

In some embodiments, the Fc domain of the binding molecule according to the invention may not bind to and/or recruit immune cells.

In some embodiments, the Fc domain of the binding molecule according to the invention may contain "LALA", "LALA-KA", "STR" or "LALA-deltaA" mutations. It should 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 receptors and/or one or more complement proteins.

In some embodiments, the Fc domain of the binding molecule according to the present invention may contain the "LALA" mutation.

In some embodiments, the Fc domain of the binding molecule according to the present invention may contain the "LALA-KA" mutation.

In some embodiments, the Fc domain of the binding molecule according to the present invention may contain the "STR" mutation.

In some embodiments, the Fc domain of the binding molecule according to the present invention may contain the "LALA-deltaA" mutation.

In some embodiments, the LALA mutation can be L234A or L235A.

In some embodiments, the LALA-KA mutation can be L234A, L235A, or K322A.

In some embodiments, the LALA-deltaA mutation (i.e., "Fc[LALA-Δa]") can be L234A, L235A, A327G, A330S, and P331S.

In some embodiments, the STR mutation can be L234S, L235T, or G236R.

In some embodiments, the amino acid codes and residues of L234A, L235A, K322A, A327G, A330S, and P331S, and L234S, L235T, and G236R may be relative to the IgG1 locations and residues described in Armour et al. (Eur. J. Immunol. 1999. 29: 2613-2624; incorporated herein by reference), or relative to the IgG constant region residues and locations according to the EU numbering system (see 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).

In some embodiments, the binding molecule according to the invention may include a heavy chain constant. In some embodiments, the heavy chain constant may be a heavy chain IgG constant. In some embodiments, the heavy chain IgG constant may be a heavy chain IgG1 constant. In some embodiments, the heavy chain IgG constant may be a heavy chain IgG2 constant. In some embodiments, the heavy chain IgG constant may be a heavy chain IgG3 constant. In some embodiments, the heavy chain IgG constant may be a heavy chain IgG4 constant.

In some embodiments, the constant heavy chain region may include a constant heavy chain 2 (CH2) region or a constant heavy chain 3 (CH3) region. In some embodiments, the constant heavy chain region may include both a constant heavy chain 2 (CH2) region and a constant heavy chain 3 (CH3) region.

In some embodiments, the binding molecule according to the invention may include a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 17 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 17. In some embodiments, the heavy chain constant region may consist of an amino acid sequence according to SEQ ID NO: 17. SEQ ID NO: 17 (huIgG1):

In some embodiments, the binding molecule according to the invention may include a light chain constant localization. In some embodiments, the light chain constant localization may be a light chain k constant localization. In some embodiments, the light chain constant localization may be a light chain λ constant localization.

In some embodiments, the binding molecule according to the invention may include a light chain isostatic region comprising an amino acid sequence according to SEQ ID NO: 18 or a variant having at least 80% sequence identity therewith. 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 with SEQ ID NO: 18. In some embodiments, the light chain isostatic region may be composed of an amino acid sequence according to SEQ ID NO: 18. SEQ ID NO: 18 (huIgK):

In some embodiments, the binding molecule according to the invention may comprise the heavy chain sequence of SEQ ID NO: 19 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 21 or a variant thereof having at least 80% sequence identity. 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 with 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 with SEQ ID NO: 21.

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

In some embodiments, the binding molecule according to the invention may comprise the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 22 or a variant thereof having at least 80% sequence identity. 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 with 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 with SEQ ID NO: 22.

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

In some embodiments, the binding molecule according to the invention may comprise the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 23 or a variant thereof having at least 80% sequence identity. 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 with 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 with SEQ ID NO: 23.

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

In some embodiments, the binding molecule according to the invention may comprise the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 24 or a variant thereof having at least 80% sequence identity. 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 with 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 with SEQ ID NO: 24.

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

The present invention provides a binding molecule that binds to the CFC1 antigen .

In some embodiments, the binding molecule according to the present invention can bind to the CFC1 antigen.

In some implementations, the CFC1 antigen may be defined according to the UniProt entry: P0CG37.

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

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

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

In some embodiments, the antibody may contain three CDRs. It should be understood that such antibodies may be single-domain antibodies (sdAbs).

In some embodiments, the antibody may contain 6 CDRs. It should be understood that such antibodies may be classical antibody molecules.

In some embodiments, the antibody or a fragment thereof may be selected from the list of the following: single-chain variable fragments (scFv), Fab, F(ab)' ₂ , Fv, single-domain antibody, nano antibody, VHH antibody, monoclonal antibody or a fragment thereof, humanized antibody or a fragment thereof, chimeric antibody or a fragment thereof, bifunctional antibody and bispecific antibody.

In some implementations, the antibody may be a non-human antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

In some implementations, the antibody may be a humanized antibody.

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.

In some embodiments, the antibody may be an IgG molecule. In some embodiments, the antibody may be an IgG1 molecule. In some embodiments, the antibody may be an IgG2 molecule. In some embodiments, the antibody may be an IgG3 molecule. In some embodiments, the antibody may be an IgG4 molecule.

In some embodiments, the antibody composition may include monoclonal antibodies or multiclonal antibodies.

In some embodiments, the antibody may be a monoclonal antibody or a fragment thereof. In some embodiments, the antibody may be a humanized monoclonal antibody or a fragment thereof.

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

In some embodiments, the antibody may be a humanized VHH antibody or a fragment thereof.

It should be understood that VHH antibodies may contain both the VH domain and the Fc domain. It should also be understood that VHH antibodies may contain the VH domain, the CH2 domain, and the CH3 domain.

The techniques used to obtain antibodies are well known in this field. For example, antibodies can be obtained by techniques involving immunizing animals with a target antigen and isolating the antibodies from the serum.

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

The term "humanized antibody" generally refers to genetically engineered antibodies produced in non-human animals. These antibodies have been engineered to reduce immunogenicity when used in humans while retaining antigen specificity. Humanized antibodies typically contain a human antibody constant domain and a non-human variable domain, which are modified to have a high sequence homology with the human variable domain.

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

The present invention provides one or more polynucleotides comprising nucleic acid sequences encoding a binding molecule or an antibody or fragment thereof according to the present invention.

As used herein, the terms "polynucleotide," "nucleotide," and "nucleic acid" are used interchangeably. Nucleic acid sequences can be RNA or DNA sequences. Nucleic acid sequences can be single-stranded or double-stranded. Nucleic acid sequences can be, for example, genomic DNA, recombinant mRNA, or cDNA. Nucleic acid sequences may contain synthetic nucleotides and/or modified nucleotides. These synthetic nucleotides and/or modified nucleotides can enhance the in vivo activity and/or stability of the polynucleotide.

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.

Due to the redundancy of genetic codes, variations can occur in the nucleic acid sequences encoding the same polypeptide. This invention covers such sequences. Therefore, it is conceivable to have multiple polynucleotides, each with a different nucleic acid sequence, but whose encoding is based on the polypeptide of this invention or another polypeptide described herein. How to design and generate such nucleic acid sequences is known in this art.

In some embodiments, the nucleic acid sequence of the polynucleotide can be codon-optimized to produce in a selected host cell.

In some embodiments, the nucleic acid sequence of the polynucleotide is operatively linked to sequences that control transcription and/or translation, such as control sequences, for example, promoter sequences, enhancer sequences, or regulatory sequences. The polynucleotide may be in the form of an expression cartridge.

Polynucleotides can be used to express themselves in prokaryotic or eukaryotic cells (such as mammalian cells).

Any promoter can be used in polynucleotides, such as strong promoters that function in prokaryotic or eukaryotic cells. Suitable promoters will be those known in this art. Promoters can be ensemble promoters. Promoters can be tissue-specific promoters.

The present invention provides a carrier comprising a polynucleotide according to the present invention.

Therefore, the carrier may contain a polynucleotide containing a nucleic acid sequence encoding a binding molecule or antibody or fragment thereof according to the present invention.

The polynucleotides according to the invention can be introduced into cells using a carrier, causing the cells to express and/or produce the binding molecules or antibodies or fragments thereof according to the invention.

As used herein, the term "vector" may be considered interchangeable with the terms "expression vector" and "expression building block." A vector may be any vector suitable for introducing and/or expressing a nucleic acid sequence in cells. A vector may contain regulatory sequences, enhancer sequences, and/or promoter sequences that promote the expression of a nucleic acid sequence in cells.

The vector according to the invention can be any reagent capable of delivering the polynucleotide according to the invention to cells and/or expressing the nucleic acid sequence of the polynucleotide according to the invention in cells. Examples of suitable vectors include (but are not limited to) plasmids, cohesporidons, bacteriophages, viruses, or artificial chromosomes.

In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the vector may be a retrotranscribing virus vector or a lentiviral vector.

The vector may be able to transfect or transduce cells.

Cells and related methods The present invention provides a cell comprising a polynucleotide according to the present invention or a carrier according to the present invention.

Polynucleotides or vectors can be introduced into cells, for example, through in vitro or in vitro transduction or transfection.

Therefore, the present invention also provides a method for generating cells according to the present invention, the method comprising the step of introducing a polynucleotide according to the present invention or a carrier according to the present invention into the cells. In some embodiments, the polynucleotide may be introduced as described herein.

In some embodiments, cells may be able to express the binding molecules or antibodies or fragments thereof according to the invention.

In some embodiments, cells may be able to produce the binding molecules or antibodies or fragments thereof according to the invention.

In some embodiments, when cells are cultured under suitable conditions, the cells may be able to express and/or produce binding molecules or antibodies or fragments thereof according to the invention.

Therefore, the present invention also provides a method for generating a binding molecule or an antibody or fragment thereof according to the present invention, wherein the method comprises the steps of: (i) introducing a polynucleotide or a carrier according to the present invention into a cell; and (ii) expressing the binding molecule or antibody or fragment thereof in the cell.

In some embodiments of the method according to the present invention, polynucleotides or carriers can be introduced into cells by in vitro or in vitro transduction or transfection.

In some embodiments of the method according to the present invention, culturing cells under suitable conditions can enable the cells to express and/or produce binding molecules or antibodies or fragments thereof.

In some embodiments, the method for producing a binding molecule or antibody or a fragment thereof may further include step (iii): harvesting the binding molecule or antibody or a fragment thereof in cells or cell culture supernatant.

It should be understood that the binding molecules or antibodies or fragments thereof according to the invention can be harvested in cells. It should also be understood that, for example, when the binding molecules, antibodies or fragments thereof are released from cells into the cell culture medium of cultured cells, the binding molecules or antibodies or fragments thereof according to the invention can be harvested in the cell supernatant.

In some embodiments, the cells may be prokaryotic or eukaryotic.

In some embodiments, the cells may be bacterial cells, fungal cells, yeast cells, plant cells, or animal cells.

In some embodiments, the cells may be mammalian cells or insect cells. In some embodiments, the cells may be human cells.

Composition The present invention provides a composition comprising a binding molecule according to the present invention, an antibody according to the present invention or a fragment thereof; and a carrier, diluent or excipient.

In some embodiments, the composition may include the binding molecule according to the invention, as well as a carrier, diluent or excipient.

In some embodiments, the composition may comprise an antibody or a fragment thereof according to the invention, as well as a carrier, diluent or excipient.

In some embodiments, the composition may comprise a polynucleotide according to the invention, as well as a carrier, diluent or excipient.

The present invention also provides an assembly comprising the carrier according to the present invention, and a carrier, diluent or excipient.

In some embodiments, the composition described herein may further include one or more of the following selected from this list: adjuvants, salts, active peptides, compounds, components, and active agents.

The composition should generally be sterile and stable under manufacturing and storage conditions. The composition according to the present invention can be manufactured using current Good Pharmaceutical Manufacturing Practices (CGMP).

As used herein, the term "carrier" can refer to a thinner, adjuvant, excipient, or mediator. Such carriers can be sterile liquids, such as saline solutions in water and oil, including petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, and sesame oil. Sterile saline solutions are preferred carriers.

Salt solutions, dextrose solutions, and glycerol solutions can also be used as liquid carriers. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicone, sodium stearate, glyceryl monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and similar substances.

When necessary, the composition may also contain small amounts of wetting agents or emulsifiers, or pH buffers. The composition of the present invention can be formulated in neutral or salt form. Salts include salts formed with free amino groups, such as salts derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc.; and salts formed with free carboxyl groups, such as salts derived from sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, iron hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

In some embodiments, the salt may contain metal cations, such as sodium salts or potassium salts.

In some embodiments, the composition may contain an aqueous diluent or solvent. In some embodiments, the aqueous diluent or solvent may be an aqueous solution of phosphate buffer, such as a sterile aqueous solution of phosphate buffer.

In some embodiments, the composition may comprise one or more vesicles, nanoparticles, liposomes (LNPs), liposomes, or polymer mixtures.

The combination enables the polynucleotides according to the invention and/or the carriers according to the invention to be delivered to cells.

The present invention also provides a composition comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding a binding molecule or an antibody or fragment thereof according to the present invention; and (ii) a vesicle, nanoparticle, liposome nanoparticle (LNP), liposome or polymer mixture; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome or polymer mixture.

If one does not wish to be bound by theory, one may use the combination to deliver a nucleic acid sequence encoding a binding molecule or an antibody or fragment thereof according to the present invention to one or more cells, such that one or more cells can express and produce the binding molecule or antibody or fragment thereof.

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

In some embodiments, in addition to (i) and (ii), the composition may further include (iii) a carrier, a diluent or an excipient.

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.

The present invention also provides a kit comprising: (i) a combination according to the present invention.

In some embodiments, the kit may include (ii) instructions for using the kit to target cells expressing CFC1 in vitro.

As used herein, the terms “identity” and “sequence identity %” refer to the percentage (expressed as a percentage) of amino acids in a peptide or protein that are identical in amino acid sequence to a reference sequence.

Therefore, the percentage of consistency is calculated by counting the number of identical (matching) paired amino acids between the two sequences (in the amino acid sequence of the peptide or protein of the present invention and in the reference sequence), dividing that number by the total number of amino acids in the paired region, and multiplying by 100.

Therefore, the consistency percentage = (number of matches divided by the length of the target region) multiplied by 100.

Insertions and deletions are not allowed when calculating the consistency percentage of amino acid sequences.

Within the scope of this invention, any method for calculating percentage consistency is permitted.

General terms and definitions This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used to practice or test embodiments of this disclosure.

The term "peptide" is used in its conventional sense, referring to a series of amino acids that are usually linked together by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids, typically L-amino acids.

The term "polypeptide" is used interchangeably with the terms "amino acid sequence", "peptide" and/or "protein".

The term "residual amino acid" is used to refer to an amino acid in an amino acid sequence.

The range of values includes the numbers that define the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

Within the range of values provided, it should be understood that, unless the context expressly specifies otherwise, interpolated values between the upper and lower limits of that range are also specifically disclosed, accurate to the tenths of the lower limit unit. This disclosure covers all smaller ranges between any stated or interpolated values within the stated range, and any other stated or interpolated value within the stated range. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and any limit, no limit, or two limit values included in any of the smaller ranges are also covered by this disclosure, but subject to any specifically excluded limit values within the stated range. Where a stated range includes one or both of the limit values, the range excluding any or both of the included limit values is also included in this disclosure.

It must be noted that, unless the context clearly indicates otherwise, the singular forms “a/an” and “the” used herein and in the scope of the accompanying patent application include multiple references.

As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," and "contains," and are inclusive or open-ended, excluding additional unlisted members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

As used herein, the term "variant" is synonymous with the term "mutant" and refers to an amino acid or nucleic acid sequence that differs from the corresponding wild-type sequence. The term "wild-type" is used to mean a protein or polynucleotide containing an amino acid sequence that is identical to a native protein or native polynucleotide (e.g., a gene). A variant may have the same function as the amino acid or nucleic acid sequence described herein, but may include one or more amino acid or nucleic acid (corresponding) substitutions, insertions, or deletions. Amino acid substitutions, insertions, and/or deletions are considered mutations. Nucleic acid substitutions, insertions, and/or deletions are considered mutations.

The disclosures discussed herein are provided only for disclosures prior to the filing date of this application. Nothing in this document should be construed as an admission that such disclosures constitute prior art within the scope of the appended patent application.

The antibody pure lines used in this study are: MAB-18-0129, MAB-20-0235, MAB-20-0240 and MAB-20-0250. sequence describe SEQ ID NO: 1 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR1 (Kabat) SEQ ID NO: 2 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR2 (Kabat) SEQ ID NO: 3 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR3 (Kabat) SEQ ID NO: 4 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR1 (Kabat) SEQ ID NO: 5 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR2 (Kabat) SEQ ID NO: 6 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR3 (Kabat) and MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR3 (IMGT) SEQ ID NO: 7 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR1 (IMGT) SEQ ID NO: 8 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR2 (IMGT) SEQ ID NO: 9 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 HCDR3 (IMGT) SEQ ID NO: 10 MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR1 (IMGT) N/A The amino acid sequence of MAB-18-0129/MAB-20-0235/MAB-20-0240/MAB-20-0250 LCDR2 (IMGT) is: DAS SEQ ID NO: 11 MAB-18-0129 VH SEQ ID NO: 12 MAB-20-0235/ MAB-20-0240/MAB-20-0250 VH SEQ ID NO: 13 MAB-18-0129 VL SEQ ID NO: 14 MAB-20-0235 VL SEQ ID NO: 15 MAB-20-0240 VL SEQ ID NO: 16 MAB-20-0250 VL SEQ ID NO: 17 Heavy chain constant region (huIgG1) SEQ ID NO: 18 Lightweight chain constant region (huIgK) SEQ ID NO: 19 Complete heavy chain sequence: SEQ ID NO: 11 + SEQ ID NO: 17 SEQ ID NO: 20 Complete heavy chain sequence: SEQ ID NO: 12 + SEQ ID NO: 17 SEQ ID NO: 21 Complete light chain sequence: SEQ ID NO: 13 + SEQ ID NO: 18 SEQ ID NO: 22 Complete light chain sequence: SEQ ID NO: 14 + SEQ ID NO: 18 SEQ ID NO: 23 Complete light chain sequence: SEQ ID NO: 15 + SEQ ID NO: 18 SEQ ID NO: 24 Complete light chain sequence: SEQ ID NO: 16 + SEQ ID NO: 18 SEQ ID NO: 25 CFC1 antigen (hidden protein):

The present invention can be described by means of the following numbered paragraphs: 1. A binding molecule comprising a CFC1 binding domain. 2. The binding molecule of paragraph 1, wherein the CFC1 binding domain binds to CFC1 and induces the internalization of the binding molecule into cells expressing CFC1. 3. The binding molecule of paragraph 1 or paragraph 2, wherein the CFC1 binding domain induces the binding molecule to be internalized at a high rate into cells expressing CFC1. 4. The binding molecule of paragraph 3, wherein the binding molecule has a high internalization rate compared to a control. 5. The binding molecule of paragraph 4, wherein the high internalization rate is determined by immunofluorescence quenching analysis. 6. The binding molecule as described in paragraph 5, wherein the high internalization rate is at least 60% at 1 hour as determined by the immunofluorescence quenching analysis, and where the high internalization rate 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. 7. A binding molecule as described in any of paragraphs 1 to 6, wherein the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementary determinant regions (HCDRs) 1 to 3 as defined by Kabat, wherein: HCDR1 comprises an amino acid sequence according to SEQ ID NO: 1 (SSDMS), HCDR2 comprises an amino acid sequence according to SEQ ID NO: 2 (IIYASDNAYYASWAKG), and HCDR3 comprises an amino acid sequence according to SEQ ID NO: 3 (LWNM); and, where appropriate, one or more of the CDRs relative to the listed sequences comprise one, two, or three amino acid mutations. 8. A binding molecule as described in any of paragraphs 1-7, wherein the CFC1 binding domain comprises a light chain variable (VL) domain; wherein the VL domain comprises light chain complementary determinant regions (LCDRs) 1 to 3 as defined by Kabat, wherein: LCDR1 comprises an amino acid sequence according to SEQ ID NO: 4 (QSSQSVYDNRLA), LCDR2 comprises an amino acid sequence according to SEQ ID NO: 5 (DASKLES), and LCDR3 comprises an amino acid sequence according to SEQ ID NO: 6 (AARYSGNIGG); and, where applicable, one or more of the CDRs relative to the listed sequences comprise one, two, or three amino acid mutations. 9. A binding molecule as described in any of paragraphs 1-6, wherein the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementary determinant regions (HCDRs) 1 to 3 as defined by IMGT, wherein: HCDR1 comprises an amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 comprises an amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and HCDR3 comprises an amino acid sequence according to SEQ ID NO: 9 (VRLWNM); and, where appropriate, one or more of the CDRs relative to the listed sequences comprise one, two, or three amino acid mutations. 10. A binding molecule as described in any 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 complementary determining regions (LCDRs) 1 to 3 as defined by IMGT, wherein: LCDR1 comprises an amino acid sequence (QSVYDNR) according to SEQ ID NO: 10, LCDR2 comprises the amino acid sequence DAS, and LCDR3 comprises an amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6; and, where appropriate, one or more of the CDRs relative to the listed sequences comprise one, two, or three amino acid mutations. 11. A molecule that is a combination of any of paragraphs 7-10, wherein the VH domain comprises (i) an amino acid sequence according to SEQ ID NO: 11 or a variant thereof having at least 80% sequence identity; or (ii) an amino acid sequence according to SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity. 12. The binding molecule as described in paragraph 11, wherein: (i) the VH domain contains an amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity therewith; and the VL domain contains an amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity therewith; or (ii) the VH domain contains an amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity therewith; and the VL domain contains an amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity therewith; or (iii) the VH domain contains an amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity therewith; and the VL domain contains an amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity therewith; and the VL domain contains an amino acid sequence according to SEQ ID NO: 14 or a variant having at least 80% sequence identity therewith; 15. An amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity; or (iv) the VH domain comprises an amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity; and the VL domain comprises an amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity. 13. A binding molecule of any of paragraphs 1-12, further comprising an Fc domain; wherein, whereby, the Fc domain is a modified Fc domain. 14. A binding molecule of paragraph 13, wherein the modified Fc domain comprises a modified hinge region. 15. A binding molecule as described in paragraph 13 or 14, wherein (i) the Fc domain has reduced or substantially no binding to one or more Fcγ receptors or C1q; and/or (ii) the Fc domain cannot bind to immune cells; and/or (iii) the Fc domain contains the LALA, LALA-KA, STR, or LALA-deltaA mutation. 16. A binding molecule as described in any of paragraphs 1-15, wherein the binding molecule comprises (i) a heavy chain constant domain comprising an amino acid sequence according to SEQ ID NO: 17 or a variant thereof having at least 80% sequence identity; and/or (ii) a light chain constant domain comprising an amino acid sequence according to SEQ ID NO: 18 or a variant thereof having at least 80% sequence identity. 17. A binding molecule as described in any of paragraphs 1-16, wherein the binding molecule comprises: (i) the heavy chain sequence of SEQ ID NO: 19 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 21 or a variant thereof having at least 80% sequence identity; (ii) the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 22 or a variant thereof having at least 80% sequence identity; (iii) the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 23 or a variant thereof having at least 80% sequence identity; or (iv) SEQ ID NO: 18. A binding molecule of any of paragraphs 1-17, wherein the CFC1 binding domain binds to the CFC1 antigen. 19. A binding molecule of paragraph 18, wherein the CFC1 antigen comprises or is composed of the amino acid sequence according to SEQ ID NO: 25 or a variant thereof having at least 80% sequence identity. 20. An antibody or an antigen-binding fragment thereof comprising a binding molecule of any of the preceding paragraphs. 21. An antibody or fragment thereof as described in paragraph 20, wherein the antibody or fragment thereof is selected from the list comprising: scFv, Fab, single-domain antibodies, nano-antibodies, VHH antibodies, monoclonal antibodies or fragments thereof, humanized antibodies or fragments thereof, chimeric antibodies or fragments thereof, and bispecific antibodies. 22. An antibody or fragment thereof as described in paragraph 20 or paragraph 21, wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof. 23. An antibody or fragment thereof as described in paragraph 20 or paragraph 21, wherein the antibody or fragment thereof is a VHH antibody or fragment thereof. 24. A polynucleotide comprising a nucleic acid sequence encoding a binding molecule as described in any of paragraphs 1 to 19 or an antibody or fragment thereof as described in any of paragraphs 20 to 23. 25. A polynucleotide as described in paragraph 24, wherein the nucleic acid sequence is an RNA sequence. 26. A vector comprising a polynucleotide as described in paragraph 24 or paragraph 25. 27. A cell comprising a polynucleotide as described in paragraph 24 or 25, or a carrier as described in paragraph 26. 28. A cell as described in paragraph 27, wherein the cell is capable of expressing a binding molecule as described in any of paragraphs 1 to 19, or an antibody or fragment thereof as described in any of paragraphs 20 to 23. 29. A method for producing a cell as described in paragraph 27 or 28, comprising the step of introducing a polynucleotide as described in paragraph 24 or 25, or a carrier as described in paragraph 26, into the cell. 30. A method for producing a binding molecule as described in any of paragraphs 1 to 19 or an antibody or fragment thereof as described in any of paragraphs 20 to 23, wherein the method comprises the steps of: (i) introducing a polynucleotide as described in paragraph 24 or 25 or a carrier as described in paragraph 26 into a cell; and (ii) expressing the binding molecule or antibody or fragment thereof in the cell; and, where appropriate, comprising: (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or a cell culture supernatant of the cell. 31. A composition comprising: a binding molecule as described in any of paragraphs 1 to 19, an antibody or fragment thereof as described in any of paragraphs 20 to 23; and a carrier, diluent, or excipient. 32. A composition comprising: (i) a polynucleotide comprising a nucleic acid sequence encoding a binding molecule as described in any of paragraphs 1 to 19 or an antibody or fragment thereof as described in any of paragraphs 20 to 23; and (ii) a vesicle, nanoparticle, liposome nanoparticle (LNP), liposome, or polymer mixture; and optionally (iii) a carrier, diluent, or excipient; wherein the polynucleotide is encapsulated within the vesicle, nanoparticle, LNP, liposome, or polymer mixture. 33. A kit comprising: (i) a composition as described in paragraph 31 or 32; and optionally (ii) instructions for using the kit to target cells expressing CFC1 in vitro.

The invention will now be further described with the aid of examples intended to help those skilled in the art to implement the invention and not intended to limit the scope of the invention in any way.

Example 1 : Generation of CFC1- Binding Monoclonal Antibodies To obtain CFC1-specific monoclonal antibodies, New Zealand white rabbits were immunized with recombinant human CFC1 extracellular domains containing the C-terminal 10xHIS protein. Individual B cells were isolated from peripheral blood using FACS and cultured to obtain monoclonal antibodies in the culture supernatant. Selected monoclonal B cell lines were dissolved in RNA extraction RLT buffer for RNA extraction of the antibody heavy and light chain variable regions, RT-PCR, and Sanger sequencing. Genes were synthesized from the variable regions of the antibody heavy and light chains, selected and colonized upstream of the hIgG1-stationary domain of the pCEP4 expression plasmid, and generated in HEK293-FreeStyle™ cells.

Example 2 : Humanization of CFC1- binding monoclonal antibodies. A computer-simulated humanization program combining CDR transplantation and antibody structure-guided modification was performed on the VL and VH region coding sequences of the MAB-18-0129 antibody. Genes were synthesized for the five heavy chain and four light chain humanized variable regions and selected to be implanted upstream of the hIgG1 constant domain of the pCEP4 expression plasmid. All possible combinations of humanized antibody heavy and light chains were generated in HEK293-FreeStyle™ cells.

Example 3 : Human CFC1 ELISA. The specificity and binding efficacy of the present invention’s anti-CFC1 chimeric and humanized antibodies against human CFC1 were determined by ELISA (see Table 1 and Figure 1).

Recombinant human CFC1 ECD (R&D Systems) was coated in PBS at a concentration of 0.6 µg/ml on 384-well Nunc MaxiSorp™ flat-bottomed plates at room temperature for 60 minutes. After washing three times with PBS 0.1% Tween (wash buffer), blocking for 60 minutes at room temperature with PBS, 2% BSA, and 0.05% Tween, and washing three more times, the chimeric and humanized antibody of this invention was added to PBS, 0.5% BSA, and 0.05% Tween (ELISA buffer) at a concentration ranging from 2,000 to 0.06 ng/ml, and the plates were incubated at room temperature for 60 minutes. As a reference antibody, control anti-CFC1 antibody was added at the same concentration range. After washing three times with wash buffer, horseradish peroxidase-coupled anti-human IgG (Fab')2 fragment (AbD Serotec) was added to the ELISA buffer at a dilution of 1:5000. The plates were incubated at room temperature for 60 minutes, washed six times with wash buffer, and then TMB solution (Thermo Fisher Scientific) was added. The colorimetric reaction was stopped with HCl, and absorbance was recorded at 450/620 nm using a Tecan Infinite M1000 reader. Data analysis was performed using IDBS XLfit, including 4PL curve fitting and EC50 calculation.

Example 4 : CHO-K1 binding between human CFC1 and CHO-K1 scythemaegi cells. High-throughput imaging analysis was used to analyze the binding efficacy of the present invention's anti-CFC1 chimeric or humanized antibody with CHO-K1 cells that translocate human or scythemaegi CFC1 (see Table 1 and Figure 2).

1,000 CHO-K1 cells were seeded into each well of a black 384-well cell culture treatment dish and incubated at 37°C and 5% CO2 for 2 hours. The chimeric or humanized antibody of this invention was diluted to 5,000 to 0.05 ng/ml. As a reference antibody, a control anti-CFC1 antibody was used at the same concentration range. After incubation overnight at 37°C and 5% CO2, the dish was washed once with PBS and 0.05% Tween 20 (cell wash buffer), and an Alexa-Fluor-448-bound anti-human IgG F(ab')2 fragment was added at a concentration of 0.8 µg/ml. The cells were incubated in the dark at 37°C and 5% CO2 for 4 hours, washed once with cell wash buffer, and then incubated for 10 minutes in medium containing 5 µg/ml Hoechst (Invitrogen). Cell-related immunofluorescence signals were recorded using a CellInsight CX5 high-throughput imaging reagent (Thermo Fisher), expressed as the mean spot intensity of all cells. Data analysis was performed using IDBS XLfit, including 4PL curve fitting and EC50 calculation. Table 1 ID ELISA Human CFC1 CHO-K1 Human CFC1 CHO-K1 Stone Crab Monkey CFC1 EC 50 [ng/mL] EC 50 [ng/mL] EC 50 [ng/mL] MAB-18-0129 14 64 14 MAB-20-0235 11 30 8 MAB-20-0240 11 47 0.1 MAB-20-0250 10 55 0.6 Control of CFC1 Ab 15 34 10

Example 5 : High-throughput imaging internalization analysis The internalization rate and intensity of the chimeric or humanized anti-CFC1 antibody of the present invention in overexpressed CHO-K1 human CFC1 cells were evaluated by high-throughput imaging (see Table 2 and Figure 3).

Cells were seeded in 384-well plates overnight and then stained with Hoechst 33342. Each antibody was added at 20 nM to the cells at 4°C for 1 hour, followed by washing and staining with 60 nM Alexafluor 488 anti-human IgG-Fab fragment (Dianova) at 4°C for 30 minutes. The plates were then incubated at 37°C in a humidified CO2 incubator for 0, 1, or 4 hours. Subsequently, a subset of the replicates were treated with unquenched cell culture medium or 100 nM anti-AlexaFluor 488-IgG quenched antibodies at 4°C for 30 minutes. Following the washing step, Hoechst and Alexa Fluor 488 staining of the measurement disc was performed in the high-throughput imaging agent CellInsight CX5 (Thermo Fisher). The SpotDetector software module was deployed for cell masking, and the average Alexa Fluor 488 intensity per well for each single cell was calculated, i.e., the average Alexa Fluor 488 spot intensity. The internalization percentage was calculated as the ratio of the average Alexa Fluor 488 intensity under quenching conditions to the average Alexa Fluor 488 intensity under non-quenching conditions. Data was plotted using GraphPad Prism. Table 2 ID Internalization [%] Antibody binding [AF488 strength] t=0h t=1h t=4h t=0 MAB-18-0129 13 88 107 1155 MAB-20-0235 17 79 98 1195 MAB-20-0240 16 70 95 1183 MAB-20-0250 13 67 86 1191 Control of CFC1 Ab 3 47 103 914

Example 6 : Cells binding to low CFC1 expression (Table 3) were seeded at 1E5 cells/well and incubated at 4°C for 1 hour with serially diluted MAB-20-0235 antibody or isotype control (anti-HIV antibody) (0-400 nM). Secondary antibody (A647 goat anti-human IgG Fcγ) (1:500 dilution) was added, and the cells were incubated at 4°C for 1 hour. Antibody binding was confirmed by FACS. Table 3 cell line Disease indications NCI-H727 Lung carcinoid tumor NCI-H810 NSCLC

MAB-20-0235 exhibited dose-dependent binding to NCI-H727 and NCI-H810 cells (see Table 4 and Figure 4). Table 4 cells antibody Maximum MFI negative blank NCI-H727 Same type 641 103.15 31.3 MAB-20-0235 1253 NCI-H810 Same type 2808 105 29.25 MAB-20-0235 3442

All disclosures mentioned in the foregoing description are incorporated herein by reference. Various modifications and variations to the methods and systems of the invention described herein will be readily apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to those specific embodiments. In fact, various modifications to the described modes for implementing the invention, which will be readily apparent to those skilled in molecular biology or related fields, are intended to fall within the scope of the following claims.

Figure 1 - ELISA was used to determine the binding of chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240, and MAB-20-0250 to human CFC1 ECD. Antibodies were diluted to concentrations ranging from 2,000 to 0.06 ng/ml. Anti-CFC1 antibodies were added as a control. Data were analyzed using nonlinear regression.

Figure 2 - High-throughput imaging study of the binding of chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240, and MAB-20-0250 to CHO-K1 of overexpressing (A) human or (B) lithocrab macaque CFC1. Antibodies were diluted to concentrations ranging from 5,000 to 0.05 ng/ml, and anti-CFC1 antibodies were added as controls. Data were analyzed by nonlinear regression.

Figure 3 - Binding and internalization of chimeric MAB-18-0129 and its humanized variants MAB-20-0235, MAB-20-0240, and MAB-20-0250 to ectopically expressed CHO-K1 human CFC1 cells using high-throughput imaging. The percentage of internalization of the 3 μg/ml anti-CFC1 antibody was determined after incubation at 37°C for 0, 1, or 4 hours. The binding of the antibody to cells was determined at time point 0 and plotted as mean total cell Alexa Fluor 488 intensity.

Figure 4 - FACS binding analysis of MAB-20-0235 and isotype control ("negative control") with (A) NCI-H810 and (B) NCI-H727 cells.

TW202542189A_113149575_SEQL.xml

Claims (15)

A binding molecule containing a CFC1 binding domain. The binding molecule of claim 1, wherein the CFC1 binding domain binds to CFC1 and induces the binding molecule to be internalized into cells expressing CFC1; where appropriate, wherein the CFC1 binding domain induces the binding molecule to be internalized into cells expressing CFC1 at a high rate. The binding molecule of claim 2, wherein the high internalization rate is at least 60% at 1 hour as determined by immunofluorescence quenching analysis, and where the high internalization rate 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 analysis. The binding molecule of any one of claims 1 to 3, wherein (a)(i) the CFC1 binding domain comprises a heavy chain variable (VH) domain; wherein the VH domain comprises heavy chain complementarity determining regions (HCDRs) 1 to 3 as defined by Kabat, 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), and HCDR3 comprises the amino acid sequence according to SEQ ID NO: 3. An amino acid sequence (LWNM); whereby one or more of the CDRs contain one, two, or three amino acid mutations relative to the listed sequences; and/or (ii) the CFC1 binding domain contains a light chain variable (VL) domain; wherein the VL domain contains light chain complementary determinant regions (LCDRs) 1 to 3 as defined by Kabat, wherein: LCDR1 contains an amino acid sequence (QSSQSVYDNRLA) according to SEQ ID NO: 4, LCDR2 contains an amino acid sequence (DASKLES) according to SEQ ID NO: 5, and LCDR3 contains an amino acid sequence (QSSQSVYDNRLA) according to SEQ ID NO: 5. The amino acid sequence of SEQ ID NO: 6 (AARYSGNIGG); whereby one or more of the CDRs contain one, two or three amino acid mutations relative to the listed sequences; or (b)(i) the CFC1 binding domain contains a heavy chain variable (VH) domain; wherein the VH domain contains heavy chain complementarity determining regions (HCDRs) 1 to 3 as defined by IMGT, wherein: HCDR1 contains the amino acid sequence according to SEQ ID NO: 7 (GFSLSSSD), HCDR2 contains the amino acid sequence according to SEQ ID NO: 8 (IYASDNA), and HCDR3 contains the amino acid sequence according to SEQ ID NO: 8 (IYASDNA). The CDR contains an amino acid sequence (VRLWNM) of SEQ ID NO: 9; whereby one or more of the CDRs contain one, two or three amino acid mutations relative to the listed sequences; and/or (ii) the CFC1 binding domain contains a light chain variable (VL) domain; wherein the VL domain contains light chain complementary determinant regions (LCDRs) 1 to 3 as defined by IMGT, wherein: LCDR1 contains an amino acid sequence (QSVYDNR) according to SEQ ID NO: 10, LCDR2 contains the amino acid sequence DAS, and LCDR3 contains an amino acid sequence (AARYSGNIGG) according to SEQ ID NO: 6; whereby one or more of the CDRs contain one, two or three amino acid mutations relative to the listed sequences. The binding molecule of claim 4, wherein the VH domain comprises (a)(i) an amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity therewith; or (ii) an amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity therewith; wherein, as appropriate, (b)(i) the VH domain comprises an amino acid sequence according to SEQ ID NO: 11 or a variant having at least 80% sequence identity therewith; and the VL domain comprises an amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity therewith; or (ii) the VH domain comprises an amino acid sequence according to SEQ ID NO: 12 or a variant having at least 80% sequence identity therewith; and the VL domain comprises an amino acid sequence according to SEQ ID NO: 13 or a variant having at least 80% sequence identity therewith; The amino acid sequence of SEQ ID NO: 14 or a variant thereof having at least 80% sequence identity; or (iii) the VH domain contains the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity; and the VL domain contains the amino acid sequence of SEQ ID NO: 15 or a variant thereof having at least 80% sequence identity; or (iv) the VH domain contains the amino acid sequence of SEQ ID NO: 12 or a variant thereof having at least 80% sequence identity; and the VL domain contains the amino acid sequence of SEQ ID NO: 16 or a variant thereof having at least 80% sequence identity. The molecule combining any of claims 1 to 5 further includes an Fc domain; where the Fc domain is a modified Fc domain. The binding molecule of any one of claims 1 to 6, wherein the binding molecule comprises (i) a heavy chain constant region comprising an amino acid sequence according to SEQ ID NO: 17 or a variant thereof having at least 80% sequence identity; and/or (ii) a light chain constant region comprising an amino acid sequence according to SEQ ID NO: 18 or a variant thereof having at least 80% sequence identity. A molecule combining any one of claims 1 to 7, wherein the molecule comprises: (i) the heavy chain sequence of SEQ ID NO: 19 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 21 or a variant thereof having at least 80% sequence identity; (ii) the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 22 or a variant thereof having at least 80% sequence identity; (iii) the heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 23 or a variant thereof having at least 80% sequence identity; or (iv) SEQ ID NO: The heavy chain sequence of SEQ ID NO: 20 or a variant thereof having at least 80% sequence identity; and the light chain sequence of SEQ ID NO: 24 or a variant thereof having at least 80% sequence identity. The binding molecule of any one of claims 1 to 8, wherein the CFC1 binding domain binds to the CFC1 antigen; wherein, if applicable, the CFC1 antigen comprises or consists of the following: an amino acid sequence according to SEQ ID NO: 25 or a variant having at least 80% sequence identity therewith. An antibody or an antigen-binding fragment thereof, comprising a binding molecule as described in any of the preceding claims. A polynucleotide comprising a nucleic acid sequence encoding a binding molecule as described in any of claims 1 to 9 or an antibody or fragment thereof as described in claim 10. A carrier comprising a polynucleotide as claimed in claim 11. A cell comprising a polynucleotide as claimed in claim 11 or a carrier as claimed in claim 12. A method for producing a binding molecule as claimed in any of claims 1 to 9 or an antibody or fragment thereof as claimed in claim 10, wherein the method comprises the steps of: (i) introducing a polynucleotide as claimed in claim 11 or a carrier as claimed in claim 12 into a cell; and (ii) expressing the binding molecule or antibody or fragment thereof in the cell; and, where appropriate, comprising: (iii) harvesting the binding molecule or antibody or fragment thereof from the cell or a cell culture supernatant of the cell. A composition comprising: a binding molecule of any one of claims 1 to 9, an antibody or fragment thereof of claim 10; and a carrier, diluent or excipient.