Antigen binding proteins

Abstract

The present invention relates to a novel antigen binding protein comprising at least two CH1 domains, wherein the at least two CH1 domains comprise a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human IgG1 CH1 domain. The invention further provides related nucleic acids, expression vectors, and host cells. The present invention also provides pharmaceutical compositions comprising said antigen binding protein. Finally, the invention also provides a method for increasing the activity of antigen binding protein.

Description

[0001] ANTIGEN BINDING PROTEINS

[0002] FIELD OF INVENTION

[0003] The present invention relates to a novel antigen binding protein comprising at least two CH1 domains, wherein the at least two CH1 domains comprise a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain. The invention further provides related nucleic acids, expression vectors, and host cells. The present invention also provides pharmaceutical compositions comprising said antigen binding protein. Finally, the invention also provides a method for increasing the activity of antigen binding protein.

[0004] BACKGROUND

[0005] Currently, therapeutic antibodies are used for treating several major diseases including autoimmune, cardiovascular, infectious diseases, cancer, and inflammatory diseases. The antibodies market size is estimated to be worth USD 236.9 billion in 2023 and is anticipated to increase at a CAGR of 13.4% from 2023 to 2033. By 2033, it is anticipated that the market for antibodies would reach USD 834.2 Billion. According to disease indications, the cancer sector is the largest shareholder in the antibodies market, owing to increased cancer prevalence and increase adoption of antibodies in cancer treatment. An increase in the number of drug pipelines, an increase in demand for biosimilar antibodies, and an increase in patient awareness of cancer treatment methods boost market expansion. With the focus on cancer research increasing, the antibodies market outlook for the upcoming decade is anticipated to remain positive.

[0006] It is the inherent characteristics of antibodies, such as high specificity and affinity, which make them an attractive source for biotherapeutic agents. However, they are not without their limitations. For example, weak and / or off-target binding of antibodies can result in altered efficacy and increased toxicity. For example, Utomilumab has relatively low efficacy, whereas, Urelumab, causes severe dose-dependent hepatotoxicity despite its antitumor efficacy.

[0007] Therefore, there is a need for alternative engineering strategies to generate improved antibodies, for example antibodies with increased activity and / or binding. The present invention aims to provide such improved antibodies and other forms of antigen binding proteins. SUMMARY OF INVENTION

[0008] In one aspect, the present invention provides an antigen binding protein comprising at least two CH1 domains, wherein the at least two CH1 domains comprise a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain.

[0009] Suitably, the antigen binding protein may comprise at least two CH1 domains and at least two corresponding CL domains.

[0010] Suitably, the antigen binding protein may comprise an antibody, or an antibody fragment.

[0011] Suitably, the antibody may be a human antibody or a humanised antibody, optionally selected from the group consisting of IgG, IgE, IgD, IgM and IgA.

[0012] Suitably, the antibody fragment may be selected from the group consisting of F(ab’)2, a Fab2, DNL-Fab3, a DNL-Fab2-scFV, a DNL-Fab2-lgG-cytokine2, a scFab-lgG(kih), a Fab-scFab- IgG(kih), a LUZ-Y scFab-IgG, a scFab-Fc(kih)-scFv2, a scFab-Fc(kih)-scFv, a TriFabs, a CODV-ig, a F(ab’)2 fusion (e.g. F(ab’)2-scFv2), and a scFv2-CH1-hinge / CL.

[0013] Suitably, the antigen binding protein may be monospecific or multispecific, optionally wherein the multispecific antigen binding protein may be bi-specific, tri-specific, tetra-specific, or pentaspecific.

[0014] Suitably, the antigen binding protein may specifically bind a cell surface receptor (such as a tumour necrosis factor receptor (TNFR)) or an immunoglobulin receptor superfamily member (IgSF), or a ligand thereof.

[0015] Suitably, the antigen binding protein may comprise an antibody selected from the group consisting of anti-CD40 (optionally LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally SAP9), anti-4-1 BB (optionally Utomilumab, Urelumab, SAP1 .3, SAP1 .3 ND, or SAP3.28), anti- CD28 (optionally TGN14-12), anti-CD27 (optionally hCD27.131A), anti-ICOS, anti-PD1 (such as Nivolumab), anti-DR4 and anti-DR5. As it will be appreciated by a person skilled in the art, herein when reference is made to known antibodies such as, for example, Utomilumab or Urelumab, these antibodies comprise the one or more mutations described herein unless required otherwise by the context (for example when reference is made to the antibodies described specifically in the art).

[0016] Suitably, at least one of the CH1 domains may comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ I D NO: 2, SEQ I D NO: 3, or SEQ I D NO: 4, optionally wherein at least one of the CH 1 domains may comprise or consist of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4. Suitably, at least two CH 1 domains may comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, optionally wherein the at least two CH1 domains may comprise or consist of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0017] Suitably, at least one of the at least two CL domains may comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain, optionally wherein the at least two CL domains may comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human IgG 1 CL domain.

[0018] Suitably, at least one of the at least two CL domains may comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 9, or SEQ ID NO: 10, optionally wherein at least one of the at least two CL domains comprises or consist of a sequence according to SEQ ID NO: 9, or SEQ ID NO: 10.

[0019] Suitably, the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 9, or SEQ ID NO: 10, optionally wherein the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9, or SEQ ID NO: 10.

[0020] In a further aspect, the present invention provides a nucleic acid encoding CH1 domain comprising a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain, and / or a CL domain comprising a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human IgG 1 CL domain.

[0021] In a further aspect, the present invention provides an expression vector comprising the nucleic acid of the invention.

[0022] In a further aspect, the present invention provides a host cell comprising the expression vector of the invention.

[0023] In a further aspect, the present invention provides a pharmaceutical composition comprising the antigen binding protein, the nucleic acid, the expression vector, and / or the host cell of the invention; and further comprising a pharmaceutically acceptable diluent, carrier or excipient.

[0024] In a further aspect, the present invention provides the antigen binding protein, the nucleic acid, the expression vector, the host cell, and / or the pharmaceutical composition of the invention for use as a medicament. In a further aspect, the present invention provides a method of increasing activity of an antigen binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into the at least two CH1 domains of the antigen binding fragment, wherein the mutation is a cysteine substitution of an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human IgG 1 CH1 domain.

[0025] In a further aspect, the present invention provides a method of increasing binding of an antigen binding protein to an antigen, the antigen binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into the at least two CH1 domains of the antigen binding fragment, wherein the mutation is a cysteine substitution of an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain.

[0026] It will be appreciated that, except for where the context requires otherwise, the considerations set out in this disclosure in relation to the antigen binding protein should be considered applicable to the methods, uses, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions described herein.

[0027] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0028] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0029] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0030] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0031] Brief description of the Figures

[0032] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0033] Figure 1 A) diagram showing the location of residues T222 and E123 within the context of the F(ab’)2 fragment of lgG2, B) diagram showing predicted disulphide linkages in T222C and E123C mutants. C) displays the crystal structure of a F(ab’)2 region and location of T222 and E123 residues in the context of the whole structure.

[0034] Figure 2 displays an SDS-PAGE performed under reducing and non-reducing conditions displaying expressed hlgG1 (hi clamp), hlgG2 (h2 clamp), and hlgG4 (h4 sp clamp) variants containing the “clamp” double mutation (222C and 123C). Under reducing conditions two bands are observed for the heavy chain and light chain at ~52 kDa and ~23 kDa, respectively. Under non-reducing conditions several bands are observed indicating some heterogeneity, however, the major band in all three isotypes is observed at -150 kDa representing the full IgG.

[0035] Figure 3 displays the results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type for hlgG1 , hlgG2, and hlgG4.

[0036] Figure 4 displays comparative immunostimulatory activity assay data of single mutants and “clamp” double mutants. This demonstrates that the 222C mutation in the heavy chain is sufficient (K222C in lgG1 , T222C in lgG2) for producing an agonistic effect. “Clamp” is with both mutations (222C and 123C) in the heavy and light chain, respectively. Data shows the equivalent activity of the 222C mutant on its own versus the full clamp.

[0037] Figure 5 displays structures of WT hlgG1 , hlgG clamp, and hlgG2 clamp crystal structures. A) The F(ab’)2 portion of WT hlgG1 as extracted from PDB: 1 HZH. B) The crystal structure of the hlgG1 clamp. The introduction of the clamp mutant results in a compaction and twisting of the two Fab arms for both hlgG1 and hlgG2. The new disulphide bond links opposing heavy constant regions in the two Fab arms. C) The disulfide bond between C222 on opposing hl gG 1 heavy chains is highlighted. D) The crystal structure of the hlgG2 clamp. E) The disulfide bond between C222 on opposing hlgG2 heavy chains is highlighted. Figure 6 displays small-angle x-ray scattering (SAXS) data of hlgG1 , hlgG2 and hlgG4 variants with the “clamp” double mutation as F(ab’)2 fragments versus WT hlgG F(ab’)2 fragments in each isotype, respectively. Information on the flexibility of a particle can be derived from a dimensionless Kratky plot. For a tightly packed globular particle, the curve will peak at the Guinier-Kratky point (shown as grey cross-hair) and return to baseline as a Gaussian-like curve / distri buton. An elevation of the peak of the curve up and to the right of the Guinier-Kratky point indicates a more flexible particle (as observed for WT hlgG1 and hlgG4), while a shift of the peak towards the Guinier-Kratky point indicates a more compact particle (as seen for hlgG1 clamp and hlgG4SP clamp). The hlgG2 is already compact in the WT form, however, the introduction of the “clamp” double mutation does result in a slight compaction. hlgG4SP clamp comprises the mutation S241 P in the hinge region, which prevents Fab arm exchange.

[0038] Figure 7 displays negative stain electron microscopy (nsEM) images of h IgG 1 WT (I gG 1 ) and hlgG1 “clamp” double mutation variant free from crystal lattice constraints. Class-averages are presented of the hlgG 1 WT (left) and hlgG 1 “clamp” double mutation variant (right). In both images the Fc region is orientated at the bottom of the squares with the Fab arms at the top. The hlgG1 WT shows a range of conformations of the Fab arms with respect to the Fc. In comparison, the orientation of the Fab arms relative to the Fc for the hlgG1 “clamp” double mutation variant is far more restricted, bringing the two Fab arms closer together.

[0039] Figure 8 displays SPR binding assay data of the hlgG1 , hlgG2, and hlgG4 “clamp” double mutants. Representative sensorgrams above show binding the WT isotypes and the “clamp” versions of the isotypes. Minimal differences are observed for the hlgG2 and hlgG4 isotypes, while in the hlgG1 isotype slightly increased maximal binding is observed for the “clamp” double mutant compared to the WT.

[0040] Figure 9 shows the analysis of the negative stain electron microscopy (nsEM) images of hlgG1 WT (lgG1) and hlgG1 “clamp” double mutation variant free from crystal lattice constraints as shown in Figure 7. Fab-Fab angles were calculated by using the angle tool in Imaged to draw a segmented line from outermost edge of one Fab domain to the centre of the particle and then to the outermost edge of the second Fab domain and then using the Analyze- >Measure tool to calculate angle measurements. The analysis shows the Fab arms in the hl gG 1 clamp display a much tighter distribution of angles than the wild type hl gG 1 with a lower mean angle and range. Figure 10 displays SPR binding assay data of the hlgG1 WT and “clamp” double mutants binding recombinant FcgRs. Representative sensorgrams show binding of the WT versus clamp versions to the different immobilised FcgRs and show retained binding of the clamp in each case.

[0041] Figure 11 displays antibody dependent cellular phagocytosis (ADCP) of hlgG 1 WT and clamp anti-CD40 mAb using hCD40 expressing Jurkat cells as targets with monocyte derived macrophages. The data shows that the clamp molecules retain the ability to trigger ADCP.

[0042] Figure 12 displays the results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type for hl gG 1 of LOB7 / 6 anti-CD40 mAbs.

[0043] Figure 13 displays small-angle x-ray scattering (SAXS) data of hl gG 1 variants with the “clamp” double or single mutations. Information on the flexibility of a particle can be derived from a dimensionless Kratky plot. For a tightly packed globular particle, the curve will peak at the Guinier-Kratky point (shown as grey cross-hair) and return to baseline as a Gaussian-like curve / distribution. An elevation of the peak of the curve up and to the right of the Guinier- Kratky point indicates a more flexible particle (as observed for WT hl gG 1 ) , while a shift of the peak towards the Guinier-Kratky point indicates a more compact particle (as seen for hlgG1 clamp and hlgG1 K222C). The hlgG1 kE123C variant displays an intermediate effect. (A) These molecules were assessed as a Fab2 fragments to allow the highest resolution study of the Fab arms in the absence of the Fc. (B) These molecules were assessed as a whole IgG fragments and so include the movement of the Fc.

[0044] Figure 14 displays results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type for hlgG1 using primary immune B cells. A) Activity is displayed with respect to cell adhesion, cell surface upregulation of CD23, CD86 and HLA-DR determined by flow cytometry and the increase in B cell proliferation as measured by3H thymidine uptake. Further data shows the clamp and single HC mutant retain equivalent activity as IgG, shown by B) CD23 expression, C) CD86 expression, D) HLA DR expression, and E) B Cell proliferation. F) provides the B cell adhesion data for the 3 donors. For each of these, h2 is included as the reference benchmark but hi is the direct comparator for the clamp.

[0045] Figure 15 displays results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type hlgG1 in vivo. Activity is displayed with respect to expansion of OVA specific CD8 T cells (OT-1 ; SIINFEKL+) after immunisation of hCD40 Tg mice with ovalbumin (OVA). The data show that the hlgG1 clamp and hlgG1 heavy chain K222C mutation have similar activity, greater than the light chain kE123C mutant.

[0046] Figure 16 displays results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type hl gG 1 in vivo. Activity is displayed with respect to induction of OVA specific antibodies after immunisation of hCD40 Tg mice with ovalbumin (OVA). The data show that the hlgG1 clamp has greater immunostimulatory activity than the parent molecule and the WT hlgG2 variant.

[0047] Figure 17 displays small-angle x-ray scattering (SAXS) data of hlgG1 variants with the “clamp” double mutations versus WT hlgG1. Information on the flexibility of a particle can be derived from a dimensionless Kratky plot. For a tightly packed globular particle, the curve will peak at the Guinier-Kratky point (shown as grey cross-hair) and return to baseline as a Gaussian-like curve / distri buton. An elevation of the peak of the curve up and to the right of the Guinier-Kratky point indicates a more flexible particle (as observed for WT hlgG1), while a shift of the peak towards the Guinier-Kratky point indicates a more compact particle (as seen for h IgG 1 clamp). NB: these molecules were assessed as Fab2 fragments to allow the highest resolution study of the Fab arms in the absence of the Fc.

[0048] Figure 18 displays data derived from small-angle x-ray scattering (SAXS) data of hlgG1 variants with the “clamp” double mutations compared to WT hlgG1. Information relating to radius of gyration (rg), maximum dimension (Dmax) is derived using the ScAtter program.

[0049] Figure 19 displays the results of the immunostimulatory activity assays of the “clamp” double mutants compared to the wild-type hlgG1 for five different mAb targeting 4-1 BB, CD27 or 0X40.

[0050] DETAILED DESCRIPTION

[0051] Previously, the inventors have linked steering disulphide shuffling in the human (h)lgG2 antibody hinge to agonistic activity using a CD40 antibody, through mediating the orientation of native disulphide bonds in the hinge region by substituting cysteine with serine. More specifically, the inventors showed that by inducing different hinge disulphide conformations in a F(ab’)2 fragment mediates agonistic activity of the hlgG2 antibody fragment (Orr et al., 2022).

[0052] The method of mediating agonistic activity previously shown by the inventors is reliant upon, and also restricted by, the extra cysteine residues present in the hlgG2 antibody hinge compared to hlgG1 and hlgG4. This is thus an unfavorable engineering strategy because among the approved IgG class mAb-related therapeutics, 74% belong to the lgG1 subclass, and 13% belonging to the lgG4 subclass. Although there is a high level of sequence homology among the IgG subclasses, they distinguish from each other by small sequence differences in the constant region of the heavy chain, the length of the hinge that connects the antigenbinding (Fab) domain to the fragment crystallizable (Fc) domain, the number of disulphide bonds between the heavy chains within the hinge region, and the disulphide bond positions between the heavy and light chains. Therefore, one cannot rely on changing disulphide bond conformation in the hinge region to mediate agonistic activity for other IgG subclasses and classes of antibodies. Accordingly, the inventors set out to find alternative engineering strategies to generate antibodies other than hlgG2 with increased activity.

[0053] The present invention is based on the inventors’ development of a method for reducing the flexibility of Fab arms on immunoglobulins other than hlgG2. Specifically, the inventors identified that a 222C mutation in each of the heavy chain CH1 domains, and optionally a 123C mutation in each of the light chain CL domains, surprisingly restricts flexibility of the Fab arms relative to each other across different IgG subclasses including hlgG1 , hlgG2, and hlgG4, by formation of a non-native disulphide bond between Fab arms through the constant region.

[0054] Surprisingly, the conformational restriction provided by the non-native disulphide bond has no negative implications on the binding affinity. Even more surprisingly, at high concentrations, the double mutant comprising a 222C mutation and 123C mutation, and single mutant 222C, in fact, increases the binding affinity relative to WT hlgG1.

[0055] Without wishing to be bound by theory, the inventors believe that the present invention may be suitable for reducing Fab-arm flexibility of all antibodies. However, in the context of antibodies for targets that are cell membrane bound, such as receptors (for example tumour necrosis factor receptors or immunoglobulin receptor superfamily members), the present invention, may be especially suitable for antibodies that bind in locations that are not proximal to the cell membrane. The inventors believe that targets that are not proximal to the cell membrane may be bound better by antibodies comprising the mutations described herein. Examples of antibodies that bind such not membrane proximal locations on targets include, but are not limited to Urelumab, Lob7 / 4, and / or MK-5890.

[0056] In one aspect, the present invention provides an antigen binding protein comprising at least two CH1 domains. The term “antigen binding protein” refers to a proteinaceous molecule (protein, protein-like or protein-containing) that is capable of binding using intermolecular interactions specific to a target molecule (i.e. an antigen). Antigens are molecules known to be specifically bound by or capable of being specifically bound by an antigen binding protein, such as an antibody or antibody fragment. As used herein, the term "specifically bound" or "specifically bound" is used with a given antigen in an amount of about 1 x 10-6M or less, such as about 1 x 10-7M or less, about 1 x 10-8M or less, about 1 x 109M or less, about 1 x 1 o10M or less, about 1 x 10’11M or less, or about 1 x 10-12M or less.

[0057] The antigen binding protein may comprise an antibody or an antibody fragment. Suitably, the antigen binding protein may consist of an antibody or an antibody fragment.

[0058] The antigen binding protein may be conjugated to a compound. For example, the antigen binding protein may be conjugated to a compound selected from the group consisting of a detectable label, a therapeutic compound (for example a small molecule), a nucleic acid, a peptide, a protein, a compound that increases the half-life of the protein. In one particular example, the antigen-binding protein is conjugated to a detectable label. An exemplary detectable label may be selected from the group consisting of: a radiolabel, a fluorescent label, an enzymatic label and an imaging agent. An exemplary protein may be for example another antigen binding protein, an immunomodulator, or a half-life extending protein.

[0059] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region typically comprises at least one of: a CH1 domain, a hinge (e.g., upper, middle, and / or lower hinge) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.

[0060] As set forth in the present disclosure, it will be understood by one of ordinary skill in the art that the heavy chain constant region may be modified such that it varies in amino acid sequence from a naturally occurring immunoglobulin molecule. Suitably, the heavy chain constant region is modified in the CH1 region. Suitably, the modified CH1 region comprises a cystine at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1. Herein, an antigen binding protein that comprise a cystine at an amino acid position corresponding to position 222 according to Kabat numbering of human I gG 1 CH1 may be referred to as “a 222C mutant” or “a 222C variant”.

[0061] As mentioned elsewhere herein, the present inventors have identified that the presence of cysteine residues at amino acid positions corresponding to position 222 according to Kabat numbering of human lgG1 CH1 results in the formation of a disulphide bond between the two CH1 domains of the antigen binding protein. This disulphide bond has been shown by the inventors to be capable of improving the activity of the antigen binding protein. The inventors believe that this is due to the disulphide bond restricting the flexibility of the CH1 domains relative to each other. As it will be appreciated by the person skilled in the art, this may further restrict the flexibility of the binding domains (such as the variable light and variable heavy regions) of the antigen binding protein, and as a result improve activity of the antigen binding protein.

[0062] As used herein the term “disulphide bond” refers to the covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulphide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulphide bond and the two heavy chains are linked by two disulphide bonds at positions corresponding to 239 and 242 using the Kabat numbering system.

[0063] The CH1 region of an antigen binding protein disclosed herein may be derived from any naturally existing immunoglobulin molecule sequence. Accordingly, the CH1 domain may be derived from an IgG, IgM, IgA, IgE or IgM CH1 sequence. Suitably, the IgG may be selected from the group consisting of hlgG1 , hlgG4, hlgG2 or hlgG3. More suitably, the hlgG may be selected from the group consisting of hlgG1 , hlgG4, or hlgG2.

[0064] The phrase “amino acid position corresponding to” as used herein refers to an amino acid position number in a human lgG1 CH1 domain. Corresponding amino acid positions in other immunoglobulins may be found by alignment of other sequences (such as other CH1 domain sequences from non-human and / or non hlgG1 CH1 domains). Sequence alignment may be performed by any one of the well known in the art alignment programs in the art. Merely by way of example, an alignment program such as ALIGN, ClustalW or similar, may be used. It is considered well-known in the art how to align a sequence or segment in a sequence and thereby determine the corresponding position in a sequence to an amino acid position according to the present invention.

[0065] The wild-type CH1 domain amino acid sequence of human lgG1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKV (SEQ ID NO:5) - the amino acid at position 222 is in bold and underlined. Sequences SEQ ID NO: 6, 7, and 8, shown below, are the wild-type sequences of the CH1 domains of human lgG2, human lgG3, and human lgG4, respectively (amino acid at position 222 is in bold and underlined): SEQ ID NO: 6: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKTV;

[0066] SEQ ID NO: 7: ASFKGPSVFPLAPCSRSTPGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSWTVPSSSLGTQTYTCNVNHKPSNTKVDKRV;

[0067] SEQ ID NO: 8: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRV.

[0068] It will be appreciated that in the context of the present disclosure, the antigen binding proteins (such as antibodies and antibody fragments) may comprise one or more CH1 domains according to SEQ ID: 1 , 2, 3 and / or 4, as shown below, respectively :

[0069] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKCV (SEQ ID NO:1);

[0070] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSNFGTQTYTCNVDHKPSNTKVDKCV (SEQ ID NO: 2);

[0071] ASFKGPSVFPLAPCSRSTPGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH TFPAVLQSSGLYSLSSWTVPSSSLGTQTYTCNVNHKPSNTKVDKCV (SEQ ID NO: 3);

[0072] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKCV (SEQ ID NO: 4).

[0073] Suitably, such CH1 domains may be variants of SEQ ID: 1 , 2, 3 and / or 4. In this context, the term “variant” means that the CH1 domains comprise a cysteine at position 97, and a least one further mutation as compared to the wild-type sequence (for example one, two, three, four, five, or more further mutations, or at least one, at least two, at least three, at least four, at least five, or more further mutations). Suitably, the variant may share at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1 , 2, 3, or 4.

[0074] The terms “sequence identity” as used herein refers to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of for example Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264- 2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al. (1997) Nucl. Acids Res. 25: 3389- 3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.

[0075] The antigen binding proteins of the present disclosure comprise at least two CH1 domains. Suitably, the at least two CH1 domains may be the same, or different (so long as they both have a cysteine at an amino acid residue corresponding to position 222 of human lgG1 CH1 domain).

[0076] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 1 .

[0077] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 2. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 2.

[0078] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 3.

[0079] In a suitable embodiment, at least one of the CH1 domain comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 4.

[0080] In a suitable embodiment, at least two CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1. Suitably, the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 1.

[0081] In a suitable embodiment, at least two CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 2. Suitably, the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 2.

[0082] In a suitable embodiment, at least two CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3. Suitably, the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 3.

[0083] In a suitable embodiment, at least two CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4. Suitably, the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 4.

[0084] Herein the amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), referred to as “numbering according to Kabat” or “Kabat numbering” herein.

[0085] In a suitable embodiment, the antigen binding protein of the invention comprises at least two CH1 domains and at least two corresponding CL domains. Such antigen binding proteins are described in more detail elsewhere herein.

[0086] As used herein, the term “light chain constant region” or “CL domain” includes amino acid sequences derived from an antibody light chain that is adjacent to the VL region. There are two types of mammalian light chains, lambda (A) and kappa (K), which have minor differences in the polypeptide sequence. Each naturally occurring antibody comprises two light chains that are identical. However, it will be appreciated that engineered antibodies, such as chimeric antibodies, can comprise two different light chains. Other types of light chains exist in nature, including those such as the iota (i) chain, which is found in lower vertebrates.

[0087] A light chain-heavy chain pair is refers to the collection of a light chain and heavy chain that can form a dimer through a disulphide bond between the CL domain of the light chain and the CH1 domain of the heavy chain. In the context of the present disclosure thus, the phrase “corresponding CL domains” refers to CL domains that form dimers through disulphide bonds with the CH1 domains.

[0088] In a suitable embodiment, the antigen binding protein of the present disclosure may comprise or consist of an antibody, or an antibody fragment.

[0089] As used herein, the term "antibody" refers to immunoglobulins or immunoglobulin-like molecules, including, but not limited to, IgG, IgD, IgE, IgA, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate (e.g., humans, goats, rabbits, alpacas, llamas, sheep, camels, and mice, and non-mammalian species such as sharks). The term “antibody” also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized antibodies), hetero-conjugate antibodies (e.g., bispecific antibodies). The antibody may comprise of one or more polypeptides. For example, mammalian IgG comprises of two identical light chains and two identical heavy chains. Such an antibody therefore comprises of two polypeptides. The resulting antibody has three functional components, two fragment binding domains (Fabs), fragment crystallizable (Fc), with the two Fabs linked to the Fc by a hinge region.

[0090] It will be appreciated that in the context of the present disclosure, when reference is made to a specific antibody type (such as IgG, IgD, IgE, IgA, or IgM) reference is made to an antibody that has a backbone of said specific antibody type but comprising a cysteine residue at an amino acid position corresponding to position 222 of human lgG1 CH1 , or has a backbone that shares at least 80% sequence identity with a backbone of said specific antibody type. By way of example, when it is said that the antibody is a human IgG 1 antibody, this means that the antibody has a human lgG1 backbone comprising a cysteine residue at position 222 of the CH1 domain, or a backbone that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with a human I gG 1 backbone. Suitably, in the context of the present invention the antibody may be a human antibody or a humanised antibody. Suitably, the human or humanised antibody may be selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. Suitably, the hlgG may be hlgG1 , hlgG2, hlgG4 or hlgG3.

[0091] In a suitable embodiment, the antibody may be Urelumab or Nivolumab. It will be appreciated that in the context of the present disclosure, when reference is made to a specific antibody, such as an anti-CD40 antibody (for example LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), the amino acid sequence of said antibody is as known in the art, with the exception that a cysteine is at position 222 of the CH1 domain.

[0092] Suitably, the antigen binding protein may specifically bind a cell surface receptor (such as a tumour necrosis factor receptor (TNFR)) or an immunoglobulin receptor superfamily member (IgSF).

[0093] Suitably, the antigen binding protein may comprise or consist of an antibody selected from the group consisting of anti-CD40 (optionally LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally SAP9), anti-4-1 bb (optionally Utomilumab, SAP1.3, SAP1.3ND, SAP3.28 and Urelumab), anti-CD28 (optionally TGN14-12), anti-CD27 (optionally hCD27.131A), anti-ICOS, anti-PD1 (such as Nivolumab), anti-DR4 and anti-DR5.

[0094] Suitably, the antigen binding protein of the invention may comprise an antibody. Non limiting examples of antigen binding proteins that comprise an antibody include an lgG2, a CovX- Body, a kappa / lamda-body common HC, an IgG(kih), an IgG(kih) common LC, a ZW1 IgG common LC, a bicionics Common LC, an orthonogal Fab IgG (kih), a duetMab, CH3 charge pairs + CH1 / CL charge pairs, a duobody, a four-in-one CrossMab (kih), a LUZ-Y common LC, a FcFc’, an lgG(kih)-Fv, an IgG(HA-TF-Fv), an lgG(kih)-scFab, a DVI-lg (four-in-one), an IgG- HC-scFv, an IgG-dAb, an IgG-taFv, an IgG-CrossFab, an IgG-orthogonal Fab, a scFv-HC- IgG, a tandem Fab-IgG (orthogonal Fab), an IgG-scFv(LC), an scFv(LC)-lgG, a Dab-IgG, a DVD-lg, a TVD-lg, a scFv4-lgG, a Zybody, a scFv4-lg, DAF, a dutaMab, a Mab2, or a DNL- Fab4-lgG.

[0095] Suitably, the antigen binding protein of the invention may comprise an antibody fragment. Non limiting examples of antigen binding proteins that comprise an antibody fragment include a F(ab’)2, a Fab2, DNL-Fab3, a DNL-Fab2-scFV, a DNL-Fab2-lgG-cytokine2, a scFab-lgG(kih), a Fab-scFab-lgG(kih), a LLIZ-Y scFab-IgG, a scFab-Fc(kih)-scFv2, a scFab-Fc(kih)-scFv, a TriFabs, a CODV-ig, a F(ab’)2 fusion (e.g. F(ab’)2-scFv2), or a scFv2-CH1-hinge / CL.

[0096] Suitably, the antigen binding protein (such as an antigen binding protein that comprises or consists of an antibody or antibody fragment) may be monospecific or multispecific.

[0097] “Specificity” refers to the selective recognition of a particular epitope of an antigen by an antigen binding protein. As used herein, "monospecific " means the antigen binding protein has one or more binding sites that each bind to the same epitope on the same antigen. A "multispecific” antigen binding protein binds to two or more different epitopes (e.g., two, three, four, or more different epitopes). The epitopes can be on the same or different antigens. The multispecific antigen binding protein may be, for example, bi-specific, tri-specific, tetraspecific, or penta-specific.

[0098] As mentioned, the antigen binding protein of the invention may comprise at least two CH1 domains and at least two corresponding CL domains. Suitably, at least one of the at least two CL domains may comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain, optionally wherein the at least two CL domains comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human IgG 1 CL domain.

[0099] The wild-type human lgG1 kappa CL domain amino acid sequence is: RTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0100] The wild-type human lgG1 lambda CL domain amino acid sequence is: GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 12).

[0101] It will be appreciated that in the context of the present disclosure, the antigen binding proteins (such as antibodies and antibody fragments) may comprise one or more CL domains according to SEQ ID: 9 or 10, as shown below, respectively: RTVAAPSVFIFPPSDCQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9);

[0102] GQPKAAPSVTLFPPSSCELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQ SNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10).

[0103] SEQ ID NO: 9 is the sequence of human lgG1 kappa CL domain (SEQ ID NO: 11) where a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain is present. By the same token, SEQ ID NO:10 is the sequence of human lgG1 kappa CL domain (SEQ ID NO: 12) where a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain is present. Herein, antigen binding proteins that comprise a cystine at an amino acid position corresponding to position 123 according to Kabat numbering of human IgG 1 CL may be referred to as “123C mutants” or “123C variants”.

[0104] Furthermore, antigen binding proteins that comprise a cystine at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL and a cystine at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain be referred to as “double mutants”, “double variants”, “123C / 222C mutants”, or “123C / 222C variants”.

[0105] Suitably, such CL domains may be variants of SEQ ID: 9 and / or 10. In this context, the term “variant” means that the CH domains comprise a cysteine at position 16 of SEQ ID NO: 9 or position 17 of SEQ ID NO: 10, and a least one further mutation as compared to the wild-type sequence (for example one, two, three, four, five, or more further mutations, or at least one, at least two, at least three, at least four, at least five, or more further mutations).

[0106] In a suitable embodiment of the antigen binding protein described herein, at least one of the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least one of the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9.

[0107] In a suitable embodiment of the antigen binding protein described herein, at least one of the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least one of the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9.

[0108] The antigen binding proteins of the present disclosure may comprise at least two CL domains. Suitably, the at least two CL domains may be the same, or different, so long as at least one or both of the CL domains have a cysteine at an amino acid residue corresponding to position 123 of human IgG 1 CL domain. In a suitable embodiment of the antigen binding protein described herein, at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 9. Suitably, at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9.

[0109] In a suitable embodiment of the antigen binding protein described herein, at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 10. Suitably, at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 10.

[0110] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1 , and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 1 , and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0111] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1 , and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 1 , and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0112] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 2, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 2, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0113] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 2, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 2, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0114] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 3, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0115] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 3, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10. In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 4, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0116] In a suitable embodiment of the antigen binding protein of the invention, at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4, and at least one of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 4, and at least one of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0117] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1 , and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 1 , and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0118] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 1 , and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 1 , and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0119] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 2, and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 2, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0120] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 21 , and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 2, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0121] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3, and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 3, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9. In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 3, and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 3, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0122] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4, and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 9. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 4, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 9.

[0123] In a suitable embodiment of the antigen binding protein of the invention, at least two of the CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity with SEQ ID NO: 4, and at least two of the CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity with SEQ ID NO: 10. Suitably, at least two of the CH 1 domains comprise or consist of a sequence according to SEQ ID NO: 4, and at least two of the CL domains comprises or consists of a sequence according to SEQ ID NO: 10.

[0124] In a further aspect, the present invention provides a nucleic acid encoding the CH1 domain and / or CL domain of the present disclosure. It will be appreciated that when reference is made to a CH1 domain of the present invention this refers to a CH1 domain comprising a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain. By the same token, when reference is made to the CL domain of the present invention, this refers to a CL domain comprising a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain.

[0125] The nucleic acid can comprise DNA, cDNA, and / or RNA.

[0126] The nucleic acid can be single or double stranded.

[0127] The nucleic acid can be naturally-occurring, synthetic, and / or recombinant.

[0128] The nucleic acid can comprise nucleotide analogues or derivatives (e.g., inosine or phosphorothioate nucleotides and the like). Silent mutations in the coding sequence result from the degeneracy (i.e., redundancy) of the genetic code, whereby more than one codon can encode the same amino acid residue. Thus, for example, leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tables showing the standard genetic code can be found in various sources (e.g., L. Stryer, 1988, Biochemistry, 3. sup. rd Edition, W.H. 5 Freeman and Co., NY).

[0129] In a further aspect, the present invention provides an expression vector comprising the nucleic acid encoding the CH1 domain and / or CL domain of the present disclosure.

[0130] The term “expression vector” as used herein refers to a nucleic acid construct that comprises a gene expression controlling region, such as a promoter or promoter component, operably linked to a nucleotide sequence coding at least one polypeptide (such as the polypeptide encoding the CH1 domain and / or the CL domain of the present disclosure). The vector may be any vector capable of transferring DNA to a cell.

[0131] Suitably, the vector is an integrating vector or an episomal vector.

[0132] Preferred integrating vectors include recombinant retroviral vectors. A recombinant retroviral vector will include DNA of at least a portion of a retroviral genome which portion is capable of infecting the target cells. The term “infection” is used to mean the process by which a virus transfers genetic material to its host or target cell. Suitably, the retrovirus used in the construction of a vector of the invention is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication-defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any retrovirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the invention. Suitably, the retroviral vector may be a lentiviral vector. Lentiviral vectors are well known in the art, and have been used to deliver genes to numerous cell types, including HeLa or B lymphoid cells. Other vectors useful in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and poxvirus vectors.

[0133] Preferred episomal vectors include transient non-replicating episomal vectors and selfreplicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1. Such integrating and episomal vectors are well known to those skilled in the art and are fully described in the body of literature well known to those skilled in the art. In particular, suitable episomal vectors are described in WO98 / 07876.

[0134] Mammalian artificial chromosomes can also be used as vectors in the present invention. The use of mammalian artificial chromosomes is discussed by Calos (1996 T rends in Genetics 72: 463-466).

[0135] In a preferred embodiment, the vector of the present invention is a plasmid. The plasmid may be a non-replicating, non-integrating plasmid.

[0136] The term “plasmid” as used herein refers to any nucleic acid encoding an expressible gene and includes linear or circular nucleic acids and double or single stranded nucleic acids. The nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by such means as methylation or the inclusion of protecting groups or cap- or tail structures.

[0137] A non-replicating, non-integrating plasmid is a nucleic acid which when transfected into a host cell does not replicate and does not specifically integrate into the host cell’s genome (i.e. does not integrate at high frequencies and does not integrate at specific sites). Replicating plasmids can be identified using standard assays including the standard replication assay of llstav et al (1991 EMBO J 10 449-457).

[0138] In a further aspect, the present invention provides a composition comprising a first nucleic acid encoding a CH1 domain and a second nucleic acid encoding the CL domain of the present disclosure, or a composition comprising a first expression vector comprising the first nucleic acid and a second expression vector comprising the second expression vector.

[0139] The present invention also provides a cell transformed or transfected with (i.e. comprising) the expression vector (or composition of expression vectors) of the present invention. Such a cell is referred to as a “host cell”. The host cell may be any cell suitable for the expression of the antigen binding proteins or fragments (such as the CH1 domain and / or the CL domain) of the invention. The host cell may be an yeast, a bacterial, an insect, a plant, or a mammalian cell. Of particular interest are bacteria such as E. coli, fungi such as Saccharomyces cerevisiae and Pichia pastoris, insect cells such as SF9, mammalian cell lines (e.g., human cell lines), as well as primary cell lines. The antigen binding proteins or fragments (such as the CH1 domain and / or the CL domain) of the invention can be produced from the cells by culturing a host cell transformed with the expression vector containing nucleic acid encoding the CH1 domain and / or the CL domain alone, or in combination with other domains typically found in antibodies and discussed elsewhere herein, under conditions, and for an amount of time, sufficient to allow expression of the proteins. Such conditions for protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation. For example, antibodies expressed in E. coli can be refolded from inclusion bodies (see, e.g., Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and methods for their use are well known in the art (see Current Protocols in Molecular Biology, Wiley & Sons, and Molecular Cloning--A Laboratory Manual--3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001)). The choice of codons, suitable expression vectors and suitable host cells will vary depending on a number of factors and may be easily optimized as needed. An antibody (or fragment thereof) described herein can be expressed in mammalian cells or in other expression systems including but not limited to yeast, baculovirus, and in vitro expression systems (see, e.g., Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0140] Suitably, the host cell is an isolated cell.

[0141] Numerous techniques are known and are useful according to the invention for delivering the vectors described herein to host cells, including the use of nucleic acid condensing agents, electroporation, complexing with asbestos, polybrene, DEAE cellulose, Dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment and direct microinjection (reviewed by Kucherlapati and Skoultchi (1984 Grit. Rev. Biochem 1&. 349- 379); Keown et al (1990 Methods Enzymol 785:527-37).

[0142] In a further aspect the present invention provides a pharmaceutical composition comprising the antigen binding protein, the nucleic acid, the expression vector, and / or cell of the invention, and a pharmaceutically acceptable diluent, carrier or excipient.

[0143] Compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.

[0144] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected nucleic acid composition, vector system or host cell without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.

[0145] Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid sequence, vector, modified cell or isolated peptide as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility. Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.

[0146] Adjuvants are pharmacological and / or immunological agents that modify the effect of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.

[0147] Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.

[0148] Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.

[0149] In another aspect, the invention provides a method of increasing activity of an antigen binding protein. Suitably, the antigen binding protein comprises at least two CH1 domains, and optionally at least two corresponding CL domains. S Suitable embodiments the antigen binding protein are described hereinabove.

[0150] The term “activity” as used herein refers to the desired function of the antigen binding protein. For example, the activity may be agonistic or antagonist.

[0151] Tumour necrosis factor receptors (TNFR) activation is an example of agonistic activity. TNFRs play important roles in immune activation and represent promising targets for the nextgeneration of cancer immuno-therapeutics. Selective TNFR activation has been shown to augment antitumor immunity and confer robust therapeutic benefits in animal models with TNFR-targeting mAbs and recombinant ligands. CD40 is a TNFR superfamily member expressed on antigen-presenting cells (APCs) and controls a key immunostimulatory pathway required for humoral and cellular immunity. Ligation of CD40 by agonistic anti-CD40 antibodies, surrogates of CD40L expressed by CD4 T cells, can effectively promote APC maturation and in turn antigen-specific CD8+ cytotoxic T-cell activation and expansion, and strengthen antitumour responses.

[0152] Activity may be typically as a result of binding of the antigen binding protein to the antigen. The inventors believe that the antigen binding proteins of the invention may have increased activity as compared to corresponding antigen binding proteins lacking the 222C, and optionally the 123C mutation described herein. In this context, the term “corresponding” means antigen binding proteins having an otherwise same amino acid sequence.

[0153] The inventors have shown that the antigenic binding proteins as described herein may have an increased binding affinity as compared to corresponding antigen binding proteins lacking the 222C, and optionally the 123C mutation described herein. It is this increased binding that the inventors believe may result in increased activity. Accordingly, the present invention also provides a method of increasing binding of an antigen binding protein. Suitably, the antigen binding protein comprises at least two CH 1 domains, and optionally at least two corresponding CL domains.

[0154] The terms "increased", "increase" or “higher” are all used herein to generally mean an increase by a statistically significant amount; for the avoidance of any doubt, the terms "increased" or "increase" means an increase of at least about 5% as compared to a reference value, for example an increase of at least about 10%, or at least about 20%, or at least 30%, or at least about 40%, or at least about 50% as compared to a reference value. For example, the increase may be of at least about 60%, or at least about 700%, or at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 400%, at least about 500%, at least about 600%, or more as compared to a reference value. The reference value, as it will be appreciated by the skilled person, refers to value of an appropriate parameter (such as activity and / or binding) by a control antigen binding protein not comprising the 222C, and optionally the 123C mutation described herein. It will be understood that such a control antigen binding protein may otherwise be identical to the antigen binding protein of the invention.

[0155] The method of the invention (i.e. the method of increasing activity and / or the method of increasing binding) comprises the step of introducing a mutation into the at least two CH1 domains of the antigen binding fragment, wherein the mutation is a cysteine substitution of an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain, and optionally introducing a mutation into at least one of the at least two CL domains, wherein the mutation is a cystine substitution of an amino acid residue at a position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain, optionally wherein the mutation is introduced into both of the at least two CL domains.

[0156] The term “introducing a mutation” as used herein means modifying a nucleotide residue (adenine nucleotide residue, guanine nucleotide residue, thymine nucleotide residue, cytosine nucleotide residue, uracil nucleotide residue) that encodes the CH1 domain, and optionally CL domain. Upon introduction of the mutation into the nucleic acid, the resulting protein will consist of a cysteine at an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain, and optionally a cystine at an amino acid residue at a position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain, optionally wherein the mutation is introduced into both of the at least two CL domains.

[0157] It will be appreciated that the method of increasing activity and / or binding as described herein, may be applied to existing (for example therapeutic) antigen binding proteins, so long as they comprise at least two CH1 domains, and optionally at least two CL domains.

[0158] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.

[0159] Aspects of the invention are demonstrated by the following non-limiting examples.

[0160] EXAMPLES

[0161] Within the following examples section “clamp” mutants refers to double mutants of 222C and 123C.

[0162] Materials and Methods

[0163] Expression of hlgG 1, hlgG2 and hlgG4 variants

[0164] Antibodies were produced using the Gibco ExpiCHO™ transient expression system. Details of antibodies are in Table 1. ExpiCHO-S™ cells cultured in ExpiCHO™ Expression Medium were transfected with DNA using ExpiFectamine™ CHO Reagent and OptiPRO™ SFM Complexation Medium. Cells were incubated at 37°C, 8% CO2, with shaking at 125 rpm. On day 1 , ExpiFectamine™ CHO Enhancer and ExpiCHO™ Feed were added to the cells. Cell supernatant was harvested after 10 days, by centrifugation of cell culture at 3230 g for 40 minutes.

[0165] Antibodies were purified from cell supernatant by Protein A affinity purification using a MabSelect SuRe protein A column (Cytiva), attached to a GE AKTA start protein purification system (Cytiva). Bound IgG was eluted using a low pH glycine buffer. All antibodies were checked by HPLC to contain <1% aggregate and purified by gel filtration using a HiLoad Superdex 200 pg 16 / 600 size exclusion chromatography column (Cytiva) if >1% aggregation was detected. Antibodies were assessed for endotoxin using an Endosafe Portable Test System (PTS) device (Charles River Laboratories) and used if determined to contain <10 endotoxin units per mg antibody.

[0166] Table 1 Antibody sequences

[0167] The Lob7-4 sequence is derived from the sequences described in US20090074711. The Utolimumab sequence is derived from the sequences described in US2015 / 014199. The Urelumab sequence is derived from the sequences described in WO2010 / 042433 A1. The TGN1412 sequence is derived from the sequences described in US7585960. The hCD27.131A sequence is derived from the sequences described in US2018 / 0086841.

[0168] Generation of F(ab’)2fragments

[0169] To generate F(ab’)2 fragments, full-length IgG was digested with pepsin to remove the Fc domain. Antibody was first dialysed into digestion buffer containing 20 mM Tris-HCI, 100 mM NaCI, 1 mM EDTA, pH 8. Antibody was then concentrated to approximately 10 mg / mL. Prior to digestion, the pH of the dialysed antibody solution was adjusted to 4.1 with 2M sodium acetate, pH 3.7, to enable optimal pepsin activity. Pepsin was prepared at a concentration of 10 mg / mL in acetate buffer (70 mM acetate, 50 mM NaCI, pH 4), and pepsin solution was added to antibody to give 3% w / w pepsin to IgG. Antibody was digested at 37°C, and digestion progress was checked using HPLC, with samples placed on ice, to pause digestion, during HPLC runs. A decrease in the IgG peak alongside an increase in the F(ab’)2 peak was observed over time. Digestion was stopped when the proportion of F(ab’)2 had stopped increasing, by adjusting the pH to 8.0 using 1M Tris. F(ab’)2 was purified by gel filtration using a HiLoad Superdex 200 pg 16 / 600 size exclusion chromatography column with 1 / 5 Tris-NaCI as the buffer, to remove undigested IgG. Pooled fractions were purified on a HiTrap 5 mL MabSelect SuRe protein A column (Cytiva), to remove any residual IgG and Fc, and then analysed by HPLC to confirm F(ab’)2 purity.

[0170] Immunostimulatory activity assays

[0171] The NF-kB / Jurkat / GFP™ cell line was transfected with pCIpuro plasmids encoding either full- length hCD40, h4-1 BB (expressing the hCD40 intracellular signalling domain), hOX40 (expressing the hCD40 intracellular domain) (Yu et al., 2021) or hCD27 (Heckel et al., 2022). Stable clones were selected with 0.25 pg / mL puromycin, and maintained in a humidified incubator at 37°C, 5% CO2, and cultured in RPMI 1640 media (Gibco, ThermoFisher) supplemented with 10% FCS, 2mM L-Glutamine, 1 mM sodium pyruvate, 100 U / mL penicillin and 100 pg / mL streptomycin (all from Thermofisher). To assess NFkB activation, Jurkat cells were incubated with serially diluted mAb or F(ab’)2 fragments for 24 hours at 37°C. The degree of NFkB activation was quantified by GFP fluorescence using flow cytometry. Flow cytometry data was acquired using a FACS Canto II (BD Biosciences), and data analysis was performed using FlowJo (BD Biosciences).

[0172] Immunostimulatory activity of anti-hCD40 mAb was also assessed using primary human B cells purified from human peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from fresh leukocyte cones by density gradient centrifugation. Blood cones were obtained from healthy adult donors through Southampton National Blood services with prior informed consent. The use of human blood for these assays was approved by the East of Scotland Research Ethics Service, Tayside, UK. Human B cells were purified from PBMCs by negative selection using a MojoSort Human B cell Isolation kit (BioLegend). B cells were incubated in vitro with anti-hCD40 mAb in 96 well round-bottom plates.

[0173] To measure homotypic adhesion, B cells were imaged 48 hours after addition of mAb with a conventional light microscope (Olympus CKX41 , running Olympus CellSens Standard software). Adhesion was observed as large macroscopic cell groupings. Upregulation of B cell activation marker expression was assessed by flow cytometry after 48 hours, using APC- labelled anti-CD23 mAb (clone EBVCS-5, BioLegend), PerCP-Cy5.5-labelled anti-CD86 mAb (clone BU63, BioLegend) and Brilliant Violet-labelled anti-MHCH mAb (clone L243, BioLegend). To assess B cell proliferation, B cells were stimulated with mAb as above for 4 days, with 1 pCi of3H — thymidine (PerkinElmer) added to each well for the last 18 hours of incubation. Cells were harvested and analysed by scintillation counting (TopCount) to measure3H-thymidine incorporation.

[0174] Surface Plasmon Resonance (SPR)

[0175] SPR was performed using a Biacore T200 instrument (Cytiva). Recombinant soluble hCD40- hFc-His (R&D Systems, Inc) was immobilised onto a CM5 sensor chip via amine coupling at a target level of 50 resonance units (RU), according to the manufacturer’s protocol. mAbs were injected through the flow cells at 100, 20, 4, 0.8, 0.16 or O nM in HBS-EP+ running buffer (0.01M HEPES, 0.15 M NaCI, 3 mM EDTA, 0.005% w / w Surfactant P20, pH 7.4) at a flow rate of 30 pL / min at 25°C with 300 seconds for association and 300 seconds for dissociation. Regeneration of the chip was performed for 30 seconds at a flow rate of 30 pL / min using 10 mM glycine, pH 1.5. All reagents, equipment and software obtained from Cytiva. The output of SPR experiments are sensorgrams showing the change in refractive index at the sensor chip surface, measured in arbitrary RU, against time. Sensorgrams were fitted with a 1 :1 binding model and ka (association rate), kd (dissociation rate) and KD (Equilibrium dissociation constant) were calculated using Biacore Bioevaluation software.

[0176] Binding of FcyRs to mAb was analysed by SPR using the Biacore T200 instrument. Briefly, anti-His antibody was immobilised onto a CM5 sensor chip via amine coupling, according to the manufacturer’s protocol. Recombinant molecules containing the different FcyR (from R&D Systems Inc.) with His tags were then flown over the chip at defined concentrations and then the mAb (e.g. hlgG 1 or hlgG 1 clamp) flown over at 200 nM (for 120 s to allow association) in HBS-EP+ running buffer (0.01M HEPES, 0.15M NaCI, 3 mM EDTA, 0.005% Surfactant P20, pH 7.4) followed by 120 s dissociation. If required, regeneration of the chip was performed for 30 s at a flow rate of 30 uL / min using 10 mM glycine, pH 1.5. Sensorgrams were generated to display the binding profiles. All reagents, equipment and software were obtained from Cytiva, unless otherwise specified.

[0177] X-ray crystallography

[0178] Protein crystallisation

[0179] F(ab’)2 samples were transferred into 50 mM Hepes, 150 mM KCI buffer, at pH 7.5, by buffer exchange using Amicon Ultra Centrifugal filters (Millipore, Sigma Aldrich). Samples were concentrated and resuspended in Hepes KCI 4-5 times, and finally concentrated to approximately 10 mg / mL for crystallisation trials. Crystallisation screens were set up using sitting drop vapour diffusion in 96 well 3 drop Intelliplates (SwissSci, Switzerland) using the Oryx8 protein crystallisation robot (Douglas Instruments, UK). Trays were incubated at 21°C in a Rumed temperature-controlled incubator. Both the TCR / pMHC optimised protein crystallisation screen (TOPS) and commercially-available MORPHEUS screen (Gorrec, 2009) were used as entry screens as they have previously provided good results with F(ab’)2 in our group. Plates were set up using a ratio of 2:1 (protein to screening condition) for the top drop, 1 :1 for the middle drop and 1 :2 for the bottom drop. Initial crystal hits were used to prepare seed stocks, with MicroSeed Beads (Molecular Dimensions) using a method adapted from (Luft and DeTitta, 1999) . Approximately 40 uL was removed from the mother liquor reservoir and a small amount dispensed onto the drop with the chosen crystal hits. Crystal hits were crushed by taking up and dispensing the liquid in the drop several times. Crystals were then transferred to a seed bead tube on ice, before vortexing. Neat and diluted tubes of seed stock were prepared. For F(ab’)2 for which initial entry screens were unsuccessful, cross-seeding was used to attempt to initiate crystal growth. Seeding and cross-seeding experiments were set up using the Oryx8 protein crystallisation robot. Crystals grew over several weeks, and trays were checked periodically for crystal growth using light and UV microscopes. Crystals were harvested by fishing with litholoops (Molecular Dimensions) of various sizes. Prior to fishing, cryoprotectant (containing mother liquor with 20% glycerol) was added to the crystal drop to protect the crystals on flash cooling. Litholoops mounted with the crystals were flash frozen in liquid nitrogen and stored in Unipucks within dewars containing liquid nitrogen, until diffraction experiments and data collection were performed.

[0180] Data collection

[0181] Standard X-ray diffraction data was collected at the European Synchrotron Radiation Facility (ESRF, France), using beamline ID30A-3. ID30A-3 is a fixed energy microfocus beamline, operating at 12.81 keV (0.978 A), with a 15 pm diameter X-ray beam. Data was collected using an Eiger X 4M detector, with samples kept under a cryostream at a temperature of 100K. Experiments were controlled using the MXCuBE3 Beamline control software. Experiments were logged in ExiMX (Extended ISPyB for MX).

[0182] Sulfur-single wavelength anomalous diffraction (S-SAD) data was collected at Diamond Light Source (DLS, Harwell, UK), using the in vacuo long-wavelength beamline I23. 123 is a tuneable energy beamline, with a range of 2.1 -11 keV (1.13 - _5.9 A). It operates in a high-vacuum environment, to prevent air absorption and scattering of X-rays, which occurs at these wavelengths, and thus samples must be prepared and transferred into vacuum using a dedicated Cryogenic Transfer System with thermally conductive materials; this facilitates sample cooling to approximately 50 K. Diffraction data was collected on a curved, semi- cylindrical Pilatus 12M detector with a multi-axis goniometer. 3600 images were collected per dataset, with 0.1 degree oscillation per image to give a 360 degree dataset. Data was collected at 4.5 keV (2.76 A). Experiments were logged in ISPyB.

[0183] Data processing

[0184] Initial data processing and phasing was performed automatically using several different pipelines (Grenoble Automatic Data processing (GrenADES), EDNA framework Fast Processing System (EDNA Autoprocessing), autoPROC, XDSAPP and XIA2_DIALS). All data manipulation, molecular replacement and refinement was carried out using the CCP4 suite of programs. Molecular replacement was performed using Molrep with PDB model 6TKE as the homologous model. Iterative model building and refinement were carried out using COOT (Emsley et al., 2010) and REFMAC5 (Murshudov et al., 2011), respectively.

[0185] Small-angle X-ray scattering (SAXS)

[0186] SAXS data was collected at the Diamond Light Source (DLS), UK, using their BioSAXS beamline, B21.B21 has an energy range of 0.5 - 14 keV, with a standard of 13 keV, and the beam size at the sample is 1102 x 240 pm. The q-range is 0.0026 - 0.34 A’1. The sample-to- detector distance is 3.7 m, and the detector is an in-vacuum Dectris Eiger 4M. Size exclusion chromatography coupled with small-angle x-ray scattering (SEC-SAXS) was used for data collection; this utilises an in-line HPLC system for purification, separating samples by size, (removing aggregates) prior to delivery into the beam. For the in-line purification a Superdex 200 Increase 3.2 column (8.6 pm pore size, 3.2 mm i.d. , 30 cm length) coupled to an Agilent 1260 HPLC system was used. 45 pL of F(ab’)2 at approximately 5 mg / mL was injected into the column at a flow rate of 0.075 mL / min, using 50 mM HEPES, 150 mM KCI, pH 7.5 as the SEC buffer. Measurements were performed at 20°C. Data was recorded from 600 three second frame exposures using the Eiger 4M detector. Data were processed and primary analysis was performed using ScAtter (version IV, R. Rambo, Diamond Light Source). Buffer subtraction was performed by automatic selection of stable buffer frames prior to the main elution peak of the SEC-SAXS signal plots. Data frames were selected from the main elution peak of the SEC-SAXS signal plots, where each point represents the integrated area of the ratio of the sample SAXS curve to the estimated background. Frames with high similarity were chosen and averaged for analysis, ensuring the scattering curve had little variation at low q.

[0187] Antibody dependent cellular phagocytosis (ADCP)

[0188] Jurkat cells stably transfected with CD40ECD (Jurkat CD40 cells) were used as targets (previously reported in Yu et al Nature 2023) with human monocyte-derived macrophages (hMDMs) as the effector cells. The hMDMs were derived by culturing monocytes in the presence of 100 ng ml-1 M-CSF (in-house) for 6 days. Two days before the phagocytosis assay, 7 x 104 hMDMs were plated onto a 96-well flat-bottom plate (ThermoFisher). On the day of the phagocytosis assay, target cells were labelled with 0.5 uM CFSE followed by opsonization with various CD40 mAb as indicated in the figure legends for 30 min at 4 °C. In total, 2 x 105 target cells were then added to each well and incubated at 37 °C for 1 hour for phagocytosis to occur. The samples were subsequently stained with anti-CD14-APC (1 :200) to identify hMDMs, and cells positive for both CFSE and CD14 as assessed by flow cytometry were classified as hMDMs that had undergone phagocytosis. The percentage of ADCP was calculated as follows: (CFSE+CD14+ cells) / (total CD14+ cells) x 100.

[0189] In vivo agonism assays

[0190] OTI expansion assay

[0191] OTI expansion assays were performed as previously described (Yu, X. et al. Isotype switching converts anti-CD40 antagonism to agonism to elicit potent antitumor activity. Cancer Cell 37, 850-866. e7 (2020)). To assess the ability of anti-CD40 monoclonal antibodies to induce OTI T cell expansion, 1 x 105 OTI cells were intravenously injected into CD40KOTg mice 1 day before the intravenous injection of 5mg OVA in combination with 100 pg of various anti-CD40 monoclonal antibodies. Mice were then bled periodically as indicated in the figure legends, and the level of OTI expansion was assessed on the basis of the proportion of CD8+ SIINFEKL tetramer-positive cells by flow cytometry.

[0192] ELISA for anti-OVA antibodies

[0193] In the same experiment as above, serum was taken periodically and then assessed for the presence of OVA-specific antibodies by ELISA as per White et al., 2010. In brief, to detect anti-OVA IgG in the serum, 96-well MaxiSorp plates were coated with 100 pg / ml OVA in coating buffer overnight at 4 °C. Plates were blocked with 1 % BSA and then serial diluted serum was added and the mouse- anti-OVA IgG mAb KB4 (in-house) was used to create a standard curve. The anti-OVA IgG bound to the plate was detected by the secondary rabbit anti-mouse IgG HRP antibody (Sigma). Optical density was measured at 490 nm using the BioTek Epoch plate reader and Gen 5 software (Agilent).

[0194] Results

[0195] The inventors identified that mutations (T222C and kE123C) result in disulphide bonds linking opposing heavy chains (Fig. 1). T222C is located within the CH1 domain and kE123C is located within the kappa CL domain.

[0196] Expression of hlgG variants

[0197] As observed in Fig. 2, the inventors successfully expressed hlgG1 , hlgG2 and hlgG4 variants containing the “clamp” double mutation.

[0198] Immunostimulatory activity

[0199] The inventors have then performed immunostimulatory activity assays of the clamp mutants compared to the wild-type for hlgG1 , hlgG2 and hlgG4 (Fig. 3).

[0200] The biological activity of the clamp double mutant variants were assessed using the NFkB / Jurkat / GFP transcriptional reporter cell line, expressing human CD40, where GFP is produced in response to receptor activity allowing for quantification by flow cytometry. hlgG 1 and hlgG4 clamp variants display a significant improvement in immunostimulatory activity when compared to wild type, while the hlgG2 clamp mutant shows similar activity to the WT hlgG2 which is already conformationally restricted.

[0201] Additionally, the inventors performed further immunostimulatory activity assays on the WT, “clamp” (double mutants, 222C AND 123C), and single mutants (222C or 123C) as displayed in Fig. 4. Surprisingly, the 222C mutation in the heavy chain (K222C in hl gG 1 , T222C in hlgG2) produced an increased agonistic effect relative to WT which was comparable with the “clamp” double mutants (222C AND 123C). These data demonstrate that only the 222C mutation is required to produce an increase in agonistic activity.

[0202] Further immunostimulatory activity assays were performed comparing the “clamp” double mutants to the wild-type for hl gG 1 of LOB7 / 6 anti-CD40 mAb. The results of these assays are displayed in Figure 12. They show that wild-type LOB7 / 6 hlgG1 has no activity in the reporter assay but that the “clamp” double mutant converts this antibody to having strong agonistic activity.

[0203] Immunostimulatory activity assays were also performed of the “clamp” double mutants compared to the wild-type for hlgG1 using primary immune B cells (Fig. 14). Activity is displayed with respect to cell adhesion, cell surface upregulation of CD23, CD86 and HLA-DR determined by flow cytometry and the increase in B cell proliferation as measured by3H thymidine uptake. Further data also shows the double clamp and single HC mutant K222C retain equivalent activity as IgG (Fig. 14 B-F). These data show clearly that the “clamp” double mutant provides the LOB7 / 4 hlgG 1 antibody with agonistic activity in each assay; it triggers homotypic adhesion (Fig. 14A; top images); upregulation of CD23, CD86 and HLA-DR on the B cell surface as well as activating them to proliferate (Fig. 14A; bottom images). In each case the wild-type LOB7 / 4 hlgG 1 antibody has no activity and is equivalent to the isotype control. Figure 14 shows that the 222C mutation in the heavy chain (K222C in hlgG1) produced an increased agonistic effect relative to WT which was comparable with the “clamp” double mutants (222C AND 123C) when measuring CD23 upregulation (Fig. 14B), CD86 upregulation (Fig. 14C) and upregulation of HLA DR (Fig. 14D) as well as proliferation (Fig. 14E). In contrast, the 123C mutant was inert in these assays, equivalent to the wild-type LOB7 / 4 hlgG1 , including in homotypic adhesion (Fig. 14F).

[0204] Assays were also performed of the “clamp” double mutants compared to the wild-type for hlgG1 for five different mAb targeting 4-1 BB, CD27 or 0X40 (Fig. 19). These show that in each case that the double clamp mutant is more agonistic than the wild-type h IgG 1 antibody.

[0205] Structural analysis

[0206] The structures of the Fab’2S of hlgG 1 clamp and hlgG2 clamp have been determined, showing the position of the disulphide bonds introduced by the mutations (Fig. 5). The introduction of the clamp mutation results in a compaction and twisting of the two Fab arms for both hlgG 1 and hlgG2. The new disulphide bond links opposing heavy constant regions in the two Fab arms. The structure of the hlgG1 is much more compact than the hlgG2, however, the inventors believe in solution the hlgG1 structure is much closer to the conformation seen in the hlgG2 crystal structure as indicated by the SAXS data and negative stain electron microscopy. The inventors are in the process of generating crystals of the hlgG4 isoform. They are also conducting sulfur single-wavelength anomalous dispersion (S-SAD) experiments at the I23 beamline, Diamond light source, UK, to definitively identify the position of the sulphur atoms within the crystal structures, thereby confirming the position of the disulphide bonds. Small-angle x-ray scattering (SAXS)

[0207] Small-angle x-ray scattering (SAXS) was performed in order to characterise the biophysical properties of the hlgG1 , hlgG2 and hlgG4 variants of the clamp mutants in solution.

[0208] This SAXS data provides information on the conformation and flexibility of these particles in solution. The data show that the variants with the clamp mutations are more rigid in solution than the wild type variants (Fig. 6). This is particularly evident for the hlgG1 and hlgG4 isotypes as they are naturally more flexible than hlgG2.

[0209] The elevation of the peak of the curve up and to the right of the Guinier-Kratky point for hl gG 1 and hlgG4 WT indicates a more flexible particle, while the shift of the peak towards the Guinier- Kratky point for hl gG 1 clamp and hlgG4SP clamp indicates a more compact particle. It can be seen that the hlgG2 is already compact in the WT form, however, the introduction of the clamp does result in a further slight compaction.

[0210] Further data (Fig. 13) displays small-angle x-ray scattering (SAXS) data of hlgG1 variants with the “clamp” double or single mutations as Fab2 fragments and IgG molecules. Information on the flexibility of a particle can be derived from a dimensionless Kratky plot. For a tightly packed globular particle, the curve will peak at the Guinier-Kratky point (shown as grey cross-hair) and return to baseline as a 5 Gaussian curve. An elevation of the peak of the curve up and to the right of the Guinier-Kratky point indicates a more flexible particle (as observed for WT hlgG 1 ), while a shift of the peak towards the Guinier-Kratky point indicates a more compact particle (as seen for hlgG 1 clamp and hlgG 1 K222C). Some molecules were assessed as a Fab2 fragment to allow the highest resolution study of the Fab arms in the absence of the Fc (Fig. 13A). Other molecules were assessed as a whole IgG fragments and so include the movement of the Fc (Fig. 13B). This data demonstrates that hlgG1 K222C (heavy chain single mutant) shows a similar Kratky plot to hlgG1 clamp, indicating that the K222C heavy chain mutation alone is sufficient for reduced flexibility and that hl gG 1 kE123C mutation has a more modest (but still measurable) effect on restricting the flexibility. Note: the differences between curves are less evident when assessing whole IgG versus the Fab2 fragments as the Fc also contributes to the flexibility and dimension of the molecules.

[0211] Further small-angle x-ray scattering (SAXS) was performed to study six antibodies (hCD27.131A, SAP1.3 ND, TGN1412, hCD27.15, Utomilumab (Uto) and Urelumab (lire)) (Fig. 17). As before, Fab2 fragments were assessed to allow the highest resolution study of the Fab arms in the absence of the Fc. In each case the hlgG 1 clamp results in a shifting of the Kratky plot to the left and down, having a peak at lower q Rg. This indicates a reduction in flexibility and increased compaction compared to the wild-type hlgG1 (explored further in Figure 18). The six antibodies show the same reduction in flexibility demonstrating the generality of the technology. Three different receptors CD27, 4-1 BB and CD28 (a non TNFRsf member) were tested, indicating the technology can be employed to target receptors of multiple classes.

[0212] Figure 18 shows data derived from small-angle x-ray scattering (SAXS) of hlgG 1 variants with the “clamp” double mutations compared to WT hl gG 1 . Information relating to radius of gyration (rg), maximum dimension (Dmax) is derived using the ScAtter program. The data shows the hlgG1 clamp has lower Rg, lower Dmax, lower Porod volume and higher Porod exponent. These are all indicative of more compact, less flexible particles. Included in the analysis is assessment of the LOB7 / 4 Fab2 fragments produced by two different methods of digestion (IdeS or Pepsin). Despite small differences in Fc cleavage site, Fab2 fragments produced by either approach show the clear effect of the double- clamp on inducing more compact, less flexible particles.

[0213] Negative stain electron microscopy (nsEM)

[0214] Negative stain electron microscopy (nsEM) was performed to investigate the conformation of the hlgG1 clamp free from crystal lattice constraints (Fig. 7). The inventors have collected negative stain electron microscopy data of the hlgG1 isotype clamp mutant and wild type version. The hl gG 1 WT shows a range of conformations of the Fab arms with respect to the Fc. In comparison, the orientation of the Fab arms relative to the Fc for the hlgG1 clamp is far more restricted, bringing the two Fab arms closer together. Additionally, the data shows a restriction in the conformations of the two Fab arms relative to one another in the variant containing the clamp mutation when compared to the wild type. Further analysis (Fig. 9) shows the Fab arms in the hlgG1 clamp display a much tighter distribution of angles than the wild type hlgG 1 with a lower mean angle and range. This means the Fab arms in the hlgG 1 clamp show very clear restriction.

[0215] Affinity / specificity using Surface Plasmon Resonance (SPR)

[0216] The inventors performed binding assays to evaluate if the introduction of the clamp mutant impacts binding. The data show that for the hl gG 1 , hlgG2 and hlgG4 “clamp” double mutants there is no negative impact on binding, with the hl gG 1 clamp showing slightly improved binding at higher concentrations compared to wild type (Fig. 8). This demonstrates that the mutation does not negatively impact binding to the antigen in the systems tested so far. hCD40 was immobilised on the sensor chip with the mAbs injected over the flow cell at a range of concentrations (100, 20, 4, 0.8, 0.16 or 0 nM).

[0217] The inventors further performed binding assays to study FcyR binding. The data (Fig. 10) shows SPR binding assay data of the hlgG1 WT and “clamp” double mutants binding recombinant FcyRs. Representative sensorgrams show binding of the WT versus clamp versions to the different immobilised FcyRs and show retained binding of the clamp in each case. This shows that the “clamp” double mutants retain binding to FcyRs, which are key effector molecules required for multiple antibody functions, including antibody dependent cellular phagocytosis (ADCP).

[0218] Antibody dependent cellular phagocytosis (ADCP)

[0219] To formally demonstrate that the clamp” double mutants retain binding to FcgRs, and can elicit antibody dependent cellular phagocytosis (ADCP), we performed ADCP assays with hlgG1 WT and clamp anti-CD40 mAb using hCD40 expressing jurkat cells as targets with monocyte derived macrophages (Fig. 11). The results show a dose dependent increase in % ADCP with both the h IgG 1 WT and clamp anti-CD40 mAb increasing % ADCP above the isotype controls. The data shows that the clamp molecules retain the ability to trigger ADCP, equivalent to that seen with WT hlgG1.

[0220] In vivo agonism assays

[0221] The inventors studied CD40 antibodies evoking agonism in vivo. They performed immunostimulatory activity assays comparing the “clamp” double mutants to the wild-type for hlgG1 in vivo (Figs 15 and 16). In one experiment, activity is displayed with respect to expansion of OVA specific (SIINFEKL+) CD8 T cells after immunisation of hCD40 Tg mice with ovalbumin (OVA). The data show that the hlgG1 clamp and hlgG1 heavy chain K222C mutation have similar activity, greater than the light chain kE123C mutant (Fig. 15). In another experiment, activity is displayed with respect to induction of OVA specific antibodies after immunisation of hCD40 Tg mice with ovalbumin (OVA). The data show that the hlgG 1 clamp has greater immunostimulatory activity than the parent molecule and the WT hlgG2 variant (Fig. 16).

[0222] Discussion

[0223] The initial data shows that the IgG antibodies tested so far containing the mutations (both double mutants (123C+222C) and single (222C) mutants) are more active compared to wild type IgG antibodies. The introduction of this disulphide bond leads to reduced flexibility of the antibody. There is no significant impact on binding or specificity, or interaction with FcyR, leading to effector functions such as ADCP. The position of these mutations within the heavy and light constant regions will make this a mutation able to be introduced into other antibodies of interest. As shown, the double mutants (123C+222C) have already been successfully incorporated into 7 different antibodies, resulting in expected reductions in Rg and Dmax.

[0224] The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

[0225] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0226] Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0227] The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

[0228] References

[0229] Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallographica Section D 66, 486-501. doi: 10.1107 / S0907444910007493.

[0230] Gorrec, F. (2009). The MORPHEUS protein crystallization screen. J Appl Crystallogr 42, 1035- 1042. 10.1107 / S0021889809042022.

[0231] Heckel, F., Turaj, A.H., Fisher, H., Chan, H.T.C., Marshall, M.J.E., Dadas, O., Penfold, C.A., Inzhelevskaya, T., Mockridge, C.I., Alvarado, D., et al. (2022). Agonistic CD27 antibody potency is determined by epitope-dependent receptor clustering augmented through Fc- engineering. Commun Biol 5, 229. 10.1038 / s42003-022-03182-6.

[0232] Luft, J.R., and DeTitta, G.T. (1999). A method to produce microseed stock for use in the crystallization of biological macromolecules. Acta Crystallogr D Biol Crystallogr 55, 988-993. 10.1107 / S0907444999002085.

[0233] Murshudov, G.N., Skubak, P., Lebedev, A. A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A. A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallographica Section D 67, 355-367. doi: 10.1107 / S0907444911001314.

[0234] Orr, C.M., Fisher, H., Yu, X., Chan, C.H., Gao, Y., Duriez, P.J., Booth, S.G., Elliott, I., Inzhelevskaya, T., Mockridge, I., et al. (2022). Hinge disulfides in human lgG2 CD40 antibodies modulate receptor signaling by regulation of conformation and flexibility. Sci

[0235] Immunol 7, eabm3723. 10.1126 / sciimmunol.abm3723.

[0236] Yu, X., James, S., Felce, J.H., Kellermayer, B., Johnston, D.A., Chan, H.T.C., Penfold, C.A., Kim, J., Inzhelevskaya, T., Mockridge, C.I., et al. (2021). TNF receptor agonists induce distinct receptor clusters to mediate differential agonistic activity. Commun Biol 4, 772. 10.1038 / S42003-021 -02309-5.

Claims

CLAIMS1. An antigen binding protein comprising at least two CH1 domains, wherein the at least two CH1 domains comprise a cysteine residue at an amino acid position corresponding to position 222 according to Kabat numbering of human IgG 1 CH1 domain.

2. The antigen binding protein according to claim 1 , wherein the antigen binding protein comprises at least two CH1 domains and at least two corresponding CL domains.

3. The antigen binding protein according to claim 1 or 2, wherein the antigen binding protein comprises an antibody, or an antibody fragment.

4. The antigen binding protein according to claim 3, wherein the antibody is a human antibody or a humanised antibody, optionally selected from the group consisting of IgG, IgE, IgD, IgM and IgA.

5. The antigen binding protein according to claim 3, wherein the antibody fragment is selected from the group consisting of F(ab’)2, a Fab2, DNL-Fab3, a DNL-Fab2-scFV, a DNL- Fab2-lgG-cytokine2, a scFab-lgG(kih), a Fab-scFab-lgG(kih), a LLIZ-Y scFab-IgG, a scFab- Fc(kih)-scFv2, a scFab-Fc(kih)-scFv, a TriFabs, a CODV-ig, a F(ab’)2 fusion (e.g. F(ab’)2- scFv2), an a scFv2-CH1-hinge / CL.

6. The antigen binding protein according to any one of claims 1 to 5, wherein the antigen binding protein is monospecific or multispecific, optionally wherein the multispecific antigen binding protein is bi-specific, tri-specific, tetra-specific, or penta-specific.

7. The antigen binding protein according to any one of claims 1 to 7, wherein the antigen binding protein specifically binds a cell surface receptor (such as a tumour necrosis factor receptor (TNFR)) or an immunoglobulin receptor superfamily member (IgSF), or a ligand thereof.

8. The antigen binding protein according to any one of claims 1 to 6, wherein the antigen binding protein comprises or consists of an antibody selected from the group consisting of anti-CD40 (optionally LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally SAP9), anti-4- 1bb (optionally Utomilumab, Urelumab, SAP1.3, SAP1.3 ND, or SAP3.28), anti-CD28 (optionally TGN14-12), anti-CD27 (optionally hCD27.131A), anti-ICOS, anti-PD1 (such as Nivolumab), anti-DR4 and anti-DR5.

9. The antigen binding protein according to any one of claims 1 to 8, wherein at least one of the CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, optionally wherein at least one of the CH1 domains comprises or consists of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

10. The antigen binding protein according to claim 9, wherein the at least two CH 1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, optionally wherein the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.11 . The antigen binding protein according to any one of claims 2 to 9, wherein at least one of the at least two CL domains comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain, optionally wherein the at least two CL domains comprise a cystine residue at an amino acid position corresponding to position 123 according to Kabat numbering of human lgG1 CL domain.

12. The antigen binding protein according to claim 11 , wherein at least one of the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ I D NO: 9, or SEQ I D NO: 10, , optionally wherein at least one of the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9, or SEQ ID NO: 10.

13. The antigen binding protein according to claim 12, wherein the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 9, or SEQ ID NO: 10, optionally wherein the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 9, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 10.

14. A nucleic acid encoding CH1 domain and / or CL domain as defined in claims 1 to 13.

15. An expression vector comprising the nucleic acid according to claim 14.

16. A host cell comprising the expression vector of claim 15.

17. A pharmaceutical composition comprising the antigen binding protein according to any one of claims 1 to 13, the nucleic acid according to claim 14, the expression vector according to claim 15, and / or the cell according to claim 16; and further comprising a pharmaceutically acceptable diluent, carrier or excipient.

18. The antigen binding protein according to any one of claims 1 to 13, the nucleic acid according to claim 14, the expression vector according to claim 15, the cell according to claim 16, and / or the pharmaceutical composition according to claim 17 for use as a medicament.

19. A method of increasing activity of an antigen binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into the at least two CH1 domains of the antigen binding fragment, wherein the mutation is a cysteine substitution of an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human lgG1 CH1 domain.

20. A method of increasing binding of an antigen binding protein to an antigen, the antigen binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into the at least two CH1 domains of the antigen binding fragment, wherein the mutation is a cysteine substitution of an amino acid residue at a position corresponding to position 222 according to Kabat numbering of human IgG 1 CH1 domain.

21. The method of claim 18 or 20, wherein the antigen binding protein comprises at least two CH1 domains and at least two corresponding CL domains.

22. The method according to any one of claims 19 to 21 , wherein the antigen binding protein comprises an antibody, or an antibody fragment.

23. The method according to claim 22, wherein the antibody is a human antibody, optionally selected from the group consisting of IgG, IgE, IgD, IgM and IgA.

24. The method according to claim 22, wherein the antibody fragment is selected from the group consisting of F(ab’)2, a Fab2, DNL-Fab3, a DNL-Fab2-scFV, a DNL-Fab2-lgG- cytokine2, a scFab-lgG(kih), a Fab-scFab-lgG(kih), a LLIZ-Y scFab-IgG, a scFab-Fc(kih)- scFv2, a scFab-Fc(kih)-scFv, a TriFabs, a CODV-ig, a F(ab’)2 fusion (e.g. F(ab’)2-scFv2), or a scFv2-CH1-hinge / CL.

25. The method according to any one of claims 18 to 24, wherein the antigen binding protein is monospecific or multispecific, optionally wherein the multispecific antigen binding protein is bi-specific, tri-specific, tetra-specific, or penta-specific.

26. The method according to any one of claims 19 to 25, wherein the antigen binding protein specifically binds a cell surface receptor (such as a tumour necrosis factor receptor (TNFR)) or an immunoglobulin receptor superfamily member (IgSF).

27. The method according to any one of claims 19 to 26, wherein the antigen binding protein comprises or consists of an antibody selected from the group consisting of anti-CD40 (optionally LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally SAP9), anti-4-1 BB (optionally Urelumab, SAP1.3, SAP1.3 ND, SAP3.28 and Utomilumab), anti-CD28 (optionally TGN14-12), anti-CD27 (optionally hCD27.131A), anti-ICOS, anti-PD1 (such as Nivolumab), anti-DR4 and anti-DR5.

28. The method according to any one of claims 19 to 27, wherein at least one of the at last two CH1 domains comprises or consists of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, optionally wherein at least one of the at least two CH1 domains comprises or consists of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

29. The method of claim 28, wherein the at least two CH1 domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, optionally wherein the at least two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

30. The method according to any one of claims 21 to 29, further comprising the step of introducing a mutation into at least one of the at least two CL domains, wherein the mutation is a cystine substation of an amino acid residue at a position corresponding to position 123 according to Kabat numbering of human IgG 1 CL domain, optionally wherein the mutation is introduced into both of the at least two CL domains.31 . The method according to claim 30, wherein at least one of the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or atleast 95% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally wherein at least one of the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

32. The method according to claim 30, wherein the at least two CL domains comprise or consist of a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, optionally wherein the at least two CL domains comprise or consist of a sequence according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

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