Antigen binding protein

Abstract

This invention relates to a novel antigen-binding protein comprising at least two CH1 domains, wherein each of the at least two CH1 domains contains a cysteine ​​residue at an amino acid position corresponding to Kabat number 222 according to the human IgG1 CH1 domain. The invention also provides related nucleic acids, expression vectors, and host cells. Furthermore, the invention provides pharmaceutical compositions comprising said antigen-binding protein. Finally, the invention provides methods for enhancing the activity of the antigen-binding protein.

Description

Technical Field

[0001] This invention relates to a novel antigen-binding protein comprising at least two CH1 domains, wherein each of the at least two CH1 domains contains a cysteine ​​residue at an amino acid position corresponding to Kabat number 222 according to the human IgG1 CH1 domain. The invention also provides related nucleic acids, expression vectors, and host cells. Furthermore, the invention provides pharmaceutical compositions comprising said antigen-binding protein. Finally, the invention provides methods for enhancing the activity of the antigen-binding protein. Background Technology

[0002] Currently, therapeutic antibodies are used to treat several major diseases, including autoimmune diseases, cardiovascular diseases, infectious diseases, cancer, and inflammatory diseases. The antibody market was estimated to be worth $236.9 billion in 2023 and is projected to grow at a CAGR of 13.4% from 2023 to 2033, reaching $834.2 billion by 2033. Based on disease indications, the cancer field is the largest segment of the antibody market due to rising cancer prevalence and increased antibody adoption in cancer treatment. Market expansion is driven by an increasing number of drug pipelines, growing demand for biosimilar antibodies, and improved patient awareness of cancer treatments. With increasing focus on cancer research, the antibody market outlook is expected to remain positive over the next decade.

[0003] It is precisely the inherent properties of antibodies, such as high specificity and affinity, that make them an attractive source of biological therapeutics. However, they are not without their limitations. For example, weak binding and / or off-target binding of antibodies can lead to altered efficacy and increased toxicity. For instance, utomilumab has relatively low efficacy, while urelumab, despite its antitumor efficacy, causes severe dose-dependent hepatotoxicity.

[0004] Therefore, alternative engineering strategies are needed to generate improved antibodies, such as those with increased activity and / or binding affinity. This invention aims to provide such improved antibodies and other forms of antigen-binding proteins. Summary of the Invention

[0005] In one aspect, the present invention provides an antigen-binding protein comprising at least two CH1 domains, wherein the at least two CH1 domains contain cysteine ​​residues at amino acid positions corresponding to Kabat number 222 according to the human IgG1 CH1 domain.

[0006] Appropriately, antigen-binding proteins may contain at least two CH1 domains and at least two corresponding CL domains.

[0007] Appropriately, antigen-binding proteins may include antibodies or antibody fragments.

[0008] Appropriately, the antibody may be a human antibody or a humanized antibody, optionally selected from the group consisting of IgG, IgE, IgD, IgM and IgA.

[0009] Suitable, the antibody fragment may be selected from the group consisting of F(ab')2, Fab2, DNL-Fab3, DNL-Fab2-scFV, DNL-Fab2-IgG-cytokine 2, scFab-IgG(kih), Fab-scFab-IgG(kih), LUZ-Y scFab-IgG, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, TriFabs, CODV-ig, F(ab')2 fusions (e.g., F(ab')2-scFv2), and scFv2-CH1-hinge / CL.

[0010] Appropriately, the antigen-binding protein may be monospecific or multispecific, and optionally, the multispecific antigen-binding protein may be bispecific, trispecific, tetraspecific, or pentaspecific.

[0011] Appropriately, antigen-binding proteins can specifically bind to cell surface receptors such as tumor necrosis factor receptor (TNFR) or members of the immunoglobulin receptor superfamily (IgSF) or their ligands.

[0012] Suitablely, 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-4-1BB (optionally, utolumab, uroselumab, 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 will be understood by those skilled in the art, when known antibodies, such as, for example, utolumab or uroselumab, are mentioned herein, unless the context otherwise requires (e.g., when referring to antibodies specifically described in the art), these antibodies contain one or more mutations described herein.

[0013] Suitablely, at least one of the CH1 domains may contain or consist of a sequence having or consisting of 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 may contain or consist of a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0014] Suitablely, at least two CH1 domains may contain or consist of sequences having 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 two CH1 domains may contain or consist of sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0015] Suitably, at least one of the at least two CL domains may contain a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain, optionally wherein the at least two CL domains may contain a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain.

[0016] Suitablely, at least one of the at least two CL domains may contain or consist of a sequence having 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 contains or consists of a sequence according to SEQ ID NO: 9 or SEQ ID NO: 10.

[0017] Suitablely, at least two CL domains contain or consist of sequences having 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 two CL domains contain or consist of sequences according to SEQ ID NO: 9 or SEQ ID NO: 10.

[0018] In a further aspect, the present invention provides a nucleic acid encoding a CH1 domain and / or a CL domain, wherein the CH1 domain contains a cysteine ​​residue at an amino acid position 222 corresponding to a Kabat number according to the human IgG1 CH1 domain, and the CL domain contains a cysteine ​​residue at an amino acid position 123 corresponding to a Kabat number according to the human IgG1 CL domain.

[0019] In a further aspect, the present invention provides an expression vector comprising the nucleic acid of the present invention.

[0020] In a further aspect, the present invention provides a host cell comprising the expression vector of the present invention.

[0021] In a further aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding protein, nucleic acid, expression vector and / or host cell of the present invention; and further comprising a pharmaceutically acceptable diluent, carrier or excipient.

[0022] In a further aspect, the present invention provides antigen-binding proteins, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions of the present invention for use as pharmaceuticals.

[0023] In a further aspect, the present invention provides a method for increasing the activity of an antigen-binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into at least two CH1 domains of the antigen-binding fragment, wherein the mutation is the substitution of an amino acid residue at position 222 corresponding to the Kabat number of the human IgG1 CH1 domain with a cysteine ​​residue.

[0024] In a further aspect, the present invention provides a method for increasing the 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 at least two CH1 domains of the antigen-binding fragment, wherein the mutation is the substitution of an amino acid residue at position 222 corresponding to the Kabat number according to the human IgG1 CH1 domain by a cysteine ​​residue.

[0025] It should be understood that, unless the context otherwise requires, considerations regarding the statements about antigen-binding proteins in this disclosure should be considered applicable to the methods, uses, nucleic acids, expression vectors, host cells, and / or pharmaceutical compositions described herein.

[0026] Throughout this specification and claims, the words “comprising” and “including”, and variations thereof, mean “including but not limited to”, and are not intended to exclude other parts, additives, components, wholes, or steps.

[0027] Unless the context otherwise requires, the singular form includes the plural form throughout the description and claims. In particular, where the indefinite article is used, unless the context otherwise requires, the description should be understood to include both the plural and singular forms.

[0028] Unless incompatible with it, the features, wholes, properties, compounds, chemical parts or groups described in connection with a particular aspect, embodiment or example of the invention shall be understood to be applicable to any other aspect, embodiment or example described herein.

[0029] The patents, scientific and technical documents cited herein establish the knowledge available to a person skilled in the art at the time of filing. The full disclosure of all published patents, published and pending patent applications, and other publications cited herein is incorporated herein by reference as if each were specifically and individually indicated to be incorporated herein by reference. In the event of any inconsistency, this disclosure shall prevail.

[0030] The various aspects of the invention are described in more detail below. Attached Figure Description

[0031] Embodiments of the invention are further described below with reference to the accompanying drawings, wherein: Figure 1 A) is a diagram showing the localization of residues T222 and E123 in the background of the F(ab')2 fragment of IgG2. B) is a diagram showing the predicted disulfide bond connections in the T222C and E123C mutants. C) shows the crystal structure of the F(ab')2 region and the localization of T222 and E123 residues in the background of the entire structure.

[0032] Figure 2 SDS-PAGE studies performed under reducing and non-reducing conditions show hIgG1 (h1 clamp), hIgG2 (h2 clamp), and hIgG4 (h4 sp clamp) variants expressing "clamp" double mutations (222C and 123C). Under reducing conditions, two bands were observed for the heavy and light chains at approximately 52 kDa and 23 kDa, respectively. Under non-reducing conditions, several bands were observed, indicating some heterogeneity; however, the dominant band observed in all three isotypes was at approximately 150 kDa, representing intact IgG.

[0033] Figure 3 The results show the immunostimulatory activity assays of the "clamp" double mutant for hIgG1, hIgG2, and hIgG4 compared to the wild type.

[0034] Figure 4Comparative immunostimulatory activity assays for single mutants and "clamp" double mutants are presented. This indicates that the 222C mutation in the heavy chain (K222C in IgG1 and T222C in IgG2) is sufficient to produce agonistic activity. The "clamp" consists of two mutations (222C and 123C) in the heavy and light chains, respectively. Data show that the activity of the 222C mutant itself is equivalent to that of the intact clamp.

[0035] Figure 5 The structures of the WT hIgG1 and hIgG clamps, as well as the crystal structure of the hIgG2 clamp, are shown. A) The F(ab')2 portion of WT hIgG1 extracted from PDB:1HZH. B) Crystal structure of the hIgG1 clamp. The introduction of the clamp mutant resulted in compression and twisting of the two Fab arms of both hIgG1 and hIgG2. New disulfide bonds connect the opposing heavy chain constant regions in the two Fab arms. C) The disulfide bond between C222 on the opposing hIgG1 heavy chain is highlighted. D) Crystal structure of the hIgG2 clamp. E) The disulfide bond between C222 on the opposing hIgG2 heavy chain is highlighted.

[0036] Figure 6 Small-angle X-ray scattering (SAXS) data of hIgG1, hIgG2, and hIgG4 variants with “clamp” double mutations as F(ab’)2 fragments compared to the WT hIgG F(ab’)2 fragment of each isotype are shown. Information about particle flexibility can be derived from the dimensionless Kratky plot. For tightly packed spherical particles, the curve will peak at the Guinier-Kratky point (shown as a gray crosshair) and return to the baseline as a Gaussian-like curve / distribution. An increase in the curve peak above and to the right of the Guinier-Kratky point indicates more flexible particles (as observed for WT hIgG1 and hIgG4), while a shift in the peak toward the Guinier-Kratky point indicates more compact particles (as seen for the hIgG1 clamp and hIgG4SP clamp). hIgG2 is already compact in the WT form; however, the introduction of the “clamp” double mutation does result in a slight compaction. The hIgG4SP clamp contains the mutation S241P in the hinge region, which prevents Fab arm exchange.

[0037] Figure 7Negative staining electron microscopy (nsEM) images of hIgG1 WT (IgG1) and the hIgG1 “clamp” double mutant variant, which are not lattice-constrained, are shown. Class-averaged plots of hIgG1 WT (left) and the hIgG1 “clamp” double mutant variant (right) are presented. In both images, the Fc region is oriented at the bottom of the box, with the Fab arms at the top. hIgG1 WT shows a series of conformations of the Fab arms relative to the Fc. In contrast, the orientation of the Fab arms relative to the Fc in the hIgG1 “clamp” double mutant variant is much more constrained, resulting in the two Fab arms being closer together.

[0038] Figure 8 SPR binding assay data for the hIgG1, hIgG2, and hIgG4 "clamp" double mutants are shown. The representative sensor plots above illustrate binding for the WT isotype and the "clamp" version of the isotype. Small differences were observed for the hIgG2 and hIgG4 isotypes, while a slight increase in maximum binding to the "clamp" double mutant was observed in the hIgG1 isotype compared to WT.

[0039] Figure 9 As shown Figure 7 The images show negative staining electron microscopy (nsEM) images of the unconstrained hIgG1 WT (IgG1) and hIgG1 “clamp” double mutant variants. Fab-Fab angles were calculated by plotting a segmented line from the outermost edge of one Fab domain to the particle center and then to the outermost edge of the second Fab domain using the angle tool in ImageJ, and then calculating the angle measurements using the Analyze->Measure tool. The analysis shows that the Fab arms in the hIgG1 clamp exhibit a tighter angular distribution than wild-type hIgG1, with a lower average angle and range.

[0040] Figure 10 SPR binding assay data for hIgG1 WT and the "clamp" double mutant bound to recombinant FcgRs are presented. Representative sensor plots illustrate the binding of WT and clamp versions to different immobilized FcgRs, and show the retained binding of the clamp in each case.

[0041] Figure 11 This study demonstrates antibody-dependent phagocytosis (ADCP) of hIgG1 WT and clamp anti-CD40 mAb using monocyte-derived macrophages as target cells, employing Jurkat cells expressing hCD40. The data show that the clamp molecule retains the ability to trigger ADCP.

[0042] Figure 12The results of an immunostimulatory activity assay for hIgG1 against LOB7 / 6 anti-CD40 mAb, compared to wild-type, are shown.

[0043] Figure 13 shows small-angle X-ray scattering (SAXS) data for hIgG1 variants with "clamp" double or single mutations. Information about particle flexibility can be derived from the dimensionless Kratky plot. For tightly packed spherical particles, the curve will peak at the Guinier-Kratky point (shown as a gray crosshair) and return to the baseline as a Gaussian-like curve / distribution. An increase in the peak above and to the right of the Guinier-Kratky point indicates more flexible particles (as observed for WThIgG1), while a shift in the peak toward the Guinier-Kratky point indicates more compact particles (as seen for hIgG1 clamp and hIgG1 K222C). The hIgG1 kE123C variant shows an intermediate effect. (A) These molecules were evaluated as Fab2 fragments to allow for the highest resolution study of the Fab arms in the absence of Fc. (B) These molecules were evaluated as whole IgG fragments, thus including Fc movement.

[0044] Figure 14 shows the results of immunostimulatory activity assays against hIgG1 in the "clamp" double mutant compared to wild-type cells when using primary immunized B cells. A) shows the cell surface regulation of cell adhesion, CD23, CD86, and HLA-DR as determined by flow cytometry, and the results of immunostimulatory activity assays as determined by flow cytometry. 3 Increased B cell proliferation activity was measured by H-thymidine uptake. Further data showed that the clamp and single HC mutants retained IgG-equivalent activity, as demonstrated by B) CD23 expression, C) CD86 expression, D) HLA-DR expression, and E) B cell proliferation. F) provides B cell adhesion data from three donors. For each of these, h2 is included as a reference baseline, but h1 is a direct control for the clamp.

[0045] Figure 15 The results of immunostimulatory activity assays of the "clamp" double mutant compared to wild-type hIgG1 are shown in vivo. The activity for the expansion of OVA-specific CD8 T cells (OT-1; SIINFEKL+) following immunization of hCD40 Tg mice with ovalbumin (OVA) is also shown. The data indicate that the hIgG1 clamp and the hIgG1 heavy chain K222C mutant have similar activity, greater than the light chain kE123C mutant.

[0046] Figure 16The results of an in vivo immunostimulatory activity assay of the "clamp" double mutant compared to wild-type hIgG1 are shown. The activity for inducing OVA-specific antibodies after immunization of hCD40-Tg mice with ovalbumin (OVA) is shown. The data indicate that the hIgG1 clamp has greater immunostimulatory activity than the parental molecule and the WT hIgG2 variant.

[0047] Figure 17 Small-angle X-ray scattering (SAXS) data for the hIgG1 variant with the "clamp" double mutation versus WT hIgG1 are shown. Information regarding particle flexibility can be derived from the dimensionless Kratky plot. For tightly packed spherical particles, the curve will peak at the Guinier-Kratky point (shown as a gray crosshair) and return to baseline as a Gaussian-like curve / distribution. Elevations in the curve peak above and to the right of the Guinier-Kratky point indicate more flexible particles (as observed with WThIgG1), while shifts in the peak toward the Guinier-Kratky point indicate more compact particles (as seen with the hIgG1 clamp). Note: These molecules were evaluated as Fab2 fragments to allow for the highest resolution study of the Fab arms in the absence of Fc.

[0048] Figure 18 Data from small-angle X-ray scattering (SAXS) data are shown for hIgG1 variants with a "clamp" double mutation compared to WT hIgG1. Information related to radius of gyration (rg) and maximum size (Dmax) was derived using the ScAtter program.

[0049] Figure 19 The results of an immunostimulatory activity assay of a "clamp" double mutant against five different mAbs targeting 4-1BB, CD27, or OX40, compared to wild-type hIgG1, are shown. Detailed Implementation

[0050] Previously, the inventors used CD40 antibodies to link steering disulfide rearrangement in the hinge region of human (h)IgG2 antibodies with agonistic activity by mediating the orientation of native disulfide bonds in the hinge region through the substitution of cysteine ​​with serine. More specifically, the inventors demonstrated that the agonistic activity of hIgG2 antibody fragments was mediated by inducing different hinge disulfide bond conformations in the F(ab')2 fragment (Orr et al., 2022).

[0051] Compared to hIgG1 and hIgG4, the inventors' previously demonstrated methods for mediating agonistic activity rely on (and are also limited by) the presence of additional cysteine ​​residues in the hIgG2 antibody hinge. This is therefore an unfavorable engineering strategy, as 74% of approved IgG mAb-related therapies belong to the IgG1 subclass and 13% to the IgG4 subclass. Despite high levels of sequence homology among IgG subclasses, they are distinguished by small sequence differences in the constant region of the heavy chain, the hinge length connecting the antigen-binding (Fab) domain and the crystallizable fragment (Fc) domain, the number of disulfide bonds between heavy chains within the hinge region, and the position of disulfide bonds between the heavy and light chains. Therefore, agonistic activity of other IgG subclasses and other antibody classes cannot be mediated by altering the disulfide bond conformation in the hinge region. Consequently, the inventors set out to find alternative engineering strategies to generate antibodies with higher activity than hIgG2.

[0052] This invention is based on a method developed by the inventors for reducing the flexibility of Fab arms on immunoglobulins other than hIgG2. In particular, the inventors identified that the 222C mutation in each heavy chain CH1 domain and optionally the 123C mutation in each light chain CL domain surprisingly restricts the flexibility of Fab arms relative to each other between different IgG subclasses (including hIgG1, hIgG2, and hIgG4) by forming non-natural disulfide bonds between constant region Fab arms.

[0053] Surprisingly, conformational restrictions provided by non-natural disulfide bonds had no negative impact on binding affinity. Even more surprisingly, at high concentrations, the double mutant containing both the 222C and 123C mutations, as well as the single mutant 222C, actually increased binding affinity relative to WT hIgG1.

[0054] Not wanting to be bound by theory, the inventors believe that this invention can be applied to reducing the Fab arm flexibility of all antibodies. However, in the context of antibodies targeting cell membrane-binding targets (such as receptors, e.g., tumor necrosis factor receptor or members of the immunoglobulin receptor superfamily)), this invention can be particularly applicable to antibodies that bind at sites not close to the cell membrane. The inventors believe that targets not close to the cell membrane can be better bound by antibodies containing the mutations described herein. Examples of antibodies that bind to targets at such membrane-free sites include, but are not limited to, uroselumab, Lob7 / 4, and / or MK-5890.

[0055] In one aspect, the present invention provides an antigen-binding protein comprising at least two CH1 domains.

[0056] The term "antigen-binding protein" refers to a protein molecule (protein, protein-like, or protein-containing) that is capable of binding to a target molecule (i.e., an antigen) using specific intermolecular interactions. An antigen is a molecule 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 "specific binding" or "specific binding" refers to a protein with a specific binding density of approximately 1 × 10⁻⁶. -6 M or less (such as about 1×10) -7 M or less, approximately 1×10 -8 M or less, approximately 1×10 -9 M or less, approximately 1×10 -10 M or less, approximately 1×10 -11 M or less, or about 1×10 -12 Used with a given antigen in amounts of M or less.

[0057] Antigen-binding proteins may include antibodies or antibody fragments. Appropriately, antigen-binding proteins may consist of antibodies or antibody fragments.

[0058] Antigen-binding proteins can be conjugated to compounds. For example, antigen-binding proteins can be conjugated to compounds selected from the group consisting of: detectable markers, therapeutic compounds (e.g., small molecules), nucleic acids, peptides, proteins, and compounds that increase the half-life of proteins. In a particular instance, the antigen-binding protein is conjugated to a detectable marker. Exemplary detectable markers can be selected from the group consisting of radioactive markers, fluorescent markers, enzyme markers, and imaging agents. Exemplary proteins can 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 an amino acid sequence derived from the immunoglobulin heavy chain. A polypeptide containing a heavy chain constant region typically includes at least one of the following: a CH1 domain, a hinge (e.g., upper hinge, middle hinge, and / or lower hinge) domain, a CH2 domain, a CH3 domain, or variants or fragments thereof.

[0060] As set forth in this disclosure, those skilled in the art will understand that the heavy chain constant region can be modified to differ in amino acid sequence from naturally occurring immunoglobulin molecules. Suitablely, the heavy chain constant region is modified in the CH1 region. Suitablely, the modified CH1 region contains a cysteine ​​residue at the amino acid position corresponding to position 222 of the Kabat number according to human IgG1 CH1. In this document, an antigen-binding protein containing a cysteine ​​residue at the amino acid position corresponding to position 222 of the Kabat number according to human IgG1 CH1 may be referred to as a “222C mutant” or “222C variant”.

[0061] As mentioned elsewhere herein, the inventors have identified that the presence of a cysteine ​​residue at amino acid position 222, corresponding to the Kabat number according to human IgG1 CH1, results in the formation of a disulfide bond between the two CH1 domains of an antigen-binding protein. The inventors have demonstrated that this disulfide bond can enhance the activity of the antigen-binding protein. The inventors believe this is because the disulfide bond restricts the flexibility of the CH1 domains relative to each other. As those skilled in the art will understand, this can further restrict the flexibility of the binding domains of the antigen-binding protein, such as the light chain variable region and the heavy chain variable region, thereby enhancing the activity of the antigen-binding protein.

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

[0063] The CH1 region of the antigen-binding protein disclosed herein can be derived from any naturally occurring immunoglobulin molecule sequence. Therefore, the CH1 domain can be derived from the CH1 sequence of IgG, IgM, IgA, IgE, or IgM. Suitablely, IgG can be selected from the group consisting of hIgG1, hIgG4, hIgG2, or hIgG3. More suitablely, hIgG can be selected from the group consisting of hIgG1, hIgG4, or hIgG2.

[0064] As used herein, the phrase "corresponding to an amino acid position" refers to the amino acid position number within the human IgG1 CH1 domain. The corresponding amino acid position in other immunoglobulins can be found by comparing it with other sequences (such as sequences from non-human and / or non-hIgG1 CH1 domains). Sequence alignment can be performed using any alignment program known in the art. By way of example only, alignment programs such as ALIGN, ClustalW, or similar programs can be used. Aligning sequences or fragments of sequences, and thereby determining the corresponding positions of the amino acid positions in the sequence according to the invention, is considered well-known in the art.

[0065] The wild-type CH1 domain amino acid sequence of human IgG1 is: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KV (SEQ ID NO:5) - The amino acid at position 222 is indicated in bold and underline. The sequences shown below, SEQ ID NO: 6, 7, and 8, are the wild-type sequences of the CH1 domain of human IgG2, human IgG3, and human IgG4, respectively (the amino acid at position 222 is indicated in bold and underline):

[0066] It should be understood that, within the context of this disclosure, antigen-binding proteins (such as antibodies and antibody fragments) may each comprise one or more CH1 domains according to SEQ ID:1, 2, 3 and / or 4, as follows:

[0067] Suitablely, such CH1 domains can be variants of SEQ ID: 1, 2, 3, and / or 4. In this context, the term "variant" means that the CH1 domain contains a cysteine ​​residue at position 97, and at least one further mutation compared to the wild-type sequence (e.g., 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). Suitablely, variants can have sequence identity with SEQ ID NO: 1, 2, 3, or 4 of 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 higher.

[0068] As used herein, the term "sequence identity" refers to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences as determined by an algorithm such as that described in Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268 (as modified by Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877). Such algorithms are integrated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. A BLAST nucleotide search is performed using the NBLAST program (score=100, word length=12) to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. A BLAST protein search is performed using the XBLAST program (score=50, word length=3) to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gap-aligned sequences for comparative purposes, use Gapped BLAST as described by Attschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. When using BLAST and Gapped BLAST programs, use the default parameters of the respective programs (e.g., XBLAST and NBLAST). Other algorithms, programs, and default settings may also be suitable, such as, but not limited to, the GCG-Sequence Analysis Package from the UK Human Genome Mapping Project Resource Centre, which includes programs for nucleotide or amino acid sequence alignment.

[0069] The antigen-binding protein disclosed herein comprises at least two CH1 domains. Appropriately, the at least two CH1 domains may be the same or different (as long as they both contain a cysteine ​​residue at position 222 corresponding to the human IgG1 CH1 domain).

[0070] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence having 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.

[0071] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence having 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.

[0072] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence having 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.

[0073] In a suitable embodiment, at least one of the CH1 domains comprises or consists of a sequence having 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.

[0074] In a suitable implementation, at least two CH1 domains comprise or consist of a sequence having 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 two CH1 domains comprise or consist of a sequence according to SEQ ID NO: 1.

[0075] In a suitable implementation, at least two CH1 domains comprise or consist of sequences having 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 two CH1 domains comprise or consist of sequences according to SEQ ID NO: 2.

[0076] In a suitable implementation, at least two CH1 domains comprise or consist of sequences having 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 sequences according to SEQ ID NO: 3.

[0077] In a suitable implementation, at least two CH1 domains comprise or consist of sequences having 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 two CH1 domains comprise or consist of sequences according to SEQ ID NO: 4.

[0078] In this paper, the amino acid positions of all constant regions and domains of the heavy and light chains are numbered according to the Kabat numbers in Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991), referred to herein as “according to Kabat numbers” or “Kabat numbers”.

[0079] In suitable embodiments, the antigen-binding protein of the present 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.

[0080] As used herein, the term "light chain constant region" or "CL domain" encompasses the amino acid sequence of the antibody light chain adjacent to the VL region. There are two types of mammalian light chains, lambda (λ) and kappa (κ), which differ slightly in their polypeptide sequences. Every naturally occurring antibody contains two identical light chains. However, it should be understood that engineered antibodies, such as chimeric antibodies, can contain two distinct light chains. Other types of light chains also exist in nature, including the iota (ι) chain found in lower vertebrates.

[0081] A light-heavy chain pair refers to a combination of a light chain and a heavy chain that can form a dimer through disulfide bonds between the CL domain of the light chain and the CH1 domain of the heavy chain. Therefore, in the context of this disclosure, the phrase "corresponding CL domain" refers to the CL domain that forms a dimer with the CH1 domain through disulfide bonds.

[0082] In appropriate embodiments, the antigen-binding proteins of this disclosure may include or consist of antibodies or antibody fragments.

[0083] 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 immune responses in any vertebrate (e.g., humans, goats, rabbits, alpacas, llamas, sheep, camels, and mice, as well as non-mammal species such as sharks). The term "antibody" also includes genetically engineered forms such as chimeric antibodies (e.g., humanized antibodies) and heterologous conjugate antibodies (e.g., bispecific antibodies). Antibodies may contain one or more polypeptides. For example, mammalian IgG contains two identical light chains and two identical heavy chains. Therefore, such antibodies contain two polypeptides. The resulting antibody has three functional components: two fragment-binding domains (Fab), a crystallizable fragment (Fc), wherein the two Fab are linked to the Fc via hinge regions.

[0084] It should be understood that, in the context of this disclosure, when referring to a specific antibody type (such as IgG, IgD, IgE, IgA, or IgM), it means an antibody having a backbone of said specific antibody type but containing a cysteine ​​residue at the amino acid position corresponding to position 222 of the CH1 domain, or having a backbone with at least 80% sequence identity to the backbone of said specific antibody type. For example, when an antibody is referred to as a human IgG1 antibody, this means that the antibody has a human IgG1 backbone containing a cysteine ​​residue at position 222 of the CH1 domain, or has a backbone with 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 to the human IgG1 backbone.

[0085] Suitablely, in the context of this invention, the antibody may be a human antibody or a humanized antibody. Suitablely, the human or humanized antibody may be selected from the group consisting of IgG, IgA, IgD, IgE, and IgM. Suitablely, hIgG may be hIgG1, hIgG2, hIgG4, or hIgG3.

[0086] In suitable embodiments, the antibody may be uroselumab or nivolumab. It should be understood that, in the context of this disclosure, when referring to a particular antibody, such as an anti-CD40 antibody (e.g., LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), the amino acid sequence of said antibody is known in the art, except that there is a cysteine ​​residue at position 222 of the CH1 domain.

[0087] Appropriately, antigen-binding proteins can specifically bind to cell surface receptors such as tumor necrosis factor receptor (TNFR) or members of the immunoglobulin receptor superfamily (IgSF).

[0088] Appropriately, the antigen-binding protein may comprise or consist of antibodies selected from the group consisting of: anti-CD40 (optionally, LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally, SAP9), anti-4-1bb (optionally, utolumab, SAP1.3, SAP1.3 ND, SAP3.28, and urerutumab), anti-CD28 (optionally, TGN14-12), anti-CD27 (optionally, hCD27.131A), anti-ICOS, anti-PD1 (such as nivolumab), anti-DR4, and anti-DR5.

[0089] Suitablely, the antigen-binding proteins of the present invention may include antibodies. Non-limiting examples of antigen-binding proteins including antibodies include IgG2, CovX-Body, κ / λ-body universal HC, IgG(kih), IgG(kih) universal LC, ZW1 IgG universal LC, bicionics universal LC, orthogonal Fab IgG(kih), duetMab, CH3 charge pair + CH1 / CL charge pair, duobody, quadruple CrossMab (kih), LUZ-Y universal LC, FcFc', IgG(kih)-Fv, IgG(HA-TF-Fv), IgG(kih)-scFab, and DVI-Ig. (Four-in-one), IgG-HC-scFv, IgG-dAb, IgG-taFv, IgG-CrossFab, IgG-OrthogonalFab, scFv-HC-IgG, Tandem Fab-IgG (OrthogonalFab), IgG-scFv(LC), scFv(LC)-IgG, Dab-IgG, DVD-Ig, TVD-Ig, scFv4-IgG, Zybody, scFv4-Ig, DAF, dutaMab, Mab2 or DNL-Fab4-IgG.

[0090] Suitable, the antigen-binding protein of the present invention may include antibody fragments. Non-limiting examples of antigen-binding proteins including antibody fragments include F(ab')2, Fab2, DNL-Fab3, DNL-Fab2-scFV, DNL-Fab2-IgG-cytokine 2, scFab-IgG(kih), Fab-scFab-IgG(kih), LUZ-Y scFab-IgG, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, TriFabs, CODV-ig, F(ab')2 fusions (e.g., F(ab')2-scFv2), or scFv2-CH1-hinge / CL.

[0091] Appropriately, antigen-binding proteins (such as antigen-binding proteins comprising antibodies or antibody fragments or composed of them) can be single-specific or multi-specific.

[0092] "Specificity" refers to the selective recognition of a specific epitope of an antigen by an antigen-binding protein. As used herein, "single-specific" means that the antigen-binding protein has one or more binding sites, each binding to the same epitope on the same antigen. "Multi-specific" antigen-binding proteins bind to two or more different epitopes (e.g., two, three, four or more different epitopes). Epitopes can be on the same or different antigens. Multi-specific antigen-binding proteins can be, for example, bispecific, trispecific, tetraspecific, or pentaspecific.

[0093] As mentioned, the antigen-binding protein of the present 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 contain a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain, optionally wherein at least two CL domains contain a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain.

[0094] The amino acid sequence of the wild-type human IgG1 κCL domain is: RTVAAPSVFIFPPSD E QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 11).

[0095] The amino acid sequence of the wild-type human IgG1 λCL domain is: GQPKAAPSVTLFPPSS EELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 12).

[0096] It should be understood that, within the context of this disclosure, antigen-binding proteins (such as antibodies and antibody fragments) may each comprise one or more CL domains according to SEQ ID:9 or 10, as follows: RTVAAPSVFIFPPSD C QLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 9); GQPKAAPSVTLFPPSS C ELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 10).

[0097] SEQ ID NO: 9 is the sequence of the human IgG1 κ CL domain (SEQ ID NO: 11), wherein a cysteine ​​residue is present at amino acid position 123 corresponding to the Kabat number according to the human IgG1 CL domain. For the same reason, SEQ ID NO: 10 is the sequence of the human IgG1 κ CL domain (SEQ ID NO: 12), wherein a cysteine ​​residue is present at amino acid position 123 corresponding to the Kabat number according to the human IgG1 CL domain. In this document, antigen-binding proteins containing a cysteine ​​residue at amino acid position 123 corresponding to the Kabat number according to the human IgG1 CL domain may be referred to as “123C mutant” or “123C variant”.

[0098] Furthermore, antigen-binding proteins containing cystine at amino acid position 123 corresponding to the Kabat number according to human IgG1 CL and amino acid position 222 corresponding to the Kabat number according to human IgG1 CH1 domain are referred to as "double mutants", "double variants", "123C / 222C mutants" or "123C / 222C variants".

[0099] Appropriately, such CL domains may be variants of SEQ ID:9 and / or 10. In this context, the term "variant" means that, compared to the wild-type sequence, the CH domain contains a cysteine ​​residue at position 16 of SEQ ID NO:9 or position 17 of SEQ ID NO:10, and at least one further mutation (e.g., 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).

[0100] In a suitable embodiment of the antigen-binding protein described herein, at least one of the at least two CL domains comprises or consists of a sequence having 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 comprises or consists of a sequence according to SEQ ID NO: 9.

[0101] In suitable embodiments of the antigen-binding protein described herein, at least one of the at least two CL domains comprises or consists of a sequence having 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 comprises or consists of a sequence according to SEQ ID NO: 9.

[0102] The antigen-binding protein disclosed herein may comprise at least two CL domains. Suitably, the at least two CL domains may be identical or different, provided that at least one or both of the CL domains contain a cysteine ​​residue at position 123 corresponding to the human IgG1 CL domain.

[0103] In a suitable embodiment of the antigen-binding protein described herein, at least two CL domains comprise or consist of a sequence having 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.

[0104] In a suitable embodiment of the antigen-binding protein described herein, at least two CL domains comprise or consist of a sequence having 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.

[0105] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0106] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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, and at least one of the CL domains comprises or consists of a sequence having 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.

[0107] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0108] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0109] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0110] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0111] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0112] In a suitable embodiment of the antigen-binding protein of the present invention, at least one of the CH1 domains comprises or consists of a sequence having 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 comprises or consists of a sequence having 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.

[0113] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 1, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 9.

[0114] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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, and at least two of the CL domains comprise or consist of sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 1, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 10.

[0115] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 2, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 9.

[0116] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 2, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 10.

[0117] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 3, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 9.

[0118] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 3, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 10.

[0119] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 4, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 9.

[0120] In a suitable embodiment of the antigen-binding protein of the present invention, at least two of the CH1 domains comprise or consist of sequences having 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 sequences having 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 CH1 domains comprise or consist of the sequence according to SEQ ID NO: 4, and at least two of the CL domains comprise or consist of the sequence according to SEQ ID NO: 10.

[0121] In a further aspect, the present invention provides a nucleic acid encoding the CH1 domain and / or CL domain of the present disclosure. It should be understood that, when referring to the CH1 domain of the present invention, this refers to a CH1 domain containing a cysteine ​​residue at amino acid position 222 corresponding to the Kabat number of the human IgG1 CH1 domain. For the same reason, when referring to the CL domain of the present invention, this refers to a CL domain containing a cysteine ​​residue at amino acid position 123 corresponding to the Kabat number of the human IgG1 CL domain.

[0122] Nucleic acids can include DNA, cDNA, and / or RNA.

[0123] Nucleic acids can be single-stranded or double-stranded.

[0124] Nucleic acids can be naturally occurring, synthetic, and / or recombinant.

[0125] Nucleic acids can contain nucleotide analogs or derivatives (e.g., inosine or phosphate-thioester nucleotides). Silent mutations in coding sequences are caused by 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, 3rd edition, WH 5 Freeman and Co., NY).

[0126] In a further aspect, the present invention provides an expression vector comprising a nucleic acid encoding the CH1 domain and / or CL domain disclosed herein.

[0127] As used herein, the term "expression vector" refers to a nucleic acid construct containing a gene expression control region (such as a promoter or promoter component) operatively linked to a nucleotide sequence encoding at least one polypeptide (such as a polypeptide encoding the CH1 domain and / or CL domain disclosed herein). A vector can be any vector capable of transferring DNA into a cell.

[0128] Appropriately, the carrier is either an integrated carrier or a free carrier.

[0129] Preferred integration vectors include recombinant retroviral vectors. The recombinant retroviral vector will comprise at least a portion of the DNA of the retroviral genome capable of infecting target cells. The term "infection" is used to refer to the process by which a virus transfers genetic material to its host or target cells. Suitablely, the retrovirus used to construct the vector of the present invention is also made replication-deficient to eliminate the influence of viral replication on the target cells. In this case, the replication-deficient viral genome can be packaged by a helper virus according to conventional techniques. Generally, any retrovirus meeting the above-described criteria for infectiousness and functional gene transfer capability can be used in the practice of the present invention. Suitablely, the retroviral vector can be a lentiviral vector. Lentiviral vectors are well known in the art and have been used to deliver genes to a variety of cell types, including HeLa or B lymphocytes. Other vectors that can be used in the present invention include adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV, and poxvirus vectors.

[0130] Preferred free vectors include transient non-replicating free vectors and self-replicating free vectors, which have functions originating from viral replication origins such as those derived from EBV, human papillomavirus (BK), and BPV-1. Such integrative and free vectors are well known to those skilled in the art and are well described in the main body of literature familiar to them. In particular, suitable free vectors are described in WO98 / 07876.

[0131] Mammalian artificial chromosomes can also be used as the vector for this invention. Calos discussed the uses of mammalian artificial chromosomes (1996 Trends in Genetics). 12 : 463-466).

[0132] In a preferred embodiment, the vector of the present invention is a plasmid. The plasmid can be a non-replicating, non-integrating plasmid.

[0133] As used herein, the term "plasmid" refers to any nucleic acid that encodes an expressible gene, including linear or circular nucleic acids as well as double-stranded or single-stranded nucleic acids. Nucleic acids can be DNA or RNA and can contain modified nucleotides or ribonucleotides, and can be chemically modified by means such as methylation or by including protective groups or cap or tail structures.

[0134] Non-replicating, non-integrating plasmids are nucleic acids that, when transfected into a host cell, do not replicate and do not specifically integrate into the host cell's genome (i.e., they do not integrate at high frequencies and do not integrate at specific sites). Replicating plasmids can be identified using standard assays, including the standard replication assay of Ustav et al. (1991 EMBO J 10: 449–457).

[0135] 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 a CL domain; or a composition comprising a first expression vector containing the first nucleic acid and a second expression vector containing the second expression vector.

[0136] The present invention also provides cells transformed or transfected with (i.e., containing) the expression vector (or a composition of expression vectors) of the present invention. Such cells are referred to as “host cells.” Host cells can be any cell suitable for expressing the antigen-binding proteins or fragments of the present invention, such as the CH1 domain and / or CL domain. Host cells can be yeast, bacteria, insects, plants, or mammalian cells. Of particular interest are bacteria (such as *Escherichia coli*), fungi (such as *Saccharomyces cerevisiae* and *Pichia pastoris*), insect cells (such as SF9), mammalian cell lines (e.g., human cell lines), and primary cell lines. The antigen-binding proteins or fragments of the present invention (such as the CH1 domain and / or CL domain) can be expressed by culturing host cells transformed with an expression vector containing a nucleic acid that individually encodes the CH1 domain and / or CL domain, or a combination thereof with other domains commonly found in antibodies and discussed elsewhere herein, under conditions and for a time sufficient to allow protein expression. Such conditions for protein expression will vary depending on the choice of expression vector and host cell and will be readily determined by those skilled in the art through routine experiments. For example, antibodies expressed in *E. coli* can be refolded from inclusion bodies (see, for example, Hou et al. (1998) Cytokine 10:319-30). Bacterial expression systems and their methods of use are well known in the art (see *Current Protocols in Molecular Biology*, Wiley & Sons, and *Molecular Cloning—A Laboratory Manual—3rd Ed.*, ColdSpring Harbor Laboratory Press, New York (2001)). The selection of codons, appropriate expression vectors, and appropriate host cells will vary depending on a number of factors and can be readily optimized as needed. The antibodies (or fragments thereof) described herein can be expressed in mammalian cells or other expression systems, including but not limited to yeast, baculoviruses, and in vitro expression systems (see, for example, Kaszubska et al. (2000) Protein Expression and Purification 18:213-220).

[0137] Appropriately, the host cell is a separate cell.

[0138] According to the present invention, various techniques are known and can be used to deliver the vectors described herein to host cells, including the use of nucleic acid agglutinants, electroporation, asbestos conjugation, polygluconine, DEAE cellulose, dextran, liposomes, cationic liposomes, lipopolyamines, polyornithine, particle bombardment, and direct microinjection (see Kucherlapati and Skoultchi (1984 Crit. Rev. Biochem 16: 349-379); Keown et al (1990 Methods Enzymol 185:527-37) reviews).

[0139] In a further aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding protein, nucleic acid, expression vector and / or cells of the present invention, and pharmaceutically acceptable diluent, carrier or excipient.

[0140] The composition may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvant immune enhancers (such as adjuvants and cytokines), and optionally other therapeutic agents or compounds.

[0141] As used herein, “pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., that the material can be administered to an individual with a selected nucleic acid composition, carrier system, or host cell without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition containing it.

[0142] Excipients are natural or synthetic substances formulated with an active ingredient (e.g., nucleic acid sequences, carriers, modified cells, or isolated peptides provided herein) for the purpose of increasing the volume of the formulation or imparting a therapeutic enhancement to the active ingredient in the final dosage form, such as promoting drug absorption or dissolution. Excipients may also be used in the manufacturing process to assist in the handling of the relevant active substance, such as by improving powder flowability or anti-sticking properties, and also to contribute to in vitro stability, such as preventing denaturation during the expected shelf life. Pharmaceutically acceptable excipients are well known in the art. Therefore, suitable excipients can be readily identified by those skilled in the art. For example, suitable pharmaceutically acceptable excipients include water, saline, aqueous glucose solutions, glycerol, ethanol, etc.

[0143] An adjuvant is a pharmacological and / or immunological agent that alters the action of other agents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. Therefore, suitable adjuvants can be readily identified by those skilled in the art.

[0144] A diluent is a diluting agent. Pharmaceutically acceptable diluents are well known in the art. Therefore, suitable diluents can be readily identified by a person skilled in the art.

[0145] At the dosage and concentration used, the carrier is non-toxic to the receptor and compatible with other components of the formulation. The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient binds to facilitate application. Pharmaceutically acceptable carriers are well known in the art. Therefore, suitable carriers can be readily identified by those skilled in the art.

[0146] In a further aspect, the present invention provides a method for increasing the 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.

[0147] The above describes the appropriate implementation methods of antigen-binding proteins.

[0148] As used herein, the term "activity" refers to the desired function of an antigen-binding protein. For example, activity can be agonistic or antagonistic.

[0149] Tumor necrosis factor receptor (TNFR) activation is an example of agonistic activity. TNFR plays a crucial role in immune activation and represents a promising target for next-generation cancer immunotherapy. Selective TNFR activation using TNFR-targeted mAbs and recombinant ligands has been shown to enhance anti-tumor immunity and confer potent therapeutic benefits in animal models. CD40 is a member of the TNFR superfamily expressed on antigen-presenting cells (APCs) and controls key immunostimulatory pathways required for both humoral and cellular immunity. Binding to CD40 with agonistic anti-CD40 antibodies (alternatives to CD40L expressed by CD4 T cells) effectively promotes APC maturation and consequently antigen-specific CD8+ cytotoxic T cell activation and expansion, thereby enhancing anti-tumor responses.

[0150] Activity is generally considered a result of antigen-binding proteins binding to antigens. The inventors believe that the antigen-binding proteins of the present invention can have increased activity compared to corresponding antigen-binding proteins lacking the 222C and optionally 123C mutations described herein. In this context, the term "corresponding" means an antigen-binding protein that has the same amino acid sequence in other respects.

[0151] The inventors have demonstrated that antigen-binding proteins as described herein can possess increased binding affinity compared to corresponding antigen-binding proteins lacking the 222C and optionally 123C mutations described herein. The inventors believe that it is this increased binding affinity that leads to increased activity. Therefore, the present invention also provides a method for increasing the binding of antigen-binding proteins. Suitably, the antigen-binding protein comprises at least two CH1 domains and optionally at least two corresponding CL domains.

[0152] As used herein, the terms “increased,” “increased,” or “higher” generally mean a statistically significant increase in quantity; for the avoidance of any doubt, the terms “increased” or “increased” mean an increase of at least about 5% 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% compared to a reference value. For example, an increase compared to a reference value could be 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 higher. As those skilled in the art will understand, a reference value refers to the value of appropriate parameters (such as activity and / or binding) of a control antigen-binding protein that does not contain the 222C and optionally 123C mutations described herein. It should be understood that such control antigen-binding proteins may otherwise be identical to the antigen-binding proteins of the present invention.

[0153] The method of the present invention (i.e., the method of increasing activity and / or the method of increasing binding) comprises: the step of introducing a mutation into at least two CH1 domains of an antigen-binding fragment, wherein the mutation is the substitution of an amino acid residue at position 222 corresponding to a Kabat number according to the human IgG1 CH1 domain with a cysteine ​​residue; and optionally introducing a mutation into at least one of at least two CL domains, wherein the mutation is the substitution of an amino acid residue at position 123 corresponding to a Kabat number according to the human IgG1 CL domain with a cysteine ​​residue, optionally wherein the mutation is introduced into each of the at least two CL domains.

[0154] As used herein, the term "introducing a mutation" refers to modifying nucleotide residues (adenine nucleotide residues, guanine nucleotide residues, thymine nucleotide residues, cytosine nucleotide residues, uracil nucleotide residues) encoding the CH1 domain and optionally the CL domain. Upon introducing a mutation into the nucleic acid, the resulting protein will consist of a cysteine ​​residue at position 222 (Kabat number according to the human IgG1 CH1 domain) and optionally a cysteine ​​residue at position 123 (Kabat number according to the human IgG1 CL domain), wherein, optionally, a mutation is introduced into each of at least two CL domains.

[0155] It should be understood that the methods for increasing activity and / or binding as described herein can be applied to existing (e.g., therapeutic) antigen-binding proteins, provided they contain at least two CH1 domains and optionally at least two CL domains.

[0156] Unless otherwise defined 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*, HarperPerennial, NY (1991) provide general dictionaries of the various terms used in this invention for those skilled in the art. Although any methods and materials similar to or equivalent to those described and used herein may be used in the practice of this invention, preferred methods and materials are described herein. Therefore, the terms defined below will be described more fully by reference to the entire specification. Furthermore, as used herein, unless the context clearly indicates otherwise, the singular terms “a,” “an,” and “the” include plural referents. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino-to-carboxyl orientation, respectively. It should be understood that the present invention is not limited to the specific methods, schemes and reagents described, as these may vary depending on the context in which those skilled in the art use them.

[0157] The aspects of the present invention are demonstrated by the following non-limiting examples.

[0158] Example

[0159] In the following examples, the "clamp" mutant refers to the double mutant of 222C and 123C.

[0160] Materials and Methods

[0161] Expression of hIgG1, hIgG2 and hIgG4 variants

[0162] Use Gibco ExpiCHO TM The transient expression system produces antibodies. Detailed antibody information is shown in Table 1. ExpiFectamine was used. TM CHO reagent and OptiPRO TM SFM complex medium, used for DNA transfection into ExpiCHO TM ExpiCHO-S cultured in expression medium TM Cells. Cells were incubated at 37°C with 8% CO2 and shaken at 125 rpm. On day 1, ExpiFectamine was added. TM CHO enhancer and ExpiCHO TM Cells were added to the feed. After 10 days, the cell culture was centrifuged at 3230 g for 40 minutes to harvest the cell supernatant.

[0163] Antibodies were purified from cell supernatant using protein A affinity purification with a MabSelect SuRe protein A column (Cytima) attached to a GE ÄKTA Initiation Protein Purification System (Cytiva). Bound IgG was eluted with a low-pH glycine buffer. All antibodies were examined by HPLC for <1% aggregates; if >1% aggregates were detected, purification was performed by gel filtration using a HiLoadSuperdex 200 pg 16 / 600 size exclusion column (Cytiva). Endotoxin levels were assessed using an Endosafe Portable Assay System (PTS) device (Charles River Laboratories), and only if <10 endotoxin units per milligram of antibody were identified.

[0164] Table 1 Antibody Sequences

[0165] The Lob7-4 sequence is derived from the sequence described in US20090074711. The utolumab sequence is derived from US2015 / The sequence described in 014199. The uroselamab sequence is derived from the sequence described in WO2010 / 042433 A1. TGN1412 sequence. The sequence is derived from the sequence described in US7585960. The hCD27.131A sequence is derived from the sequence described in US2018 / 0086841.

[0166] F(ab') 2 Fragment generation

[0167] To generate the F(ab')2 fragment, full-length IgG was digested with pepsin to remove the Fc domain. The antibody was first dialyzed into a digestion buffer (pH 8) containing 20 mM Tris-HCl, 100 mM NaCl, and 1 mM EDTA. The antibody was then concentrated to approximately 10 mg / mL. Prior to digestion, the pH of the dialyzed antibody solution was adjusted to 4.1 with 2 M sodium acetate (pH 3.7) to achieve optimal pepsin activity. Pepsin was prepared at a concentration of 10 mg / mL in acetate buffer (70 mM acetate, 50 mM NaCl, pH 4), and the pepsin solution was added to the antibody to achieve a pepsin to IgG ratio of 3% w / w. The antibody was digested at 37°C, and the digestion process was monitored by HPLC, with the sample placed on ice to pause digestion during HPLC runs. Over time, a decrease in the IgG peak and an increase in the F(ab')2 peak were observed. When the proportion of F(ab')2 stopped increasing, the cleavage was stopped by adjusting the pH to 8.0 using 1 M Tris. F(ab')2 was purified by gel filtration using a HiLoad Superdex 200 pg 16 / 600 size size column with 1 / 5 Tris-NaCl as buffer to remove undigested IgG. The combined fractions were purified on a HiTrap 5 mL MabSelect SuRe Protein A column (Cytiva) to remove any residual IgG and Fc, and then analyzed by HPLC to confirm the purity of F(ab')2.

[0168] Immunostimulatory activity assay

[0169] Transfect NF-kB / Jurkat / GFP with pCIpuro plasmids encoding full-length hCD40, h4-1BB (expressing the intracellular signaling domain of hCD40), hOX40 (expressing the intracellular domain of hCD40) (Yu et al., 2021), or hCD27 (Heckel et al., 2022). TMCell lines. Stable clones were selected with 0.25 μg / mL puromycin and incubated at 37°C in a humidified incubator with 5% CO2, then cultured in RPMI 1640 medium (Gibco, ThermoFisher) supplemented with 10% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U / mL penicillin, and 100 μg / mL streptomycin (all from ThermoFisher). To assess NFkB activation, Jurkat cells were incubated with serially diluted mAb or F(ab')2 fragments at 37°C for 24 h. The degree of NFkB activation was quantified by GFP fluorescence using flow cytometry. Flow cytometry data were acquired using FACS Canto II (BD Biosciences) and analyzed using FlowJo (BD Biosciences).

[0170] The immunostimulatory activity against 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 the National Blood Service, Southampton, with prior informed consent. The use of human blood for these assays was approved by the Research Ethics Service of East Scotland, Teyside, UK. Human B cells were purified from PBMCs by negative sorting using the MojoPort Human B Cell Isolation Kit (BioLegend). B cells were incubated in vitro with anti-hCD40 mAb in 96-well round-bottom plates.

[0171] To measure isomorphic adhesion, B cells were imaged using a conventional optical microscope (Olympus CKX41, running Olympus-CellSen standard software) 48 hours after mAb addition. Adhesion was observed as large macroscopic cell populations. 48 hours later, the upregulation of B cell activation markers was assessed by flow cytometry using APC-labeled anti-CD23 mAb (clone EBVCS-5, BioLegend), PerCP-Cy5.5-labeled anti-CD86 mAb (clone BU63, BioLegend), and Brilliant Violet-labeled anti-MHCII mAb (clone L243, BioLegend). To assess B cell proliferation, B cells were stimulated with the above mAbs for 4 days, with 1 µCi added to each well during the last 18 hours of incubation. 3 H-thymidine (PerkinElmer). Cells were collected and analyzed using scintillation counting (TopCount) to measure... 3 H-thymidine incorporation amount.

[0172] Surface plasmon resonance (SPR)

[0173] SPR was performed using a Biacore T200 instrument (Cytiva). Recombinant soluble hCD40-hFc-His (R&D Systems, Inc.) was immobilized onto a CM5 sensor chip via amine coupling, targeting a level of 50 resonance units (RU), according to the manufacturer's protocol. mAbs were injected into the flow cell at a flow rate of 30 μL / min in HBS-EP+ running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% w / w surfactant P20, pH 7.4) at 25°C with concentrations of 100, 20, 4, 0.8, 0.16, or 0 nM, for 300 seconds for binding and 300 seconds for dissociation. The chip was regenerated using 10 mM glycine (pH 1.5) at a flow rate of 30 μL / min for 30 seconds. All reagents, equipment, and software were purchased from Cytiva. The output of the SPR experiment is a sensor map, which shows the change in refractive index over time on the sensor chip surface, measured with an arbitrary RU. The sensor map is fitted with a 1:1 binding model, and ka (binding rate), kd (dissociation rate), and KD (equilibrium dissociation constant) are calculated using Biacore Bioevaluation software.

[0174] The binding of FcγRs to mAbs was analyzed by SPR using a Biacore T200 instrument. In short, anti-His antibodies were immobilized onto a CM5 sensor chip via amine coupling, according to the manufacturer's protocol. Recombinant molecules (from R&D Systems, Inc.) containing different FcγRs and His tags were then flowed onto the chip at a specified concentration, and mAbs (e.g., hIgG1 or hIgG1 clamps) were then flowed at 200 nM in HBS-EP+ running buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% surfactant P20, pH 7.4) for 120 s to allow binding, followed by 120 s of dissociation. If necessary, the chip was regenerated using 10 mM glycine (pH 1.5) at a flow rate of 30 μL / min for 30 s. A sensor map was generated to display the binding spectrum. Unless otherwise specified, all reagents, equipment and software are obtained from Cytiva.

[0175] X-ray crystallography

[0176] Protein crystallization

[0177] F(ab')2 samples were transferred to 50 mM Hepes, 150 mM KCl buffer (pH 7.5) using an Amicon ultracentrifuge filter (Millipore, Sigma Aldrich) for buffer replacement. The samples were concentrated and resuspended in HepesKCl 4–5 times, and finally concentrated to approximately 10 mg / mL for crystallization assays. Crystallization screening was set up using a 96-well, 3-drop Intelliplate (SwissSci) with a sit-drop vapor diffusion method on an Oryx8 protein crystallizer (Douglas Instruments, UK). The trays were incubated in a Rumed temperature-controlled incubator at 21°C. Both the TCR / pMHC optimized protein crystallization screen (TOPS) and the commercially available MORPHEUS screen (Gorrec, 2009) were used as licensed screens because they had previously provided good results in F(ab')2 within our team. The plates were set up using a 2:1 ratio (protein to screening conditions) for the top droplets, a 1:1 ratio for the middle droplets, and a 1:2 ratio for the bottom droplets. Seed stock was prepared using MicroSeed beads (Molecular Dimensions) adapted from a method (Luft and DeTitta, 1999), with initial hit crystals (crystal hits). Approximately 40 μL was removed from the mother liquor reservoir, and a small amount was added dropwise onto the droplets containing the selected hit crystals. The hit crystals were broken up by repeatedly aspirating and discharging the liquid from the droplets. The crystals were then transferred to seed bead tubes on ice and vortexed. Pure and diluted seed stock tubes were prepared. For F(ab')2, which did not pass initial screening, cross-seeding was used to attempt to initiate crystal growth. Seeding and cross-seeding experiments were set up using an Oryx8 protein crystallizer. Crystals grew for several weeks, and the crystal growth on the trays was checked periodically using optical and UV microscopes. Crystals are harvested using litholoops (molecular dimensions) of various sizes. Before harvesting, a cryoprotectant (a mother liquor containing 20% ​​glycerol) is added to the crystal droplets to protect them during rapid cooling. The litholoops containing the crystals are then rapidly frozen in liquid nitrogen and stored in Unipucks within Dewar flasks containing liquid nitrogen until diffraction experiments and data collection.

[0178] Data collection

[0179] Standard X-ray diffraction data were collected using beamline ID30A-3 at the European Synchrotron Radiation Facility (ESRF, France). ID30A-3 is a fixed-energy microfocal beamline operating at 12.81 keV (0.978 Å) with a 15 μm diameter X-ray beam. Data were collected using an Eiger X 4M detector, with samples held at 100 K in cryogenic flow. The experiments were controlled using MXCuBE3 beamline control software. Experiments were recorded in ExiMX (an extension of ISPyB for MX).

[0180] Sulfur single-wavelength anomalous diffraction (S-SAD) data were collected at the Diamond Light Source (DLS, Harwell, UK) using the vacuum long-wavelength beamline I23. I23 is a tunable energy beamline with a range of 2.1–11 keV (1.13–5.9 Å). It operates in a high vacuum environment to prevent air absorption and X-ray scattering that occur at these wavelengths; therefore, a dedicated cryogenic transport system with thermally conductive materials was used to prepare the sample and transfer it into the vacuum; this facilitated cooling the sample to approximately 50 K. Diffraction data were collected on a curved semi-cylindrical Pilatus 12M detector with a multiaxial goniometer. 3600 images were collected for each dataset, with 0.1 degrees of oscillation per image to obtain a 360-degree dataset. Data were collected at 4.5 keV (2.76 Å). Experiments were recorded in ISPyB.

[0181] Data processing

[0182] Initial data processing and phase determination were automated using several different pipelines: Grenoble Automated Data Processing (GrenADES), EDNA Autoprocessing, autoPROC, XDSAPP, and XIA2_DIALS. All data manipulations, molecular substitutions, and refinements were performed using the CCP4 suite of procedures. Molecular substitutions were performed using Molrep, with the PDB model 6TKE as the homology model. Iterative model construction and refinement were performed using COOT (Emsley et al., 2010) and REFMAC5 (Murshudov et al., 2011), respectively.

[0183] Small-angle X-ray scattering (SAXS)

[0184] SAXS data were collected using their BioSAXS beamline B21 at the Diamond Light Source (DLS) in the UK. B21 has an energy range of 0.5–14 keV (with 13 keV as the standard), and a beam size of 1102 × 240 μm at the sample. The range of q is 0.0026–0.34 Å. -1 The sample-to-detector distance was 3.7 m, and the detector was a vacuum Dectris Eiger 4M. Data collection was performed using size exclusion chromatography combined with small-angle X-ray scattering (SEC-SAXS); this utilizes an online HPLC system for purification, allowing for size separation of the sample (removal of aggregates) before delivery to the beam. For online purification, a Superdex 200 Increase 3.2 column (8.6 μm pore size, 3.2 mm inner diameter, 30 cm length) was used in conjunction with an Agilent 1260 HPLC system. 45 μL of F(ab')2 at a flow rate of approximately 5 mg / mL was injected into the column using 50 mM HEPES and 150 mM KCl (pH 7.5) as the SEC buffer. Measurements were performed at 20°C. 600 frames of data were recorded using an Eiger 4M detector with an exposure time of 3 seconds per frame. Data were processed and preliminarily analyzed using ScÅtter (version IV, R. Rambo, DiamondLight Source). Buffer subtraction is performed before the main elution peak of the SEC-SAXS signal map by automatically selecting stable buffer frames. Data frames are selected from the main elution peak of the SEC-SAXS signal map, where each point represents the area of ​​the integral of the sample SAXS curve to the estimated background. Frames with high similarity are selected and averaged for analysis to ensure that the scattering curves show little change at low q.

[0185] Antibody-dependent phagocytosis (ADCP)

[0186] 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 (hMDM) as effector cells. hMDM was obtained by culturing monocytes for 6 days in the presence of 100 ng / ml M-CSF (internal). Two days prior to the phagocytosis assay, 7 × 10⁶ cells were transfected with CD40ECD. 42 × 10⁶ hMDM cells were seeded on 96-well ThermoFisher plates. On the day of the phagocytosis assay, target cells were labeled with 0.5 μM CFSE and then opsonized at 4°C with various CD40 mAbs (as indicated in the figure legend) for 30 min. In total, 2 × 10⁶ hMDM cells were seeded. 5 Target cells were added to each well and incubated at 37°C for 1 hour to induce phagocytosis. Samples were then stained with anti-CD14-APC (1:200) to identify hMDM, and cells positive for both CFSE and CD14 were classified as hMDM that had undergone phagocytosis by flow cytometry. The percentage of ADCP was calculated as follows: (CFSE + CD14 + cells) / (total CD14 + cells) × 100.

[0187] In vivo agonism assay

[0188] OTI amplification assay

[0189] OTI amplification assays were performed as previously described (Yu, X. et al. Isotype switching converts anti-CD40 antagonism to agonism to elicit potent antitumor activity. CancerCell 37, 850–866.e7 (2020)). To assess the ability of anti-CD40 monoclonal antibodies to induce OTI T cell amplification, 1 × 10⁻⁶ anti-CD40 monoclonal antibodies were administered intravenously one day prior to the injection of 5 mg OVA bound to 100 µg of various anti-CD40 monoclonal antibodies. 5 One OTI cell was intravenously injected into CD40KOTg mice. Then, as indicated in the figure description, blood was collected from the mice periodically, and the OTI amplification level was assessed by flow cytometry based on the proportion of CD8+ SIINFEKL tetramer-positive cells.

[0190] ELISA of anti-OVA antibodies

[0191] In the same experiments described above, serum was collected periodically, and the presence of OVA-specific antibodies was assessed by ELISA according to White et al. (2010). Briefly, to detect anti-OVA IgG in serum, 96-well MaxiSorp plates were coated overnight at 4°C with 100 µg / ml OVA coating buffer. The plates were blocked with 1% BSA, and then serially diluted serum was added, with a standard curve established using mouse anti-OVA IgG mAb KB4 (homemade). Anti-OVA IgG bound to the plates was detected by secondary antibody rabbit anti-mouse IgG HRP antibody (Sigma). Optical density was measured at 490 nm using a BioTek Epoch plate reader and Gen 5 software (Agilent).

[0192] result

[0193] The inventors identified that the mutations (T222C and kE123C) lead to the formation of disulfide bonds connecting opposing heavy chains. Figure 1 T222C is located in the CH1 domain, and kE123C is located in the κ CL domain.

[0194] hIgG variant expression

[0195] like Figure 2 As observed, the inventors successfully expressed hIgG1, hIgG2 and hIgG4 variants containing the "clamp" double mutation.

[0196] Immunostimulatory activity

[0197] The inventors then determined the immunostimulatory activity of the clamp mutant against hIgG1, hIgG2, and hIgG4 compared to the wild type. Figure 3 ).

[0198] The bioactivity of clamp double mutant variants was evaluated using an NFkB / Jurkat / GFP transcriptional reporter cell line expressing human CD40, in which GFP is generated in response to receptor activity, thus allowing quantification by flow cytometry. When compared to wild-type, the hIgG1 and hIgG4 clamp variants showed significantly improved immunostimulatory activity, while the hIgG2 clamp mutant exhibited activity similar to that of the conformationally restricted WT hIgG2.

[0199] In addition, the inventors conducted further immunostimulatory activity assays on WT, "clamp" (double mutants, 222C and 123C), and single mutants (222C or 123C), such as... Figure 4As shown in the data, surprisingly, the 222C mutation in the heavy chain (K222C in hIgG1 and T222C in hIgG2) produced increased agonistic activity relative to WT, comparable to the "clamp" double mutants (222C and 123C). These data suggest that only the 222C mutation is required to produce an increase in agonistic activity.

[0200] Further immunostimulatory activity assays were performed, comparing the LOB7 / 6 anti-CD40 mAb hIgG1 with the "clamp" double mutant against the wild type. The results of these assays showed... Figure 12 They showed that wild-type LOB7 / 6 hIgG1 was inactive in the reporting assay, but the "clamp" double mutant transformed this antibody into one with strong agonistic activity.

[0201] Primary immune B cells were also used to assess the immunostimulatory activity of the "clamp" double mutant against hIgG1, compared to the wild type (Figure 14). Activity was shown as cell adhesion, cell surface regulation of CD23, CD86, and HLA-DR as determined by flow cytometry, and as demonstrated by... 3 Increased B-cell proliferation as measured by H-thymidine uptake. Further data showed that the double-clamp and single-HC mutant K222C retained activity comparable to IgG (Figure 14 BF). These data clearly demonstrate that the "clamp" double mutant provided agonistic activity for LOB7 / 4 hIgG1 antibody in each assay; it triggers homoadhesion ( Figure 14A (See above); Upregulation of CD23, CD86, and HLA-DR on the surface of B cells and activation of their proliferation ( Figure 14A (See figure below). In each case, the wild-type LOB7 / 4 hIgG1 antibody was inactive and comparable to the isotype control. Figure 14 shows that when CD23 upregulation was measured ( Figure 14B CD86 upregulation ( Figure 14C ) and the upregulation of HLA-DR ( Figure 14D ) and proliferation ( Figure 14E At WT, the 222C mutation in the heavy chain (K222C in hIgG1) produced an increased agonistic effect, comparable to the "clamp" double mutant (222C and 123C). Conversely, the 123C mutant was inert in these assays, comparable to wild-type LOB7 / 4 hIgG1, including in homoadhesion ( Figure 14F ).

[0202] The study also measured the "clamp" double mutant against five different mAbs targeting 4-1BB, CD27, or OX40, and against hIgG1, compared to the wild type. Figure 19These results indicate that, in each case, the double-clamp mutant is more arousing than the wild-type hIgG1 antibody.

[0203] Structural Analysis

[0204] The structures of the Fab'2 of the hIgG1 clamp and hIgG2 clamp have been determined, revealing the location of the disulfide bonds introduced by the mutation. Figure 5 The introduction of the clamp mutation resulted in a compaction and twisting of the two Fab arms of both hIgG1 and hIgG2. New disulfide bonds connect the opposing heavy chain constant regions in the two Fab arms. The structure of hIgG1 is much more compact than that of hIgG2; however, the inventors believe that in solution, as indicated by SAXS data and negative staining electron microscopy, the hIgG1 structure is closer to the conformation seen in the hIgG2 crystal structure. The inventors are currently working on generating isoforms of hIgG4. They have also conducted sulfur single-wavelength anomalous dispersion (S-SAD) experiments at the I23 beamline of the Diamond Light Source in the UK to definitively identify the positions of sulfur atoms within the crystal structure, thereby confirming the positions of the disulfide bonds.

[0205] Small-angle X-ray scattering (SAXS)

[0206] Small-angle X-ray scattering (SAXS) was performed to characterize the biophysical properties of the clamp mutants hIgG1, hIgG2, and hIgG4 variants in solution.

[0207] SAXS data provide information on the conformation and flexibility of these particles in solution. The data show that variants with clamp mutations are more rigid in solution than wild-type variants. Figure 6 This is particularly evident for hIgG1 and hIgG4 isotypes, as they are naturally more flexible than hIgG2.

[0208] The peaks of hIgG1 and hIgG4 in the WT curves, rising above and to the right of the Guinier-Kratky point, indicate more flexible particles, while the peaks of hIgG1 clamp and hIgG4SP clamp shift towards the Guinier-Kratky point, indicating more compact particles. It can be seen that hIgG2 is already compact in WT form; however, the introduction of clamps does indeed lead to a further slight compaction.

[0209] Further data (Figure 13) show small-angle X-ray scattering (SAXS) data for hIgG1 variants with "clamp" double or single mutations as Fab2 fragments and IgG molecules. Information about particle flexibility can be derived from the dimensionless Kratky plot. For tightly packed spherical particles, the curve peaks at the Guinier-Kratky point (shown as a gray crosshair) and returns to the baseline as a Gaussian curve. An increase in the peak above and to the right of the Guinier-Kratky point indicates more flexible particles (as observed for WT hIgG1), while a shift of the peak toward the Guinier-Kratky point indicates more compact particles (as seen for hIgG1 clamp and hIgG1 K222C). Some molecules were evaluated as Fab2 fragments to allow for the highest resolution study of the Fab arms in the absence of Fc. Figure 13A Other molecules were evaluated as whole IgG fragments, and therefore included the movement of Fc (…). Figure 13B This data indicates that hIgG1 K222C (heavy chain single mutant) exhibits a Kratky plot similar to that of hIgG1 clamp, suggesting that the K222C heavy chain mutation alone is sufficient to reduce flexibility, and that the hIgG1kE123C mutation has a more moderate (but still measurable) effect on limiting flexibility. Note: When evaluating intact IgG versus Fab2 fragments, the differences between the curves are less pronounced because Fc also contributes to the flexibility and size of the molecule.

[0210] Further small-angle X-ray scattering (SAXS) was performed to investigate six antibodies (hCD27.131A, SAP1.3 ND, TGN1412, hCD27.15, utolumab (Uto), and urerucumab (Ure)). Figure 17 As before, the Fab2 fragment was evaluated to allow for the highest resolution study of the Fab arm in the absence of Fc. In each case, hIgG1 clamping resulted in a leftward and downward shift in the Kratky plot, with a peak at the lower qRg. This indicates reduced flexibility and increased compactness compared to wild-type hIgG1 (in Figure 18 (Further exploration was conducted). All six antibodies exhibited the same reduction in flexibility, demonstrating the versatility of the technology. Testing of three different receptors—CD27, 4-1BB, and CD28 (non-TNFRsf members)—showed that the technology can be used to target multiple types of receptors.

[0211] Figure 18Small-angle X-ray scattering (SAXS) data from an hIgG1 variant with a "clamp" double mutation, compared to WT hIgG1, are shown. Information related to radius of gyration (rg) and maximum size (Dmax) was derived using the ScAtter program. The data show that the hIgG1 clamp has a lower Rg, a lower Dmax, a smaller Porod volume, and a higher Porod index. These all indicate more compact and less flexible particles. The analysis included an evaluation of LOB7 / 4 Fab2 fragments generated by two different enzymatic digestion methods (IdeS or pepsin). Despite small differences at the Fc cleavage site, Fab2 fragments generated by either method showed a significant effect of the double clamp on inducing more compact and less flexible particles.

[0212] Negative staining electron microscope (nsEM)

[0213] Negative staining electron microscopy (nsEM) was performed to study the conformation of hIgG1 clamps that are not lattice-constrained. Figure 7 The inventors collected negative staining electron microscopy data from the hIgG1 isotype clamp mutant and wild-type versions. hIgG1 WT showed a series of conformations of the Fab arms relative to the Fc. In contrast, the orientation of the Fab arms relative to the Fc in the hIgG1 clamp was much more restricted, resulting in the two Fab arms being closer together. Furthermore, the data showed that, compared to the wild-type, the conformation of the two Fab arms relative to each other was restricted in variants containing the clamp mutant. Further analysis (…) Figure 9 The results showed that, compared to wild-type hIgG1, the Fab arms in the hIgG1 clamp exhibited a tighter angular distribution, with a smaller average angle and range. This implies that the Fab arms in the hIgG1 clamp exhibit very significant limitation.

[0214] Affinity / specificity of surface plasmon resonance (SPR)

[0215] The inventors conducted binding assays to assess whether the introduction of clamp mutants affected binding. Data showed that the hIgG1, hIgG2, and hIgG4 clamp double mutants had no negative impact on binding, with the hIgG1 clamp showing a slightly improved binding at higher concentrations compared to the wild type. Figure 8 This indicates that, in the systems tested to date, mutations do not negatively affect binding to antigens.

[0216] hCD40 was fixed on the sensor chip, and mAb was injected into the flow cell at a series of concentrations (100, 20, 4, 0.8, 0.16 or 0 nM).

[0217] The inventors further conducted binding assays to study FcγR binding. Data ( Figure 10 The image shows SPR binding assay data for hIgG1 WT and the "clamp" double mutant bound to recombinant FcγR. Representative sensor plots show the binding of WT and clamp versions to different immobilized FcγRs, and demonstrate the retention of clamp binding in each case. This indicates that the "clamp" double mutant retains binding to FcγR, a key effector molecule required for various antibody functions, including antibody-dependent phagocytosis (ADCP).

[0218] Antibody-dependent phagocytosis (ADCP)

[0219] To formally demonstrate that the "clamp" double mutant retains binding to FcgR and can induce antibody-dependent phagocytosis (ADCP), we used hCD40-expressing jurkat cells as targets, along with monocyte-derived macrophages, to perform ADCP assays on hIgG1 WT and clamp anti-CD40 monoclonal antibodies. Figure 11 The results showed that both hIgG1 WT and clamped anti-CD40 mAb exhibited a dose-dependent increase in ADCP percentage, exceeding that of the isotype control. The data indicated that the clamped molecule retained the ability to trigger ADCP, comparable to the ability observed in WT hIgG1.

[0220] In vivo agonist activity assay

[0221] The inventors studied CD40 antibodies that elicited stimulatory effects in vivo. They performed immunostimulatory activity assays, comparing the "clamp" double mutant against hIgG1 with the wild type in vivo. Figure 15 and Figure 16 In one experiment, activity was demonstrated as the expansion of OVA-specific (SIINFEKL+) CD8T cells in hCD40 Tg mice after immunization with ovalbumin (OVA). Data showed that the hIgG1 clamp and the hIgG1 heavy chain K222C mutant exhibited similar activity, greater than the light chain kE123C mutant. Figure 15 In another experiment, activity was demonstrated by the induction of OVA-specific antibodies in hCD40-Tg mice after immunization with ovalbumin (OVA). Data showed that the hIgG1 clamp exhibited stronger immunostimulatory activity than the parental molecule and the WT hIgG2 variant. Figure 16 ).

[0222] discuss

[0223] Preliminary data show that IgG antibodies containing mutations (double mutants (123C+222C) and single mutants (222C)) tested to date are more active than wild-type IgG antibodies. This introduction of disulfide bonds leads to reduced antibody flexibility.

[0224] The mutations have no significant effect on binding or specificity, or on interaction with FcγR (leading to effector functions such as ADCP). The location of these mutations within the heavy chain constant region and light chain constant region allows them to be introduced into other antibodies of interest. As shown, the double mutant (123C+222C) has been successfully introduced into seven different antibodies, resulting in the expected reduction in Rg and Dmax.

[0225] Readers should note that all papers and documents submitted at the same time as or prior to this application, and which are made publicly available together with this specification, are incorporated herein by reference.

[0226] All features disclosed in this specification (including any appended claims, abstracts and drawings) and / or all steps of any method or process so disclosed may be combined in any combination, except that at least some of such features and / or steps are mutually exclusive combinations.

[0227] Unless otherwise expressly stated, each feature disclosed in this specification (including any appended claims, abstract, and drawings) can be replaced by alternative features for the same, equivalent, or similar purposes. Therefore, unless otherwise expressly stated, each disclosed feature is merely one example in a general series of equivalent or similar features.

[0228] This invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel feature or any novel combination of features disclosed in this specification (including any appended claims, abstract, and drawings), or any novel step or any novel combination of steps in any method or process so disclosed.

[0229] References .

Claims

1. An antigen-binding protein comprising at least two CH1 domains, wherein, The at least two CH1 domains contain cysteine ​​residues at amino acid positions corresponding to position 222 of the Kabat number according to the human IgG1 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 includes antibodies or antibody fragments.

4. The antigen-binding protein according to claim 3, wherein, The antibody is a human antibody or a humanized 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 fragments are selected from the group consisting of F(ab')2, Fab2, DNL-Fab3, DNL-Fab2-scFV, DNL-Fab2-IgG-cytokine 2, scFab-IgG(kih), Fab-scFab-IgG(kih), LUZ-Y scFab-IgG, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, TriFabs, CODV-ig, F(ab')2 fusions (e.g., F(ab')2-scFv2), and scFv2-CH1-hinge / CL.

6. The antigen-binding protein according to any one of claims 1 to 5, wherein, The antigen-binding protein is a monospecific antigen-binding protein or a multispecific antigen-binding protein, optionally wherein the multispecific antigen-binding protein is bispecific, trispecific, tetraspecific, or pentaspecific.

7. The antigen-binding protein according to any one of claims 1 to 7, wherein, The antigen-binding protein specifically binds to cell surface receptors (such as tumor necrosis factor receptor (TNFR)) or members of the immunoglobulin receptor superfamily (IgSF) or their ligands.

8. The antigen-binding protein according to any one of claims 1 to 6, wherein, The antigen-binding protein comprises or is composed of antibodies, wherein the antibodies are selected from the group consisting of anti-CD40 (optionally, LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally, SAP9), anti-4-1bb (optionally, utolumab, urerutumab, 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 a sequence having 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, or is composed of a sequence having 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 a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or is composed 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 CH1 domains comprise sequences having 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, or consist of sequences having 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 sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or consist of sequences 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 contains a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain, optionally wherein the at least two CL domains contain a cysteine ​​residue at an amino acid position corresponding to position 123 of the Kabat number according to the human IgG1 CL domain.

12. The antigen-binding protein according to claim 11, wherein, At least one of the at least two CL domains comprises a sequence having 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, or is composed of a sequence having 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 a sequence according to SEQ ID NO: 9 or SEQ ID NO: 10, or is composed 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 sequences having 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, or consist of sequences having 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 sequences according to SEQ ID NO: 9, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 10, or consist of sequences according to SEQ ID NO: 9, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO:

10.

14. A nucleic acid encoding the 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 an antigen-binding protein according to any one of claims 1 to 13, a nucleic acid according to claim 14, an expression vector according to claim 15, and / or a 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 medicine.

19. A method for increasing the activity of an antigen-binding protein comprising at least two CH1 domains, the method comprising the step of introducing a mutation into said at least two CH1 domains of the antigen-binding fragment, wherein, The mutation is the substitution of a cysteine ​​residue at position 222, corresponding to the Kabat number of the human IgG1 CH1 domain.

20. A method for increasing the binding of an antigen-binding protein to an antigen, said antigen-binding protein comprising at least two CH1 domains, said method comprising the step of introducing a mutation into said at least two CH1 domains of the antigen-binding fragment, wherein, The mutation is the substitution of a cysteine ​​residue at position 222, corresponding to the Kabat number of the human IgG1 CH1 domain.

21. The method according to 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 includes antibodies or antibody fragments.

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, Fab2, DNL-Fab3, DNL-Fab2-scFV, DNL-Fab2-IgG-cytokine 2, scFab-IgG(kih), Fab-scFab-IgG(kih), LUZ-YscFab-IgG, scFab-Fc(kih)-scFv2, scFab-Fc(kih)-scFv, TriFabs, CODV-ig, F(ab')2 fusion (e.g., F(ab')2-scFv2), or scFv2-CH1-hinge / CL.

25. The method according to any one of claims 18 to 24, wherein, The antigen-binding protein is a monospecific antigen-binding protein or a multispecific antigen-binding protein, optionally wherein the multispecific antigen-binding protein is bispecific, trispecific, tetraspecific, or pentaspecific.

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

27. The method according to any one of claims 19 to 26, wherein, The antigen-binding protein comprises or is composed of antibodies selected from the group consisting of anti-CD40 (optionally, LOB7 / 6, LOB7 / 4, or ChiLOB7 / 4), anti-OX40 (optionally, SAP9), anti-4-1BB (optionally uroselumab, SAP1.3, SAP1.3 ND, SAP3.28, and uroselumab), 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 least two CH1 domains comprises a sequence having 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, or is composed of a sequence having 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 a sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or is composed 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 according to claim 28, wherein, The at least two CH1 domains comprise sequences having 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, or consist of sequences having 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 sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4, or consist of sequences 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 the substitution of an amino acid residue at position 123, corresponding to the Kabat number according to the human IgG1 CL domain, with a cysteine ​​residue, optionally wherein the mutation is introduced into each of the at least two CL domains.

31. The method according to claim 30, wherein, At least one of the at least two CL structural domains comprises a sequence having 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, or is composed of a sequence having 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 at least one of the at least two CL structural domains comprises a sequence according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or is composed 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 sequences having 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, or are composed of sequences having 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 sequences according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or are composed of sequences according to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.

Images