Monospecific antibodies and multispecific antibodies

Abstract

Disclosed herein are monospecific and multispecific antibodies having specificity for one or more of OX40, CD40, 4-1BB, HSA, IL-22 or EGFR.

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims priority to U.S. Provisional Application No. 63 / 356,385, filed on June 28, 2022, and U.S. Provisional Application No. 63 / 434,814, filed on December 22, 2022. The entire disclosures of both applications are hereby incorporated by reference into this specification.

[0002] (Sequence Listing) The sequence listing is submitted together with this specification and is incorporated herein as a 138 KB XML file named "1959708 - 00014_Sequence_Listing.xml", created on June 27, 2023.

Summary of the Invention

[0003] (Abstract) Disclosed herein are monospecific VHH antibodies specific for OX - 40, CD40, 4 - 1BB, HSA, IL - 22, and epidermal growth factor receptor (EFGR), as well as multivalent single - chain antibodies incorporating two or more VHH domains specific for one or more of these antigens.

[0004] Some embodiments are single - domain antibodies that exclusively or predominantly contain the VHH domain of the camelid antibody. These embodiments are monospecific and monovalent.

[0005] Some embodiments include a VHH domain fused to one or more constant domains from a conventional antibody, such as the Fc region of a human IgG antibody. These embodiments are monospecific but are usually bivalent. For example, depending on the choice of the constant domain, other valencies are also possible. The Fc regions of IgA and IgM can provide higher valencies.

[0006] Some embodiments include two VHH domains that are specific for the same antigen attached to a single amino acid chain (multivalent single-chain antibodies). These embodiments are also monospecific and bivalent. Additional VHH domains can be attached to achieve higher valency.

[0007] Some embodiments include two (or more) VHH domains, each having specificity for a different antigen attached to a single amino acid chain (multivalent multispecific single-chain antibodies). These embodiments are multivalent and multispecific. In further embodiments that include three or more VHH domains, two or more VHH domains may have specificity for the same antigen, while one or more other VHH domains have specificity for a different antigen. Such constructs have a higher valency than specificity.

[0008] Each monospecific embodiment has specificity for OX-40, CD40, HSA, IL-22, 4-1BB, or EFGR. Each multispecific embodiment has specificity for one or more of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR, but may also have specificity for one or more other antigens.

[0009] In some embodiments that include multiple antigen-binding domains, antigen-binding domains obtained from a conventional VL-VH pair can be used in place of one or more (but not all) of the VHH domains in the above embodiments.

[0010] The antigen-binding domains disclosed herein that have specificity for a particular antigen may sometimes be referred to as means for binding the antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0011]

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[0012] Disclosed herein are monospecific immunoglobulin variable domains (referred to as VHH single - domain antibodies) having specificity for OX - 40, CD40, 4 - 1BB, HSA, IL - 22, and epidermal growth factor receptor (EFGR), and multivalent single - chain antibodies (MVSCA) incorporating the variable domains of two or more VHHs having specificity for one or more of these antigens.

[0013] As used herein, the term VHH refers to the variable domain of a heavy - chain antibody and is the antigen - binding fragment of only the heavy - chain antibody.

[0014] In some embodiments, the MVSCA contains two or more variable domains specific for the same antigen. That is, the MVSCA is multivalent but monospecific with respect to the antigen. In some of these embodiments, the MVSCA contains two or more repeats of the same VHH variable domain, or contains multiple VHH variable domains each specific for the same epitope. That is, they are multivalent but monospecific with respect to the epitope. Such an MVSCA binds only to one site on the antigen monomer, but can crosslink multiple copies of this monomer. In other of these embodiments, the MVSCA contains two or more VHH variable domains specific for different epitopes of the same antigen. That is, they are multivalent but multispecific with respect to the epitope. Such an MVSCA can bind to multiple sites on the antigen monomer or crosslink multiple copies of the monomer.

[0015] In some embodiments, the MVSCA contains two or more VHH variable domains specific for different antigens, i.e., the MVSCA is multivalent and multispecific with respect to the antigens. In further embodiments, the MVSCA contains multiple VHH variable domains in any combination, where an additional variable domain is identical to the first VHH variable domain, and the additional VHH variable domain is different from that of the first VHH variable domain but specific for a different epitope on the same antigen, or the additional VHH variable domain is different from that of the first VHH variable domain but specific for a different antigen.

[0016] An MVSCA containing two or more VHH variable domains may further contain a constant domain of an immunoglobulin. For example, the C-terminal VHH variable domain can retain its binding to the original VHH constant domain. Alternatively, the C-terminal VHH variable domain can be bound to a more conventional antibody, such as the constant domain or Fc region of a human antibody, such as a human IgG antibody. In some embodiments, the constant domain or the complete Fc region can confer specific functions, as is well known to those skilled in the art. In another embodiment, an MVSCA containing two or more VHH variable domains may further contain a constant domain, which is arranged between or at the N-terminus of the VHH variable domains instead of or in addition to being arranged at the C-terminus of the VHH variable domain.

[0017] antigen OX40 (CD134; TNFRSF4) is a T cell co-stimulatory molecule of the tumor necrosis factor (TNF) receptor superfamily and cooperates with other co-stimulatory factors (CD28, CD40, CD30, CD27 and 4-1BB) to control the activation of the immune response. OX40 is upregulated on antigen-activated CD4 + and CD8 + T cells upon co-stimulation by CD40-CD40 ligand and CD28-B7. OX40 promotes T cell survival, proliferation and cytokine production through its interaction with OX40 ligand on antigen-presenting cells. It also inhibits the conversion from effector T cells to regulatory T cells (Tregs) and promotes the maintenance and recall of responses in memory T cells. OX40 is constitutively expressed on Tregs and promotes the proliferation and immunosuppressive activity of Tregs. OX40-OX40 ligand signaling is involved in allergic airway inflammation, graft-versus-host disease and autoimmune diseases.

[0018] CD40, also known as TNFRSF5, is a 45–50 kDa type I transmembrane glycoprotein that is a member of the TNF receptor superfamily. Mature human CD40 consists of an extracellular domain of 173 amino acids (aa), a transmembrane domain, and a cytoplasmic domain of 62 aa. The extracellular domain of human CD40 has 58% and 56% amino acid sequence identity with mouse CD40 and rat CD40, respectively. An antagonistic soluble human CD40 splice variant has an additional sequence within the extracellular and transmembrane domains and lacks the cytoplasmic domain. CD40 is expressed on the surface of B cells, dendritic cells, macrophages, monocytes, platelets, endothelial cells, and epithelial cells. Interaction of CD40 with its ligand, CD40 ligand, results in aggregation of CD40 molecules, initiating bidirectional intracellular signaling in both CD40 and CD40 ligand-expressing cells. Ligation of CD40 by CD40 ligand promotes activation of B cells and T cell-dependent humoral responses. CD40 plays multiple functions in both hematopoietic and epithelial cancers and is a target for cancer immunotherapy.

[0019] The epidermal growth factor receptor (EGFR) is a transmembrane protein that is the receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. EGFR is a member of the ErbB family of receptors, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many cancer types, mutations that affect the expression or activity of EGFR can cause cancer. The epidermal growth factor receptor is a transmembrane protein that is activated by binding to its specific ligands (e.g., epidermal growth factor and transforming growth factor α (TGFα)). Defects in the signaling of EGFR and other receptor tyrosine kinases in humans are associated with diseases such as tumors, while on the other hand, overexpression is associated with the development of various tumors. By blocking EGFR signaling by inhibiting the EGFR binding site on the extracellular domain of the receptor or by inhibiting the intracellular tyrosine kinase activity, the growth of EGFR-expressing tumors can be inhibited and the symptoms of patients can be improved.

[0020] 4-1BB, also known as CD137 and TNFRSF9, is a ~30 kDa transmembrane glycoprotein belonging to the TNF receptor superfamily. 4-1BB functions in the proliferation and activation of multiple immune cells. Mature human 4-1BB consists of an extracellular domain (ECD) of 163 aa with four TNFR cysteine-rich repeats (SEQ ID NO: 41), a transmembrane segment of 27 aa, and a cytoplasmic domain of 42 aa. Within the ECD, human 4-1BB has 60% amino acid sequence identity with mouse and rat 4-1BB. 4-1BB is expressed on various populations of activated T cells (e.g., CD4 + , CD8 + , memory CD8 +, NKT, and regulatory T cells), similarly, on the progenitor cells of myeloid and mast cells, dendritic cells, mast cells, and bacterially infected osteoblasts, it is expressed as a disulfide-linked homodimer. It binds with high affinity to the transmembrane 4-1BB ligand / TNFSF9 expressed on antigen-presenting cells and myeloid progenitor cells. This interaction co-stimulates the proliferation, activation, and / or survival of 4-1BB-expressing cells. It can also enhance the cell death induced by the activation of repeatedly stimulated T cells. In mice lacking 4-1BB, T cell activation is enhanced, probably because 4-1BB is absent in regulatory T cells. 4-1BB binds to OX40 on activated T cells to form a complex that reacts to either ligand, inhibiting the proliferation of Treg and CD8 + T cells. Reverse signaling via the 4-1BB ligand inhibits the expression of dendritic cells, B cells, and osteoclasts, but supports the survival of mature dendritic cells and co-stimulates the proliferation and activation of mast cells. The activation of 4-1BB enhances antitumor immunity via CD8 + T cells and NK cells. Also, 4-1BB is involved in the development of inflammation in high-fat diet-induced metabolic syndrome. Soluble forms of 4-1BB and 4-1BB ligand circulate at high levels in the sera of patients with rheumatoid arthritis and blood cancers, respectively.

[0021] Human serum albumin (HSA) is the serum albumin contained in human blood. It is the most abundant protein in human plasma, accounting for about half of the serum proteins. It is produced in the liver. It is highly soluble in water and is a monomer. Albumin has functions such as transporting hormones, fatty acids and other compounds, buffering pH, and maintaining colloid osmotic pressure. Albumin is synthesized in the liver as a proalbumin precursor, which has an N-terminal peptide. This N-terminal peptide is removed before the nascent protein is released from the rough endoplasmic reticulum. The resulting proalbumin is cleaved within the Golgi vesicles to become secreted albumin. The reference range for albumin concentration in serum is about 35 - 50 g / L (3.5 - 5.0 g / dL). The half-life in serum is about 20 days. The molecular weight is 66.5 kDa. The long serum half-life of albumin is achieved by its size of 66 kDa, which prevents clearance in the kidney, and its interaction with the neonatal Fc receptor (FcRn). Fusion to anti-albumin VHH has been used to extend the half-life of anti-tumor nanobodies from 1 - 2 hours to about 10 days.

[0022] Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducible factor (IL-TIF), was first identified as a gene induced by IL-9 in mouse T cells and mast cells. Human IL-22 cDNA encodes a protein of 179 amino acid (aa) residues with a putative signal peptide of 33 amino acids (aa), and cleavage of this signal peptide generates a mature protein of 147 aa that has approximately 79% and 22% amino acid sequence identity with mouse IL-22 and human IL-10, respectively. The human IL-22 gene is located on chromosome 12q15. It exists as a single-copy gene in humans and many mouse strains, but multiple copies of the mouse IL-22 gene are present in some mouse strains including C57B1 / 6, FVB, and 129. Two mouse genes named IL-TIFα and IL-TIFβ have more than 98% sequence homology in their coding regions. IL-22 has been shown to activate STAT-1 and STAT-3 and upregulate the production of acute-phase proteins in several hepatoma cell lines. IL-22 is produced from normal T cells by anti-CD3 stimulation in humans. Mouse IL-22 expression is also induced in various organs by lipopolysaccharide injection, suggesting that IL-22 may be involved in the inflammatory response. The functional IL-22 receptor complex is composed of two receptor subunits belonging to the class II cytokine receptor family, IL-22R (an orphan receptor previously called CRF2-9) and IL-10Rβ (previously known as CRF2-4).

[0023] antibody Antibodies and the use of antibodies for the treatment of diseases are well known in the art. As used herein, the term "antibody" refers to a monomeric or multimeric protein comprising one or more polypeptide chains containing an antigen-binding site. Antibodies can specifically bind to an antigen and modulate the biological activity of the antigen. The term "antibody" as used herein can include "full-length antibodies" and "antibody fragments". As used herein, the terms "binding site" or "antigen-binding site" refer to the region of the antibody molecule to which a ligand actually binds. The term "antigen-binding site" includes the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL), or in the case of a heavy chain-only antibody, the antibody heavy chain variable region.

[0024] The specificity of an antibody refers to the selective recognition of an antibody for a specific epitope of an antigen. For example, natural antibodies are monospecific. As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites that each bind to the same epitope of the same antigen. The monospecific antibodies disclosed herein are specific for OX-40, CD40, 4-1BB, HSA, IL-22 or EFGR. In some embodiments, the monospecific antibody comprises only the VHH domain heavy chain. In another embodiment, the monospecific antibody comprises a VHH domain fused to one or more protein domains, such as the human Fc region. In yet another embodiment, the monospecific antibody comprises a VHH as the only complete protein domain, i.e., a single domain antibody. In some embodiments, the single domain antibody may further comprise a short peptide such as a His tag. The VHH domain can be shown as a means for binding a specific target (e.g., OX-40, CD40, 4-1BB, HSA, IL-22 or EFGR). Thus, any of the various antibody structures, formats or constructs disclosed herein that contain or are constructed to contain a VHH domain can be referred to as an antibody that contains a means for binding the target of interest. Some embodiments can specifically include one or more specific antibody structures, formats or constructs. In other embodiments, one or more specific antibody structures, formats or constructs can be specifically excluded.

[0025] As used herein, the terms "antibody with specificity", "antibody that recognizes", "antibody with affinity", "antibody with a binding site" and similar constructs can be used interchangeably.

[0026] The term "multispecific antibody" refers to an antibody having two or more antigen-binding specificities. The multispecific antibodies disclosed herein are specific for at least two of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR, or at least one of said specificities and at least a second specificity. In some embodiments, the multispecific antibodies disclosed herein can comprise two, three, four, or more domains capable of binding to an antigen. Further, the multispecific antibody can comprise at least two copies of the same antigen-binding sequence, or two antigen-binding sequences (biparatopic) specific for different epitopes on the same antigen, as long as the multispecific antibody has specificity for at least one of OX-40, CD40, 4-1BB, and EFGR and specificity for at least one second antigen. In some embodiments, the multispecific antibody (MVSCA) has specificity for at least two of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. In some embodiments, the multispecific antibodies disclosed herein are single-chain antibodies. Thus, some multispecific antibodies can be said to be antibodies that comprise means for binding to a first target and means for binding to a second target and the like.

[0027] The term "bispecific antibody" refers to an antibody having two different antigen-binding specificities. In some embodiments, the bispecific antibodies disclosed herein are specific for two of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. The amino acid sequences encoding the antigen-binding portions of the bispecific antibodies can be linked in various arrangements. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the bispecific antibodies are linked by a linker as disclosed herein.

[0028] "Trispecific antibody" refers to an antibody having three different antigen-binding specificities. In some embodiments, the trispecific antibodies disclosed herein are specific for three of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. The amino acid sequences encoding the antigen-binding portions of the trispecific antibodies can be linked in various arrangements. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the trispecific antibodies are linked by linkers as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0029] "Quadroma" refers to an antibody having four different antigen-binding specificities. In some embodiments, the quadromas disclosed herein are specific for four of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. The amino acid sequences encoding the antigen-binding portions of the quadromas can be linked in various arrangements. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the quadromas are linked by linkers as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0030] As used herein, the term "valence" indicates the presence of a specific number of binding sites in an antibody molecule. Thus, the terms "bivalent", "trivalent", "tetravalent", "pentavalent", "hexavalent", "heptavalent" and "octavalent" indicate the presence of 2, 3, 4, 5, 6, 7 and 8 binding sites, respectively, in an antibody molecule. The bispecific antibodies disclosed herein are "bivalent". The trispecific antibodies disclosed herein are "trivalent". The quadroma antibodies disclosed herein are "tetravalent". However, monospecific multivalent antibodies, such as bivalent, trivalent and tetravalent antibodies, are within the scope of the present disclosure, and these have multiple antigen-binding sites that bind to the same antigen. The antigen-binding sites of monospecific bivalent and trivalent antibodies (or higher valencies) can bind to either the same epitope or different epitopes on the antigen. Similarly, by combining multiple monospecific binding sites with one or more binding sites of other specificities, antibodies with a higher valency than multispecificity, such as trivalent bispecific antibodies, can be constructed.

[0031] As used herein, the term "full-length antibody" refers to the structure that constitutes the native biological form of an antibody, including the variable and constant regions. For example, in most mammals, including humans and mice, the full-length antibody of the IgG class is a tetramer, containing two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain containing the immunoglobulin domains VL and CL, and each heavy chain containing the immunoglobulin domains VH, CH1, CH2 and CH3. In certain mammals, such as camels and llamas, the IgG antibody can also be composed of only two variable heavy chains, each heavy chain containing a variable domain (VHH) bound to the Fc region (CH2 and CH3 domains).

[0032] A tetrameric antibody is usually composed of two pairs of identical polypeptide chains, each pair having one "light chain" (usually with a molecular weight of about 25 kDa) and one "heavy chain" (usually with a molecular weight of about 50 - 70 kDa). Each of the light and heavy chains is composed of two different regions called the variable region and the constant region. In immunoglobulins of the IgG class, the heavy chain consists of four immunoglobulin domains linked in the order VH-CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also denoted as VH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constant domain γ1, constant domain γ2, and constant domain γ3 respectively). The IgG light chain is composed of two immunoglobulin domains linked in the order VL-CL from the N-terminus to the C-terminus, called the light chain variable domain and the light chain constant domain respectively. The constant region has a relatively stable sequence and plays a role in binding to many natural proteins and bringing about important biochemical events.

[0033] The variable region of an antibody contains the antigen-binding determinants of the molecule and determines the specificity of the antibody for the target antigen. It is named so because it has the most different sequences from other antibodies within the same class. In the variable region, three loops gather in each of the V domains of the heavy and light chains to form the antigen-binding site. Each loop is called a complementarity-determining region (hereinafter referred to as CDR), and has the most amino acid sequence mutations. There are three CDRs each in the heavy and light chains, that is, a total of six, called VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside the CDRs is called the framework (FR) region. Although not as variable as the CDRs, there are sequence variations among different antibodies in the FR region. Overall, this characteristic structure of the antibody provides a stable scaffold (FR region), on which the immune system can explore substantial antigen-binding diversity (CDR) to obtain specificity for a wide range of antigen sequences.

[0034] Genes encoding immunoglobulin loci contain multiple V-region sequences and short nucleotide sequences designated as "D" and "J", and the combination of V, D, and J nucleotide sequences generates VH diversity.

[0035] Antibodies are classified into classes also called isotypes that are genetically determined by the constant region. Human constant light chains are classified into κ (Cκ) and λ (Cλ) light chains. Heavy chains are classified into mu (μ), delta (δ), gamma (γ), alpha (α), and epsilon (ε), and the antibody isotype is determined as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. In humans, this class includes the subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this class includes the subclasses IgG1, IgG2a, IgG2b, and IgG3. IgM has subclasses including but not limited to IgM1 and IgM2. IgA has several subclasses including but not limited to IgA1 and IgA2. Thus, as used herein, "isotype" means either a class or subclass of immunoglobulin defined by the chemical and antigenic properties of the constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. The disclosed VHH antibodies, bispecific antibodies, and multispecific antibodies can have constant regions that include all or some of the above isotypes.

[0036] Also within the scope of the present disclosure are (i) Fab fragments comprising VL, CL, VH, and CH1 domains; (ii) Fd fragments comprising VH and CH1 domains; (iii) Fv fragments comprising VL and VH domains of a single antibody; (iv) dAb fragments comprising a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments, i.e., bivalent fragments comprising two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) in which the VH domain and the VL domain are linked by a peptide linker capable of joining the two domains to form an antigen-binding site, including but not limited to these antibody fragments. Also disclosed are trivalent or tetravalent antibody fragments in which variable domains having three different specificities are linked by a cleavable or non-cleavable linker. In certain embodiments, the antibody is produced by recombinant DNA technology. In further embodiments, the antibody is produced by enzymatic or chemical cleavage of a naturally occurring antibody.

[0037] As used herein, "single-chain antibody" generally refers to a fusion protein of the antigen-binding portion (i.e., variable region) of an antibody linked by a linker peptide. Disclosed herein are multivalent monospecific and multispecific single-chain antibodies. The monospecific multivalent antibodies have specificity for at least one of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. The multispecific single-chain antibodies have at least one additional specificity in addition to at least one of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. In some embodiments, the multispecific single-chain antibodies have specificity for at least two of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR.

[0038] As used herein, a "humanized" antibody means an antibody that contains human framework regions (FRs) and one or more complementarity-determining regions (CDRs) from a non-human antibody. The non-human antibody that provides the CDRs is referred to as the "donor," and the human immunoglobulin that provides the framework is referred to as the "acceptor." In certain embodiments, humanization basically relies on grafting the donor CDRs onto the acceptor (human) VL or VH framework. This strategy is called "CDR grafting." It is often necessary to "back-mutate" selected acceptor framework residues to the corresponding donor residues in order to restore the affinity lost in the initial graft construct. A humanized antibody optimally includes at least a portion of the immunoglobulin constant region, i.e., usually the constant region of a human immunoglobulin, and often also includes the human Fc region. Humanization or other methods for reducing the immunogenicity of the non-human antibody variable region may include resurfacing. In one embodiment, the selection-based method can be used to humanize and / or affinity mature an antibody variable region, i.e., to increase the affinity of the variable region for its target antigen. Other humanization methods include methods of grafting only a portion of the CDRs, such as, but not limited to, the method described in US 6,797,492, and the disclosure of all documents regarding CDR grafting is incorporated herein by reference. The structure-based method can be used for humanization and affinity maturation, for example, as described in US 7,117,096, and all disclosures regarding humanization and affinity maturation are incorporated herein by reference.

[0039] In various embodiments of the present specification, the antibody is a VHH. Camels (camels, dromedaries and llamas) contain, in addition to conventional heavy-chain and light-chain antibodies (one antibody containing two light chains and two heavy chains), double-chain antibodies (containing only variant heavy chains). The dimeric antibody is encoded by a unique pair of VH segments called VHH genes. VH and VHH are scattered in the genome (i.e., appear to be intermixed with each other). The identification of the same D segment in the cDNA of VH and VHH suggests that the D segment is commonly used by VH and VHH. In natural VHH-containing antibodies, the entire CH1 domain of the heavy-chain constant region is deleted. The exon encoding the CH1 domain is present in the genome, but is excised by splicing because there is no functional splice acceptor sequence on the 5' side of the CH1 exon. As a result, the VDJ region is spliced onto the CH2 exon. When the VDJ region is resynthesized on such a constant region (CH2, CH3) by VHH, an antibody in which the half-antibody is single-stranded instead of a light-chain / heavy-chain pair (i.e., two heavy-chain antibodies without light-chain interaction) is produced. Binding to an antigen is different from binding to a conventional antibody, but high affinity is achieved in the same way, i.e., by high-frequency mutation of the variable region and selection of cells expressing high-affinity antibodies as described above.

[0040] In an exemplary embodiment, the disclosed VHH is produced by immunizing transgenic mice in which endogenous mouse antibody expression is eliminated and a camelid transgene is introduced. VHH mice are disclosed in US8,883,150, US8,921,524, US8,921,522, US8,507,748, US8,502,014, US2014 / 0356908, US2014 / 0033335, US2014 / 0037616, US2014 / 0356908, US2013 / 0344057, US2013 / 0323235, US2011 / 0118444 and US2009 / 0307787, which disclose heavy-chain only antibodies and their production in transgenic mice, all of which are incorporated herein by reference. The VHH mice are immunized and the resulting primed spleen cells are fused with mouse myeloma cells to form hybridomas.

[0041] In other embodiments, the VHH is obtained by immunizing a llama with a desired antigen and isolating the sequence encoding the VHH region of the resulting antigen-binding antibody. In one embodiment, the VHH is isolated using a phage display library. See, for example, WO 91 / 17271, WO 92 / 01047 and WO 92 / 06204 (each incorporated herein by reference in its entirety for the description of the preparation of phage libraries).

[0042] The present specification also discloses a multispecific antibody or a multivalent antibody in which two or more antigen-binding domains are bound to a single fusion protein. The multispecific antibody includes: (i) a multispecific Fv fragment; (ii) a heavy chain having a first specificity to which a second VH domain having a second specificity is bound (or fused); (iii) a tetrameric monoclonal antibody having a first specificity bound to a second VH domain having a second specificity (wherein the second VH domain is bound to the first VH domain); (iv) a Fab fragment (VH-CH1 / VL-CL) having a first specificity bound to a second VH domain having a second specificity. Exemplary Fab fragments include those in which a second VH sequence having a second specificity is bound to the C-terminus or N-terminus of the first VH domain, or the C-terminus or N-terminus of the first CH1 domain or the first CL domain. In a further embodiment, a VH sequence having a second and / or third specificity (or more) can be bound (or fused) to the C-terminus or N-terminus of the first VH domain, or the C-terminus or N-terminus of the first CH1 domain or the first CL domain. In various embodiments, any of these forms can include at least one of the VHH domains disclosed herein. Examples of the structure of the multispecific antibody can be found in WO2021 / 062361, which is hereby incorporated by reference in its entirety for the disclosure related thereto.

[0043] Multispecific antibodies or multivalent antibodies may include a linker sequence that links a specific antigen-binding domain (e.g., VH or VHH) to another antigen-binding domain, and this linker sequence appropriately folds the amino acid sequence to generate the desired three-dimensional conformation or antigen-binding profile. Generally, the linker sequence is a short amino acid sequence that provides sufficient space and flexibility between domains to allow the domains to fold properly. The linker may also cause steric hindrance to facilitate the binding of each domain to the target. Suitable linkers include, but are not limited to, the linkers in Table 26 (SEQ ID NOs: 84-103), EPKSCD (SEQ ID NO: 104), and ASTKGP (SEQ ID NO: 105). Additional linkers will be known to those skilled in the art.

[0044] Also within the scope of the present disclosure are variants of the amino acid sequences of the monospecific or multispecific antibodies disclosed herein. Variants of the amino acid sequence are produced by introducing appropriate nucleotide changes into the DNA encoding the antibody or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibodies of the examples herein. Any combination of deletions, insertions, and substitutions is made provided that the final construct has the desired properties to obtain the final construct. Amino acid changes may also alter the post-translational processes of humanized or variant antibodies (e.g., changes in the number or position of glycosylation sites).

[0045] A useful method for identifying specific residues or regions of an antibody that are preferred positions for mutagenesis is called "alanine scanning mutagenesis". Identify the residue or group of the target residue (e.g., charged residues such as Arg, Asp, His, Lys, and Glu), and substitute with a neutral amino acid (most preferably, alanine or polyalanine) to affect the interaction between the amino acid and the antigen. The positions of these amino acids that show functional sensitivity to the substitution are then modified by introducing another variant or other variants at the substitution site or instead of the substitution site. Thus, the site for introducing mutations in the amino acid sequence is predetermined, but it is not necessary to predetermine the nature of the mutation itself. For example, to analyze the performance of a mutation at a certain site, alanine scanning or random mutagenesis is performed on the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0046] Insertions of amino acid sequences include fusions at the amino terminus and / or carboxyl terminus in the range of polypeptide chain lengths from 1 residue to over 100 residues, as well as insertions into the sequence of 1 or more amino acid residues. Examples of terminal insertions include antibodies disclosed herein having a methionyl residue at the N-terminus or antibodies fused to an epitope tag. Other insertion-type variants of the antibody molecule include fusing an enzyme or polypeptide that increases the serum half-life of the antibody to the N-terminus or C-terminus of the antibody.

[0047] Another type of variant is the amino acid substitution variant. In these variants, at least one amino acid residue in the antibody molecule has been removed and a different residue has been inserted in its place. The most important sites for substitution mutagenesis are the hypervariable regions, but changes in the FR are also considered. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions". If the substitution results in a change in biological activity, it can be shown as "Exemplary Substitutions" in Table 1 or more substantial changes as further described below with respect to amino acid classification can be introduced and the product screened.

[0048]

Table 1

[0049] Significant modification of the biological properties of an antibody is achieved by selecting substitutions that vary widely in their effect on maintaining (a) the structure of the polypeptide backbone at the substitution site, such as sheet or helical conformations, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring residues are grouped into classes based on common side chain properties: (1) Hydrophobic residues: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic residues: Cys, Ser, Thr; (3) Acidic residues: Asp, Glu; (4) Basic residues: Asn, Gin, His, Lys, Arg; (5) Residues affecting chain direction: Gly, Pro; and (6) Aromatic residues: Trp, Tyr, Phe.

[0050] Non-conservative substitutions mean exchanging one of these classes for another.

[0051] Cysteine residues that are not involved in maintaining the proper conformation of a monospecific or multispecific antibody may also generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds can also be added to improve the stability of the antibody (especially when the antibody is an antibody fragment such as an Fv fragment).

[0052] Another type of substitution variant involves substitutions of residues in one or more hypervariable regions of a parent antibody (e.g., a humanized antibody or a camelid antibody). Generally, the resulting variants selected for further development have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6 - 7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are expressed in a monovalent manner from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The variants after phage display are screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify hypervariable region sites that are candidates for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that significantly affect antigen binding. Alternatively, in addition to mutagenesis, it may be beneficial to analyze the crystal structure of the antigen - antibody complex and identify the contact points between the antibody and the antigen. Such contact residues or residues in their vicinity are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants can be screened as described herein to select antibodies with excellent properties in one or more relevant assays for further development.

[0053] Another type of amino acid variant of an antibody modifies the native glycosylation pattern of the antibody. Modifying means deleting one or more sugar chain sites present in the antibody and / or adding one or more glycosylation sites not present in the antibody.

[0054] Antibody glycosylation is usually either N-linked or O-linked. N-linked refers to the attachment of the sugar chain moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatically attaching a sugar chain moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide forms a potential glycosylation site. O-linked glycosylation refers to the attachment of any of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0055] The addition of a glycosylation site to an antibody is readily achieved by modifying the amino acid sequence to include one or more of the above tripeptide sequences (in the case of an N-linked glycosylation site). This change can also be made by adding or substituting one or more serine or threonine residues to the sequence of the original antibody (in the case of an O-linked glycosylation site).

[0056] Nucleic acid molecules encoding amino acid sequence variants of monospecific or multispecific antibodies are produced by a variety of methods known in the art. These methods include isolation from natural sources (in the case of naturally occurring amino acid sequence variants), or production by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis and cassette mutagenesis of the antibodies disclosed herein, either pre-made variants or non-variant, although not limited to these.

[0057] Other modifications to the monospecific or multispecific antibodies are also contemplated. For example, it may be desirable to modify the antibody with respect to effector function to enhance the effectiveness of the antibody in the treatment of disease. For example, cysteine residues are introduced into the Fc region to form interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization ability and may improve complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). Homodimeric antibodies with enhanced antitumor activity can also be produced using heterobifunctional crosslinking agents. Alternatively, an antibody having two Fc regions can be produced, thereby enhancing complement lysis ability and ADCC ability.

[0058] In another embodiment, the antibody is conjugated to a "receptor" (e.g., streptavidin), and after administration of the antibody-receptor conjugate to the patient, the unbound conjugate is removed from the blood using a clearing agent, and then a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide) is administered, which can be utilized for pretargeting.

[0059] Covalent modifications of monospecific or multispecific antibodies are also within the scope of the present disclosure. These modifications can be effected, where appropriate, by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of antibodies are introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizing agent capable of reacting with selected side chains or the N-terminal or C-terminal residues. Exemplary covalent modifications of polypeptides are described in US 5,534,615, and all disclosures regarding covalent modifications of polypeptides are specifically incorporated herein by reference. Exemplary types of covalent modifications of antibodies include binding the antibody to one of various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the methods described in US 4,640,835, US 4,496,689, US 4,301,144, US 4,670,417, US 4,791,192, or US 4,179,337.

[0060] The monospecific or multispecific antibodies disclosed herein can be produced by recombinant means. Accordingly, disclosed herein are nucleic acids encoding the antibodies, expression vectors containing the nucleic acids encoding the antibodies, and cells containing the nucleic acids encoding the antibodies. Methods for recombinant production are well known in the art and include protein expression in prokaryotic and eukaryotic cells and subsequent isolation of the antibody and purification to a pharmaceutically acceptable purity. To express such an antibody in a host cell, the nucleic acid sequence encoding the antibody is inserted into an expression vector by standard methods. Expression is carried out in a suitable prokaryotic or eukaryotic host cell such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). It should be understood that any recombinant expressed protein requires an initiator methionine (or formylmethionine) or signal sequence at its N-terminus depending on whether the expression system used and the protein is expressed or secreted in the cytoplasm. Accordingly, in some embodiments, the protein sequences disclosed herein are modified with such additional amino acids at their N-terminus. In some embodiments, such N-terminal sequences are cleaved (in whole or in part) from the fully mature sequence, while in other embodiments they are retained.

[0061] Accordingly, certain embodiments disclosed herein include a method for producing a monospecific or multispecific antibody, comprising: a) transforming a host cell with at least one expression vector containing a nucleic acid molecule encoding the antibody; b) culturing the host cell under conditions capable of synthesizing the antibody molecule; and c) recovering the antibody molecule from the culture.

[0062] The antibody is appropriately separated from the culture solution by conventional immunoglobulin purification methods such as, for example, protein A-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

[0063] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and such designations include all progeny. Thus, the terms "transformant" and "transformed cell" include the original subject cell and cultures derived therefrom, regardless of the number of passages. It is understood that all progeny may not be precisely identical in DNA content due to deliberate or inadvertent mutations. Mutant progeny having the same function or biological activity as screened in the originally transformed cells are included. Where a specific designation is intended, it will be apparent from the context.

[0064] As used herein, the term "transformation" refers to the process of introducing a vector / nucleic acid into a host cell. When using a cell without a strong cell wall barrier as the host cell, transfection can be carried out, for example, by the calcium phosphate precipitation method. However, other methods for introducing DNA into cells, such as nuclear injection or protoplast fusion, can also be used. When using a prokaryotic cell or a cell containing a substantial cell wall structure, for example, one method of transfection is calcium treatment using calcium chloride.

[0065] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and / or the transcribed mRNA (also called the transcript) is subsequently translated into a peptide, polypeptide, or protein. The transcript and the encoded polypeptide are collectively referred to as the gene product. When the polynucleotide is derived from genomic DNA, expression in eukaryotic cells includes splicing of the mRNA.

[0066] A "vector" is a nucleic acid molecule that transfers an inserted nucleic acid molecule within and / or between host cells, and in particular is self-replicating. This term includes vectors that function primarily for the insertion of DNA or RNA into cells (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for the transcription and / or translation of DNA or RNA. Also included are vectors that provide multiple functions as described.

[0067] An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector that functions to produce a desired expression product.

[0068] As used herein, the term "host cell" refers to any type of cell line that can be genetically engineered to produce the antibodies disclosed herein. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0069] Suitable control sequences for prokaryotes include, for example, a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0070] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a DNA encoding a presequence or secretory leader is operably linked to a DNA encoding a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking can be accomplished by ligation at suitable restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[0071] Also disclosed herein are isolated nucleic acids encoding monospecific or multispecific antibodies, vectors and host cells containing the nucleic acids, and recombinant techniques for antibody production.

[0072] For the recombinant production of an antibody, the nucleic acid encoding the antibody can be isolated and inserted into a replicable vector for further cloning (amplification of DNA) or expression. In some embodiments, the antibody can be produced by homologous recombination, and for example, all the disclosure regarding antibody production as described in US 5,204,244 is incorporated herein by reference. The DNA encoding the antibody can be easily isolated and sequenced using conventional methods (e.g., methods using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors can be utilized. The vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, enhancer elements, a promoter, and a transcription termination sequence; for example, all the disclosure regarding protein expression as described in US 5,204,244 is specifically incorporated herein by reference.

[0073] Host cells suitable for the cloning or expression of DNA in the vectors herein are the prokaryotes, yeasts, or higher eukaryotic cells described above. Suitable prokaryotes for this purpose include true bacteria such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia (e.g., E. coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescens) and Shigella, as well as Bacillus (e.g., Bacillus subtilis and Bacillus licheniformis), Pseudomonas (e.g., Pseudomonas aeruginosa) and Streptomyces. One exemplary E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325) are also suitable. These examples are illustrative and not limiting.

[0074] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable as hosts for the cloning or expression of vectors encoding monospecific or multispecific antibodies. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful herein: for example, fission yeasts such as Schizosaccharomyces pombe; hosts of the genus Kluyveromyces, such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 16,045), Kluyveromyces wickeramii (ATCC 24,178), Kluyveromyces waltii (ATCC 56,500), Kluyveromyces drosophilarum (ATCC 36,906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070); the genus Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, the following: hosts of the genus Neurospora, Penicillium, Tolypocladium, and Aspergillus (e.g., Aspergillus nidulans and Aspergillus niger).

[0075] Host cells suitable for the expression of glycosylated monospecific or multispecific antibodies are from multicellular organisms including invertebrates such as plant cells and insect cells. Various baculovirus strains and variants from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori, and the corresponding permissive insect host cells have been identified. Various virus strains for transfection are publicly available (e.g., the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV), and such viruses can be used as the viruses herein according to the present invention, in particular, for the transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco can also be used as hosts.

[0076] However, the highest interest lies in vertebrate cells, and the growth of vertebrate cultured cells (tissue cultures) has become an everyday technique. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lines (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Sertoli cells (TM4); monkey kidney cells (CV1, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human liver tumor line (Hep G2).

[0077] The host cells are transformed with the above expression vectors for the production of monospecific or multispecific antibodies and are cultured in a conventional nutrient medium that has been modified as needed for promoter induction, selection of transformants, or gene amplification of the gene encoding the desired sequence.

[0078] Host cells used to produce monospecific or multispecific antibodies can be cultured in various media. Commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle Medium (DMEM) are suitable for culturing host cells. Further, the media described in US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO87 / 00195; or US Re.30,985 may be used as the culture medium for host cells. Any of these media may be supplemented, as necessary, with hormones and / or other growth factors (e.g., insulin, transferrin or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine or thymidine), antibiotics (e.g., GENTAMYCIN®), trace elements (usually defined as inorganic compounds present at final concentrations in the micromolar range) and glucose or an equivalent energy source. Other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those that have been used with the host cells selected for expression heretofore and will be apparent to those skilled in the art.

[0079] When using recombinant techniques, the antibody can be produced intracellularly, i.e., within the periplasmic cavity, or secreted directly into the medium. When the antibody is produced intracellularly, as a first step, any particulate fragments, host cells or lysed fragments are removed, for example, by centrifugation or ultrafiltration.

[0080] Antibody compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, and affinity chromatography is a preferred purification technique. The compatibility of protein A as an affinity ligand depends on the type and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used for the purification of antibodies based on human γ1, γ2, or γ4 heavy chains, and can also be used for the purification of antibodies without an Fc region. Protein G is useful for all mouse isotypes and human γ3. The matrix to which the affinity ligand is bound is most often agarose, but other matrices are also available. Mechanically stable matrices, such as controlled pore glass or poly(styrene divinyl) benzene, allow for faster flow rates and shorter processing times than those obtained with agarose. When the antibody contains a CH3 domain, Bakerbond ABX® resin is useful for purification. The antibodies and antibody fragments disclosed herein can also be synthesized using a histidine tag and can also be affinity purified by metal affinity chromatography.

[0081] Other techniques for protein purification, such as fractionation on an ion exchange column, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin Sepharose® chromatography on an anion or cation exchange resin (e.g., a polyaspartic acid column), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, can also be utilized depending on the antibody to be recovered.

[0082] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5 to 4.5, preferably carried out using an elution buffer at a low salt concentration (e.g., about 0 to 0.25 M salt).

[0083] Also disclosed herein is a multi-specific single-chain antibody that can be cleaved in the tumor microenvironment. In some embodiments, a tumor targeting domain (e.g., a tumor antigen binding domain) or other functional domain is cleaved by a linker to release another domain that provides a therapeutic effect when the multi-specific single-chain antibody reaches the tumor. There are a number of proteases in the tumor microenvironment that can cleave the linkers disclosed herein. Non-limiting examples of tumor proteases include matrix metalloproteases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAM (a disintegrin and metalloprotease, e.g., ADAM10 and ADAM17), kallikrein-related peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepsin (HPN), matriptase, legumain, or dipeptidyl peptidase (e.g., DDP4), but are not limited thereto.

[0084] antibody composition Also disclosed herein are pharmaceutical compositions comprising a mono-specific or multi-specific antibody, wherein the specificities include OX-40, CD40, 4-1BB, HSA, IL-22, or EFGR. Also disclosed is the use of the antibodies described herein for the manufacture of a pharmaceutical composition. Also disclosed is a method of using the disclosed antibodies and pharmaceutical compositions comprising the antibodies for the treatment of various diseases and disorders.

[0085] A pharmaceutical composition is for the purpose of treating a disease in a human and is suitable for treatment. That is, it provides an overall beneficial effect and does not contain an amount of a component or impurity that causes toxicity or other undesirable effects unrelated to the provision of the beneficial effect. A pharmaceutical composition contains one or more active agents and may further contain solvents, buffers, diluents, carriers, and other excipients to assist with the administration, solubility, absorption, or bioavailability and stability of the active agent or the composition as a whole.

[0086] The monospecific or multispecific antibodies disclosed herein can also be formulated into liposomes. Liposomes containing antibodies are produced by methods known in the art, such as those described in US4,485,045, US4,485,045, and US5,013,556. Particularly useful liposomes can be generated by the reverse-phase evaporation method using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with a defined pore size to obtain liposomes of the desired diameter. The Fab' fragment of the antibody can be conjugated to the liposome by a disulfide exchange reaction.

[0087] As used herein, "pharmaceutical carrier" includes any physiologically compatible solvent, dispersion medium, coating agent, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Preferably, the carrier is suitable for intravenous, intramuscular, intraocular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., injection or infusion). In some embodiments, the carrier is an aqueous solution.

[0088] The compositions disclosed herein can be administered by various methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. To administer the disclosed antibodies by a particular route of administration, it may be necessary to conjugate the antibody to a material or co-administer the antibody with a material to prevent inactivation of the antibody. For example, the antibody may be administered to a subject in a suitable carrier (e.g., liposome or diluent). Pharmaceutically acceptable diluents include saline and aqueous buffers. Pharmaceutical carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0089] As used herein, the terms "parenteral administration" and "administered parenterally" mean a mode of administration other than enteral administration and topical administration, usually administration by injection, including but not limited to intravenous, intramuscular, intraarterial, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, intratracheal, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0090] These compositions may contain excipients (e.g., preservatives, wetting agents, emulsifying agents and dispersing agents). By performing the above sterilization method and containing various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid), the presence of microorganisms can be prevented. It may also be desirable to include in the composition isotonicifying agents (e.g., sugars, sodium chloride). Furthermore, the absorption of injectable pharmaceuticals can be prolonged by incorporating absorption delaying agents (e.g., aluminum monostearate and gelatin).

[0091] In some embodiments, the pharmaceutical composition comprising an antibody is a lyophilized cake. The lyophilized cake can further contain bulking agents, buffers and / or salts, or other excipients as described herein. The lyophilized composition can be dissolved by adding sterile water or an aqueous buffer for administration to a patient.

[0092] Regardless of the selected route of administration, the disclosed antibodies and / or pharmaceutical compositions containing antibodies that can be used in a suitable hydrated form are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0093] The actual dosage of the active ingredient in the pharmaceutical composition can be varied to be an amount of the active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration and that is not toxic to the patient. The selected dosage is determined by a variety of pharmacokinetic factors, including the activity of the particular composition employed, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and medical history of the patient being treated, as well as similar factors well known in the medical arts.

[0094] Linker In many embodiments, the individual binding domains are not directly attached to each other, but rather a short amino acid sequence, i.e., a linker, intervenes between them. Exemplification of linkers is shown in Table 10. The length and sequence of the linker can substantially affect the expression level, structure, and binding affinity of the linked domains of MVSCA. Linkers L2 and L4 with adjustable chain lengths (see Table 10) can be used to optimize MVSCA from the perspective of these parameters. Linkers L1, L2, and L4 can be referred to as non-cleavable linker means, flexible linker means, or flexible and non-cleavable linker means.

[0095] When two copies of the same VHH domain are placed adjacent to each other in MVSCA, they frequently cause harmful interactions with each other. This can be avoided by intervening a relatively short and rigid linker between the two copies. In some embodiments, the short rigid linker has the sequence AAA (L3 in Table 10). The linker can be referred to as rigid short linker means or non-cleavable rigid short linker means.

[0096] When an anti-HSA domain-HSA complex is used to generate a prodrug with respect to the binding activity of an adjacent binding domain, a cleavable linker needs to be interposed between the two domains. L11*3 to L11*18 (see Table 10) are examples of cleavable linkers that can be used to optimize MVSCA with respect to expression level, the structure of MVSCA, the binding affinity of the linked domains, and susceptibility to cleavage by different proteases. Linkers L11*3 to L11*18 can be referred to as cleavable linker means, flexible linker means, or flexible and cleavable linker means.

[0097] MVSCA The binding domains and linkers described herein can be combined to create multifunctional MVSCA adapted for the treatment of specific diseases. They can also be combined with yet another binding domain. MVSCA can be shown to include means for achieving various functions associated with the binding domains of each component type, and / or linker means for achieving their associated functions.

[0098] use of the disclosed antibody The disclosed antibodies are useful in medicine. Terms such as "treatment" and "therapy" refer to the medical management of a patient intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes both active treatment (i.e., treatment specialized for the improvement of a disease, pathological condition, or disorder) and causal treatment (i.e., treatment directed at the removal of the cause of a related disease, pathological condition, or disorder). Further, this term includes palliative treatment (i.e., treatment aimed at alleviating symptoms rather than curing a disease, pathological condition, or disorder), prophylactic treatment (i.e., treatment aimed at minimizing or partially or completely inhibiting the onset of a related disease, pathological condition, or disorder), and supportive therapy (i.e., treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or disorder). Various embodiments may specifically include or exclude one or more of these treatment modalities.

[0099] The antibodies disclosed in this specification are also intended to be used for diagnosis and imaging.

[0100] Furthermore, the terms "treating" or "treatment" include a wide range of therapeutic activities in humans or other animals, including diagnosis, alleviation, or prevention of a disease or its manifestations, or activities that affect the structure or any function of the body of a human or other animal. Therapeutic activities include administering to a patient the pharmaceutical agents, dosage forms, and pharmaceutical compositions described herein, according to the various treatment methods disclosed herein, whether by a medical professional, the patient himself or herself, or any other person. Therapeutic activities include the orders, instructions, and advice of medical professionals such as physicians, physician assistants, nurses, etc., which are carried out by any other person, including other medical professionals or the patient himself or herself. This includes, for example, instructing a patient to undergo diagnostic procedures, such as diagnosing cancer and determining its stage, or instructing that they be carried out in a clinical laboratory, so that ultimately the patient receives appropriate treatment. This includes, for example, instructing a patient to undergo diagnostic methods such as diagnosing cancer and determining its stage, or instructing that they be carried out in a clinical laboratory, so that ultimately the patient receives appropriate treatment. In some embodiments, the orders, instructions, and advice aspects of therapeutic activities also include approving the insurance coverage of a pharmaceutical product, denying the insurance coverage of an alternative pharmaceutical product, including a pharmaceutical product in a prescription, or excluding an alternative pharmaceutical product, or providing a financial incentive to use a pharmaceutical product, so as to encourage, induce, or obligate the selection of a particular pharmaceutical product or combination thereof for the treatment of a medical condition and the actual use of the pharmaceutical product. In some embodiments, the therapeutic activities can also include encouraging, inducing, or obligating the selection of a particular pharmaceutical product for the treatment of a medical condition - and the actual use of that pharmaceutical product - according to guidelines or treatment criteria that can be established by a hospital, clinic, health maintenance organization, medical practice, or group of physicians. It is understood that all such orders, instructions, advice are conditional upon receiving the benefit of treatment by following such instructions. In some cases, following such orders, instructions, and advice also results in a financial benefit to the patient.In some cases, by complying with such orders, instructions, and advice, healthcare providers may also receive financial benefits.

[0101] cancer The disclosed monospecific VHHs and multivalent single-chain antibodies having specificity for OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR are useful for the treatment of cancer. Each antibody is designed to treat a specific classification of cancer based on the antigen-binding specificity contained in the antibody.

[0102] The present disclosure provides a method for treating cancer, comprising administering to a patient in need of said treatment an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising said antibody.

[0103] Examples of cancers that can be treated by the disclosed method include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical cancer; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer, bladder cancer; bone cancer; brainstem glioma; brain tumor; breast cancer; bronchial adenoma / carcinoid; carcinoid tumor; islet cell cancer; cancer of unknown primary origin; central nervous system lymphoma; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; endometrial cancer, epithelioma; ovarian epithelial cancer; esophageal cancer; Ewing tumor family; extracranial germ cell tumor; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; germ cell tumor; gestational trophoblastic tumor; hairy cell leukemia; head and neck cancer; hepatocellular cancer; Hodgkin lymphoma; hypopharyngeal cancer; Kaposi sarcoma; kidney cancer; laryngeal cancer; non-small cell lung cancer; small cell lung cancer; non-Hodgkin lymphoma; Waldenstrom macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell cancer; squamous cell cancer of the neck; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell neoplasms; mycosis fungoides; myelodysplastic syndrome; nasopharyngeal cancer; neuroblastoma; oral cancer; oropharyngeal cancer; osteosarcoma; pancreatic cancer; parathyroid cancer; penile cancer; pheochromocytoma; pituitary tumor; pleuropulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; soft tissue sarcoma; Sézary syndrome; skin cancer; squamous cell cancer of the neck; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and Wilms tumor.

[0104] The effectiveness of cancer treatment is usually measured from the perspective of "response". Techniques for monitoring response may be similar to the following tests used for cancer diagnosis: · Lumps or tumors including lymph nodes can be measured by external palpation during a physical examination. · Some visceral cancer tumors are displayed by X-ray examination or CT scan and can be measured with a ruler. · Blood tests, such as tests measuring organ function, can be performed. · For certain cancers, tumor marker tests can be performed.

[0105] Whether it is a blood test, a cell count, or a tumor marker test, regardless of the test used, the results are repeated at specific intervals so that they can be compared with past tests of the same type.

[0106] The efficacy of cancer treatment is defined in several ways: · Complete response - All cancer or tumors disappear. The expression level of tumor markers (if applicable) can fall within the normal range. · Partial response - The cancer has regressed by a certain percentage, but the disease remains. The level of tumor markers (if applicable) can decrease (or increase as an indicator of a decrease in tumor burden based on tumor markers), but the lesion events remain. · Stable - The cancer is neither growing nor regressing, and the amount of the lesion has not changed. Tumor markers (if applicable) have not changed significantly. · Progressive - The cancer is increasing and there are more lesions than before treatment. In a tumor marker test (if applicable), the tumor marker is rising.

[0107] Other indicators showing the effectiveness of cancer treatment include overall survival (i.e., the period from diagnosis or the start of treatment of the subject of evaluation until death from any cause), cancer - free survival (i.e., the period during which no cancer is detected after complete response), and progression - free survival (i.e., the period during which no recurrence of tumor growth can be detected after stable or partial response).

[0108] The standard methods for evaluating the treatment effect of solid tumors in terms of tumor size (tumor burden) are two: WHO and RECIST. These methods measure solid tumors to compare the current tumor with past measurements or to compare changes with future measurements in order to change the treatment regimen. In the WHO method, the long axis and short axis of the solid tumor are measured, and the product of these two measurements is calculated; if there are multiple solid tumors, the sum of all products is calculated. In the RECIST method, only the long axis is measured. If there are multiple solid tumors, the sum of all long - axis measurements is calculated. However, for lymph nodes, the short axis is measured instead of the long axis.

[0109] autoimmune disease The disclosed monospecific VHH antibodies and multivalent single-chain antibodies having specificity for OX-40, CD40, 4-1BB, HSA, IL-22 and EFGR are useful for the treatment of autoimmune diseases. Each antibody is designed for therapeutic use against a specific class of autoimmune diseases based on the antigen-binding specificity contained in the antibody.

[0110] The present disclosure provides a method of treating an autoimmune disease, comprising administering to a patient in need thereof an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising said antibody.

[0111] An autoimmune disease can be a systemic autoimmune disease or an organ-specific autoimmune disease. Non-limiting examples of autoimmune diseases that can be treated using the compounds, compositions or combinations disclosed herein include acute disseminated encephalomyelitis (ADEM), Addison's disease, allergy, allergic rhinitis, antiphospholipid antibody syndrome (APS), arthritis such as polyarthritis such as monoarthritis, pauciarticular arthritis or osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, septic arthritis, spondyloarthritis, gout, pseudogout, Still's disease, asthma, acquired immunodeficiency syndrome, acquired immunodeficiency syndrome (AIDS), autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD), type 1 diabetes mellitus (IDDM), endometriosis, gastrointestinal diseases such as inflammatory bowel diseases such as irritable bowel disease or Crohn's disease or ulcerative colitis, glomerulonephritis, Goodpasture syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial nephritis, interstitial cystitis, lupus such as discoid lupus erythematosus, drug-induced lupus erythematosus, lupus nephritis, neonatal lupus, subacute cutaneous lupus erythematosus or systemic lupus erythematosus, Marfan syndrome, multiple sclerosis (MS), myasthenia gravis, myopathy (e.g., dermatomyositis, inclusion body myositis or polymyositis, myositis), narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, recurrent disseminated encephalomyelitis, rheumatic fever, schizophrenia, scleroderma, Sjögren's syndrome, skin diseases (e.g., dermatitis, eczema, stasis dermatitis, hidradenitis suppurativa, psoriasis, pityriasis rosea or scleroderma), tenosynovitis, uveitis, vasculitis syndromes, Burger's disease, cerebral vasculitis, Churg-Strauss arteritis, cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell arteritis, Golfer's vasculitis, Henoch-Schönlein purpura, hypersensitivity vasculitis, Kawasaki disease, microscopic polyarteritis / polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid-like vasculitis, Takayasu arteritis, Wegener's granulomatosis or vitiligo.

[0112] Aspects of the present disclosure include, in part, reducing at least one symptom associated with an autoimmune disease. The actual symptoms associated with the autoimmune diseases disclosed herein are well known to those of skill in the art and can be determined considering factors including, but not limited to, the location of the autoimmune disease, the cause of the autoimmune disease, the severity of the autoimmune disease, the tissue or organ affected by autoimmunity, and the inflammation associated with the autoimmune disease. Non-limiting examples of symptoms reduced by the methods of treating autoimmune diseases disclosed herein include inflammation, fatigue, pain, cognitive impairment, neurological disorders, dizziness, malaise, fever and hyperthermia, extreme coldness of hands and feet, muscle and joint weakness, pain and / or stiffness, weight changes, digestive or gastrointestinal disorders, respiratory disorders, low or high blood pressure, hypersensitivity, anxiety or depression, infertility or decreased libido (low sex drive), blood glucose changes, and depending on the type of autoimmune disease, an increase in organ or tissue size, or organ or tissue destruction. Non-limiting examples of inflammatory symptoms reduced by the methods of treating autoimmune diseases disclosed herein include pain, loss of neurological function, loss of cognitive function, edema, congestion, erythema, bruising, tenderness, stiffness, swelling, fever, chills, congestion of airways including nose and bronchus, sinus congestion, respiratory disorders, fluid retention, thrombosis, loss of appetite, increased heart rate, granuloma, fibroma, formation of pus or non-viscous serous fluid, ulcer formation or pain.

[0113] In certain embodiments, treatment with the antibodies disclosed herein reduces at least one symptom, at least two symptoms, at least three symptoms, at least four symptoms, or at least five symptoms of an autoimmune disease.

[0114] In other embodiments, the method can be useful for treating or alleviating conditions, symptoms or disorders associated with autoimmune diseases. In some embodiments, these conditions or symptoms include, but are not limited to, anemia, asthenia, cachexia, Cushing's syndrome, fatigue, gout, periodontal disease, hematuria, hypercalcemia, hypothyroidism, internal bleeding, hair loss, mesothelioma, nausea, night sweats, neutropenia, tumor-associated syndrome, pleurisy, polymyalgia rheumatica, rhabdomyolysis, stress, swollen lymph nodes, thrombocytopenia, vitamin D deficiency or weight loss. In other embodiments, administration of the antibodies disclosed herein extends the survival of the individual being treated.

[0115] In some embodiments of the method, the mammal may experience improvement from an autoimmune disease as a result of treatment with the antibodies disclosed herein.

[0116] The following examples, sequence listings and figures are provided to assist in the understanding of the invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made to the procedures described without departing from the spirit of the invention.

Examples

[0117] Example 1. Anti-OX40 VHH Antibody Isolation of Anti-OX40 VHH Antibody from Immunized Llama Immunization: Two llamas were immunized according to the standard protocol of Abcore Inc (Ramona, CA). Recombinant human OX40-llama Fc (extracellular domain (Leu29-Ala216) accession number P43489, SEQ ID NO: 1) was mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization) (Difco, BD Biosciences). Six subcutaneous injections per llama were performed at 50 μg / dose every other week. On day 45, serum was collected from the llamas immunized with recombinant OX40-llama Fc protein to determine the antibody titer against recombinant OX40-His by ELISA. In ELISA, 96-well Maxisorp plates (Nunc) were coated with 100 ng / well of OX40-His. After blocking, diluted serum samples were added and the presence of anti-OX40 VHH antibodies was demonstrated by measuring the antibody titer of the antiserum by ELISA. 96-well Maxisorp plates (Nunc) were coated with 100 ng / well of recombinant OX40-His. After blocking, diluted serum samples were added and the presence of anti-OX40 VHH antibodies was demonstrated using horseradish peroxidase (HRP)-labeled goat anti-llama IgG antibody (Invitrogen).

Table 2

[0118] Construction and selection of the phage library: Peripheral blood mononuclear cells were prepared from a 45-day serum sample of a llama immunized with recombinant OX40 llama FC protein using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from the peripheral blood mononuclear cells using the RNeasy Midi Kit (Qiagen) according to the manufacturer's instructions and used as the starting material for RT-PCR to amplify the gene fragments encoding VHH. These fragments were cloned into a self-made phagemid vector, infected with helper phage, and then recombinant phage particles were produced. These phage particles display VHH on the phage particle surface as a gene-III fusion protein. The phage was prepared according to the standard method and stored at 4 °C after filter sterilization for further use. For selection, the phage library obtained from the llama was used. In the selection, biotinylated OX40-HI was incubated with the phage library and then bound to streptavidin dynabeads (Invitrogen). After thorough washing, the bound phage was eluted with 1 mg / ml trypsin. The eluate from this selection was rescued in Escherichia coli TG1 cells. Colonies were picked and the nucleotide sequences were determined.

[0119] The cDNA encoding the positive VHH was synthesized with an added C-terminal His tag by Atum (DNA2.0, Inc.), transiently transfected into HEK293 cells, and the positive VHH was purified by IMAC chromatography for in vitro functional assays.

[0120] Kinetic Binding Analysis by Bio-Layer Interferometry (BLI): The label-free technology of Bio-Layer Interferometry (BLI) was used to measure the binding kinetics between human OX40 hFC (R & D systems) and anti-OX40 VHH. Affinity measurements were performed using a Gator equipped with an anti-Penta-His binding (HIS1K) biosensor chip. The assay was conducted at 30 °C in 1xPBS buffer (Gibco®, PBS pH 7.2). The samples were stirred at 1000 rpm. Before analysis, the sensor was wetted for 15 minutes. The purified anti-OX40 VHH was tested for its binding ability to the HIS1K sensor chip. 20 μg / ml of anti-OX40 VHH was loaded onto the chip. Loading was performed for 300 seconds, and a binding level of 1.8 - 2 nm was obtained. Human OX40 antigen was diluted in 1xPBS to concentrations of 100, 150, 250, and 350 nM for binding analysis. After starting the binding and monitoring for 200 seconds, the chip was transferred to factor protein-free 1xPBS buffer (Gibco, PBS pH 7.2) to monitor dissociation. Sensor data was collected, processed, and analyzed using Gator data analysis software throughout the experiment.

[0121] Inhibitory assay: 100 μL of 1 μg / mL OX40L-mFc prepared with antigen coating buffer was coated overnight at 4°C on a 96-well plate, and then blocked with 2% BSA at 25°C for 1 hour. Serial dilutions of each OX40 VHH were pre-mixed with 0.025 μg of OX40-hFc at 25°C for 30 minutes, and then transferred to the plate coated with OX40L-mFc and incubated for 1 hour. After washing the plate 4 times with PBST, it was incubated with mouse anti-human IgG Fc-HRP at 25°C for 1 hour. After washing 4 times with PBST, 100 μL of TMB per well was used to develop color in the dark for 10 - 20 minutes, and 50 μL of stop solution was added to stop the reaction. The plate was read at 450 nM using a Molecular Devices Microplate reader. ELISA data was analyzed using GraphPad Prisma 9.1.

[0122] ELISA assay: OX40 Fc prepared at 1 μg / ml with coating buffer was coated at 100 μl / well overnight at 4°C on a 96-well plate, then blocked with 2% BSA at 200 μl / well at 25°C for 1 hour, and then washed 2 times with PBST. OX40 VHH supernatant was serially diluted from a maximum concentration of 500 nM to 4-fold, and added to the plate coated with OX40 Fc at 100 μl / well and incubated at 25°C for 1 hour. After washing 4 times with PBST, it was incubated with the detection antibody anti-his-HRP (1:4000 dilution) at 100 μl / well and shaken at 60 rpm for 1 hour. The plate was developed in the dark using 100 μL / well of TMB, and 50 μL of stop solution per well was added to stop the reaction. The plate was read at 450 nM using a Molecular Devices Microplate Reader. The data was analyzed using GraphPad Prisma 9.1.

[0123] Expression and purification of anti-OX40 VHH antibody: The sequences of positive phage colonies obtained from the phage library of immunized llamas were determined. The amino acid sequences are listed in Table 2 below. A cDNA sequence based on the following amino acid sequence was fused with human Fc and synthesized by Atum (DNA2.0) using the pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce a complete recombinant anti-OX40 VHH antibody. The expressed anti-OX40 VHH was purified using a HiTrap Protein A column. [Table 3] [Table 4] [Table 5]

[0124] The VHH in Table 2 includes means for binding to OX-40.

[0125] The results of the ELISA assay of the OX40 VHH antibody against recombinant human OX40-Fc are shown in Table 3.

[0126] The bio-layer interferometry (BLI) binding analysis of the anti-OX-40 VHH molecule is shown in Table 4.

[0127] [Table 6] [Table 7]

[0128] The results of the ELISA inhibition assay of the anti-OX40 VHH that inhibits the binding of OX40 to OX40 ligand are shown in Figure 1. pgXX88-10 and pgXX88-SZ-15 are each EC 50A complete inhibitor showing 0.12 nM and 0.12 nM, and ppXX88-SZ-4 is an EC 50 Partial inhibitor showing 1.98 nM.

[0129] Example 2. Anti-CD40 VHH antibody Isolation of anti-CD40 VHH antibody from immunized llamas Two llamas were immunized according to the standard protocol of Abcore Inc. Recombinant CD40-llama Fc [extracellular domain (Glu21-Arg193) accession # P25942-1, SEQ ID NO: 25, produced in-house] was mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization) as in Example 1.

[0130] On day 45, serum was collected from the llamas immunized with recombinant CD40-llama Fc protein, and the antibody titer against recombinant CD40-His was determined by ELISA. In ELISA, 96-well Maxisorp plates were coated with 100 ng / well of CD40-His. After blocking, diluted serum samples were added, and the presence of anti-CD40 VHH antibody was demonstrated using the antibody titer of the antiserum. 96-well Maxisorp plates were coated with 100 ng / well of recombinant CD40-His. After blocking, diluted serum samples were added, and the presence of anti-CD40 antibody was demonstrated using HRP-labeled goat anti-llama IgG antibody. [Table 8]

[0131] Peripheral blood mononuclear cells were prepared from a 45-day serum sample of a llama immunized with recombinant CD40 llama Fc protein using Ficoll-Paque Plus according to the manufacturer's instructions. Total RNA from the peripheral blood mononuclear cells was extracted using the RNeasy Midi Kit according to the manufacturer's instructions and used as a starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were cloned into a self-made phagemid vector, infected with helper phage, and then recombinant phage particles were produced. These phage particles display VHH on the phage particle surface as a gene-III fusion protein. The phage was prepared according to the standard method and stored at 4 °C after filter sterilization for further use. For selection, a phage library obtained from llamas was used. In the selection, biotinylated CD40-His was incubated with the phage library and then bound to streptavidin dynabeads. After thorough washing, the bound phage was eluted with 1 mg / ml trypsin. The eluate from this selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0132] The cDNA encoding the positive VHH was synthesized with an added C-terminal His tag by Atum, transiently transfected into HEK293 cells, and the positive VHH was purified by IMAC chromatography for in vitro functional assays.

[0133] The sequences of the positive phage colonies obtained from the immunized llama phage library were determined. The amino acid sequences are shown in Table 5 below. The cDNA sequences based on the following amino acid sequences were fused with human Fc and synthesized with the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce a complete recombinant anti-CD40 VHH antibody. The expressed anti-CD40 VHH was purified by a HiTrap protein A column.

Table 9

[0134] The VHH of Table 5 constitutes a means for binding to CD40.

[0135] ELISA assay CD40 Fc prepared at 1 μg / ml using coating buffer was coated on a 96-well plate at 100 μl / well at 4°C overnight. After that, it was blocked with 2% BSA at 200 μl / well at 25°C for 1 hour and then washed twice with PBST. The CD40 VHH supernatant was serially diluted 4-fold from a maximum concentration of 500 nM, and 100 μl per well was added to the plate coated with CD40 Fc and incubated at 25°C for 1 hour. After washing 4 times with PBST, 100 μl of the detection antibody anti-his-HRP (1:4000 dilution) per well was added and incubated with shaking at 60 rpm for 1 hour. The plate was developed with 100 μL of TMB per well in the dark and stopped with 50 μL of stop solution per well. The plate was read at 450 nM using a Molecular Devices Microplate Reader. The data was analyzed using GraphPad Prisma 9.1 and the results are shown in Table 6.

Table 10

[0136] Octet® kinetic binding analysis Octet® kinetic binding analysis was performed in the same manner as in Example 1. Briefly, the binding ability of the purified anti-CD40 VHH to the HIS1K sensor chip was tested. Using 20 μg / ml of anti-CD40 VHH, it was loaded onto the chip. Loading was performed for 300 seconds to achieve a binding level of 1.8 - 2 nm. Human CD40 antigen was diluted to concentrations of 100, 150, 250, and 350 nM in 1xPBS for preparation for binding analysis. After starting the binding and monitoring for 200 seconds, the chip was transferred to 1xPBS buffer without CD40 protein to monitor dissociation. The results are shown in Table 7.

[0137]

Table 11

[0138] Inhibitory assay 1 μg / mL of CD40L-mFc (100 μL) prepared with antigen coating buffer in a 96-well plate was coated overnight at 4°C and then blocked with 2% BSA for 1 hour at 25°C. Serial dilution solutions of each CD40 VHH were pre-mixed with 0.025 μg / of CD40-hFc at 25°C for 30 minutes and then transferred to the plate coated with CD40L-mFc and incubated for 1 hour. After the plate was washed 4 times with PBST, it was incubated with mouse anti-human IgG Fc-HRP at 25°C for 1 hour. After washing 4 times with PBST, 100 μL of TMB per well was used to develop color in the dark for 10 - 20 minutes, and 50 μL of stop solution was added to stop the reaction. The plate was read at 450 nM using a Molecular Devices Microplate reader. ELISA data was analyzed using GraphPad Prisma 9.1. The results are shown in Figure 2, demonstrating that pgDD40-HG-24 is a CD40 receptor inhibitor.

[0139] Example 3. Anti-4-1BB VHH antibody Isolation of anti-4-1BB VHH antibody Llamas were immunized at Abcore, Inc. as in Example 1 with recombinant human 41BB (extracellular domain (Leu24-His183), accession # Q07011, SEQ ID NO: 41) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization).

Table 12

[0140] The sequences of 4-1BB-binding phage colonies obtained from the llama phage library were determined, and the amino acid sequences of each VHH are shown in Table 8 below. cDNA sequences based on the following amino acid sequences were synthesized using the pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce a recombinant single-domain antibody (sdAb) with a his tag at the C-terminus. The expressed sdAb was purified using a HisTrap HP column.

[0141] cDNA encoding the HSA-specific VHH was synthesized with an added C-terminal His tag and transiently transfected into HEK293 cells, and the positive VHH was purified by IMAC chromatography.

[0142] [Table 13]

[0143] The VHHs in Table 4 constitute a means for binding 4-1BB.

[0144] BLI kinetic binding analysis Octet (registered trademark) kinetic binding analysis was performed in the same manner as in Example 1. Briefly, the binding ability of the purified anti-CD40 VHH to the HIS1K sensor chip was tested. Using 20 μg / ml of anti-41BB VHH, it was loaded onto the chip. Loading was performed for 300 seconds to obtain a binding level of 1.8 - 2 nm. Human 41BB antigen was diluted to concentrations of 100, 150, 250, and 350 nM in 1xPBS for preparation for the binding analysis. After starting the binding and monitoring for 200 seconds, the chip was transferred to 1xPBS buffer without 41BB protein to monitor dissociation.

[0145] BLI kinetic binding analysis was performed in the same manner as in Example 1, and the results are shown in Table 9.

[0146] [Table 14]

[0147] ELISA assay The 96-well plate was coated with 4-1BB Fc prepared at 1 μg / ml using coating buffer at 4°C overnight at 100 μl / well. After that, it was blocked with 2% BSA at 200 μl / well at 25°C for 1 hour and then washed twice with PBST. The 4-1BB VHH supernatant was serially diluted 4-fold from a maximum concentration of 500 nM and 100 μl / well was added to the plate coated with 4-1BB Fc and incubated at 25°C for 1 hour. After washing 4 times with PBST, the detection antibody anti-his-HRP (1:4000 dilution) was added at 100 μl / well and incubated at 60 rpm for 1 hour. The plate was developed with 100 μL of TMB per well in the dark and stopped with 50 μL of stop solution per well. The plate was read at 450 nM using a Molecular Devices Microplate Reader. The data was analyzed using GraphPad Prisma 9.1. The results are shown in Table 10.

Table 15

[0148] Inhibition assay Using 1 μg / mL of 41BBL-mFc (100 μL) prepared with antigen coating buffer in a 96-well plate, it was coated overnight at 4°C and then blocked with 2% BSA at 25°C for 1 hour. Serial dilution solutions of each 41BB VHH were pre-mixed with 41BB-hFc 0.025 μg / at 25°C for 30 minutes and then transferred to the plate coated with 41BBL-mFc and incubated for 1 hour. After washing the plate 4 times with PBST, it was incubated with mouse anti-human IgG Fc-HRP at 25°C for 1 hour. After washing 4 times with PBST, 100 μL of TMB per well was used to develop color in the dark for 10 - 20 minutes, and 50 μL of stop solution was added to stop the reaction. The plate was read at 450 nM using a Molecular Devices Microplate reader. ELISA data was analyzed using GraphPad Prisma 9.1. The results are shown in Figure 6. Instead of 4-1BB56, 4-1BB59 dose-dependently inhibited the binding of 41BB to 41BBL.

[0149] Example 4. Isolation of anti-EGFR VHH antibodies Llamas were immunized with recombinant human epidermal growth factor receptor (EFGR) (extracellular domain (Met1-Ser645); accession # CAA25240; SEQ ID NO: 54) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization) at Abcore, Inc., and a phage library was prepared as in Example 1. [Table 16]

[0150] To select anti-EFGR VHH, biotinylated EFGR-His was incubated with the phage library and then bound to streptavidin dynabeads. After thorough washing, the bound phage was eluted with 1 mg / ml trypsin. The eluate from this selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0151] The cDNA encoding the EGFR-binding VHH was synthesized with an added C-terminal His tag, transiently transfected into HEK293 cells, and the positive VHH was purified by IMAC chromatography.

[0152] The sequences of the EGFR-binding phage colonies obtained from the immunized llama phage library were determined, and the amino acid sequences of each VHH are shown in Table 11 below. The cDNA sequences based on the following amino acid sequences were fused with human Fc and synthesized with the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce a recombinant anti-EGFR VHH antibody. The expressed anti-EGFR VHH was purified by a HiTrap Protein A column.

[0153] Several antibodies, namely pgEG5, pgEG12, pgEG-SX40, and pgEG-SX57, were humanized based on the sequences of the human germline.

Table 17

Table 18

[0154] The VHHs in Table 11 constitute a means for binding to EGFR.

[0155] Octet (registered trademark) kinetic binding analysis was performed in the same manner as in Example 1, and the results of K D are shown in Table 12

Table 19

[0156] ELISA assay 96-well plates were coated with EGFR Fc prepared at 1 μg / ml using coating buffer at 100 μl / well overnight at 4°C. After blocking with 2% BSA at 200 μl / well for 1 hour at 25°C, the plates were washed twice with PBST. The pgEG-SX VHH supernatant was serially diluted 4-fold from a maximum concentration of 500 nM and added to the plates coated with EGFR Fc at 100 μl / well, and incubated at 25°C for 1 hour. After washing 4 times with PBST, the detection antibody anti-his-HRP (1:4000 dilution) was added at 100 μl / well and shaken at 60 rpm for 1 hour. The plates were developed with 100 μL / well of TMB in the dark and stopped with 50 μL / well of stop solution. The plates were read at 450 nM using a Molecular Devices Microplate Reader. The data were analyzed using GraphPad Prisma 9.1. The results of the ELISA assay are shown in Table 13.

[0157] Inhibition assay EGF-mFc adjusted to 1 μg / mL (28 nM) using antigen coating buffer was coated on 96-well plates at 100 μL / well. After incubation overnight at 4°C, the plates were washed 4 times with PBST and blocked with 2% BSA at 200 μL / well for 1 hour at room temperature. Purified pgEG-SX40 or 57 VHH was serially diluted from 1000 nM and pre-incubated with 0.05 nM EGFR-hFc for 30 minutes at room temperature. The plates were washed twice with PBST, and the pre-mixed samples were added to the 96-well plates and shaken at 60 rpm for 1 hour at room temperature. After washing 4 times with PBST, the plates were incubated with 100 μl / well of the detection antibody anti-His-HRP (1:4000 dilution) and shaken at 60 rpm. The plates were washed 4 times with PBST, substrate TMB was added at 100 μl / well, and then 50 μl of stop solution was added. The plates were immediately read at OD 450 by an ELISA microplate reader (Table 14).

[0158] The purified VHH antibodies SX-40 and SX-57 inhibited the binding of EGFR to EGF, while the binding of SX-3 and SX-6 was only partially inhibited (Table 14). [Table 20] [Table 21]

[0159] Example 5. Anti-HSA VHH Antibody Immunization Llamas were immunized according to the standard protocol of Abcore Inc. Recombinant human HSA (SEQ ID NO: 113) was mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization). Six subcutaneous injections per llama were performed at 50 μg / dose every other week. On day 45, serum was collected from the immunized llamas to determine the antibody titer by ELISA. In ELISA, 96-well Maxisorp plates were coated with 100 ng / well of antigen. After blocking, diluted serum samples were added, and the presence of specific antibodies was demonstrated using horseradish peroxidase (HRP)-labeled goat anti-llama IgG (H+L) antibody. [Table 22]

[0160] Construction and Selection of Phage Library Peripheral blood mononuclear cells were prepared from the serum sample of an immunized llama on day 45 using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from the peripheral blood mononuclear cells using the RNeasy Midi Kit (Qiagen) according to the manufacturer's instructions and used as the starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were cloned into a self-made phagemid vector, infected with helper phage, and then recombinant phage particles were produced. These phage particles display VHH on the surface of the phage particles as a gene-III fusion protein. The phage was prepared according to the standard method and stored at 4 °C after filter sterilization for further use.

[0161] The phage library obtained from the immunized llama was used for selection. In the selection, biotinylated HSA was incubated with the phage library and then bound to streptavidin dynabeads (Invitrogen). After thorough washing, the bound phage was eluted with 1 mg / ml trypsin. The eluate from this selection was rescued with Escherichia coli TG1 cells. Colonies were picked and sequenced at BATJ Inc.

[0162] The cDNA encoding the positive VHH was synthesized with a C-terminal His tag added by Atum (DNA2.0, Inc.), transiently transfected into HEK293 cells, and purified by IMAC chromatography for in vitro functional assays.

[0163] Octet kinetic binding analysis The label-free technology of Bio-Layer Interferometry (BLI) was used to measure the binding rate of the llama-derived positive single-domain antibody. Affinity measurements were performed using an Octet QK equipped with an anti-Penta-His binding (HIS1K) biosensor chip (ForteBio®, Menlo Park, CA, USA). The assay was carried out at 30 °C in 1x PBS buffer (Gibco®, PBS pH 7.2). The samples were stirred at 1000 rpm. Before analysis, the sensor was wetted for 15 minutes. The purified single-domain antibody was tested for its binding ability to the HIS1K sensor chip. A 20 μg / ml single-domain antibody was loaded onto the chip. Loading was performed for 300 seconds to achieve a binding level of 1.8 - 2 nm. The test antigens were diluted to concentrations of 100, 150, 250, and 350 nM in 1x PBS for binding analysis. Binding was initiated and monitored for 200 seconds, after which the chip was transferred to a factor protein-free 1x PBS buffer (Gibco, PBS pH 7.2) to monitor dissociation. Sensor data were collected, processed, and analyzed using Octet data analysis software throughout the experiment.

[0164] Results Isolation of anti-HSA single-domain antibody The sequences of the positive phage colonies obtained from the immunized llama phage library were determined. The amino acid sequences are shown in Table 15. A cDNA sequence based on the following amino acid sequence was synthesized in a pJ607 expression vector with Atum (DNA2.0). The expression plasmid was transfected into the HEK293 cell line to produce a complete recombinant single-domain antibody with a his tag at the C-terminus. The expressed VHH was purified by a HisTrapHP column. [Table 23]

[0165] [Table 24]

[0166] ELISA binding assay of MSA VHH A 96-well plate was coated with 100 μL / well of HSA or MSA (mouse serum albumin) prepared at 1 μg / ml with coating buffer at 4°C overnight. After that, it was blocked with 200 μL / well of casein at room temperature for 1 hour. MSA VHH was prepared by serial dilution and added to the plate coated with HSA or MSA and incubated for 1 hour. The plate was washed 4 times with PBST and then incubated with streptavidin conjugated with HRP at 25°C for 1 hour. After washing 3 times with 250 μL of PBST per well, the plate was developed with 100 μL of TMB per well in the dark for 10 - 20 minutes and then stopped by adding 100 μL of stop solution. The plate was read at 450 nM with a Molecular Devices Microplate reader. The results are shown in Tables 16 - 19.

[0167] [Table 25] [Table 26] [Table 27]

[0168] BLI binding rate analysis of MSA VHH The kinetic binding analysis of MSA VHH was performed using a Gator Prime system equipped with analysis software. The Gator assay protocol was employed in all experiments described in the report below. The tests were conducted at 30 °C. Before analysis, the sensor was immersed in Q buffer and shaken at 1000 rpm for 600 seconds, and then 2 μM of HIS-tagged MSA VHH was loaded onto the anti-His sensor and shaken at 400 rpm for 120 seconds. The loaded sensor was transferred to Q buffer for 60 seconds. The binding of HSA or MSA was initiated at 100 nM, shaken at 1000 rpm, monitored for 120 - 180 seconds, and finally shaken at 1000 rpm for 120 - 180 seconds to dissociate in Q buffer. Software V2.0 (Gator Bio) was used for data analysis. The on-rate (K on ) and off-rate (K d ) were determined by fitting the binding and dissociation phases of each sample. The mathematical model used assumed a 1:1 stoichiometry and was fitted such that only one analyte in solution binds to one binding site on the surface. Then, the equilibrium dissociation constant (K D ), which is an indicator of affinity, was calculated as the ratio of K d to K on . The results are shown in Tables 20 - 23.

[0169]

Table 28

Table 29

Table 30

Table 31

[0170] Example 6. Anti-IL22 VHH antibody Immunization According to the standard protocol of Abcore Inc., llamas were immunized at Abcore Inc. Recombinant human IL-22 (SEQ ID NO: 120) was mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization). Six subcutaneous injections per llama were performed at 50 μg / dose every other week. On day 45, serum was collected from the immunized llamas to determine antibody titers by ELISA. In ELISA, a 96-well Maxisorp plate was coated with 100 ng / well of antigen. After blocking, diluted serum samples were added, and the presence of specific antibodies was demonstrated using horseradish peroxidase (HRP)-labeled goat anti-llama IgG (H+L) antibody.

[0171]

Table 32

[0172] Construction and selection of phage library Peripheral blood mononuclear cells were prepared from the 45-day serum samples of immunized llamas using Ficoll-Paque Plus according to the manufacturer's instructions. Total RNA was extracted from the peripheral blood mononuclear cells using the RNeasy Midi Kit (Qiagen) according to the manufacturer's instructions and used as the starting material for RT-PCR to amplify the gene fragments encoding VHH. These fragments were cloned into a self-made phagemid vector, infected with helper phage, and then recombinant phage particles were produced. These phage particles display VHH on the phage particle surface as a gene-III fusion protein. Phages were prepared according to the standard method and stored at 4°C after filter sterilization for further use.

[0173] The phage library obtained from immunized llamas was used for selection. In the selection, biotinylated HSA was incubated with the phage library and then bound to streptavidin dynabeads (Invitrogen). After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The eluate from this selection was rescued into Escherichia coli TG1 cells. Colonies were picked and sequenced at BATJ Inc.

[0174] The cDNA encoding the positive VHH was synthesized with a C-terminal His tag added by Atum (DNA2.0, Inc.), transiently transfected into HEK293 cells, and purified by IMAC chromatography for in vitro functional assays.

[0175] Octet kinetic binding analysis The label-free technology of Bio-Layer Interferometry (BLI) was used to measure the binding kinetics of llama positive single-domain antibodies. Affinity measurements were performed using an Octet QK equipped with an anti-Penta-His binding (HIS1K) biosensor chip (ForteBio®, Menlo Park, CA, USA). eIt was performed using [the relevant method]. The assay was carried out at 30 °C in 1x PBS buffer (Gibco®, PBS pH 7.2). The sample was stirred at 1000 rpm. Before analysis, the sensor was wetted for 15 minutes. The purified single-domain antibody was tested for its binding ability to the HIS1K sensor chip. A 20 μg / ml single-domain antibody was loaded onto the chip. Loading was performed for 300 seconds to obtain a binding level of 1.8 - 2 nm. The IL22 antigen was diluted to concentrations of 100, 150, 250, and 350 nM in 1x PBS for the binding assay. After starting the binding and monitoring for 200 seconds, the chip was transferred to 1x PBS buffer without the factor protein (Gibco, PBS pH 7.2) to monitor dissociation. The sensor data was collected throughout the experiment, processed, and analyzed using Octet data analysis software 7 (Forte Bio).

[0176] Results Isolation of anti-IL22 single-domain antibody The sequences of the positive phage colonies obtained from the immunized phage library were determined. The amino acid sequences are shown in Table 24. The cDNA sequences based on the following amino acid sequences were synthesized by Atum (DNA2.0) in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce a complete recombinant single-domain antibody with a his tag at the C-terminus. The expressed VHH was purified by a HisTrap HP column. [Table 33]

[0177] The results of the Octet binding affinity assay of IL22 VHH are shown in Table 25. [Table 34]

[0178] Example 7. Multispecific single-chain antibody To construct the multispecific single-chain antibodies, one or more VHH sequences of anti-OX40, anti-CD40, anti-4-1BB, anti-EGFR, anti-IL22, anti-HSA, anti-CD47, anti-CD16, anti-PD-L1, anti-CD33 and anti-LAG3 are fused via linkers in various configurations by recombinant DNA technology. All the content disclosed in WO2021 / 062361A2 regarding the VHH sequences specific to HSA, PD-L1, CD33, CD16 and LAG3 is incorporated herein by reference.

[0179] Examples of non-cleavable and cleavable linker sequences are shown in Table 26. These constitute linker means or means for linking protein domains. These means can be further characterized as cleavable or non-cleavable.

[0180]

Table 35

[0181] The sequences of the multispecific antibodies are shown in Tables 27 and 28.

Table 36

Table 37

[0182] Also disclosed herein are mutations from proline (P) to serine (S) at specific positions to reduce the formation of dimers and aggregates.

[0183] The results of the ELISA assay regarding the binding of SM2248 to rh-CD47 and rh-CD40 are shown in FIGS. 3A-B.

[0184] Flow cytometry assay of SM2248 Cells (viability > 95%) were washed with ice-cold PBS and the cell density was 1×10 6Resuspended in staining buffer (PBS - 2% FBS) at / mL. The resuspended cells were dispensed into 96-well plates at 90 μL / well. Serial dilution solutions of the test compounds prepared using the staining buffer were added to the 96-well plates at 10 μL / well, and incubated at room temperature for 45 minutes. The 96-well plates were washed 2 - 3 times with the staining buffer, and then incubated in the dark for 25 minutes with the detection antibody conjugated with the secondary antibody. Data was acquired by a flow cytometer and analyzed by software Flowjo. Human CD47-overexpressing CHO (CHO-hCD47), human CD40-overexpressing CHO (CHO-hCD40), and two tumor cell lines, namely Raji and A431, were used in the assay. In the assay, it was found that SM2248 binds to CD47 or CD40 on the cell surface with sub-nanomolar affinity (Figures 4A - D).

[0185] CD40 reporter assay of SM2248 CHO-CD47 cells in the logarithmic growth phase (survival rate above 95%) were dissociated with 0.25% trypsin and immediately treated with RPMI1640 complete medium. After centrifugation at 150 xg for 5 minutes, the cell pellet was resuspended in RPMI1640 complete medium at a density of 2×10 6 cells / ml and added to 96-well plates at 40 μL / well. Then, the 96-well plates were incubated overnight in a 37°C CO2 incubator. SM2248 serially diluted in DMEM medium was added to the 96-well plates at 10 μL each. The 96-well plates were incubated in a 37°C CO2 incubator for 60 minutes. 293T-NFKB-CD40 reporter cells in the logarithmic growth phase (survival rate above 95%) were dissociated with 0.25% trypsin and immediately treated with RPMI1640 complete medium. After centrifugation at 150 xg for 5 minutes, the cell pellet was resuspended at 4×10 5Resuspended in RPMI1640 complete medium at a density of cells / ml, and 50 μL per well was added to a 96-well plate treated with SM2248. The 96-well plate was incubated in a 37 °C - CO2 incubator for 6 hours. After incubation, the cells in each well were transferred to a 96-well black plate. The substrate of the One-Lite Luciferase Assay System was prepared according to the manufacturer's instructions. 100 μL of the substrate aliquot was added to each well. The assay plate was measured with a GLOMAX 96 microplate luminometer using the "CellTiterGlo" program. SM2248 is a CD40 agonist bispecific antibody and activated CD40 in a dose-dependent manner (Figure 5A).

[0186] Flow Cytometry Inhibition Assay of SM2248 Jurkat cells in the logarithmic growth phase (survival rate above 95%) were washed twice with ice-cold PBS and resuspended in staining buffer (PBS + 2% FBS) at a cell density of 1x10 6 / mL, and 90 μL per well was added to a 96-well plate. SM2248 and SIRPα-his serially diluted with staining buffer were added to Jurkat cells at 10 μL per well. The final concentration of SIRPα-his in each well was 1 μg / mL. Then, the 96-well plate was incubated at room temperature for 30 minutes. After incubation, the assay plate was washed three times with 250 μL of staining buffer and centrifuged at 500 xg for 3 minutes. The cell pellet in each well was suspended in a PE anti-His detection antibody (100 μL) diluted 1:100 and allowed to stand in the dark at room temperature for 25 minutes. The 96-well plate was washed three times with staining buffer and suspended in 150 μL of cold PBS buffer. The plate was read with an Agilent, NovoCyte, and the data were analyzed with Flowjo software. From this assay, SM2248 was shown to potently inhibit the binding of SIRP to CD47 on the surface of Jurkat cells at an IC 50 50 of 2.6 nM (Figure 5B).

[0187] From these results, SM2248 was shown to be a CD47-target-dependent agonist that activates CD40 in a dose-dependent manner.

[0188] The BLI binding affinity (K D ) assay of SM2248 is summarized in Table 29. SM2248 bound to human and cynomolgus monkey (Cyno) CD40 and CD47 with similar binding affinities, but had low binding affinities for mouse and rat proteins.

Table 38

[0189] The results of the ELISA binding assay of SM2235 to recombinant human EGFR (rh-EGFR) and rh-CD16A are shown in Figure 7, indicating that SM2235 bound to recombinant human EGFR and human CD16A with sub-nanomolar affinities. Flow cytometry analysis of SM2235 binding is shown in Figures 8 and 9.

[0190] The results of the BLI binding assay of SM2235 are shown in Table 30. The BLI binding assay showed that SM2235 bound to human and monkey EGFR or CD16A with similar K D but did not bind to rat and mouse. SM2235 did not cross-react with rh-ErbB2, rh-ErbB3, and rh-ErbB4.

[0191]

Table 39

[0192] Inhibition assay CHO-EGFR in the logarithmic growth phase (survival rate of 95% or more) was washed twice with ice-cold PBS, and the cell density was 1×10 6Resuspended in staining buffer (PBS + 2% FBS) at / mL and added 90 μL to each well of a 96-well plate. Serial dilution solutions of SM2235 and EGF-his in the staining buffer were added to Jurkat cells at 10 μL per well. The final concentration of EGF-his in each well was 1 μg / mL. Subsequently, the 96-well plate was incubated at room temperature for 30 minutes. After incubation, the assay plate was washed three times with 250 μL of staining buffer and centrifuged at 500 xg for 3 minutes. The cell pellet in each well was resuspended in 100 μL of 1:100 diluted PE anti-His detection antibody and left standing in the dark at room temperature for 25 minutes. The 96-well plate was washed three times with staining buffer and resuspended in 150 μL of cold PBS buffer. The plate was read with Agilent, NovoCyte, and the data was analyzed with Flowjo software. The results are shown in Figure 10.

[0193] NK cell cytotoxicity assay Method A431 cells were resuspended in complete medium KBM581 at a cell density of 1.25×10 5 / mL. Expanded human NK (eNK) cells were thawed and resuspended in complete medium KBM581 at 1×10 6It was resuspended at cells / mL. A431 (40 μL) and eNK cells (50 μL) were dispensed into each well of a 96-well plate. The ratio of effector cells (eNK) to target cells (A431) was set at 10:1. The SM2235 stock solution was prepared by serially diluting 5-fold from a maximum concentration of 1000 nM using complete medium KBM581, and 10 μL of it was added to each well of the cell plate. The concentrations of SM2235 in the wells were 100 nM, 20 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, and 0 nM. Control groups were prepared as eNK cells alone, A431 alone, A431 lysate, and complete medium KBM581. Measurement of specific cell death was performed according to the instructions of the CytoTox-Glo® Cytotoxicity Assay Kit. Briefly, after incubating at 37 °C - 5% CO2 for 4 hours, the plate was gently mixed, and the samples from each well were transferred to a 96-well flat-bottom black plate. The AAF-Glo reagent was prepared according to the instructions and 50 μL of it was added to each well. The A431 lysate group was treated with 50 μL of the lysis reagent. The plate was incubated at room temperature in the dark for 15 minutes. The luciferase activity of each well was measured using the built-in program "CellTiterGlo" of the Promega microplate luminescence detector. The results are shown in Figure 11.

[0194] Unless otherwise indicated, all numerical values representing amounts of ingredients, properties such as molecular weights, reaction conditions, etc. used in the specification and claims are to be understood as being modified in all instances by the term "about". As used herein, the terms "about" and "approximately" mean within 10 - 15%, preferably within 5 - 10%. Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, without limiting the application of the doctrine of equivalents to the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Although the numerical ranges and parameters setting forth the broad scope of the present invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical value inherently contains certain errors necessarily resulting from the standard deviation found in each individual test measurement.

[0195] The terms "a", "an", "the" and similar reference terms used in the context of describing the present invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural forms unless specifically indicated otherwise herein or clearly contradicted by the context. The recitation of numerical ranges herein is merely intended to serve as a shorthand method for referring individually to each separate numerical value falling within the range. Unless otherwise indicated herein, each separate numerical value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by the context. The use of any example, or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not otherwise limit the scope of the claimed invention. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0196] The grouping of alternative elements or embodiments of the invention disclosed herein is not to be construed as limiting. Members of each group may be referred to individually or in any combination with other members of the group or other elements found herein and may be claimed. For reasons of convenience and / or patentability, it is also contemplated that one or more members of a group may be included in or excluded from the group. When such inclusion or exclusion occurs, the specification is considered to include the group as modified so as to satisfy the description of all Markush groups used in the appended claims.

[0197] This specification describes specific embodiments of the invention, including the best mode known to the inventors for practicing the invention. Of course, variations of these described embodiments will be apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect those of ordinary skill in the art to appropriately employ such variations, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Further, unless otherwise stated herein or clearly contradicted by context, combinations in all possible variations of the above-described elements are included in the invention.

[0198] The specific embodiments disclosed herein may be further limited in the claims by the recitation of the transitional terms "consisting of" or "consisting essentially of." When used in the claims, whether in the filing application or added by amendment, the transitional term "consisting of" excludes elements, steps, or components not specified in the claims. The transitional term "consisting essentially of" limits the scope of the claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. The embodiments of the invention recited in the claims are those essentially or explicitly described herein and are operative.

[0199] Furthermore, throughout this specification, numerous patents and printed publications are referenced. Each of the above-cited patents and printed publications is hereby incorporated by reference in its entirety into this specification, individually.

[0200] Finally, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example and not limitation, another configuration of the invention can be utilized in accordance with the teachings of this specification. Accordingly, the invention is not to be limited to that precisely illustrated and described.

Claims

**Claim 1** A variable heavy chain (VHH) domain having antigen-binding specificity for OX40, wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 2 to 24, 106, or 125 to 142. **Claim 2** A VHH domain having antigen-binding specificity for CD40, wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 26 to 40. **Claim 3** A VHH domain having antigen-binding specificity for 4-1BB, wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 42 to 53. **Claim 4** A VHH domain having antigen-binding specificity for epidermal growth factor receptor (EGFR), wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 55 to 83. **Claim 5** A VHH domain having antigen-binding specificity for human serum albumin (HSA), wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 114 to 118. **Claim 6** A VHH domain having antigen-binding specificity for IL-22, wherein the VHH domain has an amino acid sequence of any one of SEQ ID NOs: 121 to 124. **Claim 7** An antibody comprising the VHH domain according to any one of Claims 1 to 6. **Claim 8** A multispecific antibody comprising an antibody-binding domain having a first binding specificity and a second antibody-binding domain having a second binding specificity different from the first binding specificity, wherein the first binding specificity is specificity for OX40, CD40, 4-1BB, HSA, IL-22, or EGFR, and wherein (a) the OX40 binding specificity is represented by an amino acid sequence of any one of SEQ ID NOs: 2 to 24, 106, or 125 to 142; (b) the CD40 binding specificity is represented by an amino acid sequence of any one of SEQ ID NOs: 26 to 40; (c) the 4-1BB binding specificity is represented by an amino acid sequence of any one of SEQ ID NOs: 42 to 53; (d) the EGFR binding specificity is represented by an amino acid sequence of any one of SEQ ID NOs: 55 to 83; and (e) the HSA binding specificity is represented by an amino acid sequence of any one of SEQ ID NOs: 114 to 118; and (f) The IL-22 binding specificity is a multispecific antibody represented by any one of the amino acid sequences of SEQ ID NOs: 121 to 124. **Claim 9** The multispecific antibody according to claim 8, further comprising 1 to 5 additional antibody binding domains, each additional antibody binding domain being specifically directed to OX40, CD40, 4-1BB, HSA, IL-22 or EGFR. **Claim 10** The multispecific antibody according to claim 9, further comprising 1 to 4 additional antibody binding domains, each additional antibody binding domain being specific for OX40, CD40, 4-1BB, HSA, IL-22 or EGFR. **Claim 11** The multispecific antibody according to any one of claims 8 to 10, wherein the antibody is a multispecific single-chain antibody (MVSCA). **Claim 12** The multispecific antibody according to any one of claims 8 to 11, wherein the linker is selected from the linkers in Table 26. **Claim 13** The multispecific antibody according to claim 12, wherein linker L1 (SEQ ID NO: 84), L2 (SEQ ID NO: 85) or L4 (SEQ ID NO: 87) is interposed between one or more pairs of non-identical antibody binding domains. **Claim 14** The multispecific antibody according to any one of claims 8 to 12, comprising at least one pair of antibody binding domains having the same specificity. **Claim 15** The multispecific antibody according to claim 14, wherein at least one pair of antibody binding domains having the same specificity are adjacent to each other. **Claim 16** The multispecific antibody according to claim 15, wherein a linker having the amino acid sequence of linker L3 (SEQ ID NO: 86) is interposed between antibody binding domains having the same specificity. **Claim 17** The multispecific antibody according to any one of claims 8 to 16, wherein all of the antibody binding domains are VHH domains. **Claim 18** The multispecific antibody according to any one of claims 8 to 17, wherein the multispecific antibody has any one of the amino acid sequences of SEQ ID NOs: 107 to 112. **Claim 19** A pharmaceutical composition comprising the VHH domain according to any one of claims 1 to 6, the antibody according to claim 7, or the multispecific antibody according to any one of claims 8 to 18. **Claim 20** A method for treating cancer, characterized by administering the pharmaceutical composition according to claim 19 to a patient in need of treatment. **Claim 21** A method for treating an autoimmune disease, characterized by administering the pharmaceutical composition according to claim 19 to a patient in need of treatment.

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