Monospecific 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. In particular, the present disclosure provides variable heavy (VHH) domains of antibodies and related sequences that are specific for one or more of OX40, CD40, 4-1BB, HSA, IL-22, or EGFR. Further disclosed are methods of treating autoimmune diseases using monospecific or multispecific antibodies.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 356,385 filed on June 28, 2022 and U.S. Provisional Patent Application No. 63 / 434,814 filed on December 22, 2022, the entire contents of which are incorporated herein by reference.

[0003] Sequence Listing

[0004] A Sequence Listing is submitted herewith and is incorporated herein by reference as an XML file entitled "1959708-00014_Sequence_Listing.xml" created on June 27, 2023 and having a size of 138 KB.

[0005] summary

[0006] 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 comprising two or more VHH domains specific for one or more of these antigens.

[0007] Some embodiments are single domain antibodies that comprise only or predominantly the VHH domain of a camelid antibody. These embodiments are monospecific and monovalent.

[0008] Some embodiments comprise a VHH domain fused to one or more constant domains from a conventional antibody (e.g., the Fc region of a human IgG antibody). These embodiments are monospecific, but are typically bivalent. Other valencies are also possible, depending on, for example, the choice of constant domains. The Fc region of IgA and IgM can confer higher valencies.

[0009] Some embodiments comprise two VHH domains specific for the same antigen linked in a single amino acid chain (multivalent single chain antibodies). These embodiments are also monospecific and bivalent. Additional VHH domains may be linked to obtain higher valence.

[0010] Some embodiments comprise two (or more) VHH domains, wherein each VHH domain is specific for a different antigen linked in a single amino acid chain (multivalent, multispecific single chain antibodies). These embodiments are multivalent and multispecific. In further embodiments comprising three or more VHH domains, two or more VHH domains may be specific for the same antigen, while one or more other VHH domains are specific for different antigens. Such constructs have a higher valence than specificity.

[0011] Each monospecific embodiment is specific for OX-40, CD40, HSA, IL-22, 4-1BB or EFGR. Each multispecific embodiment is specific for one or more of OX-40, CD40, 4-1BB, HSA, IL-22 and EFGR, but may also be specific for one or more other antigens.

[0012] In some embodiments comprising multiple antigen binding domains, antigen binding domains derived from conventional VL-VH pairings may be used in place of one or more (but not all) VHH domains in the above embodiments.

[0013] An antigen binding domain disclosed herein that is specific for a particular antigen can be referred to as a means for binding the antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Depicted is a blocking assay of CD40 binding to CD40L by the anti-CD40 antibody pgDD40-HG-24.

[0015] Figure 2 Blockade of CD40 binding to CED40L by anti-CD40 VHHs is depicted.

[0016] Figure 3A -B depicts ELISA binding analysis of SM2248. Figure 3A Binding of SM2248 to recombinant human CD47-Fc is depicted. Figure 3B Binding of SM2248 to recombinant human CD40-Fc is depicted.

[0017] Figure 4A -D depicts the interaction between SM2248 and cancer cell line CHO-hCD47 ( Figure 4A )、CHO-hCD40( Figure 4B )、Raji( Figure 4C ) and A431( Figure 4D ) Flow cytometric binding analysis of the combined

[0018] Figure 5A -B depicts a cell-based potency assay. Figure 5A HEK-NFKB reporter gene assay is depicted. Figure 5B Depicted is the blocking of SIRP binding by SM2248 to Jurkat cells.

[0019] Figure 6 Depicted is 4-1BB59 blocking the binding of 4-1BB to 4-1BB-L.

[0020] Fig. 7A-B depicts ELISA binding analysis of SM2235-113. Fig. 7A Binding of SM2235-113 to recombinant human EGFR-Fc is depicted. Figure 7B Binding of SM2235-1113 to recombinant CD16A is depicted.

[0021] Fig. 8A -D depicts the interaction between SM2235 and cancer cell line CHO-K1-EGFR( Fig. 8A )、A431( Figure 8B )、MB231( Figure 8C ) and HCT116( Fig.8D ) Flow cytometric binding analysis of the combined

[0022] Fig.9A -B depicts the relationship between SM2235 and Jurkat-CD16A cells ( Fig.9A ) and human NK cells ( Fig. 9B ) by flow cytometry binding analysis of CD16A binding on .

[0023] Fig.10 Depicted is the blockade of EGF binding to EGFR on a CHO-EGFR overexpressing cell line.

[0024] Fig.11 Human NK cell cytotoxicity assay, 10:1 NK:A431 is depicted.

[0025] Detailed Description

[0026] Disclosed herein are monospecific immunoglobulin variable domains (referred to as VHH single domain antibodies) that are specific for OX-40, CD40, 4-1BB, HSA, IL-22, and epidermal growth factor receptor (EFGR), as well as multivalent single chain antibodies (MVSCAs) that combine the variable domains of two or more VHHs and are specific for one or more of these antigens.

[0027] As used herein, the term VHH refers to the variable domain of a heavy chain antibody, and is an antigen-binding fragment of only a heavy chain antibody.

[0028] In some embodiments, MVSCA comprises two or more variable domains that are specific to the same antigen. That is, MVSCA is multivalent, but monospecific relative to the antigen. In some of these embodiments, MVSCA comprises two or more repeated identical VHH variable domains or multiple VHH variable domains, each VHH variable domain having specificity to the same epitope. That is, they are multivalent, but monospecific relative to the epitope. Such MVSCA will only bind to a single site on an antigen monomer, but can cross-link multiple copies of monomers. In other embodiments of these embodiments, MVSCA comprises two or more VHH variable domains, each VHH variable domain having specificity to different epitopes of the same antigen. That is, they are multivalent and multispecific relative to the epitope. Such MVSCA can be bound to multiple sites on an antigen monomer or multiple copies of cross-linked monomers.

[0029] In some embodiments, the MVSCA comprises two or more VHH variable domains that are specific for different antigens, i.e., they are multivalent and multispecific with respect to the antigen. In further embodiments, the MVSCA comprises a plurality of VHH variable domains, wherein the additional variable domains are identical to the first VHH variable domain, wherein the additional VHH variable domains are different from the first VHH variable domain but are specific for different epitopes on the same antigen, or wherein the additional VHH variable domains are different from the first VHH variable domain but are specific for different antigens, in any combination.

[0030] The MVSCA comprising two or more VHH variable domains can further comprise immunoglobulin constant domains.For example, the VHH variable domain C-terminal can remain connected to its original VHH constant domain.Alternatively, the VHH variable domain C-terminal can be connected to the constant domain or Fc region of more conventional antibodies (for example human antibodies, such as human IgG antibodies).In some embodiments, constant domain or complete Fc region can confer specific functions as those skilled in the art are familiar with.In other embodiments, the MVSCA comprising two or more VHH variable domains can further comprise constant domains, wherein constant domains are located between VHH variable domains or at the N-terminal of VHH variable domains, rather than at the C-terminal of VHH variable domains, or in addition, also at the variable C-terminal of VHH.

[0031] antigen

[0032] OX40 (CD134; TNFRSF4) is a T cell co-stimulatory molecule of the tumor necrosis factor (TNF) receptor superfamily, which coordinates with other co-stimulatory factors (CD28, CD40, CD30, CD27, and 4-1BB) to manage the activation of immune responses. Under the co-stimulation of CD40-CD40 ligand and CD28-B7, OX40 activates CD4 + and CD8 + OX40 is upregulated in T cells. The interaction of OX40 with OX40 ligands on antigen presenting cells enhances T cell survival, proliferation and cytokine production. It also inhibits the transformation of effector T cells to regulatory T cells (Tregs) and can promote the maintenance and recall of memory T cell responses. OX40 is constitutively expressed on Tregs, where it promotes Treg proliferation and immunosuppressive activity. OX40-OX40 ligand signaling is involved in allergic airway inflammation, graft-versus-host disease and autoimmune diseases.

[0033] CD40, also known as TNFRSF5, is a 45-50 kDa type I transmembrane glycoprotein member of the TNF receptor superfamily. Mature human CD40 consists of an extracellular domain of 173 amino acids (aa), a transmembrane domain, and a 62 aa cytoplasmic domain. The extracellular domain of human CD40 has 58% and 56% aa sequence identity with mouse and rat CD40, respectively. Antagonistic soluble human CD40 splice variants contain alternative sequences within the extracellular and transmembrane domains and lack the cytoplasmic domain. CD40 is expressed on the surface of B cells, dendritic cells, macrophages, monocytes and platelets, as well as endothelial and epithelial cells. The interaction of CD40 with its ligand CD40 ligand results in the aggregation of CD40 molecules, thereby initiating bidirectional intracellular signaling in cells expressing CD40 and CD40 ligand. CD40 ligation with CD40 can promote B cell activation and T cell-dependent humoral responses. CD40 has multiple functions in hematopoietic and epithelial cancers and is a target for tumor immunotherapy.

[0034] Epidermal growth factor receptor (EGFR) is a transmembrane protein and a receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. EGFR is a member of the ErbB receptor family, which is 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 types of cancer, mutations that affect EGFR expression or activity can lead to cancer. The epidermal growth factor receptor is a transmembrane protein that is activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Signaling defects in EGFR and other receptor tyrosine kinases in humans are associated with diseases such as tumors, while overexpression is associated with the development of a variety of tumors. By blocking the EGFR binding site on the receptor extracellular domain, or by inhibiting the activity of the intracellular tyrosine kinase to interrupt EGFR signaling, the growth of tumors expressing EGFR can be prevented and the patient's condition can be improved.

[0035] 4-1BB, also known as CD137 and TNFRSF9, is a transmembrane glycoprotein of about 30kDa in the TNF receptor superfamily. 4-1BB plays a role in the development and activation of a variety of immune cells. Mature human 4-1BB consists of a 163aa extracellular domain (ECD) with four TNFR cysteine-rich repeats (SEQ ID NO: 41), a 27aa transmembrane segment, and a 42aa cytoplasmic domain. Within the ECD, human 4-1BB has 60% amino acid sequence identity with mouse and rat 4-1BB. 4-1BB is expressed as a disulfide-linked homodimer on various activated T cell populations, including CD4+, CD8+, memory CD8+, NKT, and regulatory T cells, as well as myeloid and mast cell progenitor cells, dendritic cells, mast cells, and bacterially infected osteoblasts. It binds to transmembrane 4-1BB ligands / TNFSF9 with high affinity, which is expressed on antigen presenting cells and bone marrow progenitor cells. This interaction co-stimulates the proliferation, activation and / or survival of cells expressing 4-1BB. It can also enhance cell death induced by repeatedly stimulated T cell activation. Mice lacking 4-1BB exhibit enhanced T cell activation, which may be due to its absence on regulatory T cells. 4-1BB can bind to OX40 on activated T cells to form a complex that responds to either ligand and inhibits the proliferation of Treg and CD8+T cells. Reverse signaling through 4-1BB ligands inhibits the development 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. 4-1BB activation enhances CD8+T cell- and NK cell-mediated antitumor immunity. It also contributes to the development of inflammation in metabolic syndrome induced by a high-fat diet. Soluble forms of 4-1BB and 4-1BB ligands circulate at elevated levels in the serum of patients with rheumatoid arthritis and hematological cancers, respectively.

[0036] Human serum albumin (HSA) is a serum albumin found in human blood. It is the most abundant protein in human plasma, accounting for about half of serum proteins. It is produced in the liver. It is soluble in water and monomers. Albumin has the functions of transporting hormones, fatty acids and other compounds, buffering pH, maintaining swelling pressure, and other functions. Albumin is synthesized in the liver as prealbumin, which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product proalbumin is in turn cleaved in Golgi vesicles to produce secreted albumin. The reference range of albumin concentration in serum is about 35-50 g / L (3.5-5.0 g / dL). Its serum half-life is about 20 days. Its molecular weight is 66.5 kDa. The long serum half-life of albumin is achieved in part by its size of 66 kDa, which prevents clearance through the kidneys, and by its interaction with the neonatal Fc receptor (FcRn). Fusion to anti-albumin VHH has been used to increase the half-life of anti-tumor nanobodies from 1-2 h to approximately 10 days.

[0037] Interleukin-22 (IL-22), also known as IL-10-related T cell-derived inducing factor (IL-TIF), was originally 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 33aa signal peptide that is cleaved to produce a 147aa mature protein that has approximately 79% and 22% aa sequence identity with mouse IL-22 and human IL-10, respectively. The human IL-22 gene is located on chromosome 12q15. Although it exists as a single copy gene in humans and many mouse strains, the mouse IL-22 gene is repeated in some mouse strains (including C57B1 / 6, FVB and 129). The two mouse genes named IL-TIFα and IL-TIFβ share greater than 98% sequence homology in their coding regions. IL-22 has been shown to activate STAT 1 and STAT 3 in several hepatoma cell lines and upregulate the production of acute phase proteins. IL-22 is produced by normal T cells in humans after anti-CD3 stimulation. IL-22 expression in mice is also induced in various organs after lipopolysaccharide injection, indicating that IL-22 may be involved in inflammatory responses. The functional IL-22 receptor complex consists of two receptor subunits, IL-22R (formerly known as the orphan receptor for CRF2-9) and IL-10Rβ (formerly known as CRF2-4), belonging to the class II cytokine receptor family.

[0038] Antibody

[0039] Antibodies and their use for treating 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 comprising an antigen binding site. Antibodies specifically bind to antigens and may be able to modulate the biological activity of the antigen. As used herein, the term "antibody" may include "full-length antibodies" and "antibody fragments". As used herein, the term "binding site" or "antigen binding site" refers to the region of an antibody molecule to which a ligand actually binds. The term "antigen binding site" includes an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or in the case of a heavy chain-only antibody, includes an antibody heavy chain variable region.

[0040] Antibody specificity refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, a natural antibody is monospecific. As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each binding site binding 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 a VHH domain heavy chain. In other embodiments, the monospecific antibody comprises a VHH domain fused to one or more protein domains, the protein domain comprising, for example, a human Fc region. In other embodiments, the monospecific antibody comprises VHH as the only complete protein domain, i.e., a single domain antibody. In some embodiments, a single domain antibody may additionally comprise a short peptide, such as a His tag. The VHH domain may be referred to as a method for binding to a specific target (such as OX-40, CD40, 4-1BB, HSA, IL-22 or EFGR). Thus, any of the various antibody structures, formats, or constructs disclosed herein that contain a VHH domain or are constructed to contain a VHH domain can be referred to as an antibody that comprises a means for binding to an indicated target. Some embodiments may specifically include one or more specific antibody structures, formats, or constructs. Other embodiments may specifically exclude one or more specific antibody structures, formats, or constructs.

[0041] As used herein, "antibody having specificity", "antibody that recognizes", "antibody having affinity", "antibody having a binding site" and similar constructs are used interchangeably.

[0042] "Multispecific antibody" refers to an antibody with two or more antigen binding specificities. The multispecific antibodies disclosed herein are specific to at least two of OX-40, CD40, 4-1BB, HSA, IL-22 and EFGR, or are specific to at least one of the aforementioned specificities and at least a second specificity. In some embodiments, the multispecific antibodies disclosed herein may include two, three, four or more domains capable of binding to antigens. In addition, the multispecific antibody may include at least two copies of the same antigen binding sequence, or two antigen binding sequences specific to different epitopes (biparatopes) on the same antigen, as long as the multispecific antibody is specific to at least one of OX-40, CD40, 4-1BB and EFGR and at least one second antigen. In some embodiments, the multispecific antibody (MVSCA) is specific to 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. Therefore, some multispecific antibodies may be referred to as antibodies comprising a means for binding to a first target and a means for binding to a second target, etc.

[0043] "Bispecific antibodies" refer to antibodies with 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 connected in various configurations. In some embodiments, the amino acid sequences encoding the antibody binding portions of the bispecific antibodies are connected by a linker as disclosed herein.

[0044] "Trispecific antibody" refers to an antibody with 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 connected in various configurations. In some embodiments, the amino acid sequences encoding the antibody binding portions of the trispecific antibodies are connected by a linker as disclosed herein. In some embodiments, two linkers are used, which can be the same or different.

[0045] "Quadruplets" refers to antibodies with four different antigen binding specificities. In some embodiments, the quadruplets 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 quadruplets can be connected in various configurations. In some embodiments, the amino acid sequences encoding the antibody binding portions of the quadruplets are connected by a linker as disclosed herein. In some embodiments, two linkers are used, which can be the same or different.

[0046] As used herein, the term "valence" indicates that a specified number of binding sites are present in an antibody molecule. Thus, the terms "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," and "octavalent" indicate that there are two binding sites, three binding sites, four binding sites, five binding sites, six binding sites, seven binding sites, and eight binding sites, respectively, in an antibody molecule. The bispecific antibodies disclosed herein are "bivalent." The trispecific antibodies disclosed herein are "trivalent." The quadruplets disclosed herein are "tetravalent." However, monospecific multivalent antibodies, such as bivalent, trivalent, and tetravalent antibodies, are within the scope of the present disclosure, wherein multiple antigen binding sites bind to the same antigen. The antigen binding sites of monospecific bivalent and trivalent (or higher) antibodies can bind to the same epitope or different epitopes on the antigen. Similarly, by combining multiple monospecific binding sites with one or more other specific binding sites, antibodies with a higher valence than multispecific, such as trivalent bispecific antibodies, can be constructed.

[0047] "Full-length antibody" herein refers to the structure of the natural biological form of the antibody, including variable regions and constant regions. For example, in most mammals (including humans and mice), IgG class full-length antibodies are tetramers and consist of two pairs of identical two immunoglobulin chains, each pair having a light chain and a heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2 and CH3. In some mammals, such as camels and llamas, IgG antibodies can also consist of only two variable heavy chains, each heavy chain comprising a variable domain (VHH) (CH2 and CH3 domains) connected to the Fc region.

[0048] Tetrameric antibodies are 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 consists of two different regions, called the variable region and the constant region. For IgG class immunoglobulins, the heavy chain consists of four immunoglobulin domains connected in the order VH-CH1-CH2-CH3 from N-terminus to C-terminus, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (also called VH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constant γ1 domain, constant γ2 domain, and constant γ3 domain, respectively). The IgG light chain consists of two immunoglobulin domains connected in the order VL-CL from N-terminus to C-terminus, referring to the light chain variable domain and the light chain constant domain, respectively. The constant region shows less sequence diversity and is responsible for binding to many natural proteins to trigger important biochemical events.

[0049] The variable region of an antibody contains the antigen-binding determinants of the molecule and therefore determines the specificity of the antibody for its target antigen. The variable region is so named because it is the most different in sequence from other antibodies of the same class. In the variable region, three loops are gathered in each V domain of the heavy and light chains to form the antigen-binding site. Each loop is called a complementarity determining region (hereinafter referred to as a "CDR"), and its amino acid sequence varies most significantly. There are six CDRs in total, three for each heavy and light chain, named VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside of the CDRs is called the framework (FR) region. Although not as diverse as the CDRs, there is indeed sequence variability in the FR region between different antibodies. Overall, this characteristic structure of antibodies provides a stable scaffold (FR region) on which the immune system can explore a large amount of antigen-binding diversity (CDRs) to obtain specificity for a wide range of antigens.

[0050] The genes encoding the immunoglobulin loci contain multiple V region sequences as well as shorter nucleotide sequences designated "D" and "J", and it is the combination of V, D and J nucleotide sequences that gives rise to VH diversity.

[0051] Antibodies are divided into several classes, also called isotypes, which are genetically determined by the constant region. Human constant light chains are classified as kappa (Cκ) and lambda (Cλ) light chains. Heavy chains are classified as mu (μ), delta (δ), gamma (γ), alpha (α) or epsilon (ε), and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA and IgE, respectively. The IgG class is most commonly used for therapeutic purposes. In humans, this class includes subclasses IgG1, IgG2, IgG3 and IgG4. In mice, this class includes subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Therefore, as used herein, "isotype" refers to any class or subclass of immunoglobulins defined by the chemical and antigenic characteristics of its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD and IgE. The disclosed VHH antibodies, bispecific and multispecific antibodies may have a constant region comprising all or part of the above isotypes.

[0052] Antibody fragments are also within the scope of the present disclosure, including but not limited to (i) Fab fragments comprising VL, CL, VH and CH1 domains, (ii) Fd fragments comprising VH and CH1 domains, (iii) Fv fragments comprising the VL and VH domains of a single antibody; (iv) dAb fragments comprising a single variable region, (v) isolated CDR regions, (vi) bivalent fragments F(ab')2 comprising two connected Fab fragments, and (vii) single-chain Fv molecules (scFv), wherein the VH domain and the VL domain are connected by a peptide linker that allows the two domains to bind to form an antigen binding site. Also disclosed are trivalent or tetravalent antibody fragments comprising variable domains having three different specificities and connected by cleavable or non-cleavable links. In certain embodiments, the antibodies are produced by recombinant DNA technology. In other embodiments, the antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies.

[0053] As used herein, "single-chain antibody" refers to a fusion protein of the antigen binding portion (i.e., variable region) of an antibody, usually connected by a linker peptide. Multivalent single-chain antibodies and multispecific single-chain antibodies are disclosed herein. The monospecific multivalent antibody is specific for at least one of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR. The multispecific single-chain antibody is specific for at least one of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR, plus at least one additional specificity. In some embodiments, the multispecific single-chain antibody is specific for at least two of OX-40, CD40, 4-1BB, HSA, IL-22, and EFGR.

[0054] As used herein, "humanized" antibodies refer to antibodies comprising a human framework region (FR) and one or more complementary determining regions (CDRs) from a non-human antibody. The non-human antibody providing the CDR is called a "donor", and the human immunoglobulin providing the framework is called an "acceptor". In certain embodiments, humanization mainly relies on transplanting the donor CDR to the acceptor (human) VL or VH framework. This strategy is referred to as "CDR transplantation". It is usually necessary to "revert" the selected acceptor framework residues to the corresponding donor residues to regain the affinity lost in the initial transplant construct. Humanized antibodies will also optimally include at least a portion of an immunoglobulin constant region, typically a constant region of a human immunoglobulin, and will typically include a human Fc region. Humanization or other methods for reducing the immunogenicity of non-human antibody variable regions may include a surface remodeling method. In one embodiment, a screening-based method can be used to humanize and / or affinity-matured antibody variable regions, i.e., increase the affinity of the variable region to its target antigen. Other humanization methods may involve the transplantation of only part of the CDR, including but not limited to the method described in US 6,797,492, all of which are incorporated herein by reference for all disclosures regarding CDR transplantation. Structure-based methods may be used for humanization and affinity maturation, such as described in US 7,117,096, all of which are incorporated herein by reference for all disclosures regarding humanization and affinity maturation.

[0055] In various embodiments herein, the antibody is a VHH. Camelids (camels, dromedaries and llamas) contain not only traditional heavy and light chain antibodies (containing 2 light chains and 2 heavy chains in one antibody), but also double-chain antibodies (containing only variant heavy chains). Dimeric antibodies are encoded by a group of different VH segments called VHH genes. VH and VHH are scattered in the genome (i.e., they seem to be mixed with each other). The identification of the same D segment in VH and VHH cDNAs shows that the D segment is used for the common use of VH and VHH. Antibodies containing natural VHH lack the entire CH1 domain of the heavy chain constant region. The exons encoding the CH1 domain are present in the genome, but are spliced ​​out due to the loss of the functional splicing acceptor sequence on the 5' side of the CH1 exon. Therefore, the VDJ region is spliced ​​to the CH2 exon. When VHH is recombined onto such constant regions (CH2, CH3), an antibody is produced in which the half antibody is a single chain rather than a light chain / heavy chain pair (i.e., an antibody with two heavy chains without light chain interactions). The binding of the antigen is different from that of a conventional antibody, but the high affinity is achieved in the same way, i.e., by hypermutation of the variable region and selection of cells expressing such high affinity antibodies.

[0056] In an exemplary embodiment, the disclosed VHHs are produced by immunizing transgenic mice in which endogenous murine antibody expression has been eliminated and a camelid transgene has been 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, all of which are incorporated herein by reference for all disclosures regarding heavy chain-only antibodies and their production in transgenic mice. VHH mice were immunized and the resulting primed splenocytes were fused with murine myeloma cells to form hybridomas.

[0057] In other embodiments, VHHs are produced by immunizing camels with the desired antigen and isolating and sequencing the resulting antigen-binding antibody VHH regions. In one embodiment, VHHs are isolated using phage display libraries. See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204 (each of which is incorporated by reference in its entirety to describe the preparation of phage libraries).

[0058] Also disclosed herein are multispecific or multivalent antibodies in which two or more antigen binding domains are connected in a single fusion protein. Multispecific antibodies can take a variety of forms, including (i) multispecific Fv fragments; (ii) a heavy chain of a first specificity having a second VH domain associated with it (or fused with it) having a second specificity; (iii) a tetrameric monoclonal antibody of a first specificity having a second VH domain associated with it having a second specificity, wherein the second VH domain is associated with the first VH domain; (iv) a Fab fragment of a first specificity (VH-CH1 / VL-CL) having a second VH domain associated with it having a second specificity. Exemplary Fab fragments include those in which a second VH sequence having a second specificity is associated with the C-terminus or N-terminus of the first VH domain, or the C-terminus or N-terminus of the first CH1 or first CL domain. In additional embodiments, the VH sequence having a second and / or third (or more) specificity can be associated (or fused) with the C-terminus or N-terminus of the first VH domain or the C-terminus or N-terminus of the first CH1 or first CL domain. In various embodiments, any of these forms may include at least one of the VHH domains disclosed herein.Examples of configurations of multispecific antibodies can be found in WO2021 / 062361, all of which is incorporated herein by reference for its disclosure regarding multispecific antigen configurations.

[0059] Multispecific or multivalent antibodies may include a linker sequence that connects a specific antigen binding domain (such as VH or VHH) to another antigen binding domain, and it allows the proper folding of the amino acid sequence to produce the desired three-dimensional conformation and antigen binding spectrum. Generally, the linker sequence will be a short amino acid sequence that provides enough space and flexibility between the domains to allow them to fold correctly. The linker may also cause steric hindrance to promote the combination with the target of each domain. Suitable linkers include, but are not limited to, linkers (SEQ ID NO: 84-103), EPKSCD (SEQ ID NO: 104) and ASTKGP (SEQ ID NO: 105) in Table 26. Other linkers are known to those skilled in the art.

[0060] Amino acid sequence variants of the monospecific or multispecific antibodies disclosed herein are also within the scope of the present disclosure. Amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the antibody encoding DNA, or by peptide synthesis. Such variants include, for example, residue deletions and / or insertions and / or substitutions within the amino acid sequences of the antibodies of the embodiments herein. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, as long as the final construct has the desired characteristics. Amino acid changes can also change the post-translational processes of the humanized or variant antibodies, such as changing the number or position of glycosylation sites.

[0061] A useful method for identifying certain residues or regions of an antibody as preferred locations for mutagenesis is called "alanine scanning mutagenesis". A residue or a group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral amino acids (most preferably alanine or polyalanine) to affect the interaction of the amino acid with the antigen. The amino acid positions that are functionally sensitive to substitution are then improved by introducing further or other variants at or for the substitution site. Therefore, although the site for introducing amino acid sequence variation is predetermined, the nature of the mutation itself does not need to be predetermined. For example, in order to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is performed at the target codon or region, and the antibody variants expressed are screened for desired activity.

[0062] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues disclosed herein or antibodies fused to epitope tags. Other insertion variants of antibody molecules include fusions to the N- or C-termini of antibodies with enzymes or polypeptides that increase the serum half-life of the antibody.

[0063] Another type of variant is an amino acid substitution variant. These variants remove at least one amino acid residue in the antibody molecule and insert different residues in its position. The most interesting sites of substitution mutations include hypervariable regions, but FR changes are also contemplated. Conservative substitutions are shown under the heading of "preferred substitutions" in Table 1. If such substitutions result in changes in biological activity, more substantial changes can be introduced, referred to as "exemplary substitutions" in Table 1, or as further described below with reference to amino acid classes, and the product is screened.

[0064] surface

[0065]

[0066]

[0067] Substantial changes in the biological properties of antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into several groups based on the common properties of the side chains:

[0068] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0069] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0070] (3) Acidic: Asp, Glu;

[0071] (4) Basic: Asn, Gln, His, Lys, Arg;

[0072] (5) Residues that affect chain orientation: Gly, Pro; and

[0073] (6) Aromatic: Trp, Tyr, Phe.

[0074] Non-conservative substitutions will entail exchanging a member of one class for another class.

[0075] Any cysteine ​​residue not involved in maintaining the proper conformation of the monospecific or multispecific antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0076] Another type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or camel antibodies). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody that produces them. A convenient way to produce such substitution variants is to use phage display for affinity maturation. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed as fusions with the M13 gene III product packaged in each particle from filamentous phage particles in a monovalent manner. Then, as disclosed herein, the biological activity (e.g., binding affinity) of the variants displayed by phage screening. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, analyzing the crystal structure of the antigen-antibody complex may be beneficial to identify the contact points between the antibody and the antigen. According to the technology set forth herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the panel of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0077] Another type of amino acid variant of an antibody alters the original glycosylation pattern of the antibody. The alteration refers to deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody.

[0078] Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate 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 the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one 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.

[0079] Addition of glycosylation sites to the antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Such alterations can also be made by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) in the sequence of the original antibody.

[0080] Nucleic acid molecules encoding amino acid sequence variants of monospecific or multispecific antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolating from natural sources (in the case of naturally occurring amino acid sequence variants) or preparing earlier prepared variants or non-variant forms of the antibodies disclosed herein by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0081] Other modifications may be considered for monospecific or multispecific antibodies. For example, it may be necessary to modify the antibody in terms of effector function in order to enhance the effectiveness of the antibody, for example, in treating a disease. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. The homodimeric antibody thus produced may have improved internalization capacity and / or enhanced complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linking agents. Alternatively, antibodies with dual Fc regions may be designed, thereby having enhanced complement lysis and ADCC capabilities.

[0082] In another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for pretargeting, where the antibody-receptor conjugate is administered to the patient, followed by the use of a clearing agent to remove unbound conjugate from the circulation, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide).

[0083] Covalent modification of monospecific or multispecific antibodies is also included in the scope of the present disclosure. If applicable, they can be prepared by chemical synthesis or by enzymatic or chemical cleavage antibodies of antibodies. By reacting the targeted amino acid residues of the antibody with an organic derivatizing agent that can react with selected side chains or N- or C-terminal residues, other types of covalent modifications of the antibody are introduced into the molecule. Exemplary covalent modifications of polypeptides are described in US5,534,615, and all contents disclosed in it about the covalent modification of polypeptides are specifically incorporated herein by reference. Exemplary types of covalent modifications of antibodies include connecting the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylene in the manner shown in US4,640,835, US4,496,689, US4,301,144, US4,670,417, US4,791,192 or US4,179,337.

[0084] The monospecific or multispecific antibodies disclosed herein can be produced by recombinant means. Therefore, disclosed herein are nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods for recombinant production are widely known in the prior art and include expressing proteins in prokaryotic and eukaryotic cells, followed by separation of antibodies and usually purification to a pharmaceutically acceptable purity. In order to express the above antibodies in host cells, nucleic acids encoding antibody sequences are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast or Escherichia coli cells, and antibodies are recovered from cells (supernatant or cells after lysis). It should be understood that any recombinantly expressed protein requires an initiator methionine (or formylmethionine) or signal sequence at its N-terminus, depending on the expression system used and whether the protein is expressed or secreted in the cytoplasm. Therefore, 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 from the full mature sequence (in whole or in part), while in other embodiments they are retained.

[0085] Accordingly, certain embodiments disclosed herein include methods for preparing monospecific or multispecific antibodies, comprising the steps of: a) transforming a host cell with at least one expression vector comprising a nucleic acid molecule encoding the antibody; b) culturing the host cell under conditions that allow synthesis of the antibody molecule; and c) recovering the antibody molecule from the culture.

[0086] The antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0087] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same, due to intentional or unintentional mutations. Variant progeny having the same function or biological activity as screened in the originally transformed cell are included. Where a different name is intended, it will be clear from the context.

[0088] As used herein, the term "transformation" refers to the process of transferring a vector / nucleic acid into a host cell. If cells without a strong cell wall barrier are used as host cells, transfection can be performed, for example, by calcium phosphate precipitation. However, other methods of introducing DNA into cells, such as by nuclear injection or by protoplast fusion, can also be used. If prokaryotic cells or cells containing substantial cell wall structures are used, for example, one transfection method is calcium treatment using calcium chloride.

[0089] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and / or the process by which the transcribed mRNA (also referred to as a transcript) is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include splicing of the mRNA.

[0090] "Vector" is a nucleic acid molecule, particularly a self-replicating one, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term includes vectors primarily used to insert DNA or RNA into cells (e.g., chromosomal integration), replication vectors primarily used for DNA or RNA replication, and expression vectors for transcription and / or translation of DNA or RNA. Also included are vectors that provide the multiple functions described.

[0091] An "expression vector" is a polynucleotide that, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell consisting of an expression vector that can function to produce a desired expression product.

[0092] As used herein, the term "host cell" refers to any kind of cell system that can be engineered to produce the antibodies disclosed herein. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0093] For example, control sequences suitable for prokaryotes include a promoter, an optional operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0094] When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the presequence or secretory leader is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the coding sequence; if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the DNA sequences being linked are continuous, and in the case of a secretory leader, continuous and in the reading frame. However, enhancers do not have to be continuous. Connections are accomplished by connecting at convenient restriction sites. If these sites do not exist, synthetic oligonucleotide adapters or joints are used according to conventional practice.

[0095] Also disclosed herein are isolated nucleic acids encoding monospecific or multispecific antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for producing the antibodies.

[0096] In order to recombinantly produce antibodies, the nucleic acid encoding it can be separated and inserted into a replicable vector for further cloning (amplification DNA) or expression. In some embodiments, antibodies can be produced by homologous recombination, for example, as described in US 5,204,244, all contents disclosed about antibody production are specifically incorporated herein by reference. The DNA encoding antibodies can be easily separated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to genes encoding antibody heavy chains and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: signal sequence, replication origin, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, for example, as described in US 5,534,615, all contents disclosed about protein expression are specifically incorporated herein by reference.

[0097] Suitable host cells for cloning or expressing the DNA in the vectors herein are the above-mentioned prokaryotes, yeast or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example Enterobacteriaceae, for example Escherichia, e.g. Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, for example Salmonella typhimurium, Serratia, for example Serratia marcescans and Shigella, and Bacilli, for example B. subtilis and B. licheniformis, Pseudomonas, for example P. aeruginosa and Streptomyces. An exemplary E. coli cloning host is E. coli 294 (ATCC 31,446), although 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.

[0098] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for monospecific or multispecific antibody encoding vectors. Saccharomyces cerevisiae or common baker's yeast is the most commonly used of the lower eukaryotic host microorganisms. However, many other genera, species and strains are generally available and can be used herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 402,226); 183,070); Candida; Trichoderma reesia (EP 244,234); Neurosporacrassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as A. nidulans and A. niger.

[0099] Suitable host cells for expressing glycosylated monospecific or multispecific antibodies are derived from multicellular organisms, including invertebrate cells such as plants and insect cells. Many baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses herein according to the present invention, particularly for transfecting Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0100] However, vertebrate cells are of greatest interest and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cell subclones 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 CCL2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0101] Host cells are transformed with the above-described expression vectors to produce monospecific or multispecific antibodies and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify the genes encoding the desired sequences.

[0102] Host cells for producing monospecific or multispecific antibodies can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10, minimum essential medium (MEM), RPMI-1640 and Dulbecco's modified Eagle medium (DMEM) are suitable for culturing host cells. In addition, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or USRe.30,985 can be used as a culture medium for host cells. Any of these culture media can be supplemented with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN TM ), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any 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 previously used to select the host cell for expression and are apparent to those of ordinary skill in the art.

[0103] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space or directly secreted into the culture medium. If the antibody is produced intracellularly, as a first step, the particulate debris (host cells or lysed fragments) is removed, for example by centrifugation or ultrafiltration.

[0104] The antibody composition prepared by the cell can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains, although protein A can be used to purify antibodies that do not have an Fc region. Protein G can be used for all mouse isotypes and human γ3. The matrix to which the affinity ligand is attached is typically agarose, but other matrices can also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl) benzene allow faster flow rates and shorter processing times than achieved with agarose. When the antibody comprises a CH3 domain, Bakerbond ABX< resin can be used for purification. The antibodies and antibody fragments disclosed herein can also be synthesized with histidine tags and affinity purified by metal affinity chromatography.

[0105] Depending on the antibody to be recovered, other protein purification techniques may also be used, such as ion exchange column fractionation, ethanol precipitation, reversed-phase high-performance liquid chromatography, silica gel chromatography, heparin agarose TMChromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

[0106] Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably at low salt concentration (e.g., about 0-0.25 M salt).

[0107] Also disclosed herein are multi-specific single-chain antibodies that can be cleaved in a tumor microenvironment. In some embodiments, once the multi-specific single-chain antibody reaches the tumor, the tumor targeting domain (such as a tumor antigen binding domain) or other functional domains are cut at the joint to release other domains that produce a therapeutic effect. The tumor microenvironment contains a variety of proteases that can cut the joints disclosed herein. Non-limiting examples of tumor proteases include, but are not limited to, matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAM (disintegrin and metalloproteinase; 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), matrix proteases, asparagine endopeptidases, or dipeptidyl peptidases (e.g., DDP4).

[0108] Antibody composition

[0109] Also disclosed herein are pharmaceutical compositions comprising monospecific or multispecific antibodies, wherein the specificity includes OX-40, CD40, 4-1BB, HSA, IL-22 or EFGR. Also disclosed are uses of the antibodies described herein in the preparation of pharmaceutical compositions. Also disclosed are methods of using the disclosed antibodies and pharmaceutical compositions comprising the antibodies to treat various diseases and conditions.

[0110] A pharmaceutical composition is a composition intended and suitable for treating a human disease. That is, it provides an overall beneficial effect without a certain amount of ingredients or contaminants that would cause toxicity or other adverse effects unrelated to providing the beneficial effect. A pharmaceutical composition will contain one or more active agents and may further contain solvents, buffers, diluents, carriers and other excipients to aid in the administration, solubility, absorption or bioavailability and / or stability of the active agent or the entire composition.

[0111] Monospecific or multispecific antibodies disclosed herein can also be formulated in liposomes. Liposomes containing antibodies are prepared by methods known in the art, as described in US4,485,045, US4,544,545 and US5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanolamine (PEG-PE). Liposomes are extruded through a filter of a specified pore size to produce liposomes with a desired diameter. The Fab' fragment of the antibody can be conjugated to the liposome via a disulfide exchange reaction.

[0112] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, intraocular, intravitreal, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is aqueous.

[0113] The compositions disclosed herein can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. In order to administer the disclosed antibodies by certain routes of administration, it may be necessary to combine the antibodies with materials or co-administer them to prevent their inactivation. For example, the antibodies can be administered to the subject in an appropriate carrier such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and medicaments for pharmaceutically active substances is known in the art.

[0114] As used herein, the phrases "parenteral administration" and "parenteral administration" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0115] These compositions may also contain excipients, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be ensured by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, etc. in the composition. In addition, extended absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0116] In some embodiments, the pharmaceutical composition comprising the antibody is a lyophilized cake. The lyophilized cake may further comprise a filler, a buffer and / or a salt or other excipient, as described herein. The lyophilized composition can be reconstituted by adding sterile water or an aqueous buffer for administration to a patient.

[0117] Regardless of the route of administration selected, the disclosed antibodies (which may be used in a suitable hydrated form) and / or pharmaceutical compositions containing the antibodies are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0118] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration and is non-toxic to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, 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 previous medical history of the patient being treated, and similar factors well known in the medical field.

[0119] Connectors

[0120] In many embodiments, the individual binding domains are not directly connected to each other, but have a short amino acid sequence, i.e., a linker, inserted between them. Examples of linkers are shown in Table 10. The length and sequence of the linker can have a substantial effect on the expression level and structure of MVSCA and the binding affinity of the connecting domain. Adjustable length linkers L2 and L4 (see Table 10) can be used to optimize MVSCA according to these parameters. Linkers L1, L2, and L4 can be referred to as non-cleavable linker methods, flexible linker methods, or flexible, non-cleavable linker methods.

[0121] When two copies of the same VHH domain are placed next to each other in MVSCA, they often adversely affect each other. This can be avoided by inserting 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). Such a linker can be referred to as a short, rigid linker or a non-cleavable short, rigid linker.

[0122] When the anti-HSA domain-HSA complex is used to generate a prodrug, a cleavable linker should be inserted between the two domains relative to the binding activity of the adjacent binding domain. L11*3 to L11*18 (see Table 10) are examples of cleavable linkers of different lengths and susceptibility to cleavage by different proteases, which can be used to optimize MVSCA in terms of expression level, structure of MVSCA, binding affinity of the connecting domains, and cleavage. Linkers L11*3 to L11*18 can be referred to as cleavable linker means, flexible linker means, or flexible, cleavable linker means.

[0123] MVSCAs

[0124] The binding domains and linkers described herein can be combined to produce multifunctional MVSCA suitable for treating specific diseases. They can also be further combined with other binding domains. MVSCA can also be referred to as comprising means for achieving various functions associated with the binding domains of each component type and / or comprising linker means for achieving their related functions.

[0125] Uses of the disclosed antibodies

[0126] The disclosed antibodies can be used in medicine. The terms "treatment", "treating", etc. refer to medical management of patients for the purpose of curing, improving, stabilizing or preventing diseases, pathological conditions or disorders. The term includes active treatment, i.e., treatment specifically for improving diseases, pathological conditions or disorders, and also includes causal treatment, i.e., treatment for eliminating the causes of related diseases, pathological conditions or disorders. In addition, the term includes palliative treatment, i.e., treatment intended to relieve symptoms rather than cure diseases, pathological conditions or disorders; preventive treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of related diseases, pathological conditions or disorders; and supportive treatment, i.e., treatment for supplementing another specific therapy intended to improve related diseases, pathological conditions or disorders. Various embodiments may specifically include or exclude one or more of these treatment modalities.

[0127] Also contemplated are uses of the antibodies disclosed herein in diagnostics and imaging.

[0128] In addition, the terms "treating" and "treatment" broadly include any type of therapeutic activity, including the diagnosis, alleviation or prevention of a disease or an aspect thereof in a human or other animal, or any activity that otherwise affects the structure or any function of the human or other animal body. Therapeutic activities include administering to a patient the drugs, dosage forms and pharmaceutical compositions described herein, particularly in accordance with the various treatment methods disclosed herein, whether by a health care professional, the patient himself / herself, or any other person. Therapeutic activities include orders, instructions and suggestions from health care professionals such as physicians, physician assistants, nurse practitioners, etc., which are then carried out by any other person including other health care professionals or the patient himself / herself. This includes, for example, directing a patient to undergo or directing a clinical laboratory to perform a diagnostic procedure, such as for cancer diagnosis and staging, so that the patient can ultimately receive beneficial appropriate treatment. In some embodiments, the commands, instructions and suggestions of treatment activities can also include encouragement, induction or mandatory selection of specific drugs or their combinations to treat the patient's condition, and the actual use of the drug, which is approved by the insurance coverage of the drug, refused to substitute the drug (including the drug on the drug formulary or exclude the insurance coverage of the substitute drug), or, provide the economic incentive of using the drug, as the insurance company or pharmacy benefit management company may do, etc. In some embodiments, treatment activities can also include encouragement, induction or mandatory selection of specific drugs to treat the patient's condition, and the actual use of the drug, which is established by the policy or practice standards such as hospitals, clinics, health maintenance organizations, medical practices or physician groups. All such orders, instructions and suggestions should be regarded as receiving treatment benefits on the condition of complying with instructions. In some cases, patients also obtain economic benefits due to compliance with such orders, instructions and suggestions. In some cases, health care professionals also obtain economic benefits due to compliance with such orders, instructions and suggestions.

[0129] cancer

[0130] The disclosed monospecific VHHs and multivalent single chain antibodies specific for OX-40, CD40, 4-1BB, HSA, IL-22 and EFGR can be used to treat cancer. Each antibody is designed to treat a specific class of cancer based on the antigen binding specificity contained in the antibody.

[0131] The present disclosure provides a method of treating cancer, comprising administering to a patient in need of such treatment an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising the antibody.

[0132] Examples of cancers that may be treated by the disclosed methods include acute lymphocytic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer, bladder cancer; bone cancer; brain stem glioma; brain tumor; breast cancer; bronchial adenoma / carcinoid; carcinoid tumor; islet cell carcinoma; primary cancer unknown; central nervous system lymphoma; cerebellar astrocytoma; brain astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; endometrial cancer, ependymoma; ovarian epithelial cancer; esophageal cancer; Ewing family of tumors; extracranial germ cell tumors; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; germ cell tumors; gestational trophoblastic tumors; capillary leukemia; head and neck cancer; hepatocellular carcinoma; Hodgkin lymphoma; hypopharyngeal cancer; Kaposi sarcoma; renal cancer; laryngeal cancer; non-small cell lung cancer; small cell lung cancer; non-Hodgkin lymphoma; Waldenstrom's macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell carcinoma; squamous neck cancer; multiple endocrine neoplasia syndrome; multiple myeloma / plasmacytoma; mycosis fungoides; myelodysplastic syndrome syndrome; nasopharyngeal carcinoma; 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; Sezary syndrome; skin cancer; squamous neck cancer; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and Wilms' tumor.

[0133] The effectiveness of cancer treatment is often measured as “response.” Techniques for monitoring response can be similar to tests used to diagnose cancer, such as, but not limited to:

[0134] A mass or tumor involving certain lymph nodes can be felt and measured externally during a physical examination.

[0135] Some internal cancer tumors will show up on an X-ray or CT scan and can be measured with a ruler.

[0136] Blood tests, including tests to measure organ function, may be done.

[0137] Tumor marker tests are available for some cancers.

[0138] Whichever test is used, whether it is a blood test, a cell count, or a tumor marker test, it is repeated at specific intervals so that the results can be compared with earlier tests of the same type.

[0139] There are several definitions of response to cancer treatment:

[0140] Complete response - All cancer or tumors disappear; no evidence of disease. Tumor marker expression levels (if applicable) may be within normal range.

[0141] Partial response - The cancer has shrunk by a certain percentage, but the disease is still there. The level of a tumor marker (if applicable) may have decreased (or increased, depending on the tumor marker, as an indication of reduced tumor burden), but evidence of disease is still there.

[0142] Stable disease - The cancer is neither growing nor shrinking; the amount of disease is not changing. Tumor markers (if applicable) have not changed significantly.

[0143] Disease progression – The cancer has grown; there is more disease now than before treatment. Tumor marker testing (if applicable) shows elevated tumor markers.

[0144] Other measures of the efficacy of cancer treatment include overall survival (ie, the time from diagnosis or the start of treatment evaluation to death from any cause), cancer-free survival (ie, the length of time after a complete response that the cancer remains undetectable), and progression-free survival (ie, the length of time after a partial response that results in undetectable disease stabilization or resumption of tumor growth).

[0145] For tumor size (tumor burden), there are two standard methods for assessing treatment response in solid cancers, the WHO and RECIST criteria. These methods measure solid tumors to compare the current tumor to past measurements, or to compare changes to future measurements and to change treatment plans. In the WHO method, the long and short axes of solid tumors are measured, and then 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.

[0146] Autoimmune diseases

[0147] The disclosed monospecific VHHs and multivalent single chain antibodies specific for OX-40, CD40, 4-1BB, HSA, IL-22 and EFGR can be used to treat autoimmune diseases. Based on the antigen binding specificity contained in the antibody, each antibody is designed to treat a specific class of autoimmune diseases.

[0148] The present disclosure provides a method of treating an autoimmune disease, comprising administering to a patient in need of such treatment an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising the antibody.

[0149] The autoimmune disorder can be a systemic autoimmune disorder or an organ-specific autoimmune disorder. Non-limiting examples of autoimmune disorders that can be treated using the compounds, compositions, or combinations disclosed herein include acute disseminated encephalomyelitis (ADEM), Addison's disease, allergies, allergic rhinitis, antiphospholipid antibody syndrome (APS), arthritis, such as monoarthritis, oligoarthritis, or polyarthritis, such as osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, pyogenic arthritis, spondyloarthropathies, gout, pseudogout, or Still's disease, asthma, acquired immunodeficiency syndrome (AIDS), autoimmune hemolytic anemia, autoimmune hepatitis, auto autoimmune inner ear disease, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD), type I diabetes mellitus (IDDM), endometriosis, gastrointestinal diseases, such as irritable bowel disease or inflammatory bowel disease, such as 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, sleep disorders, multiple sclerosis (MS), myasthenia gravis, myopathies such as dermatomyositis, inclusion body myositis or polymyositis, myositis, narcolepsy, neuromyotonia, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, relapsing disseminated encephalomyelitis, rheumatic fever, schizophrenia, scleroderma, Sjögren's syndrome, skin disorders such as dermatitis, eczema, congestion dermatitis, purulent Hidradenitis herpetiformis, psoriasis, rosacea or scleroderma, tenosynovitis, uveitis, vasculitis such as thromboangiitis obliterans, cerebral vasculitis, Churg-Strauss arteritis, cryoglobulinemia, primary cryoglobulinemia, giant cell arteritis, Golfer's vasculitis, purpuric nephritis, hypersensitivity vasculitis, Kawasaki disease, microscopic polyarteritis / polyangiitis, polyarteritis nodosa, polymyalgia rheumatica (PMR), rheumatoid vasculitis, Takayasu's arteritis, Wegener's granulomatosis, or vitiligo.

[0150] Aspects of the present disclosure include, in part, alleviating at least one symptom associated with an autoimmune disorder. Actual symptoms associated with the autoimmune disorders disclosed herein are well known and can be determined by those of ordinary skill in the art by considering the following factors, including but not limited to the location of the autoimmune disorder, the cause of the autoimmune disorder, the severity of the autoimmune disorder, the tissue or organ affected by the autoimmune disorder, and the inflammation associated with the autoimmune disorder. Non-limiting examples of symptoms alleviated by the method for treating autoimmune disorders disclosed herein include inflammation, fatigue, pain, cognitive deficits, neurological deficits, dizziness, discomfort, high fever and high body temperature, extreme sensitivity of hands and feet to cold, weakness, soreness and / or stiffness of muscles and joints, weight changes, digestion or gastrointestinal problems, respiratory problems, low or high blood pressure, irritability, anxiety or depression, infertility or decreased libido (low libido), blood sugar changes, and depending on the type of autoimmune disease, the size of an organ or tissue increases or the destruction of an organ or tissue. Non-limiting examples of inflammatory symptoms that are alleviated by the methods of treating an autoimmune disorder disclosed herein include pain, loss of neurological function, loss of cognitive function, edema, congestion, erythema, bruising, tenderness, stiffness, swelling, fever, chills, congestion of the respiratory tract including the nose and bronchial tubes, sinus congestion, breathing problems, fluid retention, blood clots, loss of appetite, increased heart rate, granulomas, formation of fibrin, pus or non-viscous serous fluid, formation of ulcers, or pain.

[0151] In certain embodiments, treatment with an antibody 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 disorder.

[0152] In other embodiments, the method can be helpful for treating or alleviating disorders, conditions or disorders associated with autoimmune diseases. In some embodiments, these disorders or symptoms may include, but are not limited to, anemia, weakness, cachexia, Cushing's syndrome, fatigue, gout, gum disease, hematuria, hypercalcemia, hypothyroidism, internal bleeding, hair loss, mesothelioma, nausea, night sweats, neutropenia, paraneoplastic syndrome, pleurisy, polymyalgia rheumatica, rhabdomyolysis, stress, lymphadenopathy, thrombocytopenia, vitamin D deficiency or weight loss. In other embodiments, the administration of the antibodies disclosed herein prolongs the survival time of the treated individual.

[0153] In some embodiments of the methods, the mammal may experience an improvement in the autoimmune disease as a result of treatment with the antibodies disclosed herein.

[0154] The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims.It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention. Example

[0155] Example 1 Anti-OX40 VHH Antibody

[0156] Isolation of anti-OX40 VHH antibodies from immunized llamas.

[0157] Immunization. Two llamas were immunized at Abcore Inc (Ramona, CA) according to their standard protocol. 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). Each llama was injected subcutaneously six times at 50 μg / dose every two weeks. On day 45, serum from llamas immunized with recombinant OX40-llama-Fc protein was collected for determination of antibody titers against recombinant OX40-His by ELISA. In the ELISA, 96-well Maxisorp plates (Nunc) were coated with 100 ng / well OX40-His. After blocking and adding diluted serum samples, the antibody titers of the antisera were determined using ELISA to demonstrate the presence of anti-OX40 VHH antibodies. 96-well Maxisorp plates (Nunc) were coated with 100 ng / well recombinant OX40-His.After blocking and addition of diluted serum samples, the presence of anti-OX40 VHH antibodies was demonstrated using horseradish peroxidase (HRP)-conjugated goat anti-llama IgG antibody (Invitrogen).

[0158] SEQ ID NO: 1—Extracellular domain of human OX40 (Leu29-Ala216) (Accession No. P43489)

[0159] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA

[0160] Construction and selection of phage libraries. Peripheral blood mononuclear cells were prepared from serum samples of llamas on day 45 immunized with recombinant OX40 llama FC protein using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from 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 VHH encoding gene fragments. These fragments were cloned into homemade phagemid vectors, which, after infection with helper phages, allowed the production of recombinant phage particles, which displayed VHH as gene-III fusion proteins on the surface of phage particles. Phages were prepared according to standard methods and stored after filter sterilization at 4°C for further use. The phage library obtained from the llama was used for selection. In the selection, biotinylated OX40-HIs were incubated with the phage library and subsequently captured on streptavidin Dynabeads (Invitrogen). After extensive washing, bound phage were eluted with 1 mg / ml trypsin. Output from the selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0161] cDNA encoding positive VHHs was synthesized with a C-terminal His-tag at Atum (DNA2.0, Inc) and transiently transfected in HEK293 cells, and the positive VHHs were purified by IMAC chromatography for in vitro functional assays.

[0162] Biolayer Interferometry (BLI) Kinetic Binding Assay. Biolayer Interferometry (BLI) is a label-free technique used to measure the binding kinetics of human OX40 hFC (R&D systems) to anti-OX40 VHH. Affinity measurements were performed with a Gator equipped with an anti-Penta-His capture (HIS1K) biosensor tip. The assay was performed at 30°C in 1× PBS buffer ( PBS pH7.2). The sample was stirred at 1000rpm. Before analysis, the sensor was humidified for 15min. The binding ability of purified anti-OX40 VHH to the HIS1K sensor tip was tested. The tip was loaded with 20μg / ml anti-OX40 VHH. The loading was performed for 300 seconds so that the capture level was between 1.8 and 2nm. Human OX40 antigen was prepared for binding analysis by diluting to a concentration of 100, 150, 250, 350nM in 1×PBS. The binding was started and monitored for 200 seconds, after which the tip was transferred to 1×PBS buffer (Gibco, PBS pH 7.2) without factor protein to monitor dissociation. Sensor data was collected throughout the experiment and processed and analyzed using Gator data analysis software.

[0163] Blocking assay. A 96-well plate was coated with 100 μL of 1 μg / mL OX40L-mFc prepared in antigen coating buffer at 4°C overnight, and then blocked with 2% BSA at 25°C for 1 hour. A series of dilutions of each OX40 VHH was premixed with OX40-hFc 0.025 μg / at 25°C for 30 min, then transferred to the OX40L-mFc-coated plate and incubated for 1 hour. The plate was washed four times with PBST and then incubated with mouse anti-human IgG Fc-HRP at 25°C for 1 hour. After washing four times with PBST, the plate was developed with 100 μL TMB / well in the dark for 10 to 20 min and then stopped by adding 50 μL of stop solution. The plate was read at 450 nm by a Molecular Devices microplate reader. ELISA data were analyzed using GraphPad Prisma 9.1.

[0164] ELISA assay. 96-well plates were coated with 100 μL / well of OX40 Fc prepared at 1 μg / ml in coating buffer at 4°C overnight, then blocked with 200 μL / well of 2% BSA at 25°C for 1 hour, followed by two washes with PBST. OX40VHH supernatant was prepared into a series of 4-fold dilutions from the highest concentration of 500 nM and added to the OX40Fc-coated plates, 100 μL per well, incubated at 25°C for 1 hour. After washing four times with PBST, the plates were incubated with detection antibody anti-his-HRP (1:4000 dilution) 100 μL / well at 60 rpm shaking for 1 hour. The plates were developed in the dark with 100 μL TMB per well and then stopped with 50 μL stop solution per well. The plates were read at 450 nm by a Molecular Devices microplate reader. Data were analyzed using GraphPadPrisma 9.1.

[0165] Expression and purification of anti-OX40 VHH antibodies. Positive phage colonies from the immunized llama phage library were sequenced. The amino acid sequences are listed in Table 2 below. The cDNA sequence based on the following amino acid sequence was fused to human Fc and synthesized by Atum (DNA2.0) in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce fully recombinant anti-OX40 VHH antibodies. The expressed anti-OX40 VHH was purified by HiTrap protein A column.

[0166] Table 2. Llama anti-OX40 VHH sequences

[0167]

[0168]

[0169]

[0170]

[0171] The VHH compositions in Table 2 bind to OX-40.

[0172] The results of ELISA assays of OX40 VHH antibodies against recombinant human OX40-Fc are presented in Table 3.

[0173] Biolayer interferometry (BLI) binding analysis of anti-OX-40 VHH molecules is presented in Table 4.

[0174]

[0175] Table 4. Binding affinity (KD) of OX40 VHH antibodies determined using BLI technology

[0176] VHH Koff(1 / s) Kon(1 / Ms) KD(M) pgXX88-SZ-4 8.73E-05 4.72E+05 1.85E-10 pgXX88-SZ-15 2.59E-04 5.39E+05 4.80E-10 pgXX88-2 2.30E-04 4.74E+05 4.85E-10 pgXX88-10 1.00E-06 6.62E+05 ＜E-12 pgXX88-60 1.00E-06 4.64E+05 ＜E-12 pgXX88-81 1.00E-06 5.49E+05 ＜E-12 pgXX88-83 2.42E-05 4.60E+05 5.26E-11 pgXX88-84 1.00E-06 4.94E+05 ＜E-12 pgXX88-88 1.00E-06 4.83E+05 ＜E-12

[0177] The results of the ELISA blocking assay of anti-OX40 VHH that blocks the binding of OX40 to OX40 ligand are presented in Figure 1 pgXX88-10 and pgXX88-SZ-15 are complete blockers, EC 50 0.12 nM and 0.12 nM, respectively. ppXX88-SZ-4 is a partial blocker, EC 50 It is 1.98nM.

[0178] Example 2. Anti-CD40 VHH Antibodies

[0179] Isolation of anti-CD40 VHH antibodies from immunized llamas

[0180] Two llamas were immunized according to standard protocols at Abcore Inc. As in Example 1, recombinant CD40-llama Fc (extracellular domain (Glu21-Arg193) Accession No. 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).

[0181] On day 45, serum was collected from llamas immunized with recombinant CD40-llama Fc protein to determine antibody titers against recombinant CD40-His by ELISA. In the ELISA, 96-well Maxisorp plates were coated with 100 ng / well CD40-His. After blocking and adding diluted serum samples, the presence of anti-CD40 VHH antibodies was demonstrated using antibodies, and the antibody titers of the antisera were tested by ELISA. 96-well Maxisorp plates were coated with 100 ng / well recombinant CD40-His. After blocking and adding diluted serum samples, the presence of anti-CD40 antibodies was demonstrated using HRP-conjugated goat anti-llama IgG antibodies.

[0182] SEQ ID NO:25—Extracellular domain of human CD40 extracellular domain (Glu21-Arg193) (Accession No. P25942-1)

[0183] EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEF

[0184] LDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHC

[0185] TSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAF

[0186] EKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLR

[0187] Peripheral blood mononuclear cells were prepared from serum samples of llamas immunized with recombinant CD40 llama Fc protein on day 45 using Ficoll-Paque Plus according to the manufacturer's instructions. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi kit according to the manufacturer's instructions and used as starting material for RT-PCR to amplify VHH encoding gene fragments. These fragments were cloned into homemade phagemid vectors, which, after infection with helper phages, allowed the production of recombinant phage particles, which displayed VHH as gene-III fusion proteins on the surface of phage particles. Phages were prepared according to standard methods and stored after filter sterilization at 4°C for further use. The phage library obtained from llama was used for selection. In the selection, biotinylated CD40-His was incubated with the phage library and subsequently captured on streptavidin Dynabeads. After extensive washing, the bound phages were eluted with 1 mg / ml trypsin. The output from the selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0188] cDNA encoding positive VHHs was synthesized in Atum with a C-terminal His-tag and transiently transfected in HEK293 cells, and the positive VHHs were purified by IMAC chromatography for in vitro functional assays.

[0189] Positive phage colonies from the immunized llama phage library were sequenced. The amino acid sequences are listed in Table 5 below. The cDNA sequence based on the following amino acid sequence was fused to human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce fully recombinant anti-CD40 VHH antibodies. The expressed anti-CD40 VHH was purified by HiTrap protein A column.

[0190] Table 5. Llama anti-CD40 VHH sequences

[0191]

[0192]

[0193] The VHH compositions in Table 5 bind to CD40.

[0194] ELISA test

[0195] 96-well plates were coated with 100 μL / well of CD40Fc prepared at 1 μg / ml in coating buffer at 4°C overnight, then blocked with 200 μL / well of 2% BSA at 25°C for 1 hour, followed by two washes with PBST. CD40 VHH supernatant was prepared into a series of 4-fold dilutions from the highest concentration of 500 nM and added to the CD40 Fc-coated plates, 100 μL per well, and incubated at 25°C for 1 hour. After washing four times with PBST, the plates were incubated with detection antibody anti-his-HRP (1:40 00 dilution) 100 μL / well at 60 rpm shaking for 1 hour. The plates were developed in the dark with 100 μL TMB per well and then stopped with 50 μL stop solution per well. The plates were read at 450 nm by a Molecular Devices microplate reader. The data were analyzed using GraphPad Prisma 9.1 and the results are presented in Table 6.

[0196] Table 6. ELISA test of CD40 VHH antibodies against rh-CD40-Fc antigen

[0197] (nM) 1000 200 40 8 1.6 0.32 0.064 0.013 0.0026 pgDD40-HZ-15 0.479 0.166 0.076 0.061 0.062 0.060 0.062 0.063 0.064 pgDD40-HZ-24 1.809 1.845 1.710 1.702 0.903 0.251 0.084 0.055 0.078 pgDD40-HZ-28 0.419 0.123 0.067 0.059 0.062 0.066 0.070 0.075 0.061 pgDD40-HZ-47 0.569 0.126 0.066 0.063 0.056 0.063 0.053 0.055 0.060 pgDD40-SZ-1 1.675 1.688 1.723 1.657 0.866 0.248 0.074 0.056 0.055 pgDD40-SZ-2 1.638 1.524 1.718 1.586 0.940 0.293 0.091 0.062 0.058 pgDD40-SZ-3 1.567 1.643 1.639 1.595 1.024 0.287 0.098 0.055 0.055 pgDD40-SZ-5 1.668 1.733 1.681 1.408 0.850 0.250 0.085 0.061 0.059 pgDD40-SZ-7 1.673 1.625 1.665 1.602 1.006 0.332 0.088 0.092 0.056 pgDD40-SZ-12 1.542 0.895 0.256 0.090 0.061 0.059 0.065 0.058 0.060

[0198] Kinetic binding analysis

[0199] As in Example 1 Kinetic binding analysis. Briefly, the ability of purified anti-CD40 VHH to bind to the HIS1K sensor tip was tested. The tip was loaded with 20 μg / ml anti-CD40 VHH. Loading was performed for 300 seconds, resulting in capture levels between 1.8 and 2 nm. Human CD40 antigen was prepared for binding analysis by diluting to concentrations of 100, 150, 250, 350 nM in 1×PBS. Binding was initiated and monitored for 200 seconds, after which the tip was transferred to 1×PBS buffer without CD40 protein to monitor dissociation. The results are presented in Table 7.

[0200] Table 7. Binding affinity (KD) analysis of CD40 VHH

[0201]

[0202]

[0203] Blocking test

[0204] A 96-well plate was coated with 100 μL of 1 μg / mL CD40L-mFc prepared in antigen coating buffer overnight at 4°C and then blocked with 2% BSA for 1 hour at 25°C. A series of dilutions of each CD40 VHH was premixed with CD40-hFc 0.025 μg / well at 25°C for 30 min and then transferred to the CD40L-mFc-coated plate for incubation for 1 hour. The plate was washed four times with PBST and then incubated with mouse anti-human IgG Fc-HRP for 1 hour at 25°C. After washing four times with PBST, the plate was developed with 100 μL TMB / well in the dark for 10 to 20 min and then stopped by adding 50 μL stop solution. The plate was read at 450 nm by a Molecular Devices microplate reader. ELISA data were analyzed using GraphPad Prisma 9.1. The results are presented in Figure 2 and demonstrated that pgDD40-HG-24 is a CD40 receptor blocker.

[0205] Example 3 Anti-4-1BB VHH Antibody

[0206] Isolation of anti-4-1BB VHH antibodies

[0207] As in Example 1, llamas were immunized with recombinant human 41BB (extracellular domain (Leu24-His183) Accession No. Q07011, SEQ ID NO: 41) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization) at Abcore, Inc.

[0208] SEQ ID NO:41—Extracellular domain of human 41BB (Extracellular domain (Leu24-His183) (Accession No. Q07011)

[0209] LQDPCSCNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCK

[0210] GVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELT

[0211] KKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVV

[0212] CGPSPADLSPGASSVTPPAPAREPGH

[0213] The phage colonies binding 4-1BB from the llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 8). The cDNA sequence based on the following amino acid sequence was synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce a recombinant single domain antibody (sdAb) with a C-terminal his-tag. The sdAb expressed was purified by HisTrap HP column.

[0214] cDNA encoding 4-1BB-specific VHHs was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells, and positive VHHs were purified by IMAC chromatography.

[0215] Table 8. Llama anti-4-1BB VHH sequences

[0216]

[0217]

[0218] The VHH compositions in Table 8 bind to 4-1BB.

[0219] BLI kinetic binding analysis

[0220] As in Example 1 Kinetic binding analysis. Briefly, the purified anti-CD40 VHHs were tested for their ability to bind to the HIS1K sensor tip. The tip was loaded with 20 μg / ml anti-41BB VHH. Loading was performed for 300 seconds, resulting in capture levels between 1.8 and 2 nm. Human 41BB antigen was prepared for binding analysis by diluting to concentrations of 100, 150, 250, 350 nM in 1× PBS. Binding was initiated and monitored for 200 seconds, after which the tip was transferred to 1× PBS buffer without 41BB protein to monitor dissociation.

[0221] BLI kinetic binding analysis was performed as in Example 1 and the results are presented in Table 9.

[0222] Table 9. BLI binding affinity assay of 41BB VHH

[0223] VHH KD(M) Koff(1 / s) Kon(1 / Ms) 4-1BB-1 3.17E-09 2.64E-03 8.34E+05 4-1BB-2 2.57E-09 2.52E-03 9.79E+05 4-1BB-3 1.82E-09 1.29E-03 7.08E+05 4-1BB-9 4.55E-09 2.76E-03 6.06E+05 4-1BB-10 6.57E-10 5.39E-04 8.20E+05 4-1BB-36 2.92E-08 9.19E-03 3.14E+05 4-1BB-49 2.63E-10 3.10E-04 1.18E+06 4-1BB-56 7.81E-10 5.52E-04 7.07E+05 4-1BB-59 2.35E-09 1.21E-03 5.12E+05

[0224] ELISA test

[0225] 96-well plates were coated overnight at 4°C with 100 μL / well of 4-1BB Fc prepared at 1 μg / ml in coating buffer, then blocked with 200 μL / well 2% BSA at 25°C for 1 hour, followed by two rounds of washing with PBST. 4-1BB VHH supernatant was prepared into a series of 4-fold dilutions from the highest concentration of 500 nM and added to the 4-1BB Fc-coated plate, 100 μL per well, incubated at 25°C for 1 hour. After four rounds of washing with PBST, the plate was incubated with detection antibody anti-his-HRP (1:40 00 dilution) 100 μl / well at 60 rpm for 1 hour. The plate was developed in the dark with 100 μL TMB per well and then terminated with 50 μL stop solution per well. The plate was read at 450 nm by a Molecular Devices microplate reader. Data were analyzed using GraphPad Prisma9.1. The results are presented in Table 10.

[0226] Table 10. ELISA test of anti-4-1BB VHH antibodies against rh-41BB

[0227]

[0228] Blocking test

[0229] A 96-well plate was coated with 100 μL of 1 μg / mL 41BBL-mFc prepared in antigen coating buffer overnight at 4°C and then blocked with 2% BSA for 1 hour at 25°C. A series of dilutions of each 41BB VHH was premixed with 41BB-hFc 0.025 μg / well at 25°C for 30 min and then transferred to the 41BBL-mFc-coated plate for incubation for 1 hour. The plate was washed four times with PBST and then incubated with mouse anti-human IgG Fc-HRP for 1 hour at 25°C. After washing four times with PBST, the plate was developed with 100 μL TMB / well in the dark for 10 to 20 min and then stopped by adding 50 μL stop solution. The plate was read at 450 nm by a Molecular Devices microplate reader. ELISA data were analyzed using GraphPad Prisma 9.1. The results are presented in Figure 6 4-1BB59, but not 4-1BB56, blocked the binding of 41BB to 41BBL in a dose-dependent manner.

[0230] Example 4 Anti-EGFR VHH Antibody

[0231] Isolation of anti-EGFR VHH antibodies

[0232] Llamas were immunized with recombinant human epidermal growth factor receptor (EFGR) (extracellular domain (Met1-Ser645); Accession No. CAA25240; SEQ ID NO:54) and a phage library prepared as in Example 1 mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization) at Abcore, Inc.

[0233] SEQ ID NO:54-Human EGFR extracellular domain (Met1-Ser645) (Accession number CAA25240)MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANK TGLKELPMRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVV TDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYAN TINWKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCKLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0234] To select anti-EFGR VHHs, biotinylated EFGR-His was incubated with the phage library and subsequently captured on streptavidin Dynabeads. After extensive washing, bound phages were eluted with 1 mg / ml trypsin. Output from the selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0235] cDNA encoding EGFR-binding VHHs was synthesized with a C-terminal His-tag and transiently transfected into HEK293 cells, and positive VHHs were purified by IMAC chromatography.

[0236] Phage colonies binding to EGFR from immune llama phage libraries were sequenced, and the amino acid sequences of each VHH are listed below (Table 11). The cDNA sequence based on the following amino acid sequence was fused to human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to produce recombinant anti-EGFR VHH antibodies. The expressed anti-EGFR VHH was purified by HiTrap protein A column.

[0237] Several antibodies, pgEG5, pgEG12, pgEG-SX40 and pgEG-SX57, were humanized based on human germline sequences.

[0238] Table 11. Llama anti-EGFR VHH sequences

[0239]

[0240]

[0241]

[0242] The VHH compositions of Table 11 bind to EGFR. Kinetic binding analysis, K D The results are presented in Table 12.

[0243] Table 12. Binding affinity of anti-EGFR VHH antibodies

[0244] VHH KD(M) Kon(1 / Ms) Kdis(1 / s) pgEG1 1.56E-08 1.56E+05 2.43E-03 pgEG5 1.08E-08 1.24E+05 1.34E-03 pgEG6 2.79E-08 4.89E+05 1.36E-02 pgEG12 1.25E-08 2.01E+05 2.51E-03 pgEG-SX-3 6.02E-10 2.75E-04 4.57E+05 pgEG-SX-6 7.98E-10 3.99E-04 5.00E+05 pgEG-SX-22 4.64E-10 2.80E-04 6.02E+05 pgEG-SX-40 1.13E-09 4.06E-04 3.58E+05 pgEG-SX-57 1.87E-09 7.15E-04 3.81E+05

[0245] ELISA test

[0246] 96-well plates were coated overnight at 4°C with 100 μL / well of EGFR Fc prepared at 1 μg / ml in coating buffer, then blocked for 1 hour at 25°C with 200 μL / well of 2% BSA, followed by two rounds of washing with PBST. The pgEG-SX VHH supernatant was prepared into a series of 4-fold dilutions from the highest concentration of 500 nM and added to the EGFR Fc-coated plates, 100 μL per well, incubated for 1 hour at 25°C. After four rounds of washing with PBST, the plates were incubated with 100 μL / well of detection antibody anti-his-HRP (1:40 00 dilution) at 60 rpm for 1 hour. The plates were developed in the dark with 100 μL TMB per well and then stopped with 50 μl stop solution per well. The plates were read at 450 nm by a Molecular Devices microplate reader. Data were analyzed using GraphPad Prisma 9.1. The results of the ELISA test are in Table 13.

[0247] Blocking test

[0248] 96-well plates were coated with 100 μL of EGF-mFc prepared at 1 μg / ml (28 nM) in coating buffer per well. After incubation at 4 °C overnight, the plates were washed four times with PBST and blocked with 200 μl / well of 2% BSA for 1 hour at room temperature. Purified pgEG-SX40 or 57VHH was made into a series of 1000 nM dilutions and pre-incubated with 0.05 nM EGFR-hFc at room temperature for 30 min. The plates were washed twice with PBST, and the premixed samples were added to the 96-well plates and shaken at 60 rpm for 1 hour at room temperature. After washing four times with PBST, the plates were incubated with detection antibody anti-his-HRP (1:40 00 dilution) 100 μL / well at 60 rpm for 1 hour. The plates were washed four times with PBST, 100 μL of substrate TMB was added to each well, and then 50 μl of stop solution was added. The plate was immediately read by ELISA reader at OD450 (Table 14).

[0249] Purified VHH antibodies SX-40 and SX-57 blocked EGFR binding to EGF, whereas SX-3 and SX-6 only partially blocked binding (Table 14).

[0250]

[0251] Table 14. Blocking of EGFR binding to EGF by pgEG-SX VHH antibodies

[0252]

[0253] Example 5 Anti-HSA VHH Antibodies

[0254] Immunization

[0255] Llamas were immunized according to their standard protocol at 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). On day 45, each llama was injected subcutaneously six times at 50 μg / dose every two weeks, and serum was collected from the immunized llama to determine the antibody titer by ELISA. In the ELISA, 96-well Maxisorp plates were coated with 100 ng / well antigen. After blocking and adding diluted serum samples, horseradish peroxidase (HRP)-conjugated goat anti-llama IgG (H+L) antibodies were used to demonstrate the presence of specific antibodies.

[0256] SEQ ID NO: 113 Human serum albumin

[0257] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERN ECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQR FPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHPDYSVVL LLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPV

[0258] SDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICT

[0259] LSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCK

[0260] ADDKETCFAEEGKKLVAASQAALGL

[0261] Construction and selection of phage libraries

[0262] Peripheral blood mononuclear cells were prepared from serum samples of day 45 of immunized llamas using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from 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 VHH encoding gene fragments. These fragments were cloned into homemade phagemid vectors, which, after infection with helper phages, allowed the production of recombinant phage particles that displayed VHH as gene-III fusion proteins on the surface of phage particles. Phages were prepared according to standard methods and stored at 4°C after filter sterilization for further use.

[0263] Phage libraries obtained from immunized llamas were used for selection. In the selection, biotinylated HSA was incubated with the phage library and subsequently captured on streptavidin Dynabeads (Invitrogen). After extensive washing, bound phages were eluted with 1 mg / ml trypsin. Output from the selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced at BATJ, Inc.

[0264] cDNA encoding positive VHHs was synthesized with a C-terminal His-tag at Atum (DNA2.0, Inc) and transiently transfected in HEK293 cells, and the positive VHHs were purified by IMAC chromatography for in vitro functional assays.

[0265] Octet kinetic binding assay

[0266] Biolayer interferometry (BLI) is a label-free technique used to measure the binding kinetics of positive llama single domain antibodies. Affinity measurements were performed using an Octet QKe( The assay was performed at 30°C in 1x PBS buffer ( PBS pH 7.2). The sample was stirred at 1000 rpm. Before analysis, the sensor was humidified for 15 minutes. The binding ability of the purified single domain antibody to the HIS1K sensor tip was tested. The tip was loaded with 20 μg / ml single domain antibody. The loading was performed for 300 seconds so that the capture level was between 1.8 and 2 nm. The test antigen was prepared for binding analysis by diluting it to a concentration of 100, 150, 250, 350 nM in 1x PBS. The binding was started and monitored for 200 seconds, after which the tip was transferred to a 1x PBS buffer (Gibco, PBS pH 7.2) without factor protein to monitor dissociation. Sensor data was collected throughout the experiment and processed and analyzed using Octet data analysis software 7 (Forte Bio).

[0267] result

[0268] Isolation of anti-HSA single domain antibodies

[0269] Positive phage colonies from the immune llama phage library were sequenced. The amino acid sequences are presented in Table 15. The cDNA sequences based on the following amino acid sequences were synthesized by Atum (DNA2.0) in the pJ607 expression vector. The Th expression plasmid was transfected into the HEK293 cell line to produce fully recombinant single domain antibodies with a C-terminal his-tag. The expressed VHHs were purified by HisTrap HP columns.

[0270] Table 15. Anti-HSA sequences

[0271]

[0272] Humanization of MSA4 based on human germline IGHV3-23*04

[0273] Humanized MSA4-1 (SEQ ID NO: 119)

[0274] EVQLVESGGGLVQPGGSLRLSCAASGRTDSAYRMGWFRQAPGKEREFVSAINWSDGRTVYLDSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAADPDSRLYYTVPQNYDYWGQGTQVTVSS

[0275] ELISA binding assay for MSA VHH

[0276] 96-well plates were coated with 100 μL / well of HSA or MSA (mouse serum albumin), which was prepared overnight at 4°C in 1 μg / ml coating buffer, and then blocked with 200 μl / well of casein at room temperature for 1 hour. MSA VHHs were made into a series of dilutions and added to HSA or MSA coated plates and incubated for 1 hour. The plates were washed four times with PBST and then incubated with HRP-conjugated streptavidin at 25°C for 1 hour. After washing three times with 250 μL PBST / well, the plates were developed in the dark with 100 μL TMB / well for 10 to 20 min and then stopped by adding 100 μl of stop solution. The plates were read on a 450nm Molecular Devices microplate reader. The results are presented in Tables 16-19.

[0277]

[0278] Table 18 ELISA results of humanized MSA4-1 binding to HSA

[0279] nM MSA4 MSA4-1 1000 0.655 0.818 200 0.417 0.363 40 0.250 0.267 8 0.135 0.156 1.6 0.024 0.053 0.32 0.009 0.003 0.064 0.028 0.009 0.0128 0.073 0.064

[0280] Table 19. ELISA results of humanized MSA4-1 binding to MSA

[0281] nM MSA4 MSA4-1 1000 1.934 1.798 200 1.668 1.274 40 1.257 0.774 8 0.673 0.347 1.6 0.149 0.100 0.32 0.083 0.059 0.064 0.051 0.029 0.0128 0.001 0.002

[0282] BLI binding kinetic analysis of MSA VHHs

[0283] Kinetic binding analysis of MSA VHHs was performed using a Gator Prime system equipped with analyze software. The Gator assay protocol was used for all experiments in this report described below. Experiments were performed at 30°C. Prior to analysis, the sensors were placed in Q buffer and shaken at 1000 rpm for 600 seconds, and then 2uM of His-tagged MSA VHHs were loaded onto the anti-His sensor and shaken at 400 rpm for 120 seconds. The loaded sensors were transferred to Q buffer for 60 seconds. Binding of HSA or MSA was initiated at 100nM and monitored for 120-180 seconds with shaking at 1000rpm, and finally dissociation was performed in Q buffer with shaking at 1000rpm for 120-180 seconds. Data analysis was performed using software V2.0 (Gator Bio). The association rate (Kon) and dissociation rate (Kd) were determined by fitting the association and dissociation phases for each sample. The mathematical model used assumed a 1:1 stoichiometry and only fit one analyte in solution to one binding site on the surface. The equilibrium dissociation constant (Kd), a measure of affinity, was then calculated as the ratio of Kd and Kon. The results are presented in Tables 20-23.

[0284] Table 20. Binding affinity of MSA VHH to HSA

[0285] <![CDATA[k off (1 / s)]]> <![CDATA[k on (1 / Ms)]]> <![CDATA[K D (M)]]> MSA-1 2.17E-02 NA NA MSA-2 2.25E-02 NA NA MSA-4 1.01E-03 8.96E+04 1.13E-08 MSA-6 1.25E-03 5.98E+04 2.09E-08 MSA-B1 5.11E-03 4.54E+05 1.13E-08

[0286] Table 21. Binding affinity of MSA VHH to MSA

[0287] <![CDATA[K off (1 / s)]]> <![CDATA[K on (1 / Ms)]]> <![CDATA[K D (M)]]> MSA-1 1.17E-03 1.76E+05 6.64E-09 MSA-2 1.26E-03 1.75E+05 7.18E-09 MSA-4 6.44E-04 1.43E+05 4.49E-09 MSA-6 2.44E-03 4.44E+04 5.49E-08 MSA-B1 6.05E-04 1.14E+05 5.32E-09

[0288] Table 22. Binding affinity of humanized MSA-4 to HSA

[0289] antigen koff(1 / s) kon(1 / Ms) KD(M) MSA-4 HSA 7.52E-04 1.05E+05 7.16E-09 hMSA4-1 HSA 5.45E-04 1.01E+05 5.38E-09

[0290] Table 23. Binding affinity of humanized MSA-4 to MSA

[0291] antigen koff(1 / s) kon(1 / Ms) KD(M) MSA-4 MSA 1.10E-03 1.25E+05 8.83E-09 hMSA4-1 MSA 6.78E-04 1.21E+05 5.58E-09

[0292] Example 6 Anti-IL22 VHH Antibody

[0293] Immunization

[0294] Llamas were immunized according to their standard protocol 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). On day 45, each llama was injected subcutaneously six times at 50 μg / dose every two weeks, and serum was collected from the immunized llama to determine the antibody titer by ELISA. In the ELISA, 96-well Maxisorp plates were coated with 100 ng / well antigen. After blocking and adding diluted serum samples, horseradish peroxidase (HRP)-conjugated goat anti-llama IgG (H+L) antibodies were used to demonstrate the presence of specific antibodies.

[0295] SEQ ID NO: 120 Human IL-22

[0296] MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFMLAKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQEVVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNACI

[0297] Construction and selection of phage libraries

[0298] Peripheral blood mononuclear cells were prepared from serum samples of day 45 of immunized llamas using Ficoll-Paque Plus according to the manufacturer's instructions. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi kit according to the manufacturer's instructions and used as starting material for RT-PCR to amplify VHH encoding gene fragments. These fragments were cloned into homemade phagemid vectors, which, after infection with helper phage, allowed the production of recombinant phage particles that displayed VHH as gene-III fusion proteins on the surface of phage particles. Phages were prepared according to standard methods and stored after filter sterilization at 4 °C for further use.

[0299] 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 captured on streptavidin Dynabeads. After extensive washing, bound phages were eluted with 1 mg / ml trypsin. The output from the selection was rescued in E. coli TG1 cells. Colonies were picked and sequenced at BATJ, Inc.

[0300] cDNA encoding positive VHHs was synthesized with a C-terminal His-tag at Atum (DNA2.0, Inc) and transiently transfected in HEK293 cells and purified by IMAC chromatography for in vitro functional assays.

[0301] Octet kinetic binding assay

[0302] Biolayer interferometry (BLI), a label-free technique, was used to measure the binding kinetics of positive llama single domain antibodies. Affinity measurements were performed using a biosensor tip equipped with an anti-Penta-His capture (HIS1K) The test was performed with Octet QKe at 30°C in 1× PBS buffer ( PBS pH7.2). The sample was stirred at 1000 rpm. Before analysis, the sensor was humidified for 15 minutes. The binding ability of the purified single domain antibody to the HIS1K sensor tip was tested. The tip was loaded with 20 μg / ml single domain antibody. The loading was performed for 300 seconds so that the capture level was between 1.8 and 2 nm. IL-22 antigen was prepared for binding analysis by diluting it to a concentration of 100, 150, 250, 350 nM in 1× PBS. The binding was started and monitored for 200 seconds, after which the tip was transferred to a 1x PBS buffer (Gibco, PBS pH 7.2) without factor protein to monitor dissociation. Sensor data was collected throughout the experiment and processed and analyzed using Octet data analysis software 7 (ForteBio).

[0303] result

[0304] Isolation of anti-IL22 single domain antibody

[0305] Positive phage colonies from the immune llama phage library were sequenced. The amino acid sequences are presented 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 plasmids were transfected into the HEK293 cell line to produce fully recombinant single domain antibodies with a C-terminal his-tag. The expressed VHHs were purified by HisTrap HP columns.

[0306] Table 24. Llama anti-IL22 VHH sequences

[0307]

[0308]

[0309] The results of the Octet binding affinity assay of IL22 VHH are presented in Table 25.

[0310] Table 25. Binding affinity (KD) of IL22 VHH to rh-IL22

[0311]

[0312] Example 7 Multispecific Single-Chain Antibody

[0313] In order to construct a multispecific single-chain antibody, one or more of the 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 VHH sequences are fused together through different configurations of linkers by recombinant DNA technology. All contents of the VHH sequences specific for HSA, PD-L1, CD33, CD16 and LAG3 disclosed in WO2021 / 062361A2 are incorporated herein by reference.

[0314] Exemplary non-cleavable and cleavable linker sequences are presented in Table 26. These constitute linker means or means for connecting protein domains. These means can be further characterized as cleavable or non-cleavable.

[0315] Table 26. Non-cleavable and cleavable linker sequences

[0316]

[0317]

[0318] The sequences of the multispecific antibodies are described in Tables 27 and 28.

[0319] Table 27. Bispecific Antibodies

[0320]

[0321]

[0322] *Stop codon

[0323] Table 28. Multispecific Antibodies

[0324]

[0325]

[0326] Also disclosed herein is the mutation of proline (P) to serine (S) at certain positions to reduce dimer and aggregate formation.

[0327] The results of ELISA test of SM2248 binding to rh-CD47 and rh-CD40 are as follows Figure 3A -B.

[0328] Flow cytometry detection of SM2248

[0329] The cells (viability > 95%) were washed with ice-cold PBS and cultured at a cell density of 1 × 10 6 / mL was resuspended in staining buffer (PBS-2% FBS). The resuspended cells were added to a 96-well plate at 90 μL per well. A series of dilutions of the test compound prepared in staining buffer were added to the 96-well plate at 10 μL per well and incubated at room temperature for 45 minutes. The 96-well plate was washed 2 to 3 times with staining buffer and then incubated with the second conjugated detection antibody in the dark for 25 minutes. Data were acquired by flow cytometry and analyzed by the software Flowjo. Human CD47 overexpressing CHO (CHO-hCD47), human CD40 overexpressing CHO (CHO-hCD40) and two tumor cell lines Raji and A431 were used for detection. The test showed that SM2248 bound to CD47 or CD40 ( Figure 4A -D).

[0330] CD40 reporter gene assay for SM2248

[0331] CHO-CD47 cells in the logarithmic growth phase (viability greater than 95%) were digested with 0.25% trypsin and immediately treated with RPMI1640 complete medium. After centrifugation at 150×g for 5 min, the cell pellet was centrifuged at 2×10 6 The cells were resuspended in RPMI1640 complete medium at a density of 100 μL / ml and added to a 96-well plate at 40 μL per well. The 96-well plate was then incubated in a 37°C CO2 incubator overnight. A series of dilutions of SM2248 in DMEM medium were added to the 96-well plate at 10 μL per well. The 96-well plate was then incubated in a 37°C CO2 incubator for 60 min. 293T-NFKB-CD40 reporter cells in the logarithmic growth phase (viability greater than 95%) were digested with 0.25% trypsin and immediately treated with RPMI1640 complete medium. After centrifugation at 150×g for 5 min, the cell pellet was centrifuged at 4×10 5Resuspended in RPMI1640 complete medium at a density of 10 cells / ml and added to SM2248-treated 96-well plates at 50 μl per well. The 96-well plates were then incubated in a 37°C-CO2 incubator for 6 hours. After incubation, cells from each well were transferred to a 96-well black plate. The substrate from the One-Lite luciferase assay system was prepared according to the manufacturer's instructions. 100 μL aliquots of the substrate were added to each well. The assay plates were measured in a GLOMAX 96 microplate luminometer using the program "CellTiterGlo". SM2248 is a CD40 agonist bispecific antibody and activates CD40 ( Figure 5A ).

[0332] Flow cytometry blocking assay of SM2248

[0333] Jurkat cells in logarithmic growth phase (viability greater than 95%) were washed twice with ice-cold PBS and cultured at a cell density of 1×10 6 / mL was resuspended in staining buffer (PBS + 2% FBS) and then applied to a 96-well plate at a concentration of 90μL per well. A series of dilutions of SM2248 in staining buffer and SIRPα-His were added to Jurkat cells, 10μL per well. The final concentration of SIRPα-His in each well was 1μg / mL. The 96-well plate was then incubated at room temperature for 30min. After incubation, the test plate was washed three times with 250μL of staining buffer and centrifuged at 500×g for 3min. The cell pellet in each well was resuspended in 100μL of 1:100 diluted PE anti-His detection antibody and stored in the dark at room temperature for 25min. The 96-well plate was washed three times with staining buffer and then resuspended in 150μl cold PBS buffer. The plate was read in Agilent, NovoCyte, and the data was analyzed by Flowjo software. The experiment showed that SM2248 effectively blocked the binding of SIRP to CD47 on the surface of Jurkat cells, IC 50 2.6nM( Figure 5B ).

[0334] These results indicate that SM2248 is a CD47 target-dependent agonist and activates CD40 in a dose-dependent manner.

[0335] BLI binding affinity (KD) experiments for SM2248 are summarized in Table 29. SM2248 binds to human and cynomolgus monkey (Cyno) CD40 and CD47 with similar binding affinities, but has lower binding affinities to mouse and rat proteins.

[0336] Table 29. BLI binding affinity (KD) detection of SM2248

[0337] antigen Koff(1 / s) Kon(1 / Ms) KD(M) rh-CD40 5.86E-05 8.28E+05 7.07E-11 Cyno-CD40* 3.36E-05 6.36E+05 5.28E-11 Rat-CD40 <![CDATA[ / 1 ]]> / / Mouse-CD40 / / / Rh-CD47 7.11E-05 8.88E+05 8.00E-11 Cyno-CD47 1.48E-03 1.95E+06 7.57E-10 Rat-CD47 / / / Mouse-CD47 / / /

[0338] 1 No binding was observed.

[0339] The results of ELISA binding assays of SM2235 against recombinant human EGFR (rh-EGFR) and rh-CD16A are presented in Figure 7, indicating that SM2235 binds to recombinant human EGFR and human CD16A with subnanomolar affinity. Flow cytometric analysis of SM2235 binding is presented in Figures 8 and 9.

[0340] The results of the BLI binding assay of SM2235 are presented in Table 30. The BLI binding assay showed that SM2235 binds to human and cynomolgus EGFR or CD16A with similar KD, but not to rat and mouse. SM2235 does not cross-react with rh-ErbB2, rh-ErbB3, and rh-ErbB4.

[0341] Table 30 BLI binding affinity test of SM2235

[0342] antigen Koff(1 / s) Kon(1 / Ms) KD(M) rh-EGFR 1.28E-04 3.69E+05 3.46E-10 Cyno-EGFR 1.70E-04 8.50E+05 2.00E-10 rh-ErbB2 / / / rh-ErbB3 / / / rh-ErbB4 / / / Mouse-EGFR / / / Rat-EGFR / / / rh-CD16A 4.28E-05 1.11E+06 3.85E-11 Cyno-CD16A 4.28E-05 1.11E+06 3.86E-10 Rat-CD16 / / / Mouse-CD16 / / /

[0343] Blocking test

[0344] CHO-EGFR cells in the logarithmic growth phase (viability greater than 95%) were washed twice with ice-cold PBS and cultured at a cell density of 1×10 6 / mL was resuspended in staining buffer (PBS + 2% FBS) and then applied to a 96-well plate at 90μL per well. A series of dilutions of SM2235 in staining buffer and EGF-his were added to Jurkat cells at 10μL per well. The final concentration of EGF-his in each well was 1μg / mL. The 96-well plate was then incubated at room temperature for 30min. After incubation, the test plate was washed three times with 250μL of staining buffer and centrifuged at 500×g for 3min. The cell pellet in each well was resuspended in 100μL of a 1:100 diluted PE anti-His detection antibody and kept in the dark at room temperature for 25min. The 96-well plate was washed three times with staining buffer and then resuspended in 150μL of cold PBS buffer. The plate was read in Agilent, NovoCyte, and the data was analyzed by Flowjo software. The results are shown in Fig.10 shown.

[0345] NK cell cytotoxicity assay

[0346] method

[0347] A431 cells were cultured at 1.25 × 10 5The expanded human NK (eNK) cells were thawed and resuspended in complete medium KBM581 at a cell density of 1×10 6 cells / mL were resuspended in complete medium KBM581. A431 (40 μL) and eNK cells (50 μL) were applied to each well of a 96-well plate. The ratio of effector cells (eNK) to target cells (A431) was 10:1. The SM2235 stock solution was prepared as a 5-fold dilution series in complete medium KBM581 and added to the cell plate at 10 μL per well from a maximum concentration of 1000 nM. 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 follows: eNK cells alone, A431 alone, A431 lysate, and complete medium KBM581. According to CytoTox-Glo TM The specific cell death was measured according to the instructions of the Cytotoxicity Assay Kit. Briefly, after incubation for 4 h at 37°C-5% CO2, the plate was gently mixed and the sample from each well was transferred to a black 96-well flat-bottom plate. The AAF-Glo reagent was prepared according to the instructions and added to each well at 50 μl. The A431 lysis group was treated with 50 μL of lysis reagent. The plate was incubated in the dark at room temperature for 15 min. The luciferase activity of each well was measured using the Promega microplate luminescence detector built-in program "CellTiterGlo". The results are presented in Fig.11 middle.

[0348] Unless otherwise stated, all quantities used in the specification and claims to indicate the amount of ingredients, molecular weight and other properties, reaction conditions, etc. should be understood to be modified by the term "about" in all cases. As used herein, the terms "about" and "approximately" refer to within 10 to 15%, preferably within 5 to 10%. Therefore, unless otherwise stated, the numerical parameters proposed in the specification and the attached claims are approximate values ​​and can be changed according to the desired characteristics sought to be obtained by the present invention. At least, and not attempting to limit the application of the principle of equivalents to the scope of the claims, each numerical parameter should at least be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques. Although the numerical ranges and parameters describing the wide range of the present invention are approximate values, the numerical values ​​described in the specific examples are reported as accurately as possible. However, any numerical value inherently contains certain errors, which are necessarily caused by the standard deviation found in the respective test measurements.

[0349] The terms "a", "an", "the" and similar indicators used in the context of describing the present invention (particularly in the context of the appended claims) should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. The restatement of the range of values ​​herein is intended only as a shorthand method for individually citing each individual value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually described herein. Unless otherwise indicated herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and is not intended to limit the scope of the present invention otherwise claimed. Any language in the specification should not be interpreted as indicating any non-claim elements that are essential to the practice of the present invention.

[0350] The grouping of the alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member can be mentioned and claimed individually or in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group can be expected to be included in the group or deleted from the group. When any such inclusion or deletion occurs, the specification is deemed to include the modified group, thereby satisfying the written description of all Markush groups used in the appended claims.

[0351] Certain embodiments of the present invention are described herein, including the best mode known to the inventor for implementing the present invention. Of course, after reading the foregoing description, variations of these described embodiments will become apparent to those of ordinary skill in the art. The inventor expects that such variations will be appropriately adopted by those skilled in the art, and the inventor intends to practice the present invention in a manner different from that specifically described herein. Therefore, the present invention includes all modifications and equivalents of the subject matter recited in the appended claims as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, the present invention encompasses any combination of the above-mentioned elements in all possible variations thereof.

[0352] Specific embodiments disclosed herein may be further limited in the claims using language consisting of or consisting essentially of. When used in a claim, whether as filed or added by amendment, the transition term "consisting of excludes any element, step, or ingredient not specified in the claim. The transition term "consisting essentially of limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics. Embodiments of the claimed invention are inherently or expressly described and enabled herein.

[0353] In addition, throughout the specification, numerous references have been made to patents and printed publications. Each of the above-cited references and printed publications is individually incorporated herein by reference in its entirety.

[0354] Finally, it should 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 also within the scope of the invention. Therefore, by way of example and not limitation, alternative configurations of the invention may be utilized according to the teachings herein. Therefore, the invention is not limited to exactly as shown and described.

Claims

1. A variable heavy (VHH) domain having antigen-binding specificity for OX40, wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 2-24, 106 or 125-142.

2. A VHH domain having antigen-binding specificity for CD40, wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 26-40.

3. A VHH domain having antigen-binding specificity for 4-1BB, wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 42-53.

4. A VHH domain having antigen-binding specificity for epidermal growth factor receptor (EGFR), wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 55-83.

5. A VHH domain having antigen-binding specificity for human serum albumin (HSA), wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 114-118.

6. A VHH domain with antigen-binding specificity for IL-22, wherein the VHH domain has an amino acid sequence of one of SEQ ID NOs: 121-124.

7. An antibody comprising the VHH domain of any one of claims 1 to 6.

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 specific for OX40, CD40, 4-1BB, HSA, IL-22 or EGFR; (a) the OX40 binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 2-24, 106 or 125-142; (b) CD40 binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 26-40; (c) 4-1BB binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 42-53; (d) EGFR binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 55-83; (e) HSA binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 114-118; and (f) IL-22 binding specificity is represented by an amino acid sequence of one of SEQ ID NOs: 121-124.

9. The multispecific antibody of claim 8, further comprising one to five additional antibody binding domains, wherein each additional antibody binding domain is individually specific for OX40, CD40, 4-1BB, HSA, IL-22 or EGFR.

10. The multispecific antibody of claim 9, further comprising one to four additional antibody binding domains, wherein each additional antibody binding domain is specific for OX40, CD40, 4-1BB, HSA, IL-22 or EGFR.

11. The multispecific antibody according to any one of claims 8 to 10, wherein the antibody is a multispecific single-chain antibody (MVSCA).

12. The multispecific antibody according to any one of claims 8 to 11, wherein the linker is selected from the linkers of Table 26.

13. The multispecific antibody according to claim 12, wherein linkers L1 (SEQ ID NO: 84), L2 (SEQ ID NO: 85) or L4 (SEQ ID NO: 87) are inserted between one or more pairs of non-identical antibody binding domains.

14. The multispecific antibody according to any one of claims 8 to 12, comprising at least one pair of antibody binding domains with the same specificity. The multispecific antibody according to claim 14 , wherein the at least one pair of antibody binding domains with the same specificity are adjacent to each other.

16. The multispecific antibody according to claim 15, wherein a linker having the amino acid sequence of linker L3 (SEQ ID NO: 86) is inserted between antibody binding domains of the same specificity.

17. The multispecific antibody according to any one of claims 8 to 16, wherein all of the antibody binding domains are VHH domains.

18. The multispecific antibody according to any one of claims 8 to 17, wherein the multispecific antibody has an amino acid sequence of one of SEQ ID NOs: 107 to 112.

19. A pharmaceutical composition comprising the VHH domain of any one of claims 1 to 6, the antibody of claim 7 or the multispecific antibody of any one of claims 8 to 18.

20. A method for treating cancer, comprising administering the pharmaceutical composition of claim 19 to a patient in need thereof.

21. A method for treating an autoimmune disease, comprising administering the pharmaceutical composition of claim 19 to a patient in need thereof.

Images