Monospecific and multispecific antibodies

Abstract

Disclosed herein are monospecific and multispecific antibodies specific for CD28 and CD3E, and one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 596,552, filed November 6, 2023, and U.S. Provisional Patent Application No. 63 / 604,398, filed November 30, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, named 1959708-00017-00017WO_Sequence_Listing.xml, which is 232KB in size and was created on November 5, 2024. The entire contents of this sequence list are incorporated herein by reference. Invention Overview

[0006] This article discloses monospecific VHH antibodies specific to CD28 and CD3E, as well as multivalent single-chain antibodies comprising two or more VHH domains, which have specificity against one or more of these antigens and bind specifically to other antibodies.

[0007] Some implementations are single-domain antibodies that contain only or primarily the VHH domain of camel antibodies. These implementations are monospecific and monovalent.

[0008] Some implementations include 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 implementations are monospecific but typically bivalent. Other valence states are possible, depending on, for example, the choice of constant domains. The Fc regions of IgA and IgM can confer higher valence states.

[0009] Some implementations include two VHH domains (multivalent single-chain antibodies) specific for the same antigen linked in a single amino acid chain. These implementations are also monospecific and bivalent. Additional VHH domains can be linked for higher valence states.

[0010] Some embodiments include two (or more) VHH domains, each specific to a different antigen, linked in a single amino acid chain (multivalent, multispecific single-chain antibody). These embodiments are multivalent and multispecific. In a further embodiment containing three or more VHH domains, two or more VHH domains may be specific to the same antigen, while one or more other VHH domains are specific to different antigens. Such constructs have a valence state higher than specificity.

[0011] Each monospecific implementation is specific to CD28 and CD3E. Each multispecific implementation is specific to one or more of CD28 and CD3E, as well as one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR, but may also be specific to one or more other antigens.

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

[0013] The antigen-binding domains that are specific to certain antigens disclosed in this article can be referred to as means for binding antigens.

[0014] Therefore, this document discloses a variable heavy chain (VHH) domain with antigen-binding specificity to CD28, wherein the VHH domain comprises a complementarity-determining region (CDR) of any one of SEQ ID NO: 2-22. In some embodiments, the VHH comprises an amino acid sequence of any one of SEQ ID NO: 2-22.

[0015] This document discloses a VHH domain with antigen-binding specificity to CD3E, wherein the VHH domain comprises the CDR of any one of SEQ ID NO: 30-44. In some embodiments, the VHH comprises the amino acid sequence of any one of SEQ ID NO: 30-44.

[0016] This article also discloses antibodies containing the CDR or VHH domains disclosed herein.

[0017] This document also discloses a multispecific antibody comprising a first 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 to CD28 or CD3E, and wherein the second binding specificity is specific to OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR; wherein: (a) the binding domain having CD28 binding specificity comprises any one of the CDRs in SEQ ID NO: 2-22; (b) the binding domain having CD3E binding specificity comprises any one of the CDRs in SEQ ID NO: 30-44; (c) the binding domain having OX40 binding specificity comprises any one of the CDRs in SEQ ID NO: 174 or 175; (d) the binding domain having CD40 binding specificity comprises any one of the CDRs in SEQ ID NO: 176-190; (e) the binding domain having 4-1BB binding specificity comprises any one of the CDRs in SEQ ID NO: 176-190. (f) A binding domain with EGFR binding specificity comprising any one of SEQ ID NO: 204-232; (g) A binding domain with HSA binding specificity comprising any one of SEQ ID NO: 107-122; (h) A binding domain with IL-22 binding specificity comprising any one of SEQ ID NO: 233-236; (i) A binding domain with PD-L1 binding specificity comprising any one of SEQ ID NO: 98-106; (j) A binding domain with CD16 binding specificity comprising any one of SEQ ID NO: 168-173; (k) A binding domain with CD47 binding specificity comprising any one of SEQ ID NO: 67-97; (l) A binding domain with CD33 binding specificity comprising any one of SEQ ID NO: 191-203; (f) A binding domain with EGFR binding specificity comprising any one of SEQ ID NO: 204-232; (g) A binding domain with HSA binding specificity comprising any one of SEQ ID NO: 107-122; (h) A binding domain with IL-22 binding specificity comprising any one of SEQ ID NO: 233-236; (i) A binding domain with PD-L1 binding specificity comprising any one of SEQ ID NO: 98-106; (j) A binding domain with CD16 binding specificity comprising any one of SEQ ID NO: 168-173; (k) A binding domain with CD47 binding specificity comprising any one of SEQ ID NO: 67-97; (l) A binding domain with CD33 binding specificity comprising any one of SEQ ID NO: 168-173; (d) A binding domain with CD47 binding specificity comprising any one of SEQ ID NO: 168-173; (f) A binding domain with CD47 binding specificity comprising any one of SEQ ID NO: 168-173; (g) A binding domain with CD33 binding specificity comprising any The CDR in any one of SEQ ID NO: 124-153; and (m) the binding domain having LAG3 binding specificity includes the CDR in any one of SEQ ID NO: 154-173; and wherein the VHH domain is linked by an amino acid linker sequence.

[0018] In some embodiments, the multispecific antibody further comprises one to five additional antibody-binding domains, wherein each additional antibody-binding domain is specifically specific to CD28, CD3E, OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

[0019] In some implementations, the multispecific antibody further includes one to four additional antibody-binding domains, each of which is specific to CD28, CD3E, OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

[0020] In some implementations, the multispecific antibody is a multispecific single-chain antibody (MVSCA).

[0021] In some embodiments, the adapter comprises an amino acid sequence of one of SEQ ID NO: 50-72. In some embodiments, the adapter is inserted between one or more pairs of distinct antibody-binding domains.

[0022] In some embodiments, the multispecific antibody comprises at least one pair of antibody-binding domains having the same specificity. In some embodiments, at least one pair of antibody-binding domains having the same specificity are adjacent to each other. In some embodiments, a linker is inserted between the antibody-binding domains having the same specificity.

[0023] In some implementations, all antibody-binding domains are VHH domains.

[0024] This article also discloses pharmaceutical compositions comprising the VHH domain disclosed herein, antibodies, or multispecific antibodies.

[0025] This document also discloses methods for treating cancer, which include administering the disclosed VHH domain, antibody, multispecific antibody, or pharmaceutical composition to a subject in need. In some embodiments, the multispecific antibody comprises the amino acid sequence of SEQ ID NO: 23.

[0026] This document also discloses the use of the VHH domain, antibodies, multispecific antibodies, or pharmaceutical compositions disclosed herein for the treatment of cancer in subjects of need. In some embodiments, the multispecific antibody comprises the amino acid sequence of SEQ ID NO: 23.

[0027] This article also discloses methods for treating autoimmune diseases, which include administering the VHH domain, antibody, multispecific antibody, or pharmaceutical composition disclosed herein to subjects in need.

[0028] This article also discloses the use of the VHH domain, antibody, multispecific antibody or pharmaceutical composition disclosed herein for the treatment of autoimmune diseases in subjects in need. Attached Figure Description

[0029] Figure 1 Two CD28-specific multispecific antibodies were described.

[0030] Figure 2 Flow cytometry analysis of two multispecific VHHs on Jurkat cells was described.

[0031] Figure 3A -G. Figure 3A Flow cytometry analysis was performed on the SM2275 CHO-hEGFR-hPD-L1 expressing cell lines MB231, A431, and H292. Figure 3B -G describes SM2275 and MB-231 ( Figure 3B A431 Figure 3C H292 Figure 3D ), CHO-hC28 ( Figure 3E ), Jurkat Figure 3F ) and human CD28+ T( Figure 3G Cell binding affinity.

[0032] Figure 4 The antibody blocking effect of PD-1 binding to A431 cells was described.

[0033] Figure 5 The Jurkat-PD1-NFAT-Luc reporter gene assay for SM2275 was characterized.

[0034] Figure 6 Cell-based functional assays of SM2275 cells using Jurkat-PD1-NFAT luciferase reporter cells were depicted.

[0035] Figure 7A -D depicts the release of human PBMC cytokines induced by SM2275 and TGN1412. Figure 7A -TNF-α; Figure 7B –IL-6; Figure 7C –IL-2; Figure 7D –INF-γ.

[0036] Figure 8 depicts cytokine release in the hCD28 / hPD-L1 humanized mouse model. Figure 8A –IL-2; Figure 8B –IL-6; Figure 8C –TNF-α; Figure 8D –INF-γ.

[0037] Figure 9 The inhibitory effect of SM2275 on tumor growth of MC38-hEGFR / hPD-L1 (mPD-L1 KO) tumors in humanized CD28 mice was demonstrated.

[0038] Figure 10The inhibitory effect of SM2275 on MC38-hEGFR / hPD-L1 tumors in hCD38 / hPD-L1 mice was described.

[0039] Figure 11 The inhibition of tumor growth in NCI-H292 tumors in NCG mice reconstituted with human PBMCs was described.

[0040] Figure 12 The pharmacokinetics of SM2272 in cynomolgus monkeys after multiple IV injections were described.

[0041] Figure 13A -B describes the ELISA binding assays for various anti-CD3E VHH.

[0042] Figure 14-C depicts the flow cytometry analysis of the binding of various anti-CD3E VHH molecules to Jurkat cells. Invention Details

[0044] This article discloses monospecific immunoglobulin variable domain antibodies (referred to as VHH monodomain antibodies) that are specific to CD28 and CD3E, as well as multivalent single-chain antibodies (MVSCA) that contain two or more VHH variable domains and are specific to one or more of these antigens.

[0045] As used herein, the term VHH refers to the variable domain of a heavy chain antibody and is the antigen-binding fragment of a heavy chain antibody only.

[0046] In some embodiments, the MVSCA comprises two or more variable domains specific to the same antigen. That is, the MVSCA is multivalent but monospecific to the antigen. In some of these embodiments, the MVSCA comprises two or more repeats of the same VHH domain, or multiple VHH domains each specific to the same epitope. That is, they are multivalent but monospecific to the epitope. Such an MVSCA will bind to only a single site on the antigen monomer, but can crosslink multiple copies of the monomer. In other of these embodiments, the MVSCA comprises two or more VHH domains, each specific to a different epitope of the same antigen. That is, they are multivalent but multispecific to the epitope. Such an MVSCA can bind to multiple sites on the antigen monomer or crosslink multiple copies of the monomer.

[0047] In some embodiments, the MVSCA comprises two or more VHH domains that are specific to different antigens; that is, they are multivalent and multispecific relative to the antigen. In further embodiments, the MVSCA comprises multiple VHH domains, wherein additional variable domains are identical to the first VHH domain, wherein additional VHH domains differ from the first VHH domain but are specific to different epitopes on the same antigen, or wherein additional VHH domains differ from the first VHH domain but are specific to different antigens; any combination of these can be used.

[0048] MVSCAs containing two or more VHH domains may further include immunoglobulin constant domains. For example, the C-terminal VHH domain may remain linked to its original VHH constant domain. Alternatively, the C-terminal VHH domain may be linked to a constant domain or Fc region of a more conventional antibody (e.g., a human antibody, such as a human IgG antibody). In some embodiments, the constant domain or intact Fc region may confer a specific function, as is known to those skilled in the art. In other embodiments, MVSCAs containing two or more VHH domains may further include constant domains located between or at the N-terminus of a VHH domain, rather than at the C-terminus of the VHH domain (or other than at the C-terminus of the VHH domain).

[0049] antigen

[0050] CD28 is a transmembrane glycoprotein expressed by T cells and some other hematopoietic cells. It is one of the proteins expressed on T cells that provides the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), T cell stimulation via CD28 can provide effective signals for the production of various interleukins, particularly IL-6. CD28 is part of the CD28 family of receptors, a group of regulatory cell surface receptors expressed on immune cells. The CD28 family of receptors is a subgroup of the immunoglobulin superfamily. Two family members, CD28 and ICOS, act as positive regulators of T cell function, while three others, BTLA, CTLA-4, and PD-1, act as repressors. The CD28 protein contains 220 amino acids and is encoded by a gene consisting of four exons. It is a glycosylated disulfide-linked homodimer with a molecular weight of 44 kDa. The structure of CD28 contains paired domains of the group V immunoglobulin superfamily (IgSF). CD28 bispecific antibodies are designed to specifically co-stimulate T cells within the tumor microenvironment. By bridging T cells to malignant cells expressing selected tumor-associated antigens (TAAs), CD28 bispecific antibodies deliver a so-called signal two to the T cells, releasing their full cytotoxic potential.

[0051] CD3E is a protein complex and T cell co-receptor involved in activating both cytotoxic T cells (CD8+ naïve T cells) and helper T cells (CD4+ naïve T cells). It consists of four distinct chains: the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3E is also known as CD3 or T3 and is primarily expressed on T cells, NK-T cells, and at varying levels on thymocytes during T cell differentiation. The CD3ε subunit of the T cell receptor complex is encoded by the CD3E gene 34. CD3 bispecific antibodies are a type of immunotherapy that has gained considerable attention in cancer treatment. They are designed to redirect T cells to recognize and kill tumor cells. These antibodies bind to both the CD3 protein complex on T cells and tumor-associated antigens (TAAs) on cancer cells. By doing so, they form an artificial immune synapse between T cells and cancer cells, leading to T cell activation and tumor cell killing. Multispecific antibodies are another class of immunotherapies that can bind to multiple targets simultaneously. They can be engineered to target different antigens on cancer cells or to connect immune cells while targeting cancer cells. Both CD3 bispecific and multispecific antibodies have shown promise in preclinical and clinical studies for the treatment of various cancers. They are being investigated as monotherapy or in combination with other immunomodulators. The goal is to enhance anti-tumor immune responses and improve patient clinical outcomes.

[0052] Multispecific antibodies may also include additional antigen specificities, including but not limited to OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR. All information disclosed in patent applications PCT / US2020 / 053064, PCT / US2023 / 069262, and PCT / US2024 / XXXXX (Attorney’s File No. 1959708.00018, filed on the same day as this application, and claiming priority to U.S. Provisional Application 63 / 596,555, filed November 6, 2023, and U.S. Provisional Application 63 / 601,931, filed November 22, 2023, regarding antibodies specific to these antigens, is incorporated herein by reference.

[0053] Antibody

[0054] Antibodies and their uses in treating diseases are well known in the art. As used herein, the term "antibody" refers to a monomeric or polymeric protein comprising one or more polypeptide chains containing antigen-binding sites. Antibodies specifically bind to antigens and can modulate the biological activity of antigens. As used herein, the term "antibody" may include "full-length antibody" and "antibody fragment." As used herein, the term "binding site" or "antigen-binding site" refers to the region of an antibody molecule to which the ligand actually binds. The term "antigen-binding site" includes the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL), or, in the case of heavy chain-only antibodies, the antibody heavy chain variable region.

[0055] Antibody specificity refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, natural antibodies are monospecific. As used herein, the term "monospecific" antibody means an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The monospecific antibodies disclosed herein are specific for CD28 or CD3E. In some embodiments, the monospecific antibody contains only a VHH domain heavy chain. In other embodiments, the monospecific antibody contains a VHH domain fused to one or more protein domains, including, for example, a human Fc region. In other embodiments, the monospecific antibody contains VHH as the only intact protein domain, i.e., a single-domain antibody. In some embodiments, the single-domain antibody may additionally contain a short peptide, such as a His tag. The VHH domain may be referred to as a construct for binding a specific target (e.g., CD28 or CD3E). Therefore, any of the various antibody structures, forms, or constructs disclosed herein containing a VHH domain, or constructed to contain a VHH domain, may be referred to as antibodies containing a construct for binding an indicator target. Some implementations may specifically include one or more particular antibody structures, forms, or constructs. Other implementations may specifically exclude one or more particular antibody structures, forms, or constructs.

[0056] As used in this article, phrases such as "antibody that is specific to...", "antibody that recognizes...", "antibody that has affinity for...", "antibody that has a binding site for...", and similar sentence structures can be used interchangeably.

[0057] A "multispecific antibody" refers to an antibody that has the specificity to bind to two or more antigens. The multispecific antibodies disclosed herein are specific to one or more of CD28 and CD3E, and one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR, or 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 antigen-binding domains. Furthermore, multispecific antibodies may include at least two copies of the same antigen-binding sequence, or two antigen-binding sequences specific to different epitopes (double complementary sites) on the same antigen, provided that the multispecific antibody is specific to at least one of CD28 and CD3E and at least one second antigen. In some embodiments, the multispecific antibodies disclosed herein are single-chain antibodies. Therefore, some multispecific antibodies may be referred to as antibodies comprising components for binding a first target and components for binding a second target, etc.

[0058] A “bispecific antibody” is an antibody that has the specificity to bind to two different antigens. In some embodiments, the bispecific antibodies disclosed herein are specific to one or both of CD28 and CD3E, and optionally one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR. The amino acid sequence encoding the antigen-binding moiety of the bispecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the bispecific antibody is linked via a linker as disclosed herein.

[0059] A “trispecific antibody” refers to an antibody that has three different antigen-binding specificities. In some embodiments, the trispecific antibodies disclosed herein are specific to one or more selected from CD28 and CD3E, and one or more selected from OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR. The amino acid sequence encoding the antigen-binding moiety of the trispecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the trispecific antibody is linked via a linker as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0060] A "tetraspecific antibody" refers to an antibody possessing four different antigen-binding specificities. In some embodiments, the tetraspecific antibodies disclosed herein are specific to four specificities selected from one or more of CD28 and CD3E, and one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR. The amino acid sequence encoding the antigen-binding moiety of the tetraspecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the tetraspecific antibody is linked via a linker as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0061] As used herein, the term "valence" indicates the presence of a specified number of binding sites in an antibody molecule. Therefore, the terms "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," and "octavalent" indicate the presence of two, three, four, five, six, seven, and eight binding sites, respectively, in an antibody molecule. The bispecific antibodies disclosed herein are "bivalent." The trispecific antibodies disclosed herein are "trivalent." The tetraspecific antibodies disclosed herein are "tetravalent." However, monospecific multivalent antibodies, such as bivalent, trivalent, and tetravalent antibodies, within the scope of this disclosure, have multiple antigen-binding sites that bind to the same antigen. The antigen-binding sites of monospecific bivalent and trivalent (or higher valence) antibodies may bind to the same 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 antibodies can be constructed, such as trivalent, bispecific antibodies.

[0062] In this article, "full-length antibody" refers to the structure of the naturally occurring biological form of the antibody, including variable and constant regions. For example, in most mammals (including humans and mice), full-length IgG antibodies are tetramers composed of two pairs of identical immunoglobulin chains, each pair having one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, such as camels and llamas, IgG antibodies may also consist of only two variable heavy chains, each containing a variable domain (VHH) attached to the Fc region (CH2 and CH3 domains).

[0063] Tetrameric antibodies typically consist of two pairs of identical polypeptide chains, each pair having a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50-70 kDa). Each of the light and heavy chains contains two distinct regions, called the variable region and the constant region. For IgG immunoglobulins, the heavy chain contains four immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VH-CH1-CH2-CH3, namely the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (also referred to as VH-Cγ1-Cγ2-Cγ3, namely the heavy chain variable domain, constant γ1 domain, constant γ2 domain, and constant γ3 domain, respectively). The IgG light chain contains two immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VL-CL, namely the light chain variable domain and the light chain constant domain. The constant region exhibits less sequence diversity and is responsible for binding many native proteins to trigger important biochemical events.

[0064] The variable region of an antibody contains the molecule's antigen-binding determinants, thus determining the antibody's specificity for its target antigen. The variable region is so named because it differs most significantly in sequence from other antibodies in its class. Within the variable region, three loops are clustered in each of the V domains of the heavy and light chains to form an antigen-binding site. Each of these loops is called a complementarity-determining region (hereinafter referred to as "CDR"), where the variation in amino acid sequence is most significant. There are a total of six CDRs, 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 the CDRs is called the frame (FR) region. Although not as diverse as the CDRs, sequence variability does exist in the FR regions between different antibodies. Overall, this characteristic architecture of an antibody provides a stable scaffold (FR region) upon which the immune system can explore rich antigen-binding diversity (CDRs) to achieve specificity against a broad array of antigens.

[0065] The gene encoding the immunoglobulin locus contains multiple V region sequences as well as shorter nucleotide sequences named “D” and “J”, and it is the combination of V, D and J nucleotide sequences that produces VH diversity.

[0066] Antibodies are classified into categories, also known as isotypes, based on their genetically determined constant regions. 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 antibody isotypes 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, and IgG3. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Therefore, as used herein, "isotype" refers to any one of the categories or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of its constant regions. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. Disclosed VHH antibodies, bispecific and multispecific antibodies may have constant regions comprising all or part of the above isotypes.

[0067] Within the scope of this disclosure are also antibody fragments, including but not limited to (i) Fab fragments containing VL, CL, VH, and CH1 domains; (ii) Fd fragments containing VH and CH1 domains; (iii) Fv fragments containing the VL and VH domains of a single antibody; (iv) dAb fragments containing a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments; bivalent fragments containing two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) wherein the VH and VL domains are linked by a peptide linker that allows the two domains to bind to form an antigen-binding site. Trivalent or tetravalent antibody fragments containing three variable domains having three different specificities linked by cleavable or non-cleavable linkers are also disclosed. In some embodiments, the antibody is generated using recombinant DNA technology. In other embodiments, the antibody is generated by enzymatic or chemical cleavage of a naturally occurring antibody.

[0068] As used herein, a “single-chain antibody” refers to a fusion protein of an antibody’s antigen-binding portion (i.e., variable region) typically linked by a linker peptide. This document discloses multivalent monospecific and multispecific single-chain antibodies. Monospecific multivalent antibodies are specific for at least one of CD28 and CD3E. Multispecific single-chain antibodies are specific for one or more of CD28 and CD3E, as well as one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

[0069] As used herein, a “humanized” antibody refers to an antibody comprising a human framework region (FR) and one or more complementarity-determining regions (CDRs) derived from a non-human antibody. The non-human antibody providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the framework is referred to as the “recipient.” In some embodiments, humanization primarily relies on transplanting the donor CDR onto the recipient (human) VL or VH framework. This strategy is referred to as “CDR transplantation.” Typically, it is necessary to “reverse mutate” selected receptor framework residues to the corresponding donor residues to regain the affinity lost in the initial transplanted construct. Humanized antibodies may also preferably contain at least a portion of the immunoglobulin constant region, typically the constant region of a human immunoglobulin, and may typically contain a human Fc region. Humanization or other methods that reduce the immunogenicity of the variable region of a non-human antibody may include surface remodeling methods. In one embodiment, a selection-based approach may be used to humanize and / or affinity-mature the antibody variable region, i.e., to increase the affinity of the variable region for its target antigen. Other humanization methods may involve transplanting only portions of the CDR, including but not limited to the methods described in US 6,797,492, which is incorporated herein by reference for all its disclosures regarding CDR transplantation. Structure-based methods can be applied for humanization and affinity maturation, such as those described in US Patent 7,117,096, which is incorporated herein by reference for all its disclosures regarding humanization and affinity maturation.

[0070] In the various embodiments described herein, the antibody is VHH. In addition to conventional heavy and light chain antibodies (two light chains and two heavy chains in one antibody), camelids (camels, dromedaries, and llamas) also possess double-chain antibodies (containing only the variable heavy chain). The dimer antibody is encoded by a unique set of VH fragments called VHH genes. VH and VHH are scattered throughout the genome (i.e., they appear mixed together). Identification of the same D fragment in the VH and VHH cDNA indicates that this D fragment is used for both VH and VHH. Antibodies containing native VHH lack the entire CH1 domain of the heavy chain constant region. The exon encoding the CH1 domain is present in the genome but has been spliced ​​out due to the loss of the functional splice acceptor sequence on the 5' side of the CH1 exon. As a result, the VDJ region is spliced ​​onto the CH2 exon. When VHH recombines into such constant regions (CH2, CH3), antibodies are produced, where the half-antibody is a single chain rather than a light / heavy chain pair (i.e., an antibody with two heavy chains but no light chain interaction). The binding of the antigen differs from that seen with conventional antibodies, but high affinity is achieved in the same way, i.e., through hypermutation of the variable region and screening of cells expressing such high-affinity antibodies.

[0071] In an exemplary embodiment, the disclosed VHH is generated by immunizing transgenic mice, wherein endogenous mouse antibody expression has been eliminated and a camelidoid transgene has been introduced. VHH mice are disclosed in US8,883,150, US8,921,524, US8,921,522, US8,507,748, US8,502,014, US 2014 / 0356908, US2014 / 0033335, US2014 / 0037616, US2014 / 0356908, US2013 / 0344057, US2013 / 0323235, US2011 / 0118444, and US2009 / 0307787, all of which disclose, in their entirety, heavy chain antibodies and their generation in transgenic mice, and are incorporated herein by reference. VHH mice were immunized, and the resulting sensitized spleen cells were fused with mouse myeloma cells to form hybridomas.

[0072] In other embodiments, VHHs are generated by immunizing the llama with the desired antigen and isolating the sequence encoding the VHH region of the resulting antigen-binding antibody. In one embodiment, VHHs are isolated using a phage display library. See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204 (each document is incorporated by reference in its entirety regarding the preparation of phage libraries).

[0073] This document also discloses multispecific or multivalent antibodies in which two or more antigen-binding domains are linked in a single fusion protein. Multispecific antibodies can take various forms, including (i) multispecific Fv fragments; (ii) heavy chains with first specificity to which a second VH domain with second specificity is bound (or fused to); (iii) tetrameric monoclonal antibodies with first specificity to which a second VH domain with second specificity is bound, wherein the second VH domain binds to the first VH domain; and (iv) Fab fragments (VH-CH1 / VL-CL) with first specificity to which a second VH domain with second specificity is bound. Exemplary Fab fragments include those in which the second VH sequence with second specificity binds to 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 another embodiment, a VH sequence having second and / or third (or more) specificity may bind (or fuse) to 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 multispecific antibody conformations can be found in WO2021 / 062361, which is incorporated herein by reference in its entirety regarding the conformations of multispecific antibodies.

[0074] Multispecific or multivalent antibodies may include a linker sequence that links a specific antigen-binding domain (such as VH or VHH) to another antigen-binding domain, and that allows for the appropriate folding of the amino acid sequence to generate the desired three-dimensional conformation and antigen-binding profile. Typically, the linker sequence can be a short amino acid sequence that provides sufficient space and flexibility between the domains for proper folding. The linker may also induce steric hindrance to facilitate binding to the target site of each domain. Suitable linkers include, but are not limited to, those listed in Table 10 (SEQ ID NO: 44-66). Other linkers are known to those skilled in the art.

[0075] Amino acid sequence variants of monospecific or multispecific antibodies disclosed herein are also within the scope of this 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, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody described herein. Any combination of deletions, insertions, and substitutions is performed to obtain a final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter the post-translational processes of humanized or variant antibodies, such as changing the number or location of glycosylation sites.

[0076] A useful method for identifying specific residues or regions of an antibody as preferred sites for mutagenesis is called "alanine scan mutagenesis." Residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral amino acids (preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Those amino acid positions that demonstrate functional sensitivity to the substitution are then refined by introducing further or other variants at or against the substitution site. Therefore, while the sites used to introduce amino acid sequence variations are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scans or random mutagenesis are performed at the target codon or region, and antibody variants expressed are screened for the desired activity.

[0077] Amino acid sequence insertions include fusions of amino and / or carboxyl terms to peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue as disclosed herein, or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions to the N-terminus or C-terminus of an antibody with an enzyme or peptide that increases the antibody's serum half-life.

[0078] Another type of variant is the amino acid substitution variant. These variants have at least one removed amino acid residue in the antibody molecule and a different residue inserted at its position. The sites of most interest for substitution mutagenesis include hypervariable regions, but FR alterations are also considered. Conservative substitutions are shown under the heading “Preferred Substitutions” in Table 1. If such substitutions result in an alteration of biological activity, more substantial changes can be introduced, named “Exemplary Substitutions” in Table 1, or as further described below regarding amino acid categories, and the products can be screened.

[0079] Table 1

[0080]

[0081]

[0082] Substantial modification of antibody biological properties is achieved by selecting substitutions that significantly differ in their role in maintaining (a) the structure of the polypeptide backbone in the substitution region, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains. Naturally occurring residues are grouped into several groups based on shared side-chain characteristics:

[0083] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0084] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0085] (3) Acidity: Asp, Glu;

[0086] (4) Alkaline: Asn, Gin, His, Lys, Arg;

[0087] (5) Residues affecting chain orientation: Gly, Pro; and

[0088] (6) Aromatics: Trp, Tyr, Phe.

[0089] Non-conservative substitution may involve exchanging members of one of these categories for another category.

[0090] Any cysteine ​​residues that do not participate in maintaining the proper conformation of monospecific or multispecific antibodies can be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0091] Another type of substitution variant involves replacing one or more hypervariable residues of the parent antibody (e.g., humanized or camelid antibody). Typically, the resulting variants selected for further development possess improved biological properties relative to the parent antibody from which they originate. A convenient method for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The resulting antibody variant is displayed monovalently from filamentous phage particles as a fusion with the M13 gene III product packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened as disclosed herein. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively, or additionally, analysis of the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and antigen may be beneficial. Such contact residues and adjacent residues are candidates for substitution according to the techniques described herein. Once such variants are generated, the variant group is screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

[0092] Another type of amino acid variant of an antibody alters its original glycosylation pattern. This alteration involves the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites not present in the antibody.

[0093] Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation involves attaching one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0094] Adding glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). This alteration can also be achieved by adding or substituting one or more serine or threonine residues (for O-linked glycosylation sites) into the sequence of the original antibody (for O-linked glycosylation sites).

[0095] Nucleic acid molecules encoding amino acid sequence variants of monospecific or multispecific antibodies are prepared using a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation from previously prepared variants or non-variant versions of antibodies disclosed herein via oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0096] Other modifications to monospecific or multispecific antibodies are considered. For example, antibodies may need to be modified in terms of effector function to enhance their potency, for example, in treating diseases. For instance, cysteine ​​residues can be introduced into the Fc region, allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibodies may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional cross-linking agents. Alternatively, antibodies with dual Fc regions can be engineered to possess enhanced complement cleavage and ADCC capabilities.

[0097] In another implementation, the antibody can be conjugated to a "receptor" (such as streptavidin) for pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by the removal of unbound conjugates from circulation using a scavenger, and then the administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide).

[0098] Covalent modifications of monospecific or multispecific antibodies are also included within the scope of this disclosure. Where applicable, they can be prepared by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of antibodies are introduced into the molecule by reacting the target amino acid residues of the antibody with an organic derivatizer capable of reacting with selected side chains or N-terminal or C-terminal residues. Exemplary covalent modifications of peptides are described in US5,534,615, all of which discloses covalent modifications of peptides and are specifically incorporated herein by reference. Exemplary types of covalent modifications of antibodies include linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in a manner as described in US4,640,835, US4,496,689, US4,301,144, US4,670,417, US4,791,192, or US4,179,337.

[0099] The monospecific or multispecific antibodies disclosed herein can be generated by recombinant means. Therefore, this document discloses 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 art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and typically purification to pharmaceutically acceptable purity. To express the antibodies as described above in host cells, nucleic acids encoding the antibody sequence are inserted into an expression vector using standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C 6 cells, yeast, or E. coli cells, and the antibody is recovered from the cells (supernatant or lysed cells). It should be understood that any recombinantly expressed protein requires an initiating methionine (or formyl-methionine) or signal sequence at its N-terminus, depending on the expression system used and whether the protein is expressed in the cytoplasm or secreted. Therefore, in some embodiments, the protein sequences disclosed herein are modified at their N-terminus with such additional amino acids. In some implementations, such N-terminal sequences are cut (completely or partially) from fully mature sequences, while in other implementations they are preserved.

[0100] Therefore, some embodiments disclosed herein include methods for preparing monospecific or multispecific antibodies, comprising the steps of: a) transforming host cells with at least one expression vector containing a nucleic acid molecule encoding an antibody; b) culturing the host cells under conditions that allow for the synthesis of antibody molecules; and c) recovering the antibody molecules from the culture.

[0101] Antibodies are appropriately separated from the culture medium using conventional immunoglobulin purification methods, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived from them, regardless of the number of passages. It should also be understood that the DNA content of all progeny may not be entirely identical due to intentional or unintentional mutations. This includes variant progeny with the same function or biological activity as those screened in the initially transformed cells. Where the intention is to distinguish the names, it will be clear from the context.

[0103] As used herein, the term "transformation" refers to the process of transferring a vector / nucleic acid into a host cell. If cells without a robust 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 the cell can also be used, such as nuclear injection or protoplast fusion. If prokaryotic cells or cells with abundant cell wall structures are used, one transfection method is calcium treatment with calcium chloride.

[0104] As used herein, “expression” refers to the process by which nucleic acids are transcribed into mRNA and / or the transcribed mRNA (also called transcript) is subsequently translated into peptides, polypeptides, or proteins. 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 the splicing of mRNA.

[0105] A “vector” is a nucleic acid molecule, particularly a self-replicating one, that transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into a cell (e.g., chromosome integration), replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose primary function is to transcribe and / or translate DNA or RNA. Vectors that provide more than one of these functions are also included.

[0106] An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce the desired expression product.

[0107] As used herein, the term "host cell" refers to any type of cell system that can be engineered to generate the antibodies disclosed herein. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0108] Control sequences applicable to prokaryotes include, for example, promoters, optional operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0109] When a nucleic acid is in a functional relationship with another nucleic acid sequence, that nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, then the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is located to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and within the reading frame. However, enhancers do not need to be contiguous. Ligation is accomplished by ligating at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0110] This article also discloses isolated nucleic acids encoding monospecific or multispecific antibodies, vectors and host cells containing such nucleic acids, and recombinant technologies for producing such antibodies.

[0111] To generate antibodies through recombination, the nucleic acid encoding it can be isolated and inserted into a reproducible vector for further cloning (DNA amplification) or for expression. In some embodiments, antibodies can be generated through homologous recombination, as described, for example, in US 5,204,244, all of which discloses information regarding antibody generation and is specifically incorporated herein by reference. The DNA encoding the antibody can be readily isolated and sequenced using conventional methods, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, as described, for example, in US 5,534,615, all of which discloses information regarding protein expression and is specifically incorporated herein by reference.

[0112] Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia coli, Enterobacter spp., Erwinia spp., Klebsiella spp., Proteus spp., Salmonella, for example, Salmonella typhimurium, Serratia spp., for example, Serratia marcescens, and Shigella spp., as well as bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas spp. and Pseudomonas aeruginosa, and Streptomyces. An exemplary Escherichia coli cloning host is Escherichia coli 294 (ATCC 31,446), although other strains such as Escherichia coli B, Escherichia coli X1776 (ATCC 31,537), and Escherichia coli W3110 (ATCC 27,325) are suitable. These examples are illustrative and not limiting.

[0113] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for single- or multi-specific antibody encoding vectors. Saccharomyces cerevisiae or Bacillus baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and are used herein, such as *Schizosaccharomyces cerevisiae*; hosts of *Kluyveromyces martensii* such as, for example, *Kluyveromyces lactis*, *Kluyveromyces brittle* (ATCC 12, 424), *Kluyveromyces bulgaricus* (ATCC 16, 045), *Kluyveromyces wickham* (ATCC 24, 178), *K. waltii* (ATCC 56, 500), *Kluyveromyces drosophila* (ATCC 36, 906), *Kluyveromyces thermostrophicus*, and *Kluyveromyces martensii*; *Yarlostomium lipolytica* (EP 402, 226); *Pichia pastoris* (EP 183, 070); *Candida* genus; *Trichoderma reesei* (EP 244, 234); *Neurospora crassa*; *Schwania* genus such as *Schwania serratifolia*; and filamentous fungi such as, for example, *Neurospora*, *Penicillium*, *Cyclophorus*, and *Aspergillus* hosts such as *Aspergillus nidus* and *Aspergillus niger*.

[0114] Suitable host cells for expressing glycosylated monospecific or multispecific antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells from hosts such as the fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm, have been identified. Several viral strains for transfection are publicly available, such as the L-1 variant of the alfalfa looper NPV and the Bm-5 strain of the silkworm NPV, and such viruses can be used as the viruses described herein according to this disclosure, particularly for transfecting fall armyworm cells. Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0115] However, people are most interested in vertebrate cells, and propagating vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of available mammalian host cell lines include: monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed with SV40; human embryonic kidney line (for 293 or 293 cell subclones grown in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Support cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumors (MMT). 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0116] The above expression vectors are used to transform host cells for the production of monospecific or multispecific antibodies, and cultured in appropriately modified conventional nutrient media for the induction of promoters, screening of transformants, or amplification of genes encoding desired sequences.

[0117] Host cells used to produce monospecific or multispecific antibodies can be cultured in a variety of media. Commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle Medium (DMEM) are suitable for culturing host cells. Additionally, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or US Re. 30,985 can be used as media for host cells. Any of these culture media may be supplemented as needed 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™), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), 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 with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0118] When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If antibodies are produced intracellularly, as a first step, particulate debris, host cell fragments, or lysed fragments are removed, for example, by centrifugation or ultrafiltration.

[0119] Cell-prepared antibody compositions can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the 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 it can also be used to purify antibodies lacking an Fc region. Protein G can be used for all mouse isotypes and human γ3. The most common matrix for linking affinity ligands is agarose, but other matrices can also be used. Mechanically stable matrices such as controlled-pore glass or poly(divinyl styrene)benzene allow for faster flow rates and shorter processing times compared to what is achievable with agarose. When the antibody contains a CH3 domain, Bakerbond ABX™ resin can be used for purification. The antibodies and antibody fragments disclosed herein can also be synthesized using histidine tags and purified by metal affinity chromatography.

[0120] Depending on the antibody to be recovered, other techniques for protein purification can also be used, such as fractionation based on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica-based chromatography, heparin SEPHAROSE™-based chromatography, chromatography based on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0121] Following any initial purification step, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH between approximately 2.5 and 4.5, preferably at a low salt concentration (e.g., approximately 0–0.25 M salt).

[0122] This document also discloses cleavable multispecific single-chain antibodies in the tumor microenvironment. In some embodiments, once the multispecific single-chain antibody reaches the tumor, a tumor-targeting domain (such as a tumor antigen-binding domain) or other functional domain is cleaved at the linker to release other domains that produce therapeutic effects. The tumor microenvironment contains a variety of proteases capable of cleaving the linkers 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), ADAMs (de-integrin and metalloproteinases; e.g., ADAM10 and ADAM17), kallikrein-associated peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepsin (HPN), matriptase, legumain, or dipeptidyl peptidases (e.g., DDP4).

[0123] antibody composition

[0124] This document also discloses pharmaceutical compositions comprising monospecific or multispecific antibodies, wherein specificity includes CD28 and CD3E, and optionally one or more of OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR. Use of the antibodies described herein for preparing pharmaceutical compositions is also disclosed. Methods for treating various diseases and conditions using the disclosed antibodies and antibody-containing pharmaceutical compositions are also disclosed.

[0125] A pharmaceutical composition is a composition intended and suited for treating a disease in a human body. That is, it provides an overall beneficial effect and does not contain any amount of any ingredient or contaminant that causes toxicity or other undesirable effects unrelated to providing the beneficial effect. A pharmaceutical composition may 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 composition as a whole.

[0126] The monospecific or multispecific antibodies disclosed herein can also be formulated in liposomes. Liposomes containing antibodies can be prepared using methods known in the art, such as those described in US4,485,045, US4,544,545, and US5,013,556. Particularly useful liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to produce liposomes with the desired diameter. The Fab' fragment of the antibody can be conjugated to the liposome via a disulfide exchange reaction.

[0127] As used herein, "drug carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. 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.

[0128] The compositions disclosed herein can be administered by a variety of methods known in the art. As those skilled in the art will understand, the route and / or mode of administration may vary depending on the desired outcome. To administer the disclosed antibodies via certain routes of administration, it may be necessary to bind the antibody to a material, or co-administer the antibody with the material, to prevent its inactivation. For example, the antibody may be administered to a subject in a suitable carrier such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Drug carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0129] As used herein, the phrases “extragastric administration” and “parenteral administration” refer to modes of administration other than enteral and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0130] These compositions may also contain excipients such as preservatives, humectants, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization procedures described above and by including various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0131] In some embodiments, the antibody-containing pharmaceutical composition is a lyophilized cake. The lyophilized cake may further contain fillers, buffers, and / or salts or other excipients, as described herein. The lyophilized composition may be reconstituted by adding sterile water or an aqueous buffer for administration to a patient.

[0132] Regardless of the chosen route of administration, the disclosed antibodies and / or antibody-containing pharmaceutical compositions that can be used in a suitable hydrated form can be formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0133] The actual dose level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and administration mode. The selected dose level may depend on a variety of pharmacokinetic factors, including the activity of the particular composition used in this disclosure, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health status, and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0134] connector

[0135] In many implementations, the individual binding domains are not directly connected to each other, but instead have short amino acid sequences inserted between them, known as linkers. Examples of linkers are shown in Table 7. The length and sequence of the linker can have a substantial impact on the expression level and structure of the MVSCA, as well as the binding affinity of the linking domains. Length-adjustable linkers L2 and L4 (see Table 7) can be used to optimize the MVSCA based on these parameters. Linkers L1, L2, and L4 may be referred to as indestructible linker members, flexible linker members, or flexible indestructible linker members.

[0136] This can be avoided by inserting a relatively short and rigid joint between the two copies. In some embodiments, the short rigid joint has a sequence AAA (L3 in Table 10). Such a joint may be referred to as a short rigid joint member or an uncut short rigid joint member.

[0137] When anti-HSA domain-HSA complexes are used to generate prodrugs with respect to the binding activity of adjacent binding domains, a cleavable linker should be inserted between the two domains. L11*3 to L11*18 (see Table 10) are examples of cleavable linkers with varying lengths and cleavage sensitivities to different proteases, which can be used to optimize MVSCA expression levels and structure, binding affinity of the linker domains, and cleavage. Linkers L11*3 to L11*18 may be referred to as cleavable linker members, flexible linker members, or flexible cleavable linker members.

[0138] MVSCAs

[0139] The binding domains and connectors described herein can be combined to create multifunctional MVSCAs suitable for treating specific diseases. They can also be further combined with other binding domains. MVSCAs may also be referred to as containing components for realizing various functions associated with the binding domains of each component type and / or containing connector components for realizing their associated functions.

[0140] Publicly disclosed uses of antibodies

[0141] The disclosed antibodies are for medical use. The terms "treatment," "treating," etc., refer to the medical management of a patient with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or symptom, and also includes etiological treatment, i.e., treatment aimed at eliminating the cause of the related disease, pathological condition, or symptom. Additionally, this term includes palliative treatment, i.e., treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or symptom; preventative treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of a related disease, pathological condition, or symptom; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving a related disease, pathological condition, or symptom. Various implementation schemes may specifically include or exclude one or more of these treatment modalities.

[0142] The use of the antibodies disclosed in this paper in diagnosis and imaging was also considered.

[0143] Furthermore, the term "treating" or "treatment" broadly encompasses any type of therapeutic activity, including any activity that diagnoses, alleviates, or prevents a disease or aspect thereof in a person or other animal, or otherwise affects the structure or any function of the body of a person or other animal. Therapeutic activities include administering the medicines, dosage forms, and pharmaceutical compositions described herein to a patient, particularly according to the various treatment methods disclosed herein, whether administered by a medical professional, the patient themselves, or any other person. Therapeutic activities include orders, instructions, and recommendations from medical professionals such as physicians, physician assistants, nurse practitioners, etc., which are then carried out by any other person, including other medical professionals or the patient themselves. This includes, for example, instructing a patient to undergo or instructing a clinical laboratory to perform diagnostic procedures, such as those used for cancer diagnosis and staging, so that the patient can ultimately receive appropriate and beneficial treatment. In some implementations, the commands, instructions, and recommendations of treatment activities may also include encouraging, guiding, or compelling the use of a specific drug (or combination thereof) to treat a condition—and that drug is actually being used—through approving insurance coverage for the drug, refusing coverage for alternative drugs, including (or excluding) the drug in a drug list, or providing financial incentives for its use; as insurance companies or pharmacy benefit management companies may do. In some implementations, treatment activities may also include encouraging, inducing, or compelling the selection of a specific drug to treat a condition—and that drug is actually being used—through policies or standards of practice that may be established by hospitals, clinics, health maintenance organizations, medical institutions, or physician groups. All such commands, instructions, and recommendations should be considered as conditions of receiving treatment benefits on the basis of compliance with the instructions. In some cases, patients also receive financial benefits for complying with such commands, instructions, and recommendations. In some cases, healthcare professionals also receive financial benefits for complying with such commands, instructions, and recommendations.

[0144] cancer

[0145] The disclosed monospecific VHH and multivalent, multispecific single-chain antibodies specific to one or more of CD28 and / or CD3E, as well as OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR, are available for the treatment of cancer. Each antibody is designed to treat a specific class of cancer based on the antigen-binding specificity contained within it.

[0146] This disclosure provides methods for treating cancer, including administering an effective amount of the disclosed antibody or a pharmaceutical composition containing the antibody to a patient in need of such treatment.

[0147] Examples of cancers treatable through the disclosed methods include: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer; bladder cancer; bone cancer; brainstem glioma; brain tumors; breast cancer; bronchial adenoma / carcinoid; carcinoid tumor; islet cell carcinoma; cancer of unknown primary site; central nervous system lymphoma; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; endometrial cancer; ependymoma; ovarian epithelial cancer; esophageal cancer; Ewing's family tumors; extracranial germ cell tumors; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; germ cell tumors; gestational trophoblastic tumors; pilonidal tumors. Leukemia; head and neck cancer; hepatocellular carcinoma; Hodgkin's lymphoma; hypopharyngeal cancer; Kaposi's sarcoma; kidney cancer; laryngeal cancer; non-small cell lung cancer; small cell lung cancer; non-Hodgkin's lymphoma; Waldenström macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell carcinoma; squamous cell carcinoma of the neck; multiple endocrine adenoma syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome; nasopharyngeal carcinoma; neuroblastoma; oral cancer; oropharyngeal cancer; osteosarcoma; pancreatic cancer; parathyroid carcinoma; penile cancer; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; soft tissue sarcoma; Cezari syndrome; skin cancer; squamous cell carcinoma of the neck; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and nephroblastoma.

[0148] The effectiveness of cancer treatments is typically measured by "response." Techniques for monitoring this response can be similar to tests used to diagnose cancer, such as, but not limited to:

[0149] A lump or tumor involving certain lymph nodes can be felt and measured externally through physical examination.

[0150] Some internal cancerous tumors can appear on X-rays or CT scans and can be measured with a ruler.

[0151] Blood tests can be performed, including tests that measure organ function.

[0152] It can be used to test tumor markers for certain cancers.

[0153] Regardless of the type of test used—whether it's a blood test, cell count, or tumor marker test—it is repeated at specific intervals so that the results can be compared with earlier tests of the same type.

[0154] There are several definitions of response to cancer treatment:

[0155] Complete response – all cancers or tumors disappear; no evidence of disease. Tumor marker expression levels (if applicable) may be within the normal range.

[0156] Partial remission – the cancer has shrunk by a certain percentage, but the disease is still present. Tumor marker levels (if applicable) may have decreased (or increased, based on tumor markers as an indicator of reduced tumor burden), but evidence of disease remains.

[0157] Disease stable - the cancer neither grows nor shrinks; the disease load remains unchanged. Tumor markers (if applicable) show no significant changes.

[0158] Disease progression - Cancer has grown; the disease is now more extensive than before treatment. Tumor marker tests (if applicable) show elevated tumor markers.

[0159] Other measures of cancer treatment efficacy include overall survival (i.e., the time to death from any cause, measured from the time of diagnosis or from the start of the evaluated treatment), cancer-free survival (i.e., the length of time after a full response when cancer remains undetectable), and progression-free survival (i.e., the length of time after disease stabilization or partial remission when tumor growth is undetectable).

[0160] There are two standard methods for assessing treatment response to solid tumors (regarding tumor size (tumor burden)): the WHO and RECIST criteria. These methods measure the solid tumor to compare the current tumor size with past measurements or to compare changes with future measurements and adjust treatment accordingly. In the WHO method, the long and short axes of the solid tumor are measured, and then the product of these two measurements is calculated; if multiple solid tumors are present, all products are summed. In the RECIST method, only the long axis is measured. If multiple solid tumors are present, all long axis measurements are summed. However, for lymph nodes, the short axis is measured instead of the long axis.

[0161] Autoimmune diseases

[0162] The disclosed monospecific VHH and multivalent single-chain antibodies specific to one or more of CD28 and / or CD3E, as well as OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR, are intended for the treatment of autoimmune diseases. Each antibody is designed to treat a specific class of autoimmune diseases based on the antigen-binding specificity contained within it.

[0163] This disclosure provides a method for treating autoimmune diseases, comprising administering an effective amount of the antibody disclosed herein or a pharmaceutical composition comprising the antibody to a patient in need of such treatment.

[0164] Autoimmune diseases can be systemic or organ-specific. Non-limiting examples of autoimmune diseases that can be treated with 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 (e.g., osteoarthritis, rheumatoid arthritis, juvenile idiopathic arthritis, suppurative arthritis, spondyloarthritis, gout, pseudogout, or Still's disease), asthma, acquired immunodeficiency syndrome (AIDS), and other autoimmune diseases. Hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, celiac disease, Chagas disease, chronic obstructive pulmonary disease (COPD), type 1 diabetes mellitus (IDDM), endometriosis, gastrointestinal diseases such as irritable bowel disease or inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), glomerulonephritis, Goodpass syndrome, Graves' disease, Guillain-Barré syndrome (GBS), Hashimoto's thyroiditis, hidradenitis suppurativa, idiopathic thrombocytopenic purpura, interstitial nephritis, interstitial cystitis, lupus such as discoid cystitis. Symptomatic lupus erythematosus, drug-induced lupus erythematosus, lupus nephritis, neonatal lupus, subacute cutaneous lupus erythematosus or systemic lupus erythematosus, scleroderma, multiple sclerosis (MS), myasthenia gravis, myopathies such as dermatomyositis, inclusion body myositis or polymyositis, myositis, narcolepsy, neuromuscular rigidity, pemphigus vulgaris, pernicious anemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, pulmonary fibrosis, relapsing disseminated encephalomyelitis, rheumatic fever, schizophrenia, scleroderma, Sjögren's syndrome, skin diseases such as dermatitis, eczema, stasis dermatitis Suppurative hidradenitis, psoriasis, rosacea or scleroderma, tenosynovitis, uveitis, vasculitis such as Burger's disease, cerebral vasculitis, Chuck-Strauss arteritis, cryoglobulinemia, idiopathic cryoglobulinemia vasculitis, giant cell arteritis, golfer's vasculitis, Henoch-Schonlein purpura, hypersensitivity vasculitis, Kawasaki disease, microscopic polyarteritis / polyangiitis, nodular polyarteritis, polymyalgia rheumatica (PMR), rheumatoid vasculitis, Takayasu arteritis, Wegener's granulomatosis, or vitiligo.

[0165] This disclosure includes, in part, the relief of at least one symptom associated with an autoimmune disease. Actual symptoms associated with the autoimmune diseases disclosed herein are well known and can be determined by a person skilled in the art by considering factors including, but not limited to, the location of the autoimmune disease, the cause of the autoimmune disease, the severity of the autoimmune disease, the tissue or organ affected by the autoimmune disease, and inflammation associated with the autoimmune disease. Non-limiting examples of symptoms relieved by the methods of treating autoimmune diseases disclosed herein include inflammation, fatigue, pain, cognitive deficits, neurological deficits, dizziness, malaise, high fever and high body temperature, extreme sensitivity of the hands and feet to cold, weakness, aches and / or stiffness of muscles and joints, weight changes, digestive or gastrointestinal problems, respiratory problems, low or high blood pressure, irritability, anxiety or depression, infertility or decreased libido (hypoactive sexual desire disorder), blood sugar fluctuations, and, depending on the type of autoimmune disease, increased size or destruction of organs or tissues. Non-limiting examples of inflammatory symptoms alleviated by the methods for treating autoimmune diseases disclosed in this article include pain, loss of neurological function, loss of cognitive function, edema, congestion, erythema, bruising, tenderness, stiffness, swelling, fever, chills, respiratory congestion including the nose and bronchi, sinus congestion, breathing problems, fluid retention, blood clots, loss of appetite, increased heart rate, granuloma formation, fibrinous, purulent or non-viscous serous fluid, ulcer formation, or pain.

[0166] In some implementations, treatment with the antibodies disclosed herein alleviates at least one, at least two, at least three, at least four, or at least five symptoms of an autoimmune disease.

[0167] In other embodiments, the method may help treat or alleviate conditions, symptoms, or symptoms associated with autoimmune diseases. In some embodiments, these conditions or symptoms may include, but are not limited to, anemia, asthenia, cachexia, Cushing's syndrome, fatigue, gout, gingival disease, hematuria, hypercalcemia, hypothyroidism, internal bleeding, alopecia, mesothelioma, nausea, night sweats, neutropenia, paraneoplastic syndrome, pleurisy, polymyalgia rheumatica, rhabdomyolysis, stress, lymphadenopathy, thrombocytopenia, vitamin D deficiency, or weight loss. In other embodiments, administration of the antibodies disclosed herein prolongs the survival of the treated individual.

[0168] In some implementations of this method, mammals can experience improvement in autoimmune diseases due to treatment with the antibodies disclosed herein.

[0169] The following embodiments, sequence listing, and drawings are provided to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the described procedures without departing from the spirit of the invention. Example

[0170] Example 1: Anti-CD28 VHH antibody and multispecific antibody

[0171] Anti-CD28 VHH antibodies were isolated from immunized llamas.

[0172] Immunization. Two llamas were immunized at Pacific Immunology, Inc (Ramona, CA) according to their standard protocol. Recombinant human / cynomolgus monkey / rhesus monkey CD28 (extracellular domain Asn19-Pro152; SEQ ID NO:1) was mixed with either full Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization) (Difco, BD Biosciences). Each llama was administered six subcutaneous injections at two-week intervals at a dose of 50 μg. On day 45, serum was collected from the llamas immunized with recombinant CD28 to define the antibody titer against CD28 by ELISA. In the ELISA, 96-well Maxisorp™ plates (Nunc) were coated with 100 ng / well of recombinant CD28 protein. After blocking and adding diluted serum samples, the presence of anti-CD28 antibodies was confirmed using horseradish peroxidase (HRP)-conjugated goat anti-llama IgG antibody (Jackson ImmunoResearch).

[0173] SEQ ID NO: 1 - Extracellular domain of human CD28 (Asn19-Pro152) (accession number P10747)

[0174] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0175] Phage library construction and selection. Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples taken on day 45 using Ficoll-Paque™ Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from PBMCs using the RNeasy Midi Kit (Qiagen) 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 a self-made phage particle vector, allowing the generation of recombinant phage particles after infection with helper phages. These phage particles displayed VHH as a gene III fusion protein on their surface. Phages were prepared according to standard methods and stored at 4°C for future use after filtration sterilization. The phage library obtained from the llamas was used for screening. In screening, biotinylated recombinant CD28 was incubated with the phage library and subsequently captured on streptavidin Dynabeads™ (Invitrogen). After extensive washing, the bound phages were eluted with 1 mg / ml trypsin. The products obtained from screening were rescued in E. coli TG1 cells. Clones were selected and sequenced.

[0176] The cDNA encoding positive VHH was fused with a C-terminal His-tag and cloned into the expression vector SVT003, which was then transiently transfected into HEK293 cells. Positive VHH was purified by IMAC chromatography for in vitro functional assays.

[0177] Biolayer Interference (BLI) Kinetic Binding Assay. Biolayer interference (BLI), a label-free technique, was used to measure the binding kinetics of anti-CD28 VHH to human CD28. Affinity was measured using an anti-pentahistidine capture (HIS1K) biosensor tip. The assay was performed at 30°C in 1x PBS buffer (Gibco®, PBS pH 7.2). The sample was stirred at 1000 rpm. The concentrations of antigen and anti-CD28 VHH were optimized for affinity measurement prior to the experiment. The sensor was humidified for 15 min prior to analysis. The binding ability of anti-CD28 VHH to the HIS1K sensor tip was detected. Loading was performed for 300 sec, resulting in capture levels between 1.8 and 2 nm. Human CD28 antigen was prepared for binding assay by dilution to concentrations of 100, 150, 250, and 350 nM in 1x PBS. Binding was initiated and monitored for 200 sec, after which the tip was transferred to 1x PBS buffer to monitor dissociation. Sensor data was collected throughout the experiment and processed and analyzed using BLI data analysis software (Forte Bio).

[0178] Flow cytometry combined with affinity analysis on the Jurkat cell line involved incubating Jurkat cells in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3) with anti-CD28 VHH antibody at concentrations ranging from 100 nM to 0.00128 nM at room temperature for 20 min, followed by incubation on ice for 45 min. Cells were washed with FACS buffer and then incubated with goat anti-human IgG Fc-FITC conjugated antibody (ThermoFisher) according to the manufacturer's instructions, followed by incubation at 4°C for 30 min. Data were acquired using an Agilent NovoCyte system.

[0179] Anti-VHH antibodies were isolated from immunized llamas. Positive phage clones from immunized llama phage libraries were sequenced. The amino acid sequences are listed in Table 2 below. A cDNA sequence based on the following amino acid sequence was fused with human Fc and cloned into the SVT003 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate fully recombinant anti-VHH antibodies. The expressed anti-VHH antibodies were purified using a HiTrap protein A column (Cytiva).

[0180] Table 2. Llama anti-CD28 VHH sequences. The CDRs for each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3].

[0181]

[0182]

[0183] The humanization of D28-12E10 is based on the IGHV-74 germline sequence, and the humanization of D28-A1 is based on the IGHV3-23 germline sequence.

[0184] The VHH in Table 2 constitutes a means for binding CD28.

[0185] The biolayer interference (BLI) binding analysis of anti-CD28 VHH molecules is presented in Table 3.

[0186] Table 3. BLT binding kinetics analysis of anti-CD28 VHH(KD)

[0187]

[0188] The multispecific antibodies against CD28 VHH included in Table 2 are presented in Figure 1 And in Table 4. Table 4 lists multispecific antibodies, including variants with different linkers.

[0189] Table 4. Multispecific antibodies with at least one specific target for CD28 (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3]).

[0190]

[0191]

[0192] Following the flow cytometry method described above, the binding affinity of the multispecific antibodies SM2275-679 and SM2275-700 was determined as follows: Figure 2 and 3 and EC in Table 5 50 .

[0193] Cell-based functional assays showed that SM2275-649 and SM2275-700 are fully functional, multispecific VHH antibodies (Table 5). Sm2275 (when the suffix is ​​omitted, it refers to SM2275-649) cross-reacts with the same KD at the cynomolgus monkey target, but does not bind to the rodent target.

[0194] Table 5. BLI binding affinity analysis of SM2275-700 and SM2275-649

[0195]

[0196] Notably, SM2275 exhibited a significantly enhanced affinity for CHO cells co-expressing EGFR and PD-L1 compared to mono-expressing cells. This resulted in PD-L1 blockade with enhanced specific affinity on the surface of EGFR+PD-L1+ double-positive cancer cells, allowing for local reactivation of tumor-infiltrating T cells without systemic immunotoxicity. In the presence of SM2275, EGFR and PD-L1 double-positive cancer cells induced significantly enhanced CD28 activation compared to cells expressing a single antigen.

[0197] As determined by flow cytometry, the monovalent EGFR and PD-L1 modules bind to cell surface targets with moderate affinity. Compared to normal tissues expressing a single target, SM2275 selectively binds to EGFR+PD-L1+ double-positive cancer cells with enhanced affinity. This enhanced affinity antibody binding allows SM2275 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells. Figure 4 Compared to CHO-hPD-L1-OKT3, the dual-target binding SM2272 showed a significantly enhanced ability to block the interaction between PD-1 and PD-L1 on CHO-Dual-OKT3. Figure 5Tumor site-specific blockade of PD-L1 / PD-1 synergistically with CD28 agonist activity to allow SM2275 to locally maximize T cell activation.

[0198] Compared to SM2275 on the cancer cell line A431, the EGFR-binding mutant SM2275 (SM2275A) showed reduced PD1 / PDL1 blockade.

[0199] SM2275 exhibits dual-target-dependent CD28 activation in synergy with PD1 / PD-L1 signaling. Figure 6 The Jurkat NFAT-PD-1 reporter cell line was used in this assay. The CHO-hEGFR-hPD-L1-OKT3 cell line was used as the target cell line. The CHO-OKT3 cell line was used as the target-free control. SM2275 showed no activity in the target-free control, indicating target-dependent CD28 activation. Compared with SM2275, SM2275(CD28-), SM2275 with a mutated and inactivated CD28 module, showed a reduced ability to inhibit PD-L1 signaling, indicating that CD28 activation significantly enhances the inhibition of PD-L1 signaling.

[0200] Compared to TGN1412 (theralizumab), SM2275 has better in vitro ( Figure 7A -D) or in the body ( Figure 8A Neither SM2275 nor TGN1412 (a CD28 superagonist) induced the release of cytokines from human PBMCs. hPD-L1 / hCD28 mice received a single dose of PBS, SM2275 (5 mg / kg, 15 mg / kg), or TGN1412 (2.5 mg / kg). Blood was collected 4 hours after administration, and serum cytokines were measured. SM2275 was well tolerated and did not induce cytokine release 4 hours after treatment. Unlike TGN1412 (a CD28 superagonist), SM2275 did not induce cytokine release in human PBMCs. Figure 7A -D) and in hPD L1 / hCD28 humanized mice ( Figure 8A -D; neither a single IV dose nor 4 hr after the administration induced cytokine release (CRS). In contrast, TGN1412 induced significant systemic cytokine release, consistent with its in vivo hyperagonist activity.

[0201] SM2275 significantly inhibited the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted, and when the tumor size reached ~80 mm³, animals were randomly assigned to groups and treated with either the drug or SM2275 every 2 days for 10 days. Body weight and tumor volume were measured on days 0, 2, 4, 6, 8, 10, 12, 14, 18, and 22 after grouping (N=6). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized CD28 mice in a dose-dependent manner. Figure 9 ).

[0202] SM2275 significantly inhibited the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 / hPD-L1 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted, and animals were randomly assigned to groups when the tumor size reached ~90 mm³. Treatment with either the drug or SM2275 was administered every 2 days for 12 days, with body weight and tumor volume measured on days 0, 2, 4, 6, 8, 10, 12, 15, and 18 (N=5). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized hCD28 / hPD-L1 mice. Figure 10 ).

[0203] SM2275 significantly inhibited the growth of NCI-H292 tumors in NCG mice reconstructed with human PBMCs. NCG mice were subcutaneously co-implanted with NCI-H292 tumor cells and human PBMCs (Shanghai Aoneng Biotechnology Co., Ltd., catalog number FPB004F-C, donor number T0035). On the day of co-implantation, mice were randomly assigned to groups and treated with either the agent or SM2275 every 3 days for 12 days. Body weight and tumor volume were measured on days 0, 4, 6, 10, 12, 17, 19, and 21 (N=6). SM2275 effectively inhibited the growth of NCI-H292 tumors in the NCG mouse model in a dose-dependent manner. Figure 11 )

[0204] The pharmacokinetics of SM2275 were evaluated in cynomolgus monkeys after multiple IV doses. Figure 12Eight healthy adult cynomolgus monkeys were randomly assigned to four groups based on body weight: a solvent control group and low, medium, and high dose groups of SM2275 (administered at doses of 5, 15, and 50 mg / kg, respectively). Each group consisted of two monkeys, with an equal number of males and females. The SM2275 administration groups received the corresponding concentration of SM2275 via intravenous infusion at a volume of 10 mL / kg, while the solvent control group received the solvent in the same manner. No dose-limiting toxicities were observed at doses up to 50 mg / kg. SM2275 exhibited favorable pharmacokinetic properties with a terminal half-life of >60 hours, consistent with the albumin-mediated FcRn cycle in cynomolgus monkeys.

[0205] Example 2 Anti-CD3E VHH antibody

[0206] Anti-CD3E VHH antibodies were isolated from immunized llamas.

[0207] Two rheas were immunized at Pacific Immunology, Inc (Ramona, CA) according to their standard protocol. As shown in Example 1, recombinant human / cynomolgus monkey / rhesus monkey CD3E (extracellular domain (23-126) accession number P07766, SEQ ID NO: 27) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization).

[0208] On day 45, serum was collected from llamas immunized with recombinant CD3E-llama CD3E protein to determine antibody titers against recombinant CD3E by ELISA. In the ELISA, 96-well Maxisorp™ plates were coated with 100 ng / well of CD3E. After blocking and adding diluted serum samples, the presence of anti-CD3E VHH antibodies was demonstrated using HRP-conjugated goat anti-llama IgG antibodies.

[0209] SEQ ID NO: 29 - Extracellular domain (23-126) of human CD3E extracellular domain (accession number P07766)

[0210] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0211] For llamas immunized with recombinant CD3E protein, peripheral blood mononuclear cells (PBMCs) were prepared from blood samples taken on day 45 using Ficoll-Paque™ Plus according to the manufacturer's instructions. Total RNA was extracted from PBMCs 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 a self-made phage vector, allowing the generation of recombinant phage particles after infection with helper phages. These phage particles displayed VHH as a gene III fusion protein on their surface. Phages were prepared according to standard methods and stored at 4°C for future use after filtration sterilization. Phage libraries obtained from llamas were used for screening. In screening, biotinylated CD3E-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 products obtained from screening were rescued in *E. coli* TG1 cells. Colonies were selected and sequenced at Genwiz, Inc.

[0212] A cDNA encoding a positive VHH with a C-terminal His tag was synthesized, cloned into the SVT003 expression vector, and transiently transfected into HEK293 cells. The positive VHH was purified by IMAC chromatography for in vitro functional assays.

[0213] Positive phage clones from an immunized llama phage library were sequenced. The amino acid sequences are listed in Table 6 below. A cDNA sequence based on the following amino acid sequence was fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate a fully recombinant anti-CD3E VHH antibody. The expressed anti-CD3E VHH was purified using a HiTrap protein A column.

[0214] Table 6. VHH sequences of llamas against CD3E (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0215]

[0216]

[0217] *These CD3E-specific VHHs cross-react with cynomolgus monkey CD3E

[0218] The VHH in Table 5 constitutes the means for binding CD3E.

[0219] ELISA. 96-well plates were coated with 100 µL of CD3E-his (Acro) per well, dissolved in coating buffer at 1 µg / mL, and incubated overnight at 4°C. The coated plates were washed three times with PBST buffer and blocked with 200 µL / well of 2% BSA at 25°C for 1 hour. Various anti-CD3E VHH clones were prepared into a series of dilutions and blotted onto 96-well plates at room temperature for 1 hour. After washing, the plates were incubated with HRP-conjugated anti-VHH secondary antibody (Jackson ImmunoResearch) at 25°C for 1 hour. The plates were washed three times with PBST buffer, and then 100 μL of substrate TMB was added to each well, followed by 50 μL of stop solution. Immediately, the plates were analyzed using an ELISA reader at OD500. 450 Read the next screen. The results are presented on... Figure 13A -B in.

[0220] Biolayer Interferometry (BLI), a label-free technique, was used to measure the binding kinetics of anti-CD3E VHH with human CD3E (R&D systems) or cynomolgus monkey CD3E (Cyno-CD3E). Affinity measurements were performed using an anti-pentahistidine capture (HIS1K) biosensor. Assays were performed at 30°C in Q-buffered saline (PBS containing 0.2% BSA and 0.02% Tween 80) with stirring at 1000 rpm. The concentrations of antigen and anti-CD3E VHH were optimized for affinity measurements prior to the experiments. The sensor was placed in Q-buffered saline for 15 min prior to analysis. Antigen CD3E-hFc was loaded onto the HIS1K sensor for 300 sec, resulting in capture levels between 1.8 and 2 nM. Anti-CD3E VHH was prepared for binding in 1x PBS. Binding was initiated and monitored for 200 sec, after which the sensor was transferred to Q-buffered saline to monitor dissociation. Sensor data were collected throughout the experiment and processed and analyzed using BLI data analysis software 7 (Forte Bio). The results are presented in Tables 7-9.

[0221] Table 7. Biolayer interference (BLI) binding affinity KD analysis of anti-CD3E VHH molecules (human CD3E)

[0222]

[0223] Table 8. Biolayer interference (BLI) binding affinity KD analysis of anti-CD3E VHH molecules (human CD3E)

[0224]

[0225] Table 9. Biolayer interference (BLI) binding affinity KD analysis of anti-CD3E VHH molecules (cynomolgus monkey CD3E, Cyno-CD3E)

[0226]

[0227] Flow cytometry combined with affinity analysis on the Jurkat cell line: Jurkat cells were seeded in 96-well plates and incubated with serially diluted anti-CD3E VHH molecules at room temperature for 30 min. Cells were washed with FACS buffer and stained with Alexa Fluor® 647 AffiniPure Fab fragment goat anti-alpaca VHH antibody (Jackson ImmunoResearch) for 30 min at room temperature. The 96-well plates were washed with FACS buffer and analyzed in an Agilent NovoCyte system. Results are presented in... Figure 14A -C

[0228] Example 3: Multispecific single-chain antibody

[0229] To construct multispecific single-chain antibodies, anti-CD28 and / or anti-CD3E VHH, as well as one or more anti-OX40, anti-CD40, anti-4-1BB, anti-EGFR, anti-IL22, anti-HSA, anti-CD47, anti-CD16, anti-PD-L1, anti-CD33 and / or anti-LAG3 VHH, were fused together via linkers of different conformations using recombinant DNA technology.

[0230] Exemplary non-cleavable and cleavable linker sequences are presented in Table 10. These constitute linker means or means for connecting protein domains. These means may be further characterized as cleavable or non-cleavable.

[0231] Table 10. Non-cuttable and cuttable joint sequences

[0232]

[0233] This paper also discloses mutations at certain positions of proline (P) to serine (S) to reduce the formation of dimers and aggregates.

[0234] Exemplary VHH domains specific to OX40, CD40, 4-1BB, EGFR, IL22, HSA, CD47, CD16, PD-L1, CD33, and LAG3 are described in Tables 11-21.

[0235] Table 11. Llama anti-CD47 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0236]

[0237]

[0238]

[0239] Table 12. Llama anti-PD-L1 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0240]

[0241] Table 13. Llama anti-HSA VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0242]

[0243]

[0244] Table 14. Llama anti-CD33 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0245]

[0246]

[0247]

[0248] Table 15 Llama anti-LAG3 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0249]

[0250] Table 16 Anti-CD16 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0251]

[0252] Table 17 Llama anti-OX40 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0253]

[0254] Table 18. Llama anti-CD40 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0255]

[0256]

[0257] * Blocking CD40:CD40L interaction

[0258] Table 19. Llama anti-4-1BB VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0259]

[0260] Table 20. Llama anti-EGFR VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0261]

[0262]

[0263] Table 21. Llama anti-IL22 VHH sequences (CDRs of each sequence are indicated by underscore [CDR1], bold [CDR2], or double underscore [CDR3])

[0264]

[0265] Unless otherwise stated, all figures describing the amount and characteristics of the expressed components, such as molecular weight, reaction conditions, etc., as used in the specification and claims should be understood to be modified in all cases by the term "about," which, as used herein, means within 10% to 15%, preferably within 5% to 10%. Therefore, unless otherwise stated, the numerical parameters set forth in the specification and appended claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained according to the invention. At least, and not intended to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying conventional rounding techniques. Although the numerical ranges and parameters that illustrate the broad scope of the invention are approximate, the values ​​set forth in the specific embodiments are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error, which is necessarily caused by the standard deviation found in their respective test measurements.

[0266] The terms “a,” “an,” “the,” and similar designations used in the context of describing the invention (particularly in the context of the appended claims) should be interpreted to cover both the singular and plural forms, unless otherwise indicated herein or clearly contradicted by the context. The description of ranges of values ​​herein is intended only as a shorthand method for individually referring to each individual value falling within that range. Each individual value is incorporated into the specification as if it were individually described herein, unless otherwise stated herein or clearly contradicted by the context. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0267] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements found herein. It is contemplated that one or more members of a group may be included in or removed from the group for convenience and / or patentability reasons. When any such inclusion or removal occurs, the specification is deemed to contain the modified group, thereby satisfying the written description of all Markush groups as used in the appended claims.

[0268] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention covers any combination of the foregoing elements in all possible variations.

[0269] The specific embodiments disclosed herein may be further limited in the claims using the language “consisting of…” or “substantially consisting of…”. When used in the claims, whether as submitted or added according to amendments, the transitional term “consisting of…” excludes any element, step, or ingredient not specified in the claims. The transitional term “substantially consisting of…” limits the scope of the claims to the specified materials or steps and those that do not substantially affect the essential and novel features. Embodiments of the claimed invention are inherently or explicitly described and implemented herein.

[0270] Furthermore, numerous references have been made to patents and print publications throughout this specification. Each of the above-cited references and print publications is incorporated herein by reference in its entirety.

[0271] 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 made are also within the scope of the invention. Therefore, alternative configurations of the invention can be utilized in accordance with the teachings herein by way of example rather than limitation. Thus, the invention is not limited to what is precisely shown and described.

Claims

1. A variable heavy chain (VHH) domain having antigen-binding specificity to CD28, wherein the VHH domain comprises a complementarity-determining region (CDR) of any one of SEQ ID NO: 2-22.

2. The VHH domain of claim 1, wherein the VHH comprises an amino acid sequence of any one of SEQ ID NO: 2-22.

3. A VHH domain having antigen-binding specificity to CD3E, wherein the VHH domain comprises a CDR of any one of SEQ ID NO: 30-44.

4. The VHH domain of claim 3, wherein the VHH comprises an amino acid sequence of any one of SEQ ID NO: 30-44.

5. An antibody comprising the CDR or VHH domain as described in claims 1-4.

6. A multispecific antibody comprising a first 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 to CD28 or CD3E, and wherein the second binding specificity is specific to OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR; wherein: (a) The binding domain with CD28 binding specificity includes the CDR in any one of SEQ ID NO: 2-22; (b) The binding domain with CD3E binding specificity includes the CDR in any one of SEQ ID NO: 30-44; (c) The binding domain having OX40 binding specificity includes the CDR in either SEQ ID NO: 174 or 175; (d) The binding domain with CD40 binding specificity includes any one of the CDRs in SEQ ID NO: 176-190; (e) The binding domain having 4-1BB binding specificity includes the CDR in any one of SEQ ID NO: 191-203; (f) The binding domain with EGFR binding specificity includes the CDR in any one of SEQ ID NO: 204-232; (g) The binding domain with HSA binding specificity includes the CDR in any one of SEQ ID NO: 107-122; (h) The binding domain having IL-22 binding specificity includes the CDR in any one of SEQ ID NO: 233-236; (i) The binding domain with PD-L1 binding specificity includes any of the CDRs in SEQ ID NO: 98-106; (j) The binding domain with CD16 binding specificity includes the CDR in any one of SEQ ID NO: 168-173; (k) The binding domain having CD47 binding specificity includes the CDR in any one of SEQ ID NO: 67-97; (l) The binding domain with CD33 binding specificity includes any one of the CDRs in SEQ ID NO: 124-153; (m) The binding domain with LAG3 binding specificity includes the CDR in any one of SEQ ID NO: 154-173.

7. The multispecific antibody of claim 6, further comprising one to five additional antibody-binding domains, wherein each additional antibody-binding domain is individually specific to CD28, CD3E, OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

8. The multispecific antibody of claim 7, further comprising one to four additional antibody-binding domains, wherein each additional antibody-binding domain is specific to CD28, CD3E, OX-40, CD40, 4-1BB, HSA, IL-22, PD-L1, CD16, CD47, CD33, LAG3, and EGFR.

9. The multispecific antibody according to any one of claims 6-8, wherein the multispecific antibody is a multispecific single-chain antibody (MVSCA).

10. The multispecific antibody of any one of claims 6-9, wherein the adapter comprises an amino acid sequence of any one of SEQ ID NO: 50-72.

11. The multispecific antibody of claim 10, wherein the adapter is inserted between one or more pairs of different antibody-binding domains.

12. The multispecific antibody according to any one of claims 6-11, comprising at least one pair of antibody-binding domains having the same specificity.

13. The multispecific antibody of claim 12, wherein the at least one pair of antibody-binding domains having the same specificity are adjacent to each other.

14. The multispecific antibody of claim 13, wherein a linker having an amino acid sequence of linker L3 (SEQ ID NO: 46) is inserted between the antibody-binding domains of the same specificity.

15. The multispecific antibody according to any one of claims 6-14, wherein the antibody-binding domain is a VHH domain.

16. A pharmaceutical composition comprising the VHH domain of any one of claims 1-4, the antibody of claim 5, or the multispecific antibody of any one of claims 6-15.

17. A method of treating cancer, the method comprising administering to a subject in need the VHH of claims 1-4, the antibody of claim 5, the multispecific antibody of any one of claims 6-15, or the pharmaceutical composition of claim 16.

18. The method of claim 17, wherein the multispecific antibody comprises the amino acid sequence of SEQ ID NO:

23.

19. Use of the VHH of claims 1-4, the antibody of claim 5, the multispecific antibody of any one of claims 6-15, or the pharmaceutical composition of claim 16 for the treatment of cancer in a subject in need of such treatment.

20. The use according to claim 19, wherein the multispecific antibody comprises the amino acid sequence of SEQ ID NO:

23.

21. A method for treating an autoimmune disease, the method comprising administering to a subject in need the VHH of claims 1-4, the antibody of claim 5, the multispecific antibody of any one of claims 6-15, or the pharmaceutical composition of claim 16.

22. Use of the VHH of claims 1-4, the antibody of claim 5, the multispecific antibody of any one of claims 6-15, or the pharmaceutical composition of claim 16 for the treatment of an autoimmune disease in a subject in need of such treatment.

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