Monospecific and multispecific antibodies

Abstract

Disclosed herein are monospecific and multispecific single chain antibodies with specificity for one or more of CD47, PD-L1, HSA, CD33, LAG3, and CD16.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority pursuant to U.S. Provisional Patent Application No. 62 / 907,275, filed on 27 September 2019, and U.S. Provisional Patent Application No. 62 / 989,327, filed on 13 March 2020, both of which are incorporated herein by reference in their entirety. [Overview of the project] [Means for solving the problem]

[0002] Disclosed herein are single-specific heavy-chain-only antibodies (HCAb) having specificity for CD47, human serum albumin (HSA), PD-L1, CD33, CD16, and LAG3, as well as multivalent single-chain antibodies incorporating two or more HCAb variable domains having specificity for one or more of these antigens.

[0003] Some embodiments are monodomain antibodies comprising, exclusively or primarily, the VHH domain of a camel antibody. These embodiments are monospecific and monovalent.

[0004] Some embodiments include an HCAb or a VHH domain fused to one or more constant domains of a conventional antibody, such as the Fc region of a human IgG antibody. These embodiments are monospecific but typically bivalent. Other titers are possible, for example, by the selection of constant domains. The Fc regions of IgA and IgM can result in high titers.

[0005] Some embodiments include two VHH domains that have specificity for the same antigen bound to a single amino acid chain (multivalent single-chain antibodies). These embodiments are also monospecific and bivalent. A higher binding titer can be obtained by attaching additional VHH domains.

[0006] Some embodiments include two (or more) VHH domains, each VHH domain having specificity for a different antigen bound to a single amino acid chain (multivalent, multispecific single-chain antibodies). These embodiments are both multivalent and polyspecific. In further embodiments including three or more VHH domains, two or more VHH domains may have specificity for the same antigen, or one or more other VHH domains may have specificity for a different antigen. Such structures have a higher-order binding titer than specificity.

[0007] Each of the monospecific embodiments has specificity for CD47, HSA, PD-L1, CD33, CD16, or LAG3. Each of the multispecific embodiments has specificity for one or more of CD47, HSA, PD-L1, CD33, CD16, and LAG3, but may also have specificity for one or more other antigens.

[0008] Some embodiments exhibit specificity for HSA and one or more other antigens. In view of these embodiments, the HSA-specific domain results in an extension of the half-life in the body, while other domains provide therapeutic effects. In another view of these embodiments, the HSA-specific domain may partially or completely inhibit the binding activity of adjacent domains. The HSA-specific domain can be bound by a cleavable linker that is cleaved by a protease present at the site of action, such as a tumor, so that cleavage mitigates the inhibition of adjacent domains. Some embodiments exhibit multispecificity, while others exhibit triple specificity.

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

[0010] Antigen-binding domains disclosed herein that have specificity for a particular antigen may be referred to as means for binding to an antigen. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the flow cytometry analysis of the binding affinity of anti-CD47 HCAb A09-10 and B6H12 to CD47-overexpressing cell lines. [Figure 2] This figure shows the competitive ELISA binding analysis of multispecific antibodies 1511 (SEQ ID NO: 156) and 3321 (SEQ ID NO: 157) that have properties against CD47. [Figure 3] This figure shows the competitive binding analysis of CD47-binding multispecific molecules 1511 and 3321 using flow cytometry in Jurkat cell lines. [Figure 4A] This figure shows a human erythrocyte (RBC) agglutination assay using CD47-binding multispecific molecules 1511 and 3321. Hu5F9 was used as a control. [Figure 4B] This figure shows the binding of 1511 and 3321 to HL-60 and human red blood cells. [Figure 5] This figure shows the antitumor activity of CD47-binding multispecific molecules 1511 and 3321 in Raja-Luc xenograft mice. [Figure 6] This report shows flow cytometry-linked analysis of anti-PD-L1 HCAbs, PL14, and PL16, in PD-L1 overexpressing CHO cells. Atezolizumab was used as a control. [Figure 7] This is a cell-based functional assay of a multispecific molecule 1511 with binding specificity to PD-L1, and atezolizumab as a control. [Figure 8A] This figure shows the inhibition of MC38-hPD-L1 tumor proliferation in B-hPD-L1 mice by the PD-L1-binding multispecific molecule 1518 (SEQ ID NO: 135). [Figure 8B] This figure shows the inhibition of MC38-hPD-L1 tumor proliferation in B-hPD-L1 mice by the PD-L1-binding multispecific molecule 1518 (SEQ ID NO: 135). [Figure 9] This figure shows the Octet® binding analysis of anti-HSA HVV antibodies. [Figure 10A] Figure showing the Octet® affinity analysis of an anti-CD33 HVV antibody. [Figure 10B] Figure showing the Octet® affinity analysis of an anti-CD33 HVV antibody. [Figure 11A] Figure showing the Octet® binding analysis of an anti-CD16A HVV molecule CD16F1. [Figure 11B] Figure showing the Octet® binding analysis of an anti-CD16A HVV molecule CD16E11. [Figure 12] Figure showing the cell-based functional assay of multispecific molecules 1511 and 3321 in a Jurkat NFAT CD16 reporter assay (ADCC) using IgG1 B6H12 and IgG4 B6H12 as a control. [Figure 13A] Figure showing the trispecific molecular composition of a molecule having an HSA binding domain and a CD47 binding domain. [Figure 13B] Figure showing the trispecific molecular composition of a molecule having an HSA binding domain and a LAG3 binding domain. [Figure 13C] Figure showing the trispecific molecular composition of a molecule having an HSA binding domain and a CD16 binding domain. [Figure 14A] Figure showing the trispecific molecular composition of pro-CD47 activated by tumor protease. [Figure 14B] Figure showing the SDS-PAGE analysis of pro-CD47 activated by tumor protease. [Figure 15] Figure showing the real-time kinetics Octet® binding analysis of PD-L1 / pro-CD47 versus PD-L1 / activated-CD47. [Figure 16] Figure showing the composition of a multispecific molecule. Mom = monovalent binding domain, BiV = bivalent binding domain containing two identical monovalent binding domains. [Figure 17]This figure shows the composition of a tetrabular (quadrispecific, Figures 14A-D). The two VHH3s can be the same VHH or different VHHs that bind to different epitopes of the same antigen. The two VHH4s can be the same VHH or different VHHs that bind to different epitopes of the same antigen. In some embodiments, VHH2 is always the HSA-binding domain. In some embodiments, VHH1 is a payload such as a CD16A agonist VHH. Figures 17C and 17D show the tetrabular in the prodrug configuration. [Figure 18] This figure shows the flow cytometry binding analysis of the CD47-binding multispecific molecules 1518-HS5 (SEQ ID NO: 173) and 1518-HS5-GS15 (SEQ ID NO: 184) in the HL60 cell line. [Figure 19] This figure shows the Octet® binding analysis of the multispecific molecule 1511. [Figure 20] This figure shows the Octet® binding analysis of the multispecific molecule 3321. [Figure 21] This figure shows the amino acid sequence alignment of the anti-CD47 VHH sequence. [Figure 22] This figure shows the amino acid sequence alignment of the anti-PD-L1 VHH sequence. [Figure 23] This figure shows the amino acid sequence alignment of the anti-HSA VHH sequence. [Figure 24] This figure shows the amino acid sequence alignment of the anti-CD33 VHH sequence. [Figure 25] This figure shows the amino acid sequence alignment of the anti-LAG3 VHH sequence. [Figure 26] This figure shows the amino acid sequence alignment of the anti-CD16A VHH sequence. [Modes for carrying out the invention]

[0012] Disclosed herein are monospecific heavy-chain-only antibodies (HCAb) having specificity for CD47, human serum albumin (HSA), PD-L1, CD33, CD16, and LAG3, or their variable domains (referred to as VHH single-domain antibodies [sdAb]), and multivalent single-chain antibodies (MVSCA) incorporating two or more variable domains of HCAb having specificity for one or more of these antigens.

[0013] In some embodiments, the MVSCA comprises two or more HCAb variable domains, each having specificity for the same antigen. That is, the MVSCA is polyvalent but monospecific with respect to the antigen. In some embodiments, the MVSCA comprises two or more repeats of the same HCAb variable domain, or multiple HCAb variable domains, each having specificity for the same epitope. That is, they are polyvalent but monospecific with respect to the epitope. Such an MVSCA binds to only a single site on the antigen monomer but can crosslink to multiple copies of the monomer. In other embodiments, the MVSCA comprises two or more HCAb variable domains, each having specificity for different epitopes of the same antigen. That is, they are polyvalent but multispecific with respect to the epitope. Such an MVSCA binds to multiple sites on the antigen monomer or crosslinks to multiple copies of the antigen monomer.

[0014] In some embodiments, the MVSCA comprises two or more HCAb variable domains having specificity for different antigens, and these domains are polyvalent and highly specific with respect to the antigen. In further embodiments, the MVSCA comprises multiple HCAb variable domains, in any combination of cases where the further variable domains are identical to the first HCAb variable domain, where the further HCAb variable domains are different from the first variable domain but have specificity for different epitopes on the same antigen, or where the further HCAb variable domains are different from the first HCAb variable domain but have specificity for different antibodies.

[0015] MVSCAs containing two or more HCAb variable domains may further include an HCAb constant domain. For example, the C-terminal HCAb variable domain may maintain binding to the original HCAb constant domain. Alternatively, the C-terminal HCAb variable domain may bind to the constant domain or Fc region of a further conventional antibody, such as a human antibody like a human IgG antibody. In some embodiments, the constant domain or entire Fc region may confer specific functionality, as is well known to those skilled in the art. In another embodiment, an MVSCA containing two or more HCAb variable domains further includes an HCAb constant domain, which is located between or at the N-terminus of the HCAb variable domains, instead of being located at the C-terminus of the HCAb variable domains, or in addition to them.

[0016] (antigen) CD47 (Cluster of Differentiation 47), also known as integrin-related protein (IAP), is a 50 kDa transmembrane protein encoded by the CD47 gene in humans. CD47 belongs to the immunoglobulin superfamily, partners with membrane integrins, and binds to ligands for thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). Thrombospondin-1 is a secreted glycoprotein with a role in angiogenesis and neovascularization, where the TSP1-CD47 interaction inhibits nitric oxide signaling in vascular cells at multiple stages. TSP-1 binding to CD47 affects several fundamental cellular functions, including cell migration and adhesion, cell proliferation, and apoptosis, playing a role in regulating angiogenesis and inflammation. Signal regulatory protein alpha is a transmembrane receptor present in myeloid cells. The CD47 / SIRPα interaction results in bidirectional signaling, leading to a variety of intercellular responses, including inhibition of phagocytosis, stimulation of cell fusion, and activation of T cells. CD47 acts as a "don't eat me" signal to macrophages in the immune system, which may make it a potential therapeutic target for some cancers.

[0017] Programmed cell death 1 (PD-1), also known as CD279, is a type I membrane protein encoded by the PDCD1 gene in humans. It has two ligands, PD-L1 and PD-L2. PD-L1, also known as CD274 or B7 homolog 1 (B7-H1), is a 40 kDa type I transmembrane protein encoded by the CD274 gene in humans. PD-L1 is expressed on the surface of active T cells and antigen-presenting cells (APCs) such as dendritic cells and macrophages. PD-L1 is also overexpressed in several tumors, including breast cancer, lung cancer, bladder cancer, head and neck cancer, and other cancers. When PD-L1 or PD-L2 binds to PD-1, an inhibitory signal is sent to T cells, reducing cytokine production and suppressing T cell proliferation.

[0018] The PD-1 pathway is a key immunosuppressive mediator of T cell exhaustion. PD-1 functions to limit the activity of peripherally activated T cells in the inflammatory response to infection, thereby limiting autoimmunity. Blocking this pathway can lead to T cell activation, expansion, and enhancement of effector function. Thus, PD-1 negatively regulates the T cell response. PD-1 has been identified as a marker of T cell exhaustion in chronic disease states, and blocking the PD-1:PD-L1 interaction has been shown to partially restore T cell function (Sakuishi et al., JEM, 207:2187-2194, 2010). Methods and compositions for the treatment of persistent infections and cancer by inhibiting the PD-1 pathway are disclosed in International Publication No. 2006 / 133396. Human monoclonal antibodies against PD-L1 are described in International Publication No. 2007 / 005874, U.S. Patent Application Publication No. 2011 / 209230, U.S. Patent No. 8,217,149, and International Publication No. 2014 / 055897.

[0019] Human serum albumin (HSA) is the most abundant protein in human serum, making up about half of it. Albumin has functions such as transporting hormones, fatty acids, and other compounds, buffering pH, and maintaining colloid osmotic pressure. Albumin is synthesized in the liver as preproalbumin, which has an N-terminal protein that is removed before the initial protein is released from the rough endoplasmic reticulum. The product, proalbumin, is then cleaved in the Golgi vesicles to produce secretory albumin. The serum half-life is approximately 20 days. Albumin's long serum half-life is achieved by its size, which prevents removal via the kidneys, and by its interaction with the fetal Fc receptor (FcRn). Fusion to anti-albumin sdAb (single-domain antibodies) has been used to increase the half-life of antitumor single-chain antibodies from 1-2 hours to approximately 10 days.

[0020] CD33, or Siglec-3 (sialic acid-binding IgG-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), is a transmembrane receptor expressed in myeloid cells. While generally considered myeloid-specific, it can also be found in some lymphoid cells. Because it binds to sialic acid, it is a member of the SIGLEC family of lectins. The extracellular portion of this receptor contains two immunoglobulin domains (one IgV domain and the other IgC2 domain), thus placing CD33 within the immunoglobulin superfamily. The intracellular portion of CD33 contains an immunoreceptor-suppressive tyrosine motif (ITIM) associated with the suppression of cellular activity. Diseases that can be treated by targeting CD33 include, but are not limited to, Alzheimer's disease, as well as retinopathy such as macular edema (e.g., diabetic macular edema) and age-related macular degeneration (AMD) (e.g., dry AMD and wet AMD).

[0021] CD33 is a target of gemtuzumab ozogamicin (Mylotarg®, Pfizer / Wyeth-Ayerst Laboratories), an antibody-drug conjugate used to treat patients with acute myeloid leukemia. CD33 is also a target of vadastuximab ptaririn (SGN-CD33A), a novel antibody-drug conjugate developed by Seattle Genetics using their ADC technology.

[0022] Lymphocyte Activator Gene 3 (LAG-3), a 503-amino acid transmembrane protein, is a checkpoint receptor protein found on the cell surface of effector T cells and regulatory T cells (Tregs), and functions to regulate T cell response, activation, and proliferation. LAG3 is a member of the immunoglobulin (Ig) superfamily. Binding of LAG3 to MHC class II molecules results in the delivery of a negative signal to LAG3-expressing cells, downregulating antigen-dependent CD4 and CD8 T cell responses. LAG3 negatively regulates T cell proliferative capacity, cytokine production capacity, and target cell lytic capacity, a phenomenon known as T cell "exhaustion." Because LAG3 plays a crucial role in tumor immunity and infectious immunity, it is an ideal target for immunotherapy. Blocking LAG3 with antagonists, including monoclonal antibodies, has been studied in the treatment of cancer and chronic viral infections.

[0023] CD16, also known as FcγRIII, is a group of differentiation-inducing molecules found on the surface of natural killer cells, neutrophils, monocytes, and macrophages. Identified as an Fc receptor, CD16 exists in two forms encoded by isolated genes: FcγRIIIa (CD16a), a transmembrane protein, and FcγRIIIb (CD16b), a GPI-anchored protein, both involved in signal transduction. Most well-studied as a membrane receptor that triggers NK cell lysis, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cell-mediated cytotoxicity (ADCC). It can be used to isolate specific immune cell populations by fluorescence-activated cell sorting (FACS) or magnetoactivated cell sorting using antibodies targeting CD16. These receptors bind to the Fc site of IgG antibodies, subsequently activating antibody-dependent cell-mediated cytotoxicity (ADCC) in human NK cells. CD16 is required for the ADCC process by human monocytes. In humans, CD16-expressing monocytes possess various ADCC properties in the presence of specific antibodies and can kill primary leukemia cells, cancer cell lines, and hepatitis B virus-infected cells. Furthermore, CD16 can directly kill some virus-infected cells and cancer cells without the need for antibodies. After binding to ligands such as the conserved portion of IgG antibodies, CD16 on human NK cells induces the transcription of genes for surface-activated molecules such as IL-2-R (CD25), as well as inflammatory cytokines such as IFN-gamma and TNF. This CD16-induced cytokine mRNA expression in NK cells is mediated by activated T cell nuclear factor (NFATp), a cyclosporine A (CsA)-sensitive factor that regulates the transcription of various cytokines. The expression of specific cytokine genes is upregulated through a CsA-sensitive and calcium-dependent mechanism.

[0024] CD16 plays a crucial role in the early activation of natural killer (NK) cells after vaccination. Furthermore, CD16 downregulation suggests the potential to mitigate NK cell responses and maintain immune homeostasis in both T cell and antibody-dependent signaling pathways. In normal, healthy individuals, crosslinking of CD16 (FcγRIII) by immune complexes induces antibody-dependent cell-mediated cytotoxicity (ADCC) in NK cells. However, this pathway can be targeted in cancer or diseased cells through immunotherapy. After influenza vaccination, CD16 downregulation is associated with significant upregulation of influenza-specific plasma antibodies and shows a positive correlation with NK cell degranulation.

[0025] CD16 is commonly used as an additional marker to reliably identify different subsets of human immune cells. Several other CD molecules, such as CD11b and CD33, have traditionally been used as markers for human myeloid-derived suppressor cells (MDSCs). However, since these markers are also expressed in NK cells and all other myeloid-derived cells, other markers such as CD14 and CD15 are needed. CD14 is low and CD15 is high in neutrophils, while CD14 is high and CD15 is low in monocytes. These two markers are sufficient to distinguish between neutrophils and monocytes, while eosinophils express CD15 as well as neutrophils. Therefore, CD16 is used as an additional marker to identify neutrophils. CD16 is high in mature neutrophils and low in both eosinophils and monocytes. CD16 allows for the distinction between these two types of granulocytes. Furthermore, CD16 expression differs between different stages of neutrophil development. CD16 levels are low in neutrophil progenitor cells with differentiation potential, while CD16 expression is increased in metamyelocytes, band neutrophils, and mature neutrophils, respectively.

[0026] Due to its expression in neutrophils, CD16 has potential as a target in cancer immunotherapy. Margetuximab, an Fc-optimized monoclonal antibody that identifies human epidermal growth factor receptor 2 (HER2) expressed in tumor cells of breast cancer, bladder cancer, and other solid tumors, targets CD16A rather than CD16B. Furthermore, CD16 may play a role in antibody-targeted cancer therapies. Bispecific antibody fragments, such as anti-CD19 / CD16, enable the targeting of antimicrobial agents to cancer cells. Anti-CD19 / CD16 bispecific antibodies have been shown to enhance the natural killer cell response to B-cell lymphoma. In addition, targeting of exogenous factors such as FasL or TRAIL to the tumor cell surface triggers cell death receptors, inducing apoptosis through both autocrine and paracrine processes.

[0027] (antibody) Antibodies and their use for the treatment of diseases are well known in the field. As used herein, the term “antibody” means a monomeric or multimeric protein comprising one or more polypeptide chains containing an antigen-binding site. Antibodies can specifically bind to an antigen and modulate the biological activity of the antigen. 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” means the region of the 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 an antibody consisting only of a heavy chain, the antibody heavy chain variable region.

[0028] An antibody specifically refers to the selective identification of an antibody against a particular epitope of an antigen. Natural antibodies, for example, are monospecific. As used herein, the term “monospecific” means an antibody that has one or more binding sites, each binding to the same epitope of the same antigen. The monospecific antibodies disclosed herein are specific to CD47, HSA, PD-L1, CD33, CD16, or LAG3. In some embodiments, the monospecific antibody is a heavy-chain-only antibody (HCAb). In another embodiment, the monospecific antibody contains one or more protein domains, for example, a VHH domain fused to a human Fc region. In yet another embodiment, the monospecific antibody contains VHH as the sole complete protein domain, i.e., a single-domain antibody. In some embodiments, the single-domain antibody may further contain a short peptide, such as a His tag. The terms “VHH domain” and “HCAb variable domain” are used interchangeably. The VHH domain may be referred to as a means for binding to a specific target (CD47, HSA, PD-L1, CD33, CD16, or LAG3). Any of the various antibody structures, configurations, or constructions disclosed herein that include or are configured to include a VHH domain can be considered antibodies that have means for binding to an indicated target. Some embodiments may specifically include the structure, configuration, or construction of one or more specific antibodies. Other embodiments may specifically exclude the structure, configuration, or construction of one or more specific antibodies.

[0029] As used herein, the terms "antibody having specificity for," "antibody that identifies," "antibody having affinity for," "antibody having a binding site for," and similar constructs are used interchangeably.

[0030] A “multispecific antibody” means an antibody having two or more antigen-binding specificities. The multispecific antibodies disclosed herein are specific to at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3, or have at least one of the above specificities and at least one second specificity. In some embodiments, the multispecific antibodies disclosed herein may contain two, three, four, or more domains capable of binding to an antigen. Furthermore, a multispecific antibody may contain at least two copies of the same antigen-binding sequence, or two antigen-binding sequences (biparatopics) having specificity to different epitopes on the same antigen, insofar as it has specificity to at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3, and at least one second antigen. In some embodiments, a multispecific antibody (MVSCA) has properties for at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3. In some embodiments, the multispecific antibodies disclosed herein are single-chain antibodies. Accordingly, some multispecific antibodies may be referred to as antibodies comprising means for binding to a first target, means for binding to a second target, and so on.

[0031] A “bispecific antibody” means an antibody having two different antigen-binding specificities. In some embodiments, the bispecific antibodies disclosed herein are specific to two of the following: CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequences encoding the antigen-binding portions of a bispecific antibody can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portions of a bispecific antibody are linked by linkers such as those disclosed herein.

[0032] A “triply specific antibody” means an antibody having three different antigen-binding specificities. In some embodiments, the triply specific antibodies disclosed herein are specific to three of the following: CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequences encoding the antigen-binding portions of the triply specific antibody can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the triply specific antibody are linked by linkers as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0033] A "quadrispecific antibody" refers to an antibody having four different antigen-binding specificities. In some embodiments, the quadruspecific antibodies disclosed herein are specific to four of the following: CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequences encoding the antigen-binding portions of the quadruspecific antibody can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the quadruspecific antibody are linked by linkers as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0034] As used herein, the term “valence” refers to the presence of a specific number of binding sites in an antibody molecule. Therefore, “divalent,” “trivalent,” “tetravalent,” “pentavalent,” “hexavalent,” “heptavalent,” and “octavalent” refer to the presence of two, three, four, five, six, seven, and eight binding sites in the antibody molecule, respectively. A bispecific antibody disclosed herein is “divalent.” A trispecific antibody disclosed herein is “trivalent.” A tetraspecific antibody disclosed herein is “tetravalent.” However, monospecific polyvalent antibodies, such as bivalent, trivalent, and tetravalent antibodies, are within the scope of this disclosure, and they have multiple antigen-binding sites that bind to the same antigen. The antigen-binding sites of monospecific bivalent and trivalent (or higher-valence) antibodies can bind to either the same epitope or different epitopes of the antigen. Similarly, by combining multiple single-specificity binding sites with one or more other specificity binding sites, it is possible to construct antibodies with higher binding titers than multi-specific antibodies, such as trivalent bispecific antibodies.

[0035] As used herein, “full-length antibody” refers to the structure that constitutes the natural biological form of an antibody, including a variable region and a constant region. For example, in most mammals, including humans and mice, a full-length antibody of the IgG class is a tetramer, consisting of two identical pairs of two immunoglobulin chains, each pair having one light chain and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains HV, CH1, CH2, and CH3. In some mammals, such as camels and llamas, an IgG antibody may also consist of only two heavy chains (HCAb), each heavy chain containing a variable domain bound to an Fc region (CH2 and CH3 domains).

[0036] Tetrameric antibodies typically consist of two identical polypeptide chains, each pair comprising one "light" chain (usually with a molecular weight of approximately 25 kDa) and one "heavy" chain (usually with a molecular weight of approximately 50-70 kDa). Each of the light and heavy chains is composed of two distinct regions called the variable region and the constant region. In the IgG class of immunoglobulins, the heavy chain consists of four immunoglobulin domains linked in the order VH-CH1-CH2-CH3 from the N-terminus to the C-terminus, referred to as the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (sometimes referred to as VH-Cγ1-Cγ2-Cγ3, referred to as the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain, respectively). The IgG light chain consists of two immunoglobulin domains linked in a VL-CL order from the N-terminus to the C-terminus, known as the light chain variable domain and the light chain constant domain, respectively. The constant domain exhibits less sequence diversity and can trigger important biochemical events in response to the binding of numerous native proteins.

[0037] The variable region of an antibody contains the antigen-binding determinants of the molecule and therefore determines the antibody's specificity against a target antigen. The variable region is so named because its sequence is most different from that of other antibodies in the same class. In the variable region, three loops come together in the V domains of the heavy and light chains to form the antigen-binding site. Each loop is called a complementarity-determining region (hereinafter referred to as "CDR" in this specification), and the difference in amino acid sequence is most pronounced. There are a total of six CDRs, three in the heavy chain and three in the light chain: VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. Outside the CDRs of the variable region is called the framework (FR) region. Although not as diverse as the CDRs, there is sequence variation in the FR region between different antibodies. Overall, this characteristic structure of antibodies provides a stable scaffold (FR region), upon which substantial antigen-binding diversity (CDR) can be explored by the immune system to acquire specificity against a wide range of antigens.

[0038] The gene encoding the immunoglobulin locus contains multiple V regions along with short nucleotide sequences named "D" and "J," and the combination of V, D, and J nucleotide sequences gives rise to the diversity of VH.

[0039] Antibodies are classified into classes, also called isotypes, which are genetically determined by their constant region. Human constant light chains are classified into kappa (Cκ) and lambda (Cλ) light chains. Heavy chains are classified into mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. The IgG class is the most commonly used for therapeutic purposes. In humans, this class includes the IgG1, IgG2, IgG3, and IgG4 subclasses. In mice, this class includes the IgG1, IgG2a, IgG2b, and IgG3 subclasses. IgM has, but is not limited to, the IgM1 and IgM2 subclasses. IgA has, but is not limited to, several subclasses, including IgA1 and IgA2. Therefore, as used herein, “isotype” means a class or subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant region. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. The disclosed HCAb antibodies, bispecific and polyspecific antibodies may include all or some of the above-mentioned isotypes.

[0040] The scope of this disclosure also includes, but is not limited to, antibody fragments containing (i) Fab fragments containing VL, CL, VH, and CH1 domains, (ii) Fd fragments containing VH1 and CH1 domains, (iii) Fv fragments containing VL and VH domains of a single antibody, (iv) dAb fragments containing a single variable region, (v) a separated CDR region, (vi) a bivalent fragment F(ab')2 fragment containing two linked Fab fragments, and (vii) an antibody fragment containing a single-stranded Fv molecule (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 variable regions linked by linkers that have three different specificities and are cleavable or non-cleavable are also disclosed. In some embodiments, the antibody is produced by recombinant DNA technology. In further embodiments, the antibody is produced by enzymatically or chemically cleaving a naturally occurring antibody.

[0041] As used herein, “single-chain antibody” refers to a fusion protein of the antigen-binding portion (i.e., variable region) of an antibody, generally linked by a linker peptide. Disclosed herein are polyvalent single- or multispecific single-chain antibodies. A single-specific polyvalent antibody has specificity for at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3. A multispecific single-chain antibody has specificity for at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3, and at least one further specificity. In some embodiments, a multispecific single-chain antibody has specificity for at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3.

[0042] As used herein, a “humanized” antibody means an antibody that contains a human framework region (FR) and a complementarity-determining region (CDR) of one or more non-human antibodies. The non-human providing the CDR is called the “donor,” and the human immunoglobulin providing the framework is called the “acceptor.” In some embodiments, humanization is primarily achieved by grafting a donor CDR onto the VL or VH framework of an acceptor (human). This method is called a “CDR graft.” To regain the affinity lost in the initial graft structure, it may be necessary to “reverse mutagenerate” framework residues of a selected acceptor to corresponding donor residues. An optimal humanized antibody will likely contain at least a portion of the immunoglobulin constant region, typically that of a human immunoglobulin, and often the human Fc region. Other methods of humanization, or reducing the immunogenicity of a non-human antibody variable region, may include resurfacing methods. In some embodiments, selection-based methods may be employed to humanize and / or affinity mature an antibody variable region, i.e., to increase the affinity of the variable region to its target antigen. Other humanization methods may include, but are not limited to, grafts of only a portion of the CDR, the method described in U.S. Patent No. 6,797,492, which is incorporated herein by reference for all disclosures relating to CDR transplantation. Structure-based methods may be employed to humanize and achieve affinity maturation, for example, as described in U.S. Patent No. 7,117,096, which is incorporated herein by reference for all disclosures relating to humanization and affinity maturation.

[0043] In various embodiments of this specification, the antibody is a heavy-chain-only antibody (HCab). Camel species (camels, dromedaries, and llamas) include double-chain antibodies (containing only variable heavy chains) in addition to conventional heavy-chain and light-chain antibodies (two light chains and two heavy chains in one antibody). Dimeric antibodies are encoded by a different set of VH segments called the VHH gene. VH and VHH are dispersed throughout the genome (i.e., they coexist with each other). The identification of identical D segments in the cDNA of VH and VHH suggests a shared D segment in VH and VHH. Antibodies containing native VHH lack the entire CH1 domain of the constant region of the heavy chain. The exon encoding the CH1 domain is present in the genome but is excised due to the deletion of the functional splicing acceptor sequence on the 5' end of the CH1 exon. As a result, the VDJ region is spliced ​​onto the CH2 exon. When VHH is recombined into such constant regions (CH2, CH3), antibodies are produced in which the semi-antibodies are single-chain rather than light-chain / heavy-chain pairs (i.e., antibodies with two heavy chains that do not interact with the light chain). Antigen binding is different from that seen with conventional antibodies, but high affinity is achieved in a similar manner, namely through hypermutation of the variable region and selection of cells that express such high-affinity antibodies.

[0044] In exemplary embodiments, the disclosed HCAb is produced by immunizing transgenic mice that have been genetically modified with camelid genes and in which endogenous mouse antibody expression has been eliminated. HCAb mice are disclosed in U.S. Patent Nos. 8,883,150, 8,921,524, 8,921,522, 8,507,748, 8,502,014, U.S. Patent Publication Nos. 2014 / 0356908, 2014 / 0033335, 2014 / 0037616, 2014 / 0356908, 2013 / 0344057, 2013 / 0323235, 2011 / 0118444, and 2009 / 0307787, all of which are incorporated herein by reference with respect to the disclosure of heavy-chain-only antibodies and their production in transgenic mice. The spleen cells obtained by immunizing with HCAb are fused with mouse myeloma cells to form hybridomas.

[0045] In other embodiments, HCAb was prepared by immunizing llamas with a desired antigen and isolating the sequence encoding the VHH region of the resulting antigen-binding antibody. In one embodiment, VHH was isolated using a phage display library. See, for example, International Publication Nos. 91 / 17271, 92 / 01047, and 92 / 06204 (each of which, for descriptions of the preparation of a phage library, is incorporated herein by reference in its entirety).

[0046] Also disclosed herein are multispecific or multivalent antibodies in which two or more antigen-binding domains are bound to a single fusion protein. Multispecific antibodies can take many forms, including (i) a multispecific Fv fragment, (ii) a heavy chain of first specificity bound to (or fused to) a second VH domain having second specificity, (iii) a tetrameric monoclonal antibody having first specificity bound to a second VH domain having second specificity, wherein the second VH domain is bound to the first VH domain, and (iv) a Fab fragment of first specificity (VH-CH1 / VL-CL) bound to a second VH domain having second specificity. Exemplary Fab fragments include those in which a second VH sequence having second specificity is bound to the C-terminus or N-terminus of the first VH domain, or to the C-terminus or N-terminus of the first CH1 or first CL domain. In further embodiments, a VH sequence having a second and / or third (or more) specificity may bind to (or fuse with) the C-terminus or N-terminus of the first VH domain, or the C-terminus or N-terminus of the first CH1 or first CL domain. In various embodiments, any of these configurations may include at least one of the HCAb variable domains disclosed herein.

[0047] Multispecific or polyvalent antibodies may contain linker sequences that link specific antigen-binding domains (e.g., VH or VHH) to other antigen-binding domains, allowing the amino acid sequences to fold appropriately to produce a desired three-dimensional structure and antigen-binding profile. Generally, linker sequences are short amino acid sequences that provide sufficient space and flexibility between domains for proper folding. Linkers may also cause steric hindrance to facilitate the binding of each domain to its target. Suitable linkers include, but are not limited to, those listed in Table 15 (SEQ ID NOs. 100-119), EPKSCD (SEQ ID NOs. 224), and ASTKGP (SEQ ID NOs. 225). Further linkers will be known to those skilled in the art.

[0048] Furthermore, the scope of this disclosure includes amino acid sequence variants of monospecific or multispecific antibodies disclosed herein. Amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the DNA encoding the antibody or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues into the amino acid sequence of the antibodies of the examples herein. Any combination of deletions, insertions, and substitutions can lead to the final structure, as long as the final structure retains the desired properties. Amino acid changes can also cause post-translational process changes in humanized or variant antibodies, such as changes in the number or position of glycosylation sites.

[0049] A useful method for identifying specific residues or regions at favorable locations for antibody mutagenesis is called "alanine scanning mutation." A single residue or group of target residues is identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced with an uncharged amino acid (most preferably alanine or polyalanine) to influence the interaction between the amino acid and the antigen. The locations of those amino acids that are functionally sensitive to the substitution are then improved by introducing further or other variants at the substitution site. Therefore, while the sites for introducing mutations in the amino acid sequence 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, an alanine scan or random mutagenesis is performed at the target codon or region, and the expressed antibody variant is screened for desired activity.

[0050] Amino acid sequence insertions include fusions to amino and / or carboxyl terminals ranging in length from one residue to polypeptides containing 100 or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies disclosed herein having an N-terminal methionyl residue, or antibodies fused with an epitope tag. Other insertion variants of antibody molecules include fusions of enzymes or polypeptides to the N-terminus or C-terminus of the antibody that increase the serum half-life of the antibody.

[0051] Another type of variant is the one-amino acid substitution variant. These variants involve the removal of at least one amino acid residue within the antibody molecule and the insertion of another residue in its place. While the most important sites for substitutional mutagenesis are the high-frequency variable regions, modifications of the FR (Frequency-Range) are also possible. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." If such substitutions result in changes in biological activity, the product should be screened by introducing more substantial modifications, such as those shown in Table 1 under "Exemplary Substitutions," or further described below in relation to amino acid classes.

[0052] [Table 1]

[0053] Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the polypeptide backbone at the substitution site, e.g., a sheet or helical structure, (b) the electrification or hydrophobicity of the molecule at the target site, or (c) the majority of the side chain. Naturally occurring residues are grouped based on their common side chain properties. (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile (2) Neutral hydrophilicity: Cys, Ser, Thr (3) Acidic: Asp, Glu (4) Basicity: Asn, Gin, His, Lys, Arg (5) Residues that affect chain orientation: Gly, Pro (6) Aromatic: Trp, Tyr, Phe

[0054] Non-conservative substitution means swapping members of these classes with those of other classes.

[0055] To improve the oxidative stability of the molecule and prevent abnormal crosslinking, any cysteine ​​residues that do not contribute to maintaining the proper structure of monospecific or multispecific antibodies may generally be substituted with serine. Conversely, cysteine ​​bonds may be added to the antibody to improve stability (especially if the antibody is an antibody fragment such as an Fv fragment).

[0056] Another type of substitution variant involves substituting residues in one or more high-frequency variable regions of the parent antibody (e.g., a humanized antibody or a camelid antibody). Generally, the resulting variants selected for further development will have improved biological properties compared to the parent antibody from which they were generated. A convenient method for generating such substitution variants is affinity maturation using phage display. Briefly, several high-frequency variable regions (e.g., 6-7 sites) are mutated to produce all possible amino residues at each site. The antibody variants thus generated are presented in a monovalent form as fusions from filamentous phage particles to the M13 gene III product packaged within each particle. The phage display variants are then screened by their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate high-frequency variable regions for modification, alanine scanning mutations can be performed to identify high-frequency variable region residues that significantly contribute to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and adjacent residues are candidates for substitution by the techniques detailed herein. Once such variants are generated, a panel of variants is subjected to screening as described herein, and antibodies exhibiting superior properties in one or more relevant assays may be selected for further development.

[0057] Another type of amino acid variant in an antibody alters the antibody's original glycosylation pattern. This alteration involves the removal of one or more carbohydrate sites present in the antibody and / or the addition of one or more glycosylation sites that were not present in the antibody.

[0058] Antibody glycosylation is typically either N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for enzymatically attaching the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either tripeptide sequence in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid; 5-hydroxyproline or 5-hydroxylysine may also be used, but serine or threonine are the most common.

[0059] The addition of a glycosylation site to an antibody can be conveniently achieved by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). This alteration may also be created by adding or substituting one or more serine or threonine residues into the original antibody sequence (O-linked glycosylation sites).

[0060] Nucleic acid molecules encoding amino acid sequence variants of monospecific or multispecific antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural resources (in the case of naturally occurring amino acid sequence variants), or mutagenesis via (or site-directed) oligonucleotides of previously prepared variants or non-variants of the antibodies disclosed herein, mutagenesis by PCR, and cassette mutagenesis.

[0061] Other modifications to monospecific or multispecific antibodies should be considered. For example, modifying antibodies in terms of effector function may be desirable to enhance their efficacy in treating diseases. For instance, introducing cysteine ​​residues into the Fc region may enable the formation of interchain disulfide bonds in this region. The resulting dimerized antibodies may have improved internal capacity and / or enhance complement-mediated cell death and antibody-dependent cell-mediated cytotoxicity (ADCC). Alternatively, dimerized antibodies with enhanced antitumor activity may be prepared using heterobifunctional crosslinkers. Or, antibodies with two Fc regions that can thereby improve complement lysis and ADCC activity can be designed.

[0062] In other embodiments, the antibody may be pre-targeted by being conjugated to a "receptor" (such as streptavidin), the antibody-receptor conjugate is administered to the patient, the unconjugated conjugate is then removed from circulation using a clearing agent, and then a "ligand" (such as avidin) conjugated to a cytotoxic agent (such as a radionuclide) is administered.

[0063] Furthermore, covalent modifications of monospecific or multispecific antibodies are also within the scope of this disclosure. These are sometimes produced by chemical synthesis or by enzymatic or chemical cleavage of antibodies. Other types of covalent modifications of antibodies involve introducing target amino acid residues of the antibody into the molecule by reacting them with an organic derivatizing agent that can react with a selected side chain or N-terminal or C-terminal residue. Exemplary covalent modifications of polypeptides are described in U.S. Patent No. 5,534,615, and all disclosures relating to covalent modifications of polypeptides are incorporated herein by reference. Exemplary types of covalent modifications of antibodies include linking an antibody to one of various non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, by the methods described in U.S. Patents No. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337.

[0064] The monospecific or multispecific antibodies disclosed herein may be produced by recombinant means. Therefore, disclosed herein are nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods for recombinant production are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, subsequent isolation of the antibody, and purification for pharmaceutically acceptable purity. For antibody expression in host cells as described above, the nucleic acid encoding the antibody sequence is inserted into the expression vector by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). It is understood that recombinant expression proteins require an initiation methionine (or formylmethionine) or signal sequence at the 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 have their N-terminuses modified with amino acids. In some embodiments, such N-terminal sequences are cleaved (in whole or in part) from the complete, mature sequence, while in other embodiments they are retained.

[0065] Accordingly, certain embodiments disclosed herein include a method for preparing a monospecific or multispecific antibody, comprising: 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 the antibody molecule; and c) recovering the antibody molecule from the culture.

[0066] Antibodies can be appropriately isolated from the culture medium by conventional immunoglobulin purification procedures such as protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0067] Where used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny cells. Therefore, “transformed organism” and “transformed cell” include the original target cell and numerous cultures derived therefrom without transformation. It is also understood that, due to intentional or unintentional mutations, not all progeny may be exactly identical in DNA content. This also includes progeny of variants that have the same function or biological activity as those screened for the original transformed cell. Where a different name is intended, it will be evident from the context.

[0068] As used herein, the term "transformation" refers to the process of transferring a vector / nucleic acid into a shriveled cell. When cells without a cell wall barrier are used as host cells, transfection can be performed, for example, by calcium phosphate precipitation. However, other methods for introducing DNA into cells may also be used, for example, by nucleic acid injection or by protoplast fusion. When prokaryotic cells or cells containing a rigid cell wall structure are used, for example, one method of transfection is calcium treatment with calcium chloride.

[0069] As used herein, “expression” refers to the process by which a nucleic acid is transcribed into mRNA, and / or the process by which the transcribed mRNA (also called the transcript) is subsequently translated into peptides, polypeptides, or proteins. The transcript and the polypeptide it encodes are collectively referred to as the gene product. When polynucleotides are derived from genomic DNA, expression in eukaryotic cells may involve mRNA splicing.

[0070] A "vector" is a self-replicating nucleic acid molecule that moves an inserted nucleic acid molecule into and / or between host cells. This term includes vectors that primarily insert DNA or RNA into cells (e.g., chromosome integration), replication vectors that primarily replicate DNA or RNA, and expression vectors that perform DNA or RNA transcription and / or translation. It also includes vectors that provide two or more of the above functions.

[0071] 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" usually refers to a suitable host cell containing an expression vector capable of functioning to obtain the desired expression product.

[0072] As used herein, “host cells” means any cell line designed to produce the antibodies disclosed herein. In some embodiments, HEK293 cells and CHO cells are used as host cells.

[0073] Appropriate regulatory sequences for prokaryotes include, for example, promoters, further operator sequences, and ribosome-binding sites. In eukaryotic cells, promoters, enhancers, and polyadenylation signals are known to be utilized.

[0074] Nucleic acids are "operably ligated" when they are placed in a functional relationship with other nucleic acid sequences. For example, pre-sequence or secretion leader DNA is operably ligated to the DNA for a polypeptide if it is expressed as a preprotein involved in polypeptide secretion; promoters or enhancers are operably ligated to coding sequences if they affect the transcription of the sequence; or ribosome binding sites are operably ligated to coding sequences if they are located in a site that facilitates translation. Generally, "operably ligated" means that the ligated DNA sequences are adjacent, and in the case of secretion leaders, adjacent and within the reading frame. However, enhancers do not need to be adjacent. Ligation is achieved by ligation at convenient restriction sites. If such sites are unavailable, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

[0075] Also disclosed herein are isolated nucleic acids, vectors, and host cells encoding single-specific or multi-specific antibodies, including nucleic acids and recombinant technologies for antibody production.

[0076] For recombinant antibody products, the nucleic acid encoding it can be isolated and inserted into a replication vector for further cloning (DNA amplification) or expression. In some embodiments, antibody production may be carried out by homologous recombination, as described in U.S. Patent No. 5,204,244, incorporated herein by reference for all disclosures relating to antibody production. The DNA encoding the antibody is readily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The components of a vector generally include, but are not limited to, one or more of the following, as described in U.S. Patent No. 5,534,615, incorporated herein by reference for all disclosures relating to protein expression: a signal sequence, an origin of replication, one or more marker genes, an enhancer sequence, a promoter, and a transcription termination sequence.

[0077] Suitable host cells for cloning or expressing DNA in a vector as described herein are prokaryotic cells, yeast, or the higher eukaryotic cells mentioned above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive bacteria, such as the Enterobacteriaceae family including Escherichia (e.g., Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacillus (e.g., B. subtilis, B. licheniformis), Pseudomonas (e.g., Pseudomonas), and Streptomyces. One exemplary Escherichia coli cloning host is Escherichia coli 294 (ATCC31,446), but other strains such as Escherichia coli B, Escherichia coli X1776 (ATCC31,537), and Escherichia coli W3110 (ATCC27,325) are also suitable. These examples are illustrative and not limiting.

[0078] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable for cloning or expressing vectors encoding single-specific or multi-specific antibodies. Among lower eukaryotic host microorganisms, budding yeast or common baker's yeast are the most commonly used. However, fission yeasts such as K. lactis, K. fragilis (ATCC12, 424), K. bulgaricus (ATCC16, 045), K. wickeramii (ATCC24, 178), K. waltii (ATCC56, 500), K. drosophilarum (ATCC36, 906), K. thermolerans, and K. marxianus are also suitable hosts for the genus Kruywelomyces; Yarrowia (EP402, 226); Pichia pastoris (EP183, 070); Candida; Trichoderma reesia (EP244, 234); and Neurospora crassa; Schwanniomyces Many other genera, species, and subgenus of filamentous fungi, such as the genera Schwanniomyces (e.g., occidentalis), and filamentous fungi such as the genera Neurospora, Penicillium, Tolypocladium, and Aspergillus (e.g., A. nidulan and A. niger), are commonly available and useful herein.

[0079] Host cells suitable for the expression of glycosylated single-specific or multispecific antibodies are derived from multicellular organisms and include cells from non-vertebrates such as plant cells and insect cells. Numerous baculovirus strains and mutants, as well as corresponding permissible insect host cells, have been identified from hosts such as Spodoptera frugiperda (larva), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyxmori. Various virus strains for transfection, for example, viruses for transfection of Spodoptera frugiperda cells are known, and such viruses can be used as the viruses of this specification according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0080] However, interest in vertebrate cells is highest, and the proliferation of vertebrate cells in tissue culture is a routine procedure. Useful mammalian host cell lines include SV40-transformed monkey kidney CV1 (COS-7, ATCC CRL 1651), human embryonic kidney cells (293 or 293 cells subcloned for proliferation in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / -DHFR (CHO), mouse Sertoli cells (TM4), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human embryonic cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), and mouse mammary tumor cells (MMT). The cells used were 060562 (ATCC CCL51), TRI cells, MRC5 cells, FS4 cells, and a human hepatocellular carcinoma cell line (Hep G2).

[0081] Host cells are transformed with the aforementioned expression vectors to produce single-specific or multi-specific antibodies, cultured in a conventional nutrient medium modified for promoter induction, and transformants are selected or genes encoding desired sequences are amplified.

[0082] Host cells for producing monospecific or multispecific antibodies can be cultured in a variety of media. Commercial media such as Ham F10 (sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Furthermore, U.S. Patents 4,767,704, 4,657,866, 4,927,762, 4,560,655, 5,122,469, International Publication No. 90 / 03430, International Publication No. 87 / 00195, or U.S. Reissue Patent No. 30,985 can be used as culture media for host cells. If necessary, 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 GENTAAMYCIN®), trace elements (usually defined as inorganic compounds whose final concentration is in the micromolar range), and glucose or equivalent energy sources may be added to any of these media if needed. Other necessary auxiliary substances may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those conventionally used in host cells selected for expression and will be apparent to those skilled in the art.

[0083] When recombinant technology is used, antibodies can be produced intracellularly, in the perimembrane space, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove certain debris, which may be host cells or lysed fragments, by, for example, centrifugation or ultrafiltration.

[0084] Antibody compositions prepared from cells are purified using methods such as hydroxyl apatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies that lack an Fc region, but it can also be used to purify human γ1, γ2, or γ4 heavy-chain-based antibodies. Protein G is useful for all mouse isotypes and human γ3. The matrix to which the affinity ligand binds is almost always agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Bakerbond ABX® resin is useful for purification when the antibody contains a CH3 domain. Furthermore, the antibodies and antibody fragments disclosed herein can be synthesized with histidine tags and affinity-purified by metal affinity chromatography.

[0085] Other techniques for protein purification, such as preparative separation by ion exchange column, ethanol precipitation, reverse-phase HPLC, silica chromatography, heparin-SEPHAROSE™ chromatography, anion or cation exchange resin chromatography (e.g., polyaspartate column), isoelectric focusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0086] Following any preliminary purification steps, the mixture containing the antibody of interest and any contaminants may 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 to 0.25 M salt).

[0087] Also disclosed herein are multispecific single-chain antibodies that can be cleaved in the tumor microenvironment. In some embodiments, a tumor-targeting domain (such as a tumor antigen-binding domain) or other functional domains (such as an anti-HSA domain that can extend the half-life in the body) are cleaved by a linker upon the multispecific single-chain antibody reaching the tumor to release other domains that contribute to the therapeutic effect. The tumor microenvironment contains numerous proteases that can cleave the linker disclosed herein. Non-limiting examples of tumor proteases include, but are not limited to, matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAM (a disintegrin and metalloprotease, e.g., ADAM10 and ADAM17), kallikrein-related peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepsin (HPN), matryptase, regmaine, or dipeptidyl peptidase (e.g., DDP4).

[0088] (Antibody composition) Also disclosed herein are pharmaceutical compositions comprising monospecific or multispecific antibodies having specificity to CD47, HSA, PD-L1, CD33, CD16, or LAG3, etc. Also disclosed is the use of the antibodies disclosed herein for the manufacture of pharmaceutical compositions. Also disclosed is the use of the disclosed antibodies and pharmaceutical compositions comprising the antibodies for the treatment of various diseases and disorders.

[0089] A pharmaceutical composition is intended for and suitable for the treatment of diseases in humans. That is, it provides a beneficial effect as a whole and does not contain any components or contaminants that cause toxicity or other undesirable effects unrelated to the provision of a beneficial effect. A pharmaceutical composition contains one or more active agents and may further contain solvents, buffers, diluents, carriers, and other additives to assist the dosability, solubility, absorption or bioavailability, and / or stability of the active agents or the composition as a whole.

[0090] Furthermore, the monospecific or multispecific antibodies disclosed herein may be formulated in liposomes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in U.S. Patents 4,485,045, 4,544,545, and 5,013,556. Particularly useful liposomes are lipid compositions containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE), which are produced by reverse-phase evaporation. The liposomes are extruded through a filter of a specified pore size to obtain liposomes of the desired diameter. The Fab' fragment of the antibody may be bound to the liposome by disulfide interconversion.

[0091] As used herein, “pharmaceutical carrier” includes any and all physiologically compatible solvents, dispersions, coatings and antifungal agents, isotonic agents and absorption retarders. 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.

[0092] The compositions disclosed herein can be administered by various methods known in the art. As will be apparent to those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. To administer the disclosed antibodies by a particular route of administration, it may be necessary to associate the antibody with a substance to prevent inactivation, or to co-administer the antibody with such a substance. For example, the antibody may be administered to a target in a suitable carrier, e.g., liposomes, or diluents. Pharmaceutically acceptable diluents include physiological saline and aqueous buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersants and sterile powders for the immediate preparation of sterile injectable solutions or dispersants. The use of such media and agents for pharmaceutically active substances is known in the art.

[0093] As used herein, the phrases “parenteral administration” and “administered parenterally” generally mean, but are not limited to, methods of administration other than enteral and topical administration, mostly by infusion, including injections and infusions into veins, muscles, arteries, the brain, capsules, orbits, hearts, eyes, vitreous, skin, abdominal, trachea, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal cord, epidural, and sternal regions.

[0094] These compositions may also contain additives such as preservatives, humectants, emulsifiers, and dispersants. Prevention of microbial presence can be ensured by both the aforementioned sterilization procedures and the inclusion of various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, the extension of absorption in injectable composition forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0095] 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 additives as described herein. The lyophilized composition is reconstituted for administration to a patient by the addition of sterile water or aqueous buffer.

[0096] Regardless of the selected route of administration, the disclosed antibodies, and / or pharmaceutical compositions containing antibodies, which can be used in appropriate hydrate form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0097] The actual dose of the active ingredient in a pharmaceutical composition may vary depending on the specific patient, composition, and method of administration, in order to obtain an amount of the active ingredient that is effective in achieving the desired therapeutic response and is not toxic to the patient. The selected dose level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention adopted, the route of administration, the time of administration, the elimination rate of the particular composition adopted, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition adopted, the age, sex, weight, symptoms, general health status, and medical history of the patient being treated, and other factors well known in the medical field.

[0098] (Disclosed MVSCAs and their constituent antibody domains, and linker functions) (Anti-HSA HVV) The primary function of the anti-HSA domain in MVSCA is to bind to HSA, thereby extending the half-life of MVSCA in the body. The presence of the anti-HSA domain can extend the half-life from just a few hours to over a week. In most cases, one anti-HSA domain is sufficient for this purpose. Therefore, the anti-HSA domain constitutes a means of extending the half-life of MVSCA.

[0099] Upon binding to HSA, the anti-HSA domain can mediate partial or complete blockade of adjacent binding domains, thereby inhibiting or modulating their activity (effective affinity). Whether the blockade is substantially complete or partial depends on the length of the linker between the two domains; a shorter linker leads to more complete blockade of antigen binding. Partial blockade is often observed as a decrease in the apparent or effective affinity of VHH to antigens. In some cases, partial blockade is observed as an increase in the specificity of VHH, because the domain continues to bind to antigens with higher affinity but shows no significant binding to antigens with lower affinity. Thus, the anti-HSA domain constitutes a means of inhibiting the binding activity of adjacent binding domains.

[0100] Furthermore, the blockade may be reversible. By positioning the anti-HSA domain at the terminal site of MVSCA and attaching a cleavable linker, the anti-HSA domain can be removed, and the full binding activity of the adjacent binding domain is restored. Such an antibody structure is an effective prodrug. For example, if the linker is cleaved by a protease present at the desired site of action, MVSCA circulates in the body with the adjacent binding site inactive. However, upon reaching the site of action (e.g., a tumor), the linker is cleaved, the anti-HSA domain is released, and the inhibition of the binding activity of the adjacent binding domain is released. Therefore, when the anti-HSA domain is paired with a cleavable linker, it constitutes a means of reversibly inhibiting the binding activity of the adjacent binding domain in MVSCA or a multispecific antibody.

[0101] (anti-CD47) The function of the anti-CD47 domain is to inhibit the "don't eat me" signal of CD47 on tumor cells, allowing it to be phagocytosed by macrophages. CD47 is widely expressed, and anti-CD47 activity can be problematic if it involves substantial binding to normal, healthy cells. This can be avoided in several ways. CD47 has multiple structures, and the structure commonly found on tumor cells differs from, for example, that found on RBCs. As shown in Example 1, the VHH disclosed herein binds to CD47 expressed on tumor cells but not to CD47 expressed on RBCs. It is also important to avoid binding to RBCs so that MVSCA is not taken up into the bloodstream and prevented from reaching its target.

[0102] Another way to avoid undesirable or harmful rare binding of MVSCA to CD47 can be achieved, as described above, by positioning the anti-HSA domain adjacent to the anti-HSA domain in a manner that reduces or interferes with its binding to CD47. Once MVSCA binds to tumor cells via another binding domain, and the anti-HSA domain is cleaved and released by local proteases, the anti-CD47 domain can bind to CD47 and interfere with phagocytosis inhibitory interaction with macrophages.

[0103] Therefore, the anti-CD47 domain constitutes a means of reducing the inhibition of phagocytosis.

[0104] (anti-CD16) The function of the anti-CD16 domain is to upregulate the ADCC activity of NK cells. CD16B is widely distributed in tissues, but CD16A is specifically expressed in NK cells. Antibodies specific to CD16A are preferable because they bind only to the desired target, NK cells. However, antibodies that bind to both CD16A and CD16B, and antibodies that bind only to CD16, are both agonists that can promote the ADCC activity of NK cells.

[0105] CD16 typically interacts with the Fc portion of antibodies. When CD16A on NK cells binds to the Fc portion of an antibody, the cytolytic activity of the NK cell is directed towards the cell or microorganism to which the antibody's variable domain is bound. However, there are multiple Fc sequences and multiple types of Fc receptors, resulting in multiple effects that can be mediated by the Fc region. By using an anti-CD16 domain instead of the Fc region, MVSCA can specifically recruit NK-mediated ADCCs to targets of other specificities. Therefore, the anti-CD16 domain constitutes a means of recruiting NK-mediated ADCCs.

[0106] (anti-PD-L1) The anti-PD-L1 domain functions as both an immune checkpoint inhibitor and an antitumor antigen antibody. The anti-PD-L1 domain acts as a PD-1 binding antagonist. By blocking PD-L1 (e.g., on tumor cells) from binding to PD-1 (e.g., on T cells), the anti-PD-L1 domain inhibits the relevant immune checkpoint, releasing the T-cell-mediated immune response. Blocking PD-1 using anti-PD-1 or anti-PD-L1 antibodies is a well-known cancer treatment. Therefore, the anti-PD-L1 domain constitutes a means of blocking PD-1, or a means of deactivating the PD-1 immune checkpoint.

[0107] The anti-PD-L1 domain, as an antitumor antigen antibody, can mediate the binding of MVSCA to tumor cells. If MVSCA also contains an anti-CD16 domain, NK-mediated ADCC is promoted. MVSCA containing an anti-CD47 domain promotes macrophage-mediated phagocytosis. Multivalent binding to tumor cells improves binding affinity and ADCC. This is achieved by having multiple copies of the anti-PD-L1 domain and / or one or more binding domains targeting other tumor antigens. Therefore, the anti-PD-L1 domain constitutes a means of binding to tumor cells, a means of binding to tumor antigens, or a means of binding to the PD-L1 tumor antigen.

[0108] (anti-LAG3) The anti-LAG3 domain functions as an immune checkpoint inhibitor. It acts as an antagonist to the binding of LAG3 to class II MHC proteins. By blocking LAG3 on T cells from binding to class II MHC on tumor cells, the anti-LAG3 domain inhibits the relevant immune checkpoint, releasing the T cell-mediated immune response. Therefore, the anti-LAG3 domain constitutes a means of deactivating the LAG3 immune checkpoint.

[0109] (anti-CD33) The anti-CD33 domain can be used in two ways. It is a tumor antigen because it is expressed in myeloid cells and some lymphocytes, and in some hematological malignancies such as acute myeloid leukemia (AML). As a tumor antigen antibody, the anti-CD33 domain can mediate the binding of MVSCA to tumor cells. If MVSCA also contains an anti-CD16 domain, NK-mediated ADCC is promoted. MVSCA also containing an anti-CD47 domain promotes macrophage-mediated phagocytosis. Multivalent binding to tumor cells improves binding affinity and ADCC. This is achieved by having multiple copies of the anti-CD33 domain and / or one or more binding domains targeting other tumor antigens. Therefore, the anti-CD33 domain constitutes a means of binding to tumor cells, a means of binding to tumor antigens, or a means of binding to the CD33 tumor antigen.

[0110] Furthermore, for example, when CD33 binds to sialic acid residues in β-amyloid or other glycolipid-depositing proteins, an inhibitory signaling cascade leads to inhibition of phagocytic activity. Antibodies possessing an anti-CD33 domain function as CD33-stimulating antagonists, thereby promoting phagocytic activity and removing it for the treatment of Alzheimer's disease. Also, in retinal diseases such as dry age-related macular degeneration (AMD), insoluble deposits are present and can be removed by microglial phagocytosis. Therefore, antibodies possessing an anti-CD33 domain may also be useful in the treatment of dry AMD and other retinal diseases. Thus, the anti-CD33 domain constitutes a means of promoting phagocytic activity (in CD33-expressing cells), a means of promoting β-amyloid removal, or a means of removing insoluble deposits.

[0111] MVSCAs suitable for the treatment of Alzheimer's disease and retinal diseases are preferably divalent with respect to CD33 and contain an anti-HSA domain to improve half-life. These further contain an FC5 nanobody domain (Rissiek et al., Front. Cell. Neurosci. 8:344, 2014) to facilitate cross-human blood-brain barrier transport.

[0112] (Linker) In many embodiments, the individual binding domains are not directly bound to each other but have linkers, which are short amino acid sequences sandwiched between them. Examples of linkers are shown in Table 15. The length and sequence of the linkers can have a significant impact on the expression level and structure of MVSCA, as well as the binding affinity of the linked domains. MVSCA can be optimized to take these parameters into account using linkers L2 and L4 (see Table 15), whose lengths can be adjusted. Linkers L1, L2, and L4 may be referred to as non-cleavable linkers, flexible linkers, or flexible and non-cleavable linkers.

[0113] When two copies of the same VHH domain are placed in close proximity to each other within an MVSCA, they often interact in a detrimental manner. This can be avoided by inserting a relatively short, non-bending linker between the two copies. In some embodiments, the short, non-bending linker has sequence AAA (see L3 in Table 15). Such linkers may be referred to as short, non-bending linker means, or non-cuttable short, non-bending linker means.

[0114] When an anti-HSA domain-HSA complex is used to generate a prodrug related to the binding activity of adjacent binding domains, a cleavable linker should be sandwiched between the two domains. L11*3 to L11*18 (see Table 15) are examples of cleavable linkers of various lengths and susceptibility to cleavage by various proteases that can be used to optimize MVSCA in terms of expression level and structure, binding affinity of the binding domain, and cleavage. Linkers L11*3 to L11*18 may be referred to as cleavable linker means, flexible linker means, or flexible and cleavable linker means.

[0115] (MVSCA) The binding domains and linkers described herein can be combined to create multifunctional MVSCAs adapted for the treatment of specific diseases. They may be further combined with other binding domains. Furthermore, an MVSCA can be said to include means for realizing various functions associated with each type of binding domain, and / or include linker means for realizing those associated functions. An exemplary structure is briefly described below.

[0116] HSA / CD47 / PD-L1: This structure is suitable for treating PD-L1-expressing tumors, promoting phagocytosis, deactivating the PD-1 immune checkpoint, and extending its half-life in the body. In various embodiments, MVSCA may be divalent for the anti-CD47 and / or anti-PD-L1 binding domains. Depending on the linker used, the anti-HSA domain (once HSA is bound) may or may not inhibit binding to CD47, and if inhibition occurs, it can be restored by cleaving a cleavable linker. In some embodiments, the binding domains are arranged in a different order, but the anti-HSA domain should be at the terminal position if cleaved. In addition to describing this configuration of MVSCA as a means of realizing one or more functions of its components, MVSCA can also be considered a means of promoting phagocytosis (and deactivating the PD-1 immune checkpoint) of PD-L1-expressing tumors. Some embodiments of this structure are shown in Example 7.

[0117] HSA / LAG3 / PD-L1: This structure is suitable for treating tumors expressing PD-L1, and will deactivate LAG3 and PD-1 immune checkpoints and extend their half-lives in the body. In various embodiments, MVSCA may be divalent for anti-LAG3 and / or anti-PD-L1 binding domains. In some embodiments, the binding domains are arranged in a different order. In addition to describing this configuration of MVSCA as a means of realizing one or more functions of its components, MVSCA can also be described as a means of recruiting T effector cells to PD-L1 expressing tumors (and deactivating LAG3 and PD-1 immune checkpoints). Some embodiments of this structure are shown in Example 7.

[0118] CD16A / CD47 / PD-L1: This structure is suitable for treating PD-L1-expressing tumors and will promote phagocytosis, recruit NK cells to mediate ADCC, deactivate the PD-1 immune checkpoint, and extend its half-life in the body. In various embodiments, MVSCA may be bivalent for the anti-CD-47 and / or anti-PD-L1 binding domains. In some embodiments, the binding domains are arranged in a different order, but the anti-HSA domain should be located at the terminal position if cleaved. Depending on the linker used, the anti-HSA domain (once HSA is bound) may or may not inhibit binding to CD47, and if inhibition occurs, it can be restored by cleaving a cleavable linker. In addition to describing this configuration of MVSCA as a means of realizing one or more functions of its components, MVSCA can also be described as a means of promoting phagocytosis and recruiting NK-mediated ADCC to PD-L1-expressing tumors (and deactivating the PD-1 immune checkpoint). Some embodiments of this structure are shown in Example 8.

[0119] CD16A / CD47 / CD33: This structure is suitable for treating tumors expressing CD33, promoting phagocytosis, recruiting NK cells to mediate ADCC, and extending its half-life in the body. In various embodiments, MVSCA may be bivalent for the anti-CD-47 and / or anti-CD33 binding domains. In some embodiments, the binding domains are arranged in a different order, but the anti-HSA domain should be located at the terminal position if cleaved. Depending on the linker used, the anti-HSA domain (once HSA is bound) may or may not inhibit binding to CD47, and if inhibition occurs, it can be restored by cleaving a cleavable linker. In addition to describing this configuration of MVSCA as a means of realizing one or more functions of its components, MVSCA can also be considered a means of promoting phagocytosis and recruiting NK-mediated ADCC to CD33-expressing tumors. Some embodiments of this structure are shown in Example 8.

[0120] Divalent anti-CD33MVSCA: These structures are suitable for treating diseases associated with the deposition of insoluble substances by, for example, blocking the inhibition of phagocytosis by microglial cells. Such diseases include Alzheimer's disease and dry AMD. The HSA / CD33 / CD33 structure will have a prolonged circulating half-life. The FC5 / CD33 / CD33 structure will cross the blood-brain barrier. The FC5 / CD33 / CD33 / HAS structure will have a prolonged circulating half-life and cross the blood-brain barrier. The simple CD33 / CD33 structure is suitable for local injection into the eye or brain, in which case the ability to prolong the circulating half-life or cross the blood-brain barrier is negligibly important. In some embodiments, the binding domains are arranged in a different order. In addition to this description of MVSCA configurations as means of realizing one or more functions of the components, MVSCA can also be considered a means of promoting phagocytosis (by microglia) of insoluble deposits. Several embodiments of this structure are shown in Example 9.

[0121] (Use of disclosed antibodies) The disclosed antibodies are useful in medicine. Terms such as “treatment” and “to treat” refer to the medical management of a patient with the intention of treating, improving, stabilizing or preventing a disease, condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, condition, or disorder, and causal treatment, i.e., treatment aimed at eliminating the cause associated with the disease, condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment for the relief of symptoms rather than a cure for the disease, condition, or disorder, preventive treatment, i.e., treatment aimed at minimizing, partially or completely inhibiting, the onset of the associated disease, condition, or disorder, and adjunct treatment, i.e., treatment employed in conjunction with other specific treatments aimed at improving the associated disease, condition, or disorder.

[0122] Furthermore, the use of the antibodies disclosed herein in diagnosis and imaging is also intended.

[0123] Furthermore, the terms “to treat” or “to cure” broadly include any type of therapeutic act, including the diagnosis, mitigation, or prevention of disease or a condition in humans or other animals, or any act that affects any structure or function of the body of a human or other animal. Therapeutic acts include the administration of pharmaceuticals, dosage forms, and pharmaceutical compositions described herein to a patient by a medical professional, the patient themselves, or any other person, particularly in the various ways disclosed herein. Therapeutic acts include orders, instructions, and advice from medical professionals such as physicians, physician assistants, and clinical nurses, which are carried out by any other person, including other medical professionals or the patient themselves. This includes, for example, instructing a patient to undergo or to perform diagnostic procedures, such as cancer diagnosis and staging, so that the patient may ultimately receive appropriate treatment. In some embodiments, the command, instruction, and advice aspects of a therapeutic act may include encouraging, inducing, or mandating the selection of a particular drug or combination of drugs for the treatment of a condition, and ensuring that the drug is actually used, such as by an insurance company or pharmacy benefits management company, by approving insurance coverage for a drug, refusing the use of an alternative drug, including a drug in a prescription or excluding an alternative drug, or providing a financial incentive for using the drug. In some embodiments, a therapeutic act may also include encouraging, inducing, or mandating the selection of a particular drug for the treatment of a condition and ensuring that the drug is actually used, such as by a policy or practice standard that may be established by a hospital, clinic, health maintenance organization, medical practice, or group of physicians. All such commands, instructions, and advice are considered to be therapeutic benefits subject to compliance with those instructions. In some cases, a patient may receive a financial benefit by complying with such commands, instructions, and advice. In some cases, a healthcare professional may receive a financial benefit by complying with such commands, instructions, and advice.

[0124] The disclosed monospecific HCAb, as well as multivalent single-chain antibodies with specificity for CD47, HSA, PD-L1, CD33, CD16, and LAG3, are useful in the treatment of cancer. Each antibody is designed to treat a specific class of cancer based on the antigen-binding specificity it possesses.

[0125] This disclosure provides a method for treating cancer, comprising administering an effective amount of the antibody disclosed herein, or a pharmaceutical composition containing the antibody, to a patient in need of such treatment.

[0126] Examples of cancers that can be treated by the disclosed method include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-associated lymphoma, AIDS-associated malignancies, anal cancer, astrocytoma, cholangiocarcinoma, bladder cancer, osteosarcoma, brainstem glioma, brain tumor, breast cancer, bronchial adenoma / bronchial carcinoid, carcinoid tumor, islet cell carcinoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, ovarian epithelial carcinoma, esophageal cancer, Ewing's tumor family, extracranial germ cell tumor, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, germ cell tumor, gestational trophoblastoma, and hirsutism. Alveolar leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, non-small cell lung cancer, small cell lung cancer, non-Hodgkin lymphoma, Waldenström macroglobulinemia, malignant mesothelioma, malignant thymoma, medulloblastoma, melanoma, Merkel cell carcinoma, cervical squamous cell carcinoma, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytic neoplasm, mycosis fungoides, bone This includes myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, pancreatic cancer, parathyroid cancer, penile cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, Sézary syndrome, skin cancer, cervical squamous cell carcinoma, testicular cancer, thymoma, thyroid cancer, trophoblastoma, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[0127] The effectiveness of cancer treatment is typically measured using the term "remission." While there are no limitations on the techniques used to monitor remission, • Some lumps or tumors, including those containing lymph nodes, can be felt and measured externally during a physical examination. • Some internal cancerous tumors can be seen on X-ray or CT scans and measured with a ruler. • Blood tests, including those measuring organ function, can be performed. • Tumor marker tests can be performed for specific types of cancer. This is similar to the tests used for diagnosing cancers such as [list of cancers].

[0128] Regardless of the test used—whether it's a blood test, cell count, or tumor marker test—the results are repeated at regular intervals so that they can be compared to the same test performed previously.

[0129] Cancer remission is defined by several conditions. • Complete remission - All cancer or tumors disappear, leaving no trace of the disease. Tumor marker levels (if applicable) may decrease to the normal range. • Partial remission - The cancer has shrunk by a certain percentage, but the disease remains. Tumor marker levels (if applicable) have decreased (or have increased based on tumor markers, indicating a reduction in the tumor burden), but traces of the disease remain. • Stable disease - The cancer is neither growing nor shrinking, and there is no change in the amount of disease. There are no significant changes in tumor markers (if applicable). • Disease progression - Cancer is growing, and the disease is worse than before treatment. Tumor marker tests (if applicable) show an increase in tumor markers.

[0130] Other measures of cancer treatment effectiveness include overall survival (time from diagnosis or initiation of the evaluated treatment to death from any cause), cancer-free survival (length of time from complete remission until cancer is no longer detectable), and progression-free survival (length of time from disease stabilization or partial remission until no recurrence of tumor growth is detected).

[0131] There are two criteria for evaluating remission of solid tumors in terms of tumor size (tumor burden): the WHO criteria and the RECIST criteria. These methods measure solid tumors and compare current tumor measurements with past measurements or with future measurements to adjust treatment strategies. The WHO method measures the long and short axes of solid tumors and calculates the product of these two measurements. If there are multiple solid tumors, all products are added together. The RECIST method measures only the long axis. If there are multiple solid tumors, all long axis measurements are added together. However, for lymph nodes, the short axis is measured instead of the long axis.

[0132] This disclosure provides a method for treating eye disorders, comprising administering the antibodies disclosed herein to a patient in need of such treatment. Examples of eye disorders include age-related macular degeneration (AMD), such as wet AMD and dry AMD, or macular edema, such as diabetic macular edema. In some embodiments, the eye disorder is a retinal disorder.

[0133] Furthermore, this disclosure also provides, but is not limited to, methods for treating neurodegenerative diseases including Alzheimer's disease, Lewy body disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, leukodystrophy, progressive supranuclear palsy, neuroinflammation, inflammatory demyelinating diseases, dementia, or neuropathy. In one embodiment, the neurodegenerative disease is Alzheimer's disease.

[0134] The following examples, sequence listings, and drawings are provided to aid in understanding the present invention, and its true scope is defined in the appended claims.

[0135] (List of specific embodiments) The following list of embodiments is illustrative of various embodiments relating to the width, combinations and subcombinations, classes of invention, etc., described herein, but is not intended to be an exhaustive list of all embodiments supported herein.

[0136] Embodiment 1. A variable heavy chain (VHH) domain with antigen-binding specificity for CD47.

[0137] Embodiment 2. A VHH domain of Embodiment 1 having one amino acid sequence of SEQ ID NOs: 2-29 or 223.

[0138] Embodiment 3. A variable heavy chain (VHH) domain with antigen-binding specificity for PD-L1.

[0139] Embodiment 4. The VHH domain of Embodiment 3 having one amino acid sequence of SEQ ID NOs. 31-38.

[0140] Embodiment 5. A variable heavy chain (VHH) domain with antigen-binding specificity for human serum albumin (HSA).

[0141] Embodiment 6. The VHH domain of Embodiment 5 having one amino acid sequence of SEQ ID NOs. 40-48.

[0142] Embodiment 7. A variable heavy chain (VHH) domain with antigen-binding specificity for CD33.

[0143] Embodiment 8. The VHH domain of Embodiment 7 having one amino acid sequence of SEQ ID NOs. 50-78.

[0144] Embodiment 9. A variable heavy chain (VHH) domain with antigen-binding specificity for LAG3.

[0145] Embodiment 10. The VHH domain of Embodiment 9 having one amino acid sequence of SEQ ID NOs. 80-93.

[0146] Embodiment 11. A variable heavy chain (VHH) domain with antigen-binding specificity for CD16.

[0147] Embodiment 12. The VHH domain of Embodiment 11 having one amino acid sequence of SEQ ID NOs. 96-99.

[0148] Embodiment 13. A heavy-chain-only antibody (HCAb) containing one of the VHH domains from Embodiments 1 to 12.

[0149] Embodiment 14. An antibody comprising one or more constant domains and means for binding to CD47, HSA, PD-L1, CD33, CD16, or LAG3.

[0150] Embodiment 15. A multispecific antibody comprising one or more VHH domains of Embodiments 1 to 12, or means for binding to CD47, HSA, PD-L1, CD33, CD16, or LAG3.

[0151] Embodiment 16. One or more further resistance original A multispecific antibody of embodiment 15, further comprising a binding domain.

[0152] Embodiment 17. Further resistance original The binding domain is the multispecific antibody of Embodiment 16, which includes FC5 (SEQ ID NO: 222).

[0153] Embodiment 18. Further resistance original The binding domain is a multispecific antibody of Embodiment 16, comprising Fv or Fab.

[0154] Embodiment 19. A multispecific single-chain antibody (MVSCA), one of the multispecific antibodies described in Embodiments 15 to 17.

[0155] Embodiment 20. 2, 3, 4, 5, or 6 resistance original MVSCA of Embodiment 19, including a binding domain.

[0156] Embodiment 21. 1, 2, 3, or 4 resistance original MVSCA of embodiment 20 having binding specificity.

[0157] Embodiment 22. Anti-HSA, CD47, and PD-L1 recognition original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0158] Embodiment 23. Anti-hatching that recognizes HSA, CD47, and CD33 original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0159] Embodiment 24. Anti-HSA, LAG3, and PD-L1 recognition original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0160] Embodiment 25. Anti-HSA, LAG3, and CD33 original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0161] Embodiment 26. Anti-CD16, HSA, and PD-L1 recognition original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0162] Embodiment 27. Anti-CD16, HSA, and CD33 original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0163] Embodiment 28. Anti-CD16, HSA, CD47, and PD-L1 recognition originalAny one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0164] Embodiment 29. Anti-CD16, HSA, CD47, and CD33 original Any one of the MVSCAs of embodiments 19 to 21, including a binding domain.

[0165] Embodiment 30. Anti-CD16, preferably CD16A, is recognized by original An MVSCA of any one of embodiments 14-21 or 26-29, including a binding domain.

[0166] Embodiment 31. Two adjacent antibodies with identical specificity original Any one of the MVSCAs of embodiments 19 to 30, including a binding domain.

[0167] Embodiment 32. Two adjacent antibodies with identical specificity original The MVSCA of embodiment 31 has binding domains having short, non-bending linker means sandwiched between them.

[0168] Embodiment 33. The short, non-bendable linker means is the MVSCA of Embodiment 31, which consists of the amino acid sequence AAA (SEQ ID NO: 102).

[0169] Embodiment 34. Two adjacent anchors original The binding domain is one of the MVSCAs from embodiments 31 to 33 that binds to CD33.

[0170] Embodiment 35. Antibody that recognizes HSA original An MVSCA of embodiment 34 further including a binding domain.

[0171] Embodiment 36. An MVSCA of embodiment 34 or 35, further including FC5.

[0172] Embodiment 37. Two adjacent anchors original The binding domain is one of the MVSCAs from embodiments 31 to 33 that binds to PD-L1.

[0173] Embodiment 38. Two adjacent anchors original The binding domain is one of the MVSCAs from embodiments 31 to 33 that binds to LAG3.

[0174] Embodiment 39. Two adjacent anchors original The binding domain is one of the MVSCAs from embodiments 31 to 33 that binds to CD16.

[0175] Embodiment 40. Two adjacent anchors original The binding domain is one of the MVSCAs from embodiments 31 to 33 that binds to CD47.

[0176] Embodiment 41. Adjacent original Any one of the MVSCAs of embodiments 19 to 30, including a linker between the bonded domains.

[0177] Embodiment 42. The MVSCA of Embodiment 41 has a linker sandwiched between non-identical antigen-binding domains, which is L1 (SEQ ID NO: 100), L2 (SEQ ID NO: 101), or L4 (SEQ ID NO: 103).

[0178] Embodiment 43. Any one of the MVSCAs of Embodiments 19 to 30, comprising a flexible, non-cleavable linker means sandwiched between non-identical antigen-binding domains.

[0179] Embodiment 44. Antibody located at the N-terminus or C-terminus that binds to HSA. original Any one of the MVSCAs of embodiments 19 to 30, including a binding domain.

[0180] Embodiment 45. When it binds to HSA, it acts as an anti-HSA agent. original Binding domain and adjacent anti original The binding domain is an MVSCA of Embodiment 44, in which binding to the antigen is inhibited.

[0181] Embodiment 46. Anti-HSA original Binding domain and adjacent anti original The binding domain is the MVSCA of embodiment 45, which recognizes CD47.

[0182] Embodiment 47. A severable linker binds to the HSA. original Binding domain and adjacent anti original MVSCA of Embodiment 45 or 46, wherein the linkers sandwiched between the binding domains and capable of cleavage are L11*3 (SEQ ID NO: 104), L11*4 (SEQ ID NO: 105), L11*5 (SEQ ID NO: 106), L11*6 (SEQ ID NO: 107), L11*7 (SEQ ID NO: 108), L11*8 (SEQ ID NO: 109), L11*9 (SEQ ID NO: 110), L11*10 (SEQ ID NO: 111), L11*11 (SEQ ID NO: 112), L11*12 (SEQ ID NO: 113), L11*13 (SEQ ID NO: 114), L11*14 (SEQ ID NO: 115), L11*15 (SEQ ID NO: 116), L11*16 (SEQ ID NO: 117), L11*17 (SEQ ID NO: 118), or L11*18 (SEQ ID NO: 119).

[0183] Embodiment 48. A severable linker means bonds with the HSA. original Binding domain and adjacent anti original An MVSCA of embodiment 45 or 46, sandwiched between binding domains.

[0184] Embodiment 49. All resistance original Any one of the MVSCAs from Embodiments 19 to 48, wherein the binding domain is a VHH domain.

[0185] Embodiment 50. A pharmaceutical composition comprising any one of the VHH domains or antibodies from Embodiments 1 to 49.

[0186] Embodiment 51. A pharmaceutical composition comprising means for binding to HSA, means for extending the in vivo half-life of a multispecific antibody or MVSCA, and means for reversibly inhibiting the binding activity of adjacent binding domains.

[0187] Embodiment 52. A pharmaceutical composition comprising means for binding to CD or means for reducing the inhibition of phagocytosis.

[0188] Embodiment 53. A pharmaceutical composition comprising means for binding to CD16 or CD16A, or means for recruiting ADCC via NK.

[0189] Embodiment 54. A pharmaceutical composition comprising means for binding to PD-L1, means for binding to a PD-L1 tumor antigen, means for blocking PD-1, or means for deactivating a PD-1 immune checkpoint.

[0190] Embodiment 55. A pharmaceutical composition comprising means for binding to a tumor antigen, means for binding to a PD-L1 tumor antigen, or means for binding to a CD33 tumor antigen.

[0191] Embodiment 56. A pharmaceutical composition comprising means for binding to LAG3, or means for deactivating a LAG3 immune checkpoint.

[0192] Embodiment 57. A pharmaceutical composition comprising means for deactivating an immune checkpoint, means for deactivating a PD-1 immune checkpoint, or means for deactivating a LAG3 immune checkpoint.

[0193] Embodiment 58. A pharmaceutical composition comprising means for binding to CD33, means for binding to a CD33 tumor antigen, means for promoting the removal of β-amyloid, or means for removing insoluble deposits.

[0194] Embodiment 59. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1.

[0195] Embodiment 60. A pharmaceutical composition comprising means for recruiting a T effector to a tumor expressing PD-L1.

[0196] Embodiment 61. A pharmaceutical composition comprising means for promoting phagocytosis of a tumor expressing PD-L1, and means for recruiting NK-mediated ACDD to a tumor expressing PD-L1.

[0197] Embodiment 62. A pharmaceutical composition comprising means for promoting phagocytosis of a tumor expressing CD33, and means for recruiting NK-mediated ACDD to a tumor expressing CD33.

[0198] Embodiment 63. A method for treating cancer, comprising administering one antibody from any of Embodiments 1 to 48, or one pharmaceutical composition from any of Embodiments 49 to 61, to a patient in need of treatment.

[0199] Embodiment 64. A method for treating Alzheimer's disease or retinal disease, comprising administering to a patient in need of treatment an antibody comprising a CD33-binding domain, which is one of the antibodies in Embodiments 7-8, 13-21, 34-36, or 48-49, or the pharmaceutical composition of Embodiment 58.

[0200] Embodiment 65. Antibodies recognize CD47, PD-L1, LAG3, or CD16. original The method of embodiment 64, which does not include a binding domain.

[0201] Embodiment 66. The retinal disease is dry AMD, according to the method of embodiment 64 or 65.

[0202] Each of embodiments 63 to 66 has a corresponding embodiment for use in therapy, for use in the manufacture of pharmaceuticals, for use in therapy, and for use in the manufacture of pharmaceuticals.

[0203] (Examples) (Example 1) Anti-CD47HCAb antibody Isolation of anti-CD47HCAb antibodies from immunized llamas.

[0204] (Immunity conferred) Two llamas were immunized with Abcore Inc (Ramona, CA) according to a standard protocol. Recombinant human CD47 (extracellular domains 19-139, SEQ ID NO: 1) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization) (Difco, BD Biosciences). 50 μg per llama was administered subcutaneously every other week for six doses. On day 45, serum was collected from llamas immunized with recombinant CD47 protein, and antibody titers against hCD47 were measured by ELISA. For ELISA, 96-well maxisoape plates (Nunc) were coated with 100 ng / well of hCD47. After blocking and adding diluted serum samples, the presence of anti-CD47 antibodies was indicated using horseradish peroxidase (HRP)-conjugated goat anti-llama IgG (H+L) antibody (Invitrogen).

[0205] Sequence ID 1: Extracellular domain of human CD47 (19-139, Q08722) QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP

[0206] (Construction and selection of phage libraries) Peripheral blood mononuclear cells were prepared from 45-day-old blood samples of llamas immunized with recombinant CD47 protein using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi Kit (Qiagen) according to the manufacturer's instructions and used as a starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were cloned into phagemid vectors to enable the production of recombinant phage particles that, after infection with helper phages, display VHH as a gene-III fusion protein on the surface of the phage particles. The phages were prepared by standard methods, filtered, and stored at 4°C for further use.

[0207] For the selection of CD47-conjugated phages, biotinylated CD47 was incubated with a phage library and then captured with streptavidin Dynabeads (Invitrogen). After thorough washing, conjugated phages were eluted with 1 mg / ml trypsin. Selective phages were rescued into E. coli TG1 cells. Colonies were picked and sequenced at BATJ, Inc. (San Diego, CA).

[0208] cDNA encoding CD47-binding VHH was synthesized at Atum (Newark, CA) with a histidine tag at the C-terminus, transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0209] CD47-conjugated phage colonies from an immunized llama phage library were sequenced, and the amino acid sequences for each VHH were determined as listed below (Table 2). The cDNA sequences based on these amino acid sequences were fused with human Fc cells and synthesized using a pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce recombinant anti-CD47HCAb antibodies. The expressed anti-CD47HCAb was purified using a HiTrap Protein A column.

[0210] A09-10 VHH was humanized based on the IGHV3-23 human germline sequence.

[0211] [Table 2-1] [Table 2-2]

[0212] In Table 2, VHH constitutes a means for coupling to CD47.

[0213] (Octet® binding analysis of anti-CD47HCAb molecules)

[0214] To measure the binding kinetics of human CD47 (R&D systems) and anti-CD47VHH, label-free Bio-Layer Interferometry (BLI) was used. An Octet® QK equipped with an Anti-Penta-His capture (HIS1K) biosensor chip (ForteBio®) was used. eAffinity measurements were performed. The assay was carried out at 30°C in 1× PBS buffer (Gibco®, PBS pH 7.2). The sample was agitated at 1000 rpm. Before analysis, the sensor was moistened for 15 minutes. The binding ability of purified anti-CD47VHH to the HIS1K sensor tip was tested. The tip was loaded with 20 μg / ml anti-CD47VHH. A capture level of 1.8–2 nm was obtained after 300 seconds of loading. For binding analysis, human CD47 antigen was prepared by diluting it in 1× PBS to concentrations of 100, 150, 250, and 350 nM. To monitor dissociation, association was initiated and monitored for 200 seconds, after which the tip was transferred to 1× PBS buffer (Gibco, PBS pH 7.2). Sensor data was collected and processed throughout the experiment and analyzed using Octet® data analysis software 7 (ForteBioR).

[0215] Table 3 shows the Octet® kinetic analysis of the binding affinity of anti-CD47 HCAbs. HCAbs A09-04, A09-06, A09-08, and A09-10 show pM binding affinity.

[0216] [Table 3]

[0217] (Flow cytometry analysis of the binding affinity of anti-CD47HCAb to CD47-overexpressing CHO cell lines)

[0218] 1 × 10⁶ units in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3) 6CD47-overexpressing CHO cells at [x] cells / ml were incubated with anti-CD47 HCAb at concentrations ranging from 100 nM to 0.00128 nM, or with B6H12 anti-CD47 antibody as a control, and incubated on ice for 45 minutes. The cells were washed with FACS buffer, and goat anti-human IgG Fc, FITC-conjugated antibody (ThermoFisher) was added according to the manufacturer's instructions, and then incubated at 4°C for 30 minutes. Data were acquired using a Guava EasyCyte HT system.

[0219] According to the above flow cytometry method, the binding affinity for anti-CD47 HCAb was measured as EC 50 as shown in Figure 1.

[0220] (Competitive ELISA binding assay of multispecific molecules with anti-CD47 domains)

[0221] A competitive ELISA binding assay was performed to screen CD47-binding multispecific molecules 1511 (SEQ ID NO: 156, CD16F-L1-HSA-L1-CD47-L3-CD47-L1-PDL1-L3-PDL1) and 3321 (SEQ ID NO: 159, CD16F-L1-HSA-L1-CD47-L1-CD33-L3-CD33), both of which contain anti-CD47 VHH A09-10 that competitively blocks the binding of CD47 antigen to its receptor SIPPα. Multispecific antibodies are identified by their binding domains (e.g., CD47) and linkers (e.g., L1 identified in Table 15) that separate the binding domains. On a 96-well plate, 100 ng of CD47-Fc (R&D systems) was coated per well, pre-incubated with 10 nM biotinylated human SIRPα and multispecific molecules 1511 and 3321 at different concentrations, and then HRP-conjugated streptavidin was added. Multispecific molecules 1511 and 3321 competitively block the binding of CD47 to its receptor SIRPα at EC50 as shown in Figure 2.

[0222] (Competitive Flow Cytometry Binding Analysis of Multispecific Molecules with Anti-CD47 Domains)

[0223] A competitive flow cytometry assay was performed to confirm that the multispecific molecules 1511 and 3321 blocked the binding of the CD47 antigen to its receptor SIRPα on cells that naturally express CD47. 1×10 6 cells / ml of Jurkat cells (ATCC) in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3) were incubated with 1511 or 3321 at concentrations ranging from 100 nM to 0.00128 nM, incubated on ice for 45 minutes, and then 25 nM of SIRPα-Fc (R&D systems) was added and incubated for an additional 45 minutes. The cells were washed with FACS buffer, and a goat anti-human VHH FITC-conjugated antibody (Jackson Immuno Research) was added according to the manufacturer's instructions and then incubated at 4°C for 30 minutes. Data were acquired using a Guava EasyCyte HT system. The multispecific molecules 1511 and 3321 competitively blocked the binding of CD47 to its receptor SIRPα on the surface of Jurkat cells with an EC50, as shown in Figure 3.

[0224] (Human RBC Agglutination Assay of Multispecific Molecules with Anti-CD47 Domains)

[0225] Human blood samples were provided by healthy donors. Whole blood was centrifuged at 3000 rpm (1800 rcf) for 5 minutes to remove serum and buffy coat. The red blood cells were resuspended in approximately twice the volume of normal saline (0.9% NaCl) of the red blood cells, and the tubes were inverted and mixed. The red blood cells were centrifuged at 2000 rpm for an additional 20 minutes, and the red blood cells were mixed with normal saline to obtain a 6% (v / v) cell suspension. Next, the red blood cells were added to a 96-well round-bottom plate and mixed with different amounts of antibody (0 - 10 μg / ml). The plate was incubated at 37°C for 2 hours. Unlike the Hu5F9 anti-CD47 control antibody, the multispecific molecules 1511 and 3321 did not induce RBC agglutination, as shown in Figure 4.

[0226] Flow cytometry binding assays confirmed that the multispecific molecules 1511 and 3321, containing anti-CD47VHH, can selectively bind to the surface of tumor cells natively expressing CD47, but cannot bind to CD47 on the surface of RBC cells. (1 × 10⁶ molecules in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3)) 6 HL60 cells (ATCC) at a concentration of cells / ml or 10% washed human RBC cells (Rockland Immunochemicals, Inc.) were incubated with 1511 or 3321 at concentrations ranging from 500 nM to 0.00128 nM on ice for 45 minutes. Cells were washed with FACS buffer, and goat anti-human VHH FITC-conjugated antibody (Jackson Immuno Research) was added according to the manufacturer's instructions, followed by incubation at 4°C for 30 minutes. Data were acquired using the Guava EasyCyte HT system. The multispecific molecules 1511 and 3321 were EC as shown in Figure 4B. 50 It selectively bound to the surface of tumor cells natively expressing CD47, but did not bind to RBC cells.

[0227] (Antitumor activity of multispecific molecules containing an anti-CD47 domain)

[0228] 1E6 Raji-Luc cells were intravenously inoculated into NSG mice. Mice were treated daily with IV injections of either 10 mg / kg of the multispecific molecule 3321 or a PBS control. Representative bioluminescence images of Raji tumors are shown at the start of treatment (day 0), mid-experiment (day 3), and end of the experiment (day 7). The multispecific molecule 3321 protected xenotransplanted mice from human leukemia, as shown in Figure 5.

[0229] (Example 2) (Anti-PD-L1 HHCab antibody) (Isolation of anti-PD-L1 HCAb antibodies from immunized llamas)

[0230] Two llamas were immunized with Abcore Inc. according to a standard protocol. Recombinant human PD-L1 (extracellular domains 19-238, SEQ ID NO: 30) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). 50 μg per llama was administered subcutaneously every other week for six doses. On day 45, serum was collected from llamas immunized with recombinant PD-L1 protein, and antibody titers against PD-L1 were measured by ELISA. For ELISA, 96-well maxisorp plates were coated with 100 ng / well of PD-L1. After blocking and adding diluted serum samples, the presence of anti-PD-L1 antibodies was indicated using HRP-conjugated goat anti-llama IgG (H+L) antibody.

[0231] Sequence ID 30: Extracellular domain of human PD-L1 (19-238, Q9NZQ7) FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0232] Peripheral blood mononuclear cells were prepared from 45-day-old blood samples of llamas immunized with recombinant PD-L1 using Ficoll-Paque+ according to the manufacturer's instructions. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi Kit according to the manufacturer's instructions and used as a starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were recombinant into phagemid vectors to enable the production of recombinant phage particles that display VHH as a gene-III fusion protein on the surface of the phage particles after infection with helper phages. The phages were prepared by standard methods, filtered, and stored at 4°C for further use.

[0233] For the selection of PD-L1-bound VHH phages, biotinylated PD-L1 was incubated with a phage library and then captured with streptavidin Dynabeads. After thorough washing, bound phages were eluted with 1 mg / ml trypsin. Selective phages were rescued into E. coli TG1 cells. Colonies were picked and sequenced using BATJ, Inc.

[0234] cDNA encoding PD-L1-binding VHH was synthesized with a histidine tag at its C-terminus, transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0235] PD-L1-binding phage colonies from an immunized llama phage library were sequenced. The amino acid sequences for each VHH are listed below (Table 4). cDNA sequences based on these amino acid sequences were fused with human Fc cells and synthesized using the pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce recombinant anti-PD-L1HCAb antibody. The expressed anti-PD-L1HCAb antibody was purified using a HiTrap Protein A column. VHH cells PL14 and PL16 from two llamas were humanized based on the IGHV3-23 human germline sequence.

[0236] [Table 4]

[0237] The VHH in Table 4 constitutes a means for binding to PD-L1.

[0238] (Octet® Kinetic Coupling Analysis)

[0239] Octet (registered trademark) kinetic binding analysis was performed in the same manner as in Example 1. Briefly, the binding ability of the purified anti-PD-L1 VHH to the HIS1K sensor chip was tested. The chip was loaded with 20 μg / ml of anti-PD-L1 VHH. A capture level of 1.8 - 2 nm was obtained by loading for 300 seconds. For the binding analysis, human PD-L1 antigen was prepared by diluting it to concentrations of 100, 150, 250, and 350 nM in 1×PBS. To monitor dissociation, association was initiated and monitored for 200 seconds, after which the chip was transferred to a 1×PBS buffer without PD-L1 protein.

[0240] The Octet (registered trademark) kinetic analysis of the binding affinity of anti-CD47 HCAb is shown in Table 5. The analysis showed that PL14, PL16, and PL17 exhibited binding affinities in the pM range.

[0241] [Table 5]

[0242] (Flow cytometry binding analysis of anti-PD-L1 HCAb)

[0243] 1×10 6 cells / ml of PD-L1 overexpressing CHO cells were incubated with anti-PD-L1 HCAb in the range of 100 nM to 0.00128 nM and incubated on ice for 45 minutes. The cells were washed with FACS buffer, and goat anti-human IgG Fc-FITC conjugated antibody (ThermoFisher) was added, followed by incubation at 4°C for 30 minutes. Data was acquired using a Guava EasyCyte HT system. The binding affinity of anti-PD-L1 HCAb to PD-L1 of PD-L1 overexpressing CHO cells was measured as EC 50 as shown in Figure 6.

[0244] (Cell-based functional assay of multi-specific molecules having a PD-L1 binding domain)

[0245] APC / CHO-K1 cells expressing PD-L1 were seeded at 100K per well in a 96-well plate and incubated at 37 °C for 16 hours. Next, the multispecific molecule 1511 and the control antibody atezolizumab were serially diluted 1:3 starting from 100 nM and added to the cell wells at 25 μl / well. Finally, PD-1 effector cells (cells expressing PD-1 and luciferase) were added and incubated at 37 °C for 6 hours. After 6 hours, 75 μl of Bio-Glo™ luciferase assay reagent was added and luminescence was measured using a VICTOR multiplate reader. Data analysis was performed using GraphPad Prism software.

[0246] Cell-based functional data showed that the multispecific molecule 1511 and the control antibody atezolizumab completely blocked PD-L1 activity at the EC50, as shown in Figure 7.

[0247] (Antitumor activity of the multispecific molecule having a PD-L1 binding domain)

[0248] Mouse colorectal cancer MC38-hPD-L1 cells (Biocytogen Co., Ltd; 5×10 5 ) were implanted subcutaneously into syngeneic B-hPD-L1 mice (female, 6 weeks old, n = 6). When the tumor volume reached approximately 100 mm 3 , the mice were grouped and at that time, treated with the multispecific molecule 1518 (SEQ ID NO: 157, CD16F-L1-HSA-L1-CD47-L3-CD47-L1-PDL1-L3-PDL1) at the doses and schedule shown in Figure 8A. Changes in body weight during the treatment period are shown in Figure 8B. As shown in Figure 8A, the multispecific molecule 1518A1 had an effect of controlling tumor growth in B-hPD-L1 mice. Values are represented as mean ± SEM.

[0249] (Example 3) (Anti-HSA HCAb antibody) (Isolation of anti-HSA antibody)

[0250] Similar to Example 1, llamas were immunized by Abcore Inc. with recombinant human HSA (SEQ ID NO: 39) mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization).

[0251] Human serum albumin (SEQ ID NO: 39) MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0252] For the selection of anti-HSA VHH phages, biotinylated HSA was incubated with a phage library and then captured with streptavidin Dynabeads (Invitrogen). After thorough washing, bound phages were eluted with 1 mg / ml trypsin. Selective phages were rescued into E. coli TG1 cells. Colonies were picked and sequenced at BATJ, Inc. (San Diego, California).

[0253] cDNA encoding HSA-specific VHH was synthesized with a histidine tag at its C-terminus, transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0254] HSA-conjugated phage colonies from a llama phage library were sequenced, and the amino acid sequences for each VHH were listed below (Table 6). cDNA sequences based on these amino acid sequences were synthesized in a pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce recombinant single-domain antibodies (sdAbs) with a histidine tag at the C-terminus. The expressed sdAbs were purified using HisTrap HP.

[0255] Two Lama VHH cells, HS5 and HS10, were humanized based on the IGHV3-23 human germline sequence.

[0256] [Table 6]

[0257] VHH constitutes a means for coupling with HSA.

[0258] (Octet® Kinetic Coupling Analysis)

[0259] Octet® kinetic coupling analysis was performed in the same manner as in Example 1, and the results are shown in Table 7 and Figure 9. HS5, HS6, HS12, and HS27 clones showed affinity for HSA. Cross-species activity was confirmed and is shown in Table 8.

[0260] [Table 7]

[0261] [Table 8]

[0262] (Example 4) (Anti-CD33HCAb antibody) (Isolation of CD33VHH antibody)

[0263] Similar to Example 1, llamas were immunized with recombinant human CD33 (SEQ ID NO: 49) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization) at Abcore Inc. to prepare a phage library.

[0264] Human CD33 (Sequence ID 49, P20138, 18-259) DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHV TDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH

[0265] For the selection of anti-CD33VHH phages, biotinylated CD33 was incubated with a phage library and then captured with streptavidin Dynabeads. After thorough washing, bound phages were eluted with 1 mg / ml trypsin. Selective outputs were rescued into E. coli TG1 cells. Colonies were picked and sequenced in BATJ, Inc.

[0266] cDNA encoding CD33-bound VHH was synthesized with a histidine tag at its C-terminus, transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0267] CD33-conjugated phage colonies from an immunized llama phage library were sequenced, and the amino acid sequences for each VHH were listed below (Table 9). cDNA sequences based on these amino acid sequences were fused with human Fc cells and synthesized using a pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce recombinant anti-CD33HCAb antibodies. The expressed anti-CD33HCAb antibodies were purified using a HiTrap Protein A column.

[0268] 33-14, one of the Lama VHH strains, was humanized based on the IGHV3-23 human germline.

[0269] [Table 9-1] [Table 9-2]

[0270] VHH in Table 9 constitutes a means for coupling to CD33.

[0271] Octet® kinetic coupling analysis was performed in the same manner as in Example 1, and K D The results are shown in Figure 10 and Table 10.

[0272] [Table 10]

[0273] (Example 5) (anti-LAG3VHH) (Isolation of anti-LAG3VHH antibody)

[0274] Llamas were immunized with Abcore Inc. according to a standard protocol. Recombinant human LAG3 (extracellular domains 19-238, SEQ ID NO: 79) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). 50 μg per llama was administered subcutaneously every other week for six doses. On day 45, serum was collected from llamas immunized with recombinant human LAG3 protein, and antibody titers against human LAG3 were determined by ELISA. For ELISA, 96-well maxisorp plates were coated with 100 ng / well of LAG3. After blocking and adding diluted serum samples, the presence of anti-LAG31 antibodies was indicated using antibodies whose antiserum titers were determined by ELISA. 96-well maxisorp plates were coated with 100 ng / well of hLAG3. After blocking and adding diluted serum samples, the presence of anti-LAG31 antibodies was indicated using HRP-conjugated goat anti-llama IgG(H+L) antibody.

[0275] Extracellular domain of human LAG3 (SEQ ID NO: 79, P18627, 23-450) VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFS LWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPW GCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNA TVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL

[0276] Phage libraries were prepared in the same manner as in Examples 1-4. cDNA encoding LAG3-binding VHH was synthesized with a histidine tag at the C-terminus, transiently transfected into HEK293 cells, and purified of LAG3-binding VHH by IMAC chromatography.

[0277] LAG3-conjugated phage colonies from an immunized llama phage library were sequenced, and the amino acid sequences for each VHH were listed below (Table 11). cDNA sequences based on these amino acid sequences were fused with human Fc cells and synthesized using a pJ607 expression vector. This expression plasmid was transfected into HEK293 cell lines to produce recombinant anti-LAG3HCAb antibodies. The expressed anti-LAG3HCAb antibodies were purified using a HiTrap Protein A column.

[0278] LG9, one of the Lama VHH strains, was humanized based on the IGHV3-23 human germline.

[0279] [Table 11]

[0280] The VHH in Table 11 constitutes a means for binding to LAG3.

[0281] Octet® binding analysis of anti-LAG3VHH was performed in the same manner as in Examples 1-4, and the results are shown in Table 12.

[0282] [Table 12]

[0283] (Example 6) (anti-CD16VHH) (Isolation of anti-CD16VHH antibody)

[0284] Llamas were immunized with Abcore Inc. according to a standard protocol. Recombinant human CD16A (SEQ ID NO: 94) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). 50 μg per llama was administered subcutaneously every other week for six doses. On day 45, serum was collected from the immunized llamas, and antibody titers were determined by ELISA. For ELISA, 96-well maxisorp plates were coated with 100 ng / well of antigen. After blocking and adding diluted serum samples, the presence of specific antibodies was indicated using HRP-conjugated goat anti-llama IgG (H+L) antibody.

[0285] Human CD16A (SEQ ID NO: 94) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETTVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0286] Human CD16B (SEQ ID NO: 95) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRW VFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI

[0287] A phage library was prepared in the same manner as in Example 1. The cDNA encoding CD16A-binding VHH was synthesized with a histidine tag at the C-terminus, transiently transfected into HEK293 cells, and purified of CD16A-binding VHH by IMAC chromatography.

[0288] CD16A-conjugated phage colonies from an immunized llama phage library were sequenced, and the amino acid sequences for each VHH were listed below (Table 13). cDNA sequences based on these amino acid sequences were fused with human Fc cells and synthesized using a pJ607 expression vector. This expression plasmid was transfected into the HEK293 cell line to produce recombinant anti-CD16HCAb antibodies. The expressed anti-CD16HCAb antibodies were purified using a HiTrap Protein A column.

[0289] CD16F1, one of the Lama VHH strains, was humanized based on the IGHV3-23 human germline.

[0290] [Table 13]

[0291] VHH in Table 13 constitutes a means for coupling to CD16.

[0292] Octet® binding analysis of anti-CD16VHH was performed in the same manner as in Example 1, and the results are shown in Table 14 and Figure 11.

[0293] [Table 14]

[0294] CD16-F1 is selective for CD16A, while CD16-E11 couples to both CD16A and CD16B.

[0295] Both CD16F1 and CD16E11 are agonists of the anti-CD16VHH antibody and activated CD16A in the Jurkat-Lucia NFAT-CD16 ADCC reporter assay (Invivogen).

[0296] Functional assays of multispecific molecules 1511 and 3321 (including anti-CD16A VHH CD16F1), as well as control anti-CD47 antibodies, B6H12 IgG1, and B6H12 IgG4, were performed using the Jurkat-Lucia NFAT-CD16 reporter assay (Invivogen), and the results are shown in Figure 12. The data showed that CD16F1 is a potent CD16A agonist.

[0297] (Example 7) (Triple-specific single-chain antibody (HSA / CD47 / PD-L1 or HSA / LAG3 / PD-L1)) To construct triple-specific single-chain antibodies, anti-HSA, anti-CD47, and anti-PD-L1 or anti-CD33 VHH sequences, or anti-HSA, anti-LAG3, and anti-PD-L1 or anti-CD33 VHH sequences, were fused via linkers in six different ways using recombinant DNA technology (Figure 13). Figure 13 shows the structures of exemplary triple-specific molecules and anti-HSA / CD47 / PD-L1, anti-HSA / CD47 / CD33, and anti-HSA / LAG3 / PD-L1 and anti-HSA / LAG37 / CD33 antibodies. Exemplary non-cleavable and cleavable linker sequences are shown in Table 15. These constitute linker means, or means for linking protein domains. These means are further characterized as cleavable or non-cleavable. The amino acid sequences of the exemplary triple-specific molecules are shown in Table 16. Linker sequences are underlined in Tables 16 and 17.

[0298] [Table 15]

[0299] [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4] [Table 16-5] [Table 16-6]

[0300] The anti-HSA domain that binds to HSA can extend the half-life of MVSCA in the body. It can also inhibit the activity of other domains, which may be desirable for MVSCA distributed throughout the body in some cases, but undesirable if MVSCA is located at the site of intended action, such as a tumor. Therefore, the anti-HSA domain and other antigen-binding domains are preferably linked by a cleavable linker that can be cleaved at the site of intended action. In this way, MVSCA can act as a prodrug. MVSCAs having a linker containing a protease-cleavable sequence used between HSA VHH and CD47 VHH or LAG3 VHH, and other linkers used to link CD47 VHH and PD-L1 or LAG3 VHH and PD-L1 are shown in Figure 13 and Table 9. Figure 14B shows the SDS-PAGE analysis of the antibody in Figure 14A after protease digestion.

[0301] (Protease analysis)

[0302] (1) MMP-9 activity assay Recombinant human MMP-9 (rhMMP-9, R&D Systems) was diluted to 100 μg / ml in assay buffer (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij-35, pH 7.5). Then, rhMMP-9 was activated by adding APMA (ρ-aminophenylmercury acetate, Sigma) to a final concentration of 1 mM and incubating at 37°C for 24 hours. The activated rhMMP-9 was titrated with an equal volume of 20 μM antibody in the assay buffer and incubated at room temperature for 1 hour. The resulting digested substrate was analyzed by SDS-PAGE.

[0303] (2) u-plasminogen activator (uPA, urokinase) activity assay The substrate was diluted to 200 μM in assay buffer (50 mM Tris, 0.01% Tween 20, pH 8.5) and titrated with an equal volume of recombinant human u-plasminogen activator (rhuPA, R&D Systems) in the assay buffer. The reaction mixture was incubated at room temperature for 1-2 hours, and the resulting digested substrate was analyzed by SDS-PAGE.

[0304] (3) Matryptase activity assay The substrate was diluted to 200 μM in assay buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween20) and titrated with an equal volume of recombinant human matryptase (R&D Systems) in the assay buffer. The reaction mixture was incubated at room temperature for 1–2 hours, and the resulting digested substrate was analyzed by SDS-PAGE.

[0305] Polyacrylamide gel electrophoresis (SDS-PAGE). Denatured SDS-PAGE was performed according to the specifications of Invitrogen NuPAGE®. Briefly, 7.5 μL of protein sample (3 μg protein) was mixed with 2.5 μL of 4× LDS sample loading buffer (Invitrogen) and heated at 70°C for 10 minutes. Then, the sample was loaded onto a precast NuPAGE Novex 4-12% Bis-Tris 1.0 mm minigel (Invitrogen). Next, 5 μL of pre-stained SDS-PAGE standard (Bio-Rad) was loaded onto each gel run. Using a constant voltage (200 V), electrophoresis was performed at room temperature for approximately 45 minutes in a solution of 1× NuPAGE MOPS SDS running buffer (Invitrogen) until the dye front reached the edge of the 60 mm gel. The gel was stained with SimplyBlue SafeStain (Invitrogen).

[0306] Figure 15 shows real-time kinetic binding analysis of PD-L1 / pro-CD47 (HSA-CD47-PD-L1 antibody) versus PD-L1 / activated CD47 (the same antibody with the HSA-binding domain cleaved) in the presence of 10 mg / ml HSA. PD-L1 / pro-CD47 did not bind to CD47 at all or very little, while PD-L1 / activated CD47 showed robust binding to CD47. There was no difference or effect on PD-L1 binding in this assay.

[0307] (Example 8) (Multispecific molecules including CD16A / HSA / CD47 / (PD-L1 or CD33)) To construct multispecific molecules, anti-CD16A, anti-HSA, anti-CD47, and anti-PD-L1 or CD33 VHH sequences were fused with linkers in eight different ways using recombinant DNA technology. Figure 16 shows the structures of exemplary multispecific molecules for anti-CD16A, anti-HSA, anti-CD47, and anti-PD-L1 or CD33 VHH. The amino acid sequences of the exemplary multispecific molecules are shown below (Table 17).

[0308] Figure 17A compares the lengths of the linkers G4SG3S(L1, SEQ ID NO: 100) versus (G4S)3(L4, SEQ ID NO: 103) between VHH2 and VHH3 in a flow cytometry binding assay in HL60 cells. Since HL60 cells express CD47 but not PD-L1, the binding of 1518-HS5 (SEQ ID NO: 173) and 1518-HS5-GS15 (SEQ ID NO: 184) indicated that two molecules were binding to a single CD47 on the surface of HL60 cells. Longer linkers such as (G4S)3 (GS15 being a 15-amino acid linker) versus G4SG3S (9 amino acids) improved CD47 binding, with EC50 values ​​of 8.4 versus 26 nM.

[0309] [Table 17-1] [Table 17-2] Table 17-3 Table 17-4 Table 17-5 Table 17-6 Table 17-7 Table 17-8 Table 17-9 Table 17-10 Table 17-11 Table 17-12 Table 17-13 Table 17-14 Table 17-15 Table 17-16 Table 17-17 Table 17-18 Table 17-19 [Table 17-20]

[0310] Octet® binding analysis of multispecific molecules was performed in the same manner as in Example 8, and the results are shown in Figures 17 and 18.

[0311] (Example 9) (MVSCA containing anti-CD33 domain for the treatment of Alzheimer's disease and retinal diseases)

[0312] [Table 18]

[0313] Each of the four MVSCAs in Table 18 contains an anti-CD33 pair linked by linker L3 (sequence AAA; SEQ ID NO: 102). The first in Table 18 is hHS5-L1-H33-14-L3-H33-14 and contains an N-terminal anti-HSA domain to increase its half-life in the body. The second in Table 18 is FC5-L1-H33-14-L3-H33-14 and contains an N-terminal FC5 nanobody domain to facilitate cross-brain barrier passage. The third in Table 18 is FC5-L1-H33-14-L3-H33-14-L1-hHS5 and contains an N-terminal FC5 nanobody domain to facilitate cross-brain barrier passage and a C-terminal anti-HSA domain to increase its half-life in the body. These configurations are generally suitable for systemic administration, such as intravenous or subcutaneous injection or infusion. The fourth item in Table 18 is H33-14-L3-H3314, a bivalent, single-specific MVSCA with specificity only for CD33. Its smaller size makes it more suitable for local injection into the brain or eye.

[0314] The amino acid sequence of the FC5 nanobody domain is: This is EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSS (Sequence ID 222).

[0315] (Example 10) (Alignment of multiple sequences) Figures 21-26 show Clustal O(1.2.4) alignments of multiple sequences for each specificity of the VHH sequences disclosed herein, allowing for easy identification of identical, conserved, and highly variable positions. Below each alignment position, symbols are displayed, where an asterisk indicates identity, a colon indicates high conservation, a period indicates low conservation, and a space indicates near-non-conservation between aligned sequences.

[0316] Unless otherwise specified, all figures representing quantities such as component amounts, molecular weights, and reaction conditions used in the specification and claims should be understood in all cases to be added or subtracted by the term "approximately." As used herein, the terms "approximately" and "about" mean within 10-15%, preferably within 5-10%. Thus, unless otherwise indicated, the numerical parameters shown herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by the invention. At the very least, without attempting to limit the application as an equivalent to the claims, each numerical parameter should be interpreted at least by applying normal rounding means in light of the number of significant figures reported. Although the numerical ranges and parameters representing the broad scope of the invention are approximations, the numerical values ​​shown in specific examples are reported as accurately as possible. However, any numerical value inherently contains errors that inevitably arise from the standard deviation observed in each test measurement.

[0317] The terms “a,” “an,” “the,” and similar references used in the context describing the present invention (particularly in the context of the following claims) should be construed to cover both singular and plural forms unless otherwise stated or unless the context clearly contradicts this. The enumeration of value ranges in this specification is intended simply as abbreviation for referring individually to each individual value contained within the range. Unless otherwise stated herein, individual values ​​are incorporated herein as if they were individually listed herein. All methods described herein may be performed in any suitable order unless otherwise stated herein or unless the context clearly contradicts this. All embodiments or use of exemplary language (e.g., “like”) provided herein are intended to better illustrate the present invention and do not limit the scope of the invention as otherwise stated in the claims. Nothing described herein should be construed as indicating non-claimed elements essential to the practice of the present invention.

[0318] The grouping of alternative elements or embodiments of the present invention disclosed herein should not be construed as limitation. Each group's components may be referenced and claimed individually or in any combination with other components of the group or other elements found herein. For convenience or patentability reasons, it is anticipated that one or more components of a group may be included in or removed from the group. If such inclusion or removal occurs, this specification shall be deemed to include the modified group and shall satisfy all descriptions of the Markush group used in the appended claims.

[0319] This specification describes several embodiments of the invention, including the best mode for carrying out the invention as known to the inventor. Of course, variations of these described embodiments will be obvious to those skilled in the art by reading the preceding description. The inventor expects those skilled in the art to use such variations appropriately, and the inventor intends to carry out the invention in ways other than those specifically described herein. Accordingly, the invention includes all modifications and equivalents of the subject matter described in the appended claims, as permitted by applicable law. Furthermore, any combination of the above elements with any conceivable modifications is included in the invention unless otherwise stated herein or unless it is clearly inconsistent with the context.

[0320] Some of the embodiments disclosed herein may be further limited in the claims by the use of the phrases "consisting of" or "essentially consisting of." Where used in a claim, whether at the time of filing or added by amendment, the transitional phrase "consisting of" excludes elements, steps, or components not specified in the claim. The transitional phrase "essentially consisting of" limits the scope of the claim to those that do not substantially affect a particular material or step and its basic and novel features. Embodiments of the invention claimed in this manner are essentially or expressly described and enabled herein.

[0321] Furthermore, numerous patents and printed publications are referenced throughout this specification. Each of the above references and printed publications is incorporated herein by reference in its entirety.

[0322] Finally, it should be understood that the embodiments of the present invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be adopted are within the scope of the invention. Therefore, alternative configurations of the present invention can be utilized in accordance with the teachings herein, not as examples but as limitations. Accordingly, the present invention is not limited exactly as shown and described.

[0323] (Note) (Note 1) (a) CD47, wherein the VHH domain has one amino acid sequence of sequence numbers 2-29 or 223, CD47, (b) PD-L1, wherein the VHH domain has one amino acid sequence of SEQ ID NOs. 31-38, (c) Human serum albumin (HSA), wherein the VHH domain has one amino acid sequence of sequence numbers 40-48, (d) CD33, wherein the VHH domain has one amino acid sequence of sequence numbers 50-78, CD33, (e) LAG3, wherein the VHH domain has one amino acid sequence of sequence numbers 80-93, or (f) CD16, wherein the VHH domain has one amino acid sequence of sequence numbers 96-99, CD16, A variable heavy chain (VHH) domain that has antigen-binding specificity for one of the following.

[0324] (Note 2) A heavy-chain-only antibody (HCAb) containing the VHH domain as described in Appendix 1.

[0325] (Note 3) Antibody having first binding specificity original A binding domain, and a second antibody having a second binding specificity different from the first binding specificity. original A multispecific antibody containing a binding domain, The first binding specificity has specificity for CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The CD47 binding specificity is represented by one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The PD-L1 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 31-38, (c) The HSA binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 40-48, (d) The CD33 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 50 to 78, (e) The LAG3 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 80-93, (f) The CD16 binding specificity is represented by a single amino acid sequence of sequence numbers 96-99. Multispecific antibodies.

[0326] (Note 4) The second resistance original The binding domain has specificity for CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The CD47 binding specificity is represented by one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The PD-L1 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 31-38, (c) The HSA binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 40-48, (d) The CD33 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 50 to 78, (e) The LAG3 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 80-93, (f) The CD16 binding specificity is represented by a single amino acid sequence of sequence numbers 96-99. The multispecific antibodies described in Appendix 3.

[0327] (Note 5) 1-5 additional resistances original A multispecific antibody further comprising a binding domain, The aforementioned further resistance original Each binding domain individually possesses specificity for CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The CD47 binding specificity is represented by one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The PD-L1 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 31-38, (c) The HSA binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 40-48, (d) The CD33 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 50 to 78, (e) The LAG3 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 80-93, (f) The CD16 binding specificity is represented by a single amino acid sequence of sequence numbers 96-99. The multispecific antibodies described in Appendix 3.

[0328] (Note 6) 1-4 further resistance original A multispecific antibody further comprising a binding domain, The aforementioned further resistance original Each binding domain has specificity for CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The CD47 binding specificity is represented by one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The PD-L1 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 31-38, (c) The HSA binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 40-48, (d) The CD33 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 50 to 78, (e) The LAG3 binding specificity is represented by a single amino acid sequence of SEQ ID NOs. 80-93, (f) The CD16 binding specificity is represented by a single amino acid sequence of sequence numbers 96-99. The multispecific antibodies described in Appendix 4.

[0329] (Note 7) A multispecific single-chain antibody (MVSCA), as described in any one of the appendices 3 to 6.

[0330] (Note 8) Linker L1 (SEQ ID NO: 100), Linker L2 (SEQ ID NO: 101), or Linker L4 (SEQ ID NO: 103) are not identical. original A multispecific antibody, as described in any one of appendices 3 to 7, sandwiched between one or more pairs of binding domains.

[0331] (Note 9) Antibody with the same specificity original A multispecific antibody as described in any one of appendices 3 to 8, comprising at least one pair of binding domains.

[0332] (Note 10) Antibody with the same specificity original The multispecific antibody according to Appendix 9, wherein at least one pair of binding domains are adjacent to each other and a linker L3 (SEQ ID NO: 102) is sandwiched between them.

[0333] (Note 11) Antibody located at the N or C terminus, which has specificity for HSA original A binding domain, and adjacent to it, an anti original A linker that can be cleaved is placed between the binding domain and the anti original A multispecific antibody, including a binding domain, as described in any one of the appendices 3 to 10.

[0334] (Note 12) The multispecific antibody as described in Appendix 11, wherein the cleavable linker is L11*3 (sequence number 104), L11*4 (sequence number 105), L11*5 (sequence number 106), L11*6 (sequence number 107), L11*7 (sequence number 108), L11*8 (sequence number 109), L11*9 (sequence number 110), L11*10 (sequence number 111), L11*11 (sequence number 112), L11*12 (sequence number 113), L11*13 (sequence number 114), L11*14 (sequence number 115), L11*15 (sequence number 116), L11*16 (sequence number 117), L11*17 (sequence number 118), or L11*18 (sequence number 119).

[0335] (Note 13) All said resistance original A multispecific antibody described in any one of the appendices 3 to 12, wherein the binding domain is a VHH domain.

[0336] (Note 14) Anti-HSA, CD47, and PD-L1 recognition original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0337] (Note 15) Anti-hatching that recognizes HSA, CD47, and CD33 original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0338] (Note 16) Anti-HSA, LAG3, and PD-L1 recognition original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0339] (Note 17) Anti-HSA, LAG3, and CD33 original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0340] (Note 18) Anti-CD16, HSA, and PD-L1 recognition original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0341] (Note 19) Anti-CD16, HSA, and CD33 original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0342] (Note 20) Anti-CD16, HSA, CD47, and PD-L1 recognition original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0343] (Note 21) Anti-CD16, HSA, CD47, and CD33 original A multispecific antibody containing a binding domain, as described in any one of the appendices 3 to 13.

[0344] (Note 22) Antibody with specificity for CD33 original A multispecific antibody containing a pair of binding domains.

[0345] (Note 23) Antibody specific to HSA, FC5 nanobody (SEQ ID NO: 222), or both original A multispecific antibody as described in Appendix 22, further comprising a binding domain.

[0346] (Note 24) A pharmaceutical composition comprising the VHH domain described in Appendix 1, the HCAb described in Appendix 2, or a multispecific antibody described in any one of Appendix 3 to 21.

[0347] (Note 25) A method for treating cancer, comprising administering the pharmaceutical composition described in Appendix 24 to a patient in need of treatment.

[0348] (Note 26) A pharmaceutical composition comprising the multispecific antibody described in Appendix 22 or 23.

[0349] (Note 27) A method for treating Alzheimer's disease or retinal disease, comprising administering the pharmaceutical composition described in Appendix 24 to a patient in need of treatment.

[0350] (Note 28) The retinal disease is dry age-related macular degeneration, as described in Appendix 27.

Claims

1. A VHH domain present in a heavy chain antibody having antigen-binding specificity for CD47, wherein the VHH domain has one amino acid sequence of SEQ ID NOs: 2-29 or 223.

2. A heavy chain-only antibody (HCAb) containing the VHH domain described in claim 1.

3. A multispecific antibody comprising an antigen-binding domain having a first binding specificity and a second antigen-binding domain having a second binding specificity different from the first binding specificity, The first binding specificity is the binding specificity to CD47, The antigen-binding domain having the first binding specificity has one amino acid sequence of SEQ ID NOs: 2-29 or 223. Multispecific antibodies.

4. The second antigen-binding domain has binding specificity to PD-L1, HSA, CD33, LAG3, or CD16. (a) The second antigen-binding domain having binding specificity to PD-L1 has one amino acid sequence of SEQ ID NOs: 31 to 38, (b) The second antigen-binding domain having binding specificity to the HSA has one amino acid sequence of SEQ ID NOs: 40 to 48, (c) The second antigen-binding domain having binding specificity to CD33 has one amino acid sequence of SEQ ID NOs. 50 to 78, (d) The second antigen-binding domain having binding specificity to LAG3 has one amino acid sequence of SEQ ID NOs: 80 to 93, (e) The second antigen-binding domain having binding specificity to CD16 has one amino acid sequence of SEQ ID NOs: 96-99 The multispecific antibody according to claim 3.

5. A multispecific antibody further comprising 1 to 5 additional antigen-binding domains, Each of the further antigen-binding domains individually has binding specificity to CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The further antigen-binding domain having binding specificity to CD47 has one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The further antigen-binding domain having binding specificity to PD-L1 has one amino acid sequence of SEQ ID NOs: 31-38, (c) The further antigen-binding domain having binding specificity to the HSA has one amino acid sequence of SEQ ID NOs: 40-48, (d) The further antigen-binding domain having binding specificity to CD33 has one amino acid sequence of SEQ ID NOs: 50 to 78, (e) The further antigen-binding domain having binding specificity to LAG3 has one amino acid sequence of SEQ ID NOs: 80 to 93, (f) The further antigen-binding domain having binding specificity to CD16 has one amino acid sequence of SEQ ID NOs: 96-99 The multispecific antibody according to claim 3.

6. A multispecific antibody further comprising 1 to 4 additional antigen-binding domains, Each of the further antigen-binding domains has binding specificity to CD47, PD-L1, HSA, CD33, LAG3, or CD16. (a) The further antigen-binding domain having binding specificity to CD47 has one amino acid sequence of SEQ ID NOs: 2-29 or 223, (b) The further antigen-binding domain having binding specificity to PD-L1 has one amino acid sequence of SEQ ID NOs: 31-38, (c) The further antigen-binding domain having binding specificity to the HSA has one amino acid sequence of SEQ ID NOs: 40-48, (d) The further antigen-binding domain having binding specificity to CD33 has one amino acid sequence of SEQ ID NOs: 50 to 78, (e) The further antigen-binding domain having binding specificity to LAG3 has one amino acid sequence of SEQ ID NOs: 80 to 93, (f) The further antigen-binding domain having binding specificity to CD16 has one amino acid sequence of SEQ ID NOs: 96-99 The multispecific antibody according to claim 4.

7. A multispecific single-chain antibody (MVSCA) according to any one of claims 3 to 6.

8. A multispecific antibody according to any one of claims 3 to 7, wherein linker L1 (SEQ ID NO: 100), linker L2 (SEQ ID NO: 101), or linker L4 (SEQ ID NO: 103) is sandwiched between one or more pairs of non-identical antigen-binding domains.

9. A multispecific antibody according to any one of claims 3 to 8, comprising at least one pair of antigen-binding domains having the same binding specificity.

10. The multispecific antibody according to claim 9, wherein at least one pair of antigen-binding domains having the same binding specificity are adjacent to each other and a linker L3 (SEQ ID NO: 102) is sandwiched between them.

11. A multispecific antibody according to any one of claims 3 to 10, comprising an antigen-binding domain located at the N or C terminus, having binding specificity to HSA, wherein a cleavable linker is sandwiched between it and an adjacent antigen-binding domain.

12. The multispecific antibody according to claim 11, wherein the cleavable linker is L11*3 (SEQ ID NO: 104), L11*4 (SEQ ID NO: 105), L11*5 (SEQ ID NO: 106), L11*6 (SEQ ID NO: 107), L11*7 (SEQ ID NO: 108), L11*8 (SEQ ID NO: 109), L11*9 (SEQ ID NO: 110), L11*10 (SEQ ID NO: 111), L11*11 (SEQ ID NO: 112), L11*12 (SEQ ID NO: 113), L11*13 (SEQ ID NO: 114), L11*14 (SEQ ID NO: 115), L11*15 (SEQ ID NO: 116), L11*16 (SEQ ID NO: 117), L11*17 (SEQ ID NO: 118), or L11*18 (SEQ ID NO: 119).

13. A multispecific antibody according to any one of claims 3 to 12, wherein all of the antigen-binding domains are VHH domains.

14. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize HSA, CD47, and PD-L1.

15. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize HSA, CD47, and CD33.

16. A multispecific antibody according to any one of claims 3 to 13, comprising an antigen-binding domain that recognizes HSA, LAG3, and PD-L1.

17. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize HSA, LAG3, and CD33.

18. A multispecific antibody according to any one of claims 3 to 13, comprising an antigen-binding domain that recognizes CD16, HSA, and PD-L1.

19. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize CD16, HSA, and CD33.

20. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize CD16, HSA, CD47, and PD-L1.

21. A multispecific antibody according to any one of claims 3 to 13, comprising antigen-binding domains that recognize CD16, HSA, CD47, and CD33.

22. A multispecific antibody according to any one of claims 3 to 13, comprising a pair of antigen-binding domains having binding specificity to CD33.

23. The multispecific antibody according to claim 22, further comprising an antigen-binding domain having binding specificity to HSA, FC5 nanobody (SEQ ID NO: 222), or both.

24. A pharmaceutical composition comprising the VHH domain described in claim 1, the HCAb described in claim 2, or the multispecific antibody described in any one of claims 3 to 21.

25. The pharmaceutical composition according to claim 24, for use in the treatment of cancer, to be administered to a patient in need of treatment.

26. A pharmaceutical composition comprising the multispecific antibody described in claim 22 or 23.

27. The pharmaceutical composition according to claim 24, for use in the treatment of Alzheimer's disease or retinal disease, administered to a patient in need of treatment.

28. The pharmaceutical composition according to claim 27, wherein the retinal disease is dry age-related macular degeneration.

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