Monospecific and multispecific antibodies

Abstract

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

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 907,275, filed September 27, 2019, and U.S. Provisional Patent Application No. 62 / 989,327, filed March 13, 2020, the entire contents of which are incorporated herein by reference. Summary of the Invention

[0003] This article discloses monospecific heavy chain-only antibodies (HCAbs) that are specific to CD47, human serum albumin (HSA), PD-L1, CD33, CD16, and LAG3; and multivalent single-chain antibodies that incorporate two or more variable HCAb domains that are specific to one or more of these antigens.

[0004] Some implementations are single-domain antibodies that specifically or primarily contain the VHH domain of camel-family antibodies. These implementations are monospecific and monovalent.

[0005] Some implementations are HCAbs, or VHH domains (e.g., the Fc region of human IgG antibodies) fused with one or more constant domains from conventional antibodies. These implementations are monospecific but are typically bivalent. Depending on, for example, the choice of constant domain, other titers are also possible. The Fc regions of IgA and IgM can confer higher titers.

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

[0007] Some embodiments include two (or more) VHH domains, each of which is specific to different antigens that bind in a single amino acid chain (multivalent, multispecific single-chain antibodies). These embodiments are multivalent and multispecific. In other embodiments containing three or more VHH domains, two or more VHH domains may be specific to the same antigen, while one or more other VHH domains may be specific to different antigens. Such constructs have a higher order of potency than specificity.

[0008] Each of the single-specific embodiments will be specific to CD47, HSA, PD-L1, CD33, CD16, or LAG3. Each of the multi-specific embodiments will be specific to one or more of CD47, HSA, PD-L1, CD33, CD16, and LAG3, but may also be specific to one or more other antigens.

[0009] Some implementations are specific to HSA and one or more other antigens. In one aspect of these implementations, the HSA-specific domain confers an extended in vivo half-life, while other domains provide a therapeutic effect. In another aspect of these implementations, the HSA-specific domain can partially or completely inhibit the binding activity of adjacent domains. The HSA-specific domain can bind via a cleavable linker, which is cleaved by a protease present at the intended site of action (e.g., in a tumor), such cleavage relieves the inhibition of adjacent domains. Some multispecific implementations are trispecific.

[0010] In some embodiments that include multiple antigen-binding domains, antigen-binding domains derived from conventional VL-VH pairings may be used to replace one or more (but not all) VHH domains in the embodiments described above.

[0011] The antigen-binding domains that are specific to certain antigens disclosed in this article can be referred to as means for binding antigens. Attached Figure Description

[0012] Figure 1 Flow cytometry analysis was performed to depict the binding affinity of anti-CD47 HCAb A09-10 and B6H12 to cell lines overexpressing CD47.

[0013] Figure 2 Competitive ELISA binding assays were described for the multispecific antibodies 1511 (SEQ ID NO:156) and 3321 (SEQ ID NO:157) that have binding specificity to CD47.

[0014] Figure 3 The competitive binding analysis of the multispecific molecules 1511 and 3321 binding to CD47 using flow cytometry on the Jurkat cell line is described.

[0015] Figure 4A A human erythrocyte (RBC) hemagglutination assay was described using the multispecific molecules 1511 and 3321 that bind CD47. Hu5F9 was used as a control. Figure 4B The binding of 1511 and 3321 to HL-60 cells and human RBCs was depicted.

[0016] Figure 5 The antitumor activity of the multispecific molecule 3321, which binds to CD47, in Raja-Luc xenograft mice was described.

[0017] Figure 6 Flow cytometry binding analyses of anti-PD-L1 HCAb, PL14, and PL16 were depicted on CHO cells overexpressing PD-L1. Atezolizumab was used as a control.

[0018] Figure 7 Cell-based functional assays of PD-L1 binding-specific multispecific molecule 1511 and atezolizumab as a control.

[0019] Figure 8 illustrates the inhibition of MC38-hPD-L1 tumor growth in B-hPD-L1 mice by the multispecific molecule 1518 (SEQ ID NO:135) binding to PD-L1.

[0020] Figure 9 Octet depicts the anti-HSA VHH antibody ® Combined analysis.

[0021] Figures 10A to 10B Octet describes the anti-CD33 VHH antibody ® Combined with affinity analysis.

[0022] Figures 11A to 11B The anti-CD16A VHH molecule CD16F1 ( Figure 11A ) and CD16E11 ( Figure 11B Octet ® Combined analysis.

[0023] Figure 12 Cell-based functional assays of multispecific molecules 1511 and 3321 were performed in the Jurkat NFAT CD16 reporter gene assay (ADCC assay) using IgG1 B6H12 and IgG4 B6H12 as controls.

[0024] Figures 13A to 13C Molecules with HSA-binding domains and CD47-binding domains were depicted. Figure 13A Molecules possessing both HSA-binding and LAG3-binding domains ( Figure 13B ) and molecules with HSA-binding domains and CD16-binding domains ( Figure 13C The three specific molecular forms of ).

[0025] Figures 14A to 14B The three-specific molecular forms were described ( Figure 14A SDS-PAGE analysis of pro-CD47 activated by tumor proteases ( Figure 14B ).

[0026] Figure 15 Octet depicted the real-time kinetics of PD-L1 / pro-CD47 versus PD-L1 / active CD47. ® Combined analysis.

[0027] Figure 16 The forms of multispecific molecules are described. Mon = monovalent binding domain; BiV = bivalent binding domain containing two identical monovalent binding domains.

[0028] Figure 17 The forms of four-specific antibodies are depicted (four specificities, from left to right). Two VHH3s can be the same VHH binding different epitopes to the same antigen or different VHHs. Two VHH4s can be the same VHH binding different epitopes to the same antigen or different VHHs. In some embodiments, VHH2 is always the HSA-binding domain. In some embodiments, VHH1 is the payload, such as a CD16A agonist VHH. The first and second right figures show four-specific antibodies in prodrug form.

[0029] Figure 18 Flow cytometry binding analysis of the multispecific molecules 1518-HS5 (SEQ ID NO: 173) and 1518-HS5-GS15 (SEQ ID NO: 184) that bind to CD47 is described in the HL60 cell line.

[0030] Figure 19 Octet depicts the multispecific molecule 1511 ® Combined analysis.

[0031] Figure 20 Octet depicts the multispecific molecule 3321 ® Combined analysis.

[0032] Figure 21 The amino acid sequence alignment of the anti-CD47 VHH sequence is described.

[0033] Figure 22 The amino acid sequence alignment of the anti-PD-L1 VHH sequence was depicted.

[0034] Figure 23 The amino acid sequence alignment of the anti-HSA VHH sequence was depicted.

[0035] Figure 24 The amino acid sequence alignment of the anti-CD33 VHH sequence was depicted.

[0036] Figure 25 The amino acid sequence alignment of the anti-LAG3 VHH sequence was depicted.

[0037] Figure 26 The amino acid sequence alignment of the anti-CD16A VHH sequence is described. Detailed Implementation

[0038] This document discloses monospecific heavy chain-only antibodies (HCAbs) or their variable domains (referred to as VHH single-domain antibodies [sdAbs]) that are specific to CD47, human serum albumin (HSA), PD-L1, CD33, CD16, and LAG3; and multivalent single-chain antibodies (MVSCAs) that incorporate the variable domains of two or more HCAbs, which are specific to one or more of these antigens.

[0039] In some embodiments, the MVSCA comprises two or more HCAb variable domains that are specific to the same antigen. That is, the MVSCA is multivalent but single-specific with respect to the antigen. In some of these embodiments, the MVSCA comprises two or more repeating identical HCAb variable domains or multiple HCAb variable domains, each HCAb variable domain being specific to the same epitope. That is, they are multivalent but single-specific with respect to the epitope. Such an MVSCA will bind to only a single site on the antigen monomer, but can crosslink multiple copies of that monomer. In other embodiments of these embodiments, the MVSCA comprises two or more HCAb variable domains that are specific to different epitopes of the same antigen. That is, they are multivalent but multi-specific with respect to the epitope. Such an MVSCA can bind to multiple sites on the antigen monomer or crosslink multiple copies of that monomer.

[0040] In some embodiments, the MVSCA comprises two or more HCAb variable domains that are specific to different antigens; that is, they are multivalent and multispecific with respect to antigens. In further embodiments, the MVSCA comprises any combination of multiple HCAb variable domains, wherein additional variable domains are identical to the first HCAb variable domain, wherein additional HCAb variable domains differ from the first HCAb variable domain but are specific to different epitopes on the same antigen, or wherein additional HCAb variable domains differ from the first HCAb variable domain but are specific to different antigens.

[0041] A MVSCA containing two or more HCAb variable domains may further include HCAb constant domains. For example, the C-terminal HCAb variable domain may remain attached to its original HCAb constant domain. Alternatively, the C-terminal HCAb variable domain may attach to a constant region or Fc region of a more conventional antibody (e.g., a human antibody, such as human IgG antibody). In some embodiments, as will be known to those skilled in the art, the constant domain or intact Fc region may confer specific functionality. In other embodiments, a MVSCA containing two or more HCAb variable domains may further include HCAb constant domains, wherein the HCAb constant domain is located between the HCAb variable domains or at the N-terminus of the HCAb variable domain, rather than at the C-terminus of the HCAb variable domain, or elsewhere than at the C-terminus of the HCAb variable domain.

[0042] antigen

[0043] CD47 (Cluster 47), also known as integrin-associated protein (IAP), is a 50 kDa transmembrane protein encoded by the CD47 gene in humans. CD47 belongs to the immunoglobulin superfamily, works with membrane integrins, and also binds to ligands platelet-reactive protein-1 (TSP-1) and signal regulatory protein α (SIRPα). Platelet-reactive protein-1 is a secreted glycoprotein that plays a role in angiogenesis and vascular development, and in the latter capacity, the TSP1-CD47 interaction inhibits nitric oxide signaling at multiple levels in vascular cells. The binding of TSP-1 to CD47 affects several fundamental cellular functions, including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation. Signal regulatory protein α is a transmembrane receptor found on bone marrow cells. The CD47 / SIRPα interaction leads to bidirectional signal transduction, resulting in different cell-to-cell responses, including inhibition of phagocytosis, stimulation of cell-to-cell fusion, and T cell activation. CD47 acts as a signaling receptor for macrophages in the immune system. No Want to eat me This signal makes CD47 a potential therapeutic target in some cancers.

[0044] Programmed cell death protein 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-1 is expressed on the surface of activated T cells, while PD-L1 is expressed on the surface of 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 transmitted to T cells, which reduces cytokine production and inhibits T cell proliferation.

[0045] The PD-1 pathway is a key immunosuppressive mediator of T cell exhaustion. PD-1 acts to limit the activity of activated T cells in the periphery during the inflammatory response to infection, thereby limiting autoimmunity. Blocking this pathway leads to T cell activation, expansion, and enhanced effector function. Therefore, PD-1 negatively regulates T cell responses. PD-1 has been identified as a marker of exhausted T cells 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 treating persistent infections and cancer by inhibiting the PD-1 pathway are disclosed in WO 2006 / 133396. Human monoclonal antibodies against PD-L1 are described in WO 2007 / 005874, US2011 / 209230, US8,217,149, and WO2014 / 055897.

[0046] Human serum albumin (HSA) is the most abundant protein in human plasma; it constitutes approximately half of serum proteins. Albumin transports hormones, fatty acids, and other compounds, buffers pH, maintains turgor pressure, and performs other functions. Albumin is synthesized in the liver as prealbuminogen, which has an N-terminal peptide that is removed before naïve albumin is released from the rough endoplasmic reticulum. The product, prealbumin, is then cleaved in Golgi vesicles to produce secretory albumin. Its serum half-life is approximately 20 days. The long serum half-life of albumin is achieved in part by its size, which prevents clearance by the kidneys, and by its interaction with nascent Fc receptors (FcRn). Fusion with anti-albumin sdAbs (single-domain antibodies) has been used to increase the half-life of antitumor single-chain antibodies from 1–2 hours to approximately 10 days.

[0047] CD33, or Siglec-3 (sialic acid-binding immunoglobulin-like lectin 3, SIGLEC3, SIGLEC-3, gp67, p67), is a transmembrane receptor expressed on myeloid cells. It is generally considered bone marrow-specific, but it can also be found on some lymphoid cells. It binds to sialic acid and is therefore a member of the SIGLEC family of lectins. The extracellular portion of this receptor contains two immunoglobulin domains (one IgV and one IgC2 domain), placing CD33 within the immunoglobulin superfamily. The intracellular portion of CD33 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM), which is associated with the inhibition of cellular activity. Diseases that can be treated by targeting CD33 include, but are not limited to, Alzheimer's disease and retinal diseases such as macular edema (e.g., diabetic macular edema) and age-related macular degeneration (AMD) (e.g., dry AMD and wet AMD).

[0048] CD33 is a target of gemtuzumab ozomicin (Mylotarg®; Pfizer / Wyeth-Ayerst Laboratories), an antibody-drug conjugate (ADC) used to treat patients with acute myeloid leukemia. CD33 is also a target of vasutuximab taglilin (SGN-CD33A), a novel antibody-drug conjugate developed by Seattle Genetics using the company's ADC technology.

[0049] Lymphocyte activation gene 3 (LAG-3), a 503-amino acid transmembrane protein, is an immune checkpoint receptor protein present on the cell surface of effector T cells and regulatory T cells (Tregs) and functions to control T cell responses, activation, and growth. 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 and downregulation of antigen-dependent CD4 and CD8 T cell responses. LAG3 negatively regulates T cell proliferation, cytokine production, and target cell lysis, a phenomenon known as T cell 'exhaustion'. Due to its important role in tumor and infection immunity, LAG3 is an ideal target for immunotherapy. Blocking LAG3 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections.

[0050] CD16, also known as FcγRIII, is a differentiation cluster molecule present on the surface of natural killer cells, neutrophils, monocytes, and macrophages. CD16 is identified as an Fc receptor, existing in two forms encoded by separate genes: FcγRIIIa (CD16a), a transmembrane protein; and FcγRIIIb (CD16b), a GPI-anchored protein; both involved in signal transduction. The most well-studied membrane receptor associated with NK cell-triggered lysis, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to separate specific populations of immune cells using antibodies against CD16 via fluorescent-activated cell sorting (FACS) or magnetically activated cell sorting. These receptors bind to the Fc portion of IgG antibodies, which subsequently activates antibody-dependent cell-mediated cytotoxicity (ADCC) in human NK cells. CD16 is essential for ADCC processes in human monocytes. In humans, monocytes expressing CD16 possess various ADCC capabilities in the presence of specific antibodies and can kill primary leukemia cells, cancer cell lines, and cells infected with hepatitis B virus. Furthermore, CD16 can mediate the direct killing of some virus-infected cells and cancer cells in the absence of antibodies. Upon binding to ligands (such as conserved fragments of IgG antibodies), CD16 on human NK cells induces the transcription of surface-activating molecules such as IL-2-R (CD25) and inflammatory cytokines such as IFN-γ and TNF. The expression of these CD16-induced cytokine mRNAs 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 upregulation of specific cytokine genes occurs via CsA-sensitivity and calcium-dependent mechanisms.

[0051] CD16 plays a crucial role in the early activation of natural killer (NK) cells following vaccination. Furthermore, CD16 downregulation represents a possible mechanism for regulating NK cell responses and maintaining immune homeostasis in both T cell and antibody-dependent signaling pathways. In healthy individuals, antibody-dependent cytotoxicity (ADCC) is induced in NK cells by immune complex cross-linking of CD16 (FcγRIII). However, this pathway can also target cancer cells or diseased cells via immunotherapy. Following influenza vaccination, CD16 downregulation was associated with a significant upregulation of influenza-specific plasma antibodies and was positively correlated with NK cell degranulation.

[0052] CD16 is frequently used as an additional marker for reliably identifying 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 on NK cells and all other cells derived from myeloid cells, additional markers, such as CD14 and CD15, are needed. Neutrophils have been found to be low on CD14 and high on CD15, while monocytes are high on CD14 and low on CD15. While these two markers are sufficient to distinguish neutrophils from monocytes, eosinophils have similar CD15 expression to neutrophils. Therefore, CD16 has been used as a further marker for identifying neutrophils: mature neutrophils are high on CD16, while eosinophils and monocytes are low on CD16. CD16 allows for differentiation between these two types of granulocytes. Furthermore, CD16 expression varies between different stages of neutrophil development: CD16 is low in neutrophil progenitor cells with differentiation capacity, while CD16 expression increases progressively in promyelocytes, band cells, and mature neutrophils.

[0053] Due to its expression on neutrophils, CD16 represents a potential target for cancer immunotherapy. Margetuximab is an Fc-optimized monoclonal antibody that recognizes human epidermal growth factor receptor 2 (HER2) expressed on tumor cells in breast, bladder, and other solid tumor cancers, targeting CD16A preferentially over CD16B. Furthermore, CD16 can play a role in antibody-targeted cancer therapy. Bispecific antibody fragments, such as anti-CD19 / CD16, allow immunotherapeutic drugs to target cancer cells. Anti-CD19 / CD16 bispecific antibodies have been shown to enhance natural killer cell responses to B-cell lymphoma. Additionally, targeting exogenous factors such as FasL or TRAIL to the surface of tumor cells triggers death receptors, thereby inducing apoptosis through autocrine and paracrine processes.

[0054] Antibody

[0055] Antibodies and their use in treating diseases are well known in the art. As used herein, the term "antibody" refers to a monomeric or polymeric protein comprising one or more polypeptide chains containing antigen-binding sites. Antibodies bind specifically to antigens and may be able to modulate the biological activity of antigens. As used herein, the term "antibody" may include "full-length antibody" and "antibody fragment." The term "binding site" or "antigen-binding site" as used herein refers to 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 heavy chain-only antibodies, the antibody heavy chain variable region.

[0056] Antibody specificity refers to the selective recognition of a specific epitope of an antigen by an antibody. Natural antibodies are, for example, monospecific. As used herein, the term "monospecific" antibody means an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. The monospecific antibodies disclosed herein are specific for CD47, HSA, PD-L1, CD33, CD16, or LAG3. In some embodiments, the monospecific antibody is a heavy-chain-only antibody (HCAb). In other embodiments, the monospecific antibody comprises a VHH domain fused to one or more protein domains, including, for example, the human Fc region. In other embodiments, the monospecific antibody comprises VHH as the only intact protein domain, i.e., a single-domain antibody. In some embodiments, the single-domain antibody may additionally comprise a short peptide, such as a His tag. The terms "VHH domain" and "HCAb variable domain" are used interchangeably. The VHH domain can be referred to as a means of binding a specific target (e.g., CD47, HSA, PD-L1, CD33, CD16, or LAG3). Therefore, any antibody structure, form, or construct disclosed herein containing a VHH domain, or constructed to contain a VHH domain, can be referred to as an antibody comprising a means of binding a specified target. Some embodiments may specifically include one or more specific antibody structures, forms, or constructs. Other embodiments may specifically exclude one or more specific antibody structures, forms, or constructs.

[0057] As used herein, “antibody that is specific to…”, “antibody that recognizes…”, “antibody that has affinity for…”, “antibody that has a binding site for…”, and similar structures can be used interchangeably.

[0058] "Multispecific antibody" refers to an antibody that has the specificity to bind to two or more antigens. The multispecific antibodies disclosed herein are specific to at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3, or specific to at least one of the aforementioned specificities and at least a second specificity. In some embodiments, the multispecific antibodies disclosed herein may include two, three, four, or more antigen-binding domains. Furthermore, multispecific antibodies may include at least two copies of the same antigen-binding sequence, or two antigen-binding sequences (double complementary sites) specific to different epitopes on the same antigen, provided that the multispecific antibody is specific to at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3 and at least one second antigen. In some embodiments, the multispecific antibody (MVSCA) is specific to at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3. In some embodiments, the multispecific antibodies disclosed herein are single-chain antibodies. Therefore, some multispecific antibodies can be described as antibodies that include means for binding a first target and means for binding a second target, etc.

[0059] A “bispecific antibody” is an antibody that has the specificity to bind to two different antigens. In some embodiments, the bispecific antibodies disclosed herein are specific to two of CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequence encoding the antigen-binding moiety of the bispecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the bispecific antibody is linked via a linker as disclosed herein.

[0060] A "trispecific antibody" refers to an antibody that has the specificity for binding to three different antigens. In some embodiments, the trispecific antibodies disclosed herein are specific for three of CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequence encoding the antigen-binding moiety of the trispecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the trispecific antibody is linked via a linker as disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0061] A "tetraspecific antibody" refers to an antibody that has the specificity for binding to four different antigens. In some embodiments, the tetraspecific antibodies disclosed herein are specific to four of CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequence encoding the antigen-binding moiety of the tetraspecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the tetraspecific antibody is linked via a linker disclosed herein. In some embodiments, two linkers are used, which may be the same or different.

[0062] As used herein, the term "valence" indicates the presence of a specified number of binding sites in an antibody molecule. Therefore, the terms "bivalent," "trivalent," "tetravalent," "pentavalent," "hexavalent," "heptavalent," and "octavalent" indicate the presence of two, three, four, five, six, seven, and eight binding sites in an antibody molecule, respectively. The bispecific antibodies disclosed herein are "bivalent." The trispecific antibodies disclosed herein are "trivalent." The tetraspecific antibodies disclosed herein are "tetravalent." However, monospecific multivalent antibodies, such as bivalent, trivalent, and tetravalent antibodies, within the scope of this disclosure, have multiple antigen-binding sites that bind to the same antigen. The antigen-binding sites of monospecific bivalent and trivalent (or higher) antibodies may bind to the same or different epitopes on the antigen. Similarly, antibodies with a higher valence than multispecific antibodies, such as trivalent bispecific antibodies, can be constructed by combining multiple monospecific binding sites with binding sites for one or more other specificities.

[0063] In this paper, "full-length antibody" refers to the structure of the naturally occurring biological form of the antibody, including variable and constant regions. For example, in most mammals, including humans and mice, full-length IgG antibodies are tetramers and consist of two pairs of identical immunoglobulin chains, each pair containing one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and C1, and each heavy chain contains immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, such as camels and llamas, IgG antibodies may also consist of only two heavy chains (HCAbs), each containing variable domains (CH2 and CH3 domains) attached to the Fc region.

[0064] Tetrameric antibodies typically consist of two pairs of identical polypeptide chains, each pair having a "light chain" (usually with a molecular weight of about 25 kDa) and a "heavy chain" (usually with a molecular weight of about 50-70 kDa). Each of the light and heavy chains comprises two distinct regions called the variable and constant regions. For IgG immunoglobulins, the heavy chain consists of four immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VH-CH1-CH2-CH3. These four immunoglobulin domains are the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (also known as VH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constant γ1 domain, constant γ2 domain, and constant γ3 domain, respectively). The IgG light chain consists of two immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VL-CL. These two immunoglobulin domains are the light chain variable domain and the light chain constant domain. Constant regions exhibit less sequence diversity and are responsible for binding many natural proteins to trigger important biochemical events.

[0065] The variable region of an antibody contains the molecule's antigen-binding determinants and thus determines the antibody's specificity for its target antigen. The variable region is so named because it is most distinct in sequence from other antibodies of the same class. Within the variable region, three loops are assembled from each V domain of the heavy and light chains to form an antigen-binding site. Each of these loops is called a complementarity-determining region (hereinafter referred to as "CDR"), where the amino acid sequence variation is most pronounced. There are a total of six CDRs, three for each heavy and light chain, named VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside the CDRs is called the backbone (FR) region. While not as diverse as the CDRs, there is indeed sequence variability in the FR regions between different antibodies. Overall, this characteristic architecture of an antibody provides a stable scaffold (FR region) upon which the immune system can explore a large number of antigen-binding diversity (CDRs) to obtain specificity for a variety of antigens.

[0066] The gene encoding the immunoglobulin locus contains multiple V region sequences as well as shorter nucleotide sequences named “D” and “J”, and it is the combination of V, D and J nucleotide sequences that produces VH diversity.

[0067] Antibodies are classified into several classes, also known as isotypes, as determined by the genetic characteristics of constant regions. Human constant light chains are classified as κ (Cκ) and λ (Cλ) light chains. Heavy chains are classified as μ (µ), delta (δ), gamma (γ), alpha (α), or epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. In humans, this class includes subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this class includes subclasses IgG1, IgG2a, IgG2b, and IgG3. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Therefore, as used herein, "isotype" refers to any class or subclass of immunoglobulin defined by the chemical and antigenic properties of the constant regions of the immunoglobulin. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. Disclosed HCAb antibodies, bispecific antibodies, and multispecific antibodies may have constant regions encompassing all or some of the aforementioned isotypes.

[0068] The scope of this disclosure includes, but is not limited to, the following antibody fragments: (i) Fab fragments containing VL, CL, VH, and CH1 domains; (ii) Fd fragments containing VH and CH1 domains; (iii) Fv fragments containing a single antibody with VL and VH domains; (iv) dAb fragments containing a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments, a bivalent fragment containing two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) wherein the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site. Trivalent or tetravalent antibody fragments containing three different specificities linked by cleavable or non-cleavable linkers are also disclosed. In some embodiments, antibodies are generated using recombinant DNA technology. In other embodiments, antibodies are generated by enzymatic or chemical cleavage of naturally occurring antibodies.

[0069] As used herein, "single-chain antibody" refers to a fusion protein of an antibody whose antigen-binding portion (i.e., variable region) is typically linked by a linker peptide. This document discloses multivalent monospecific and multispecific single-chain antibodies. Monospecific multivalent antibodies are specific for at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3. Multispecific single-chain antibodies are specific for at least one of CD47, HSA, PD-L1, CD33, CD16, and LAG3, plus at least one further specificity. In some embodiments, multispecific single-chain antibodies are specific for at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3.

[0070] As used herein, a “humanized” antibody refers to an antibody comprising a human backbone region (FR) and one or more complementarity-determining regions (CDRs’) derived from a non-human antibody. The non-human antibody providing the CDRs’ is referred to as the “donor,” and the human immunoglobulin providing the backbone is referred to as the “recipient.” In some embodiments, humanization primarily relies on transplanting the donor CDR onto the recipient (human) VL or VH backbone. This strategy is referred to as “CDR transplantation.” Typically, selected recipient backbone residues are “reverted” to the corresponding donor residues to regain the affinity lost in the originally transplanted construct. Humanized antibodies will also preferably contain at least a portion of the immunoglobulin constant region (typically the constant region of human immunoglobulins) and will typically contain a human Fc region. Humanization or other methods for reducing the immunogenicity of the variable region of a non-human antibody may include surface remodeling methods. In one embodiment, a selection-based approach may be used to humanize and / or affinity-mature the antibody variable region, i.e., to increase the affinity of the variable region for its target antigen. Other humanization methods may involve the transplantation of only a portion of the CDR, including but not limited to the method described in US 6,797,492, all of which discloses information regarding CDR transplantation and is incorporated herein by reference. Structure-based methods may be employed for humanization and affinity maturation, such as those described in US 7,117,096, all of which discloses information regarding humanization and affinity maturation and is incorporated herein by reference.

[0071] In the various embodiments described herein, the antibodies are heavy-chain-only antibodies (HCAbs). In addition to conventional heavy-chain and light-chain antibodies (one antibody containing two light chains and two heavy chains), camelids (camels, dromedaries, and llamas) also contain double-chain antibodies (containing only variant heavy chains). The dimer antibody is encoded by a unique set of VH regions called VHH genes. VH and VHH are scattered throughout the genome (i.e., they appear to be mixed together). The identification of the same D region in VH and VHH cDNA indicates that VH and VHH share the D region. Naturally VHH-containing antibodies lack the entire CH1 domain of the heavy-chain constant region. The exon encoding the CH1 domain is present in the genome but is spliced ​​out due to the absence of a functional splice acceptor sequence on the 5' side of the CH1 exon. As a result, the VDJ region is spliced ​​onto the CH2 exon. When VHH recombines into such constant regions (CH2, CH3), antibodies are produced in which the hapten is a single chain rather than a light / heavy chain pair (i.e., an antibody with two heavy chains but no light chain interaction). The binding to the antigen differs from that seen with conventional antibodies, but the high affinity is achieved in the same way, through hypermutation of the variable region and selection of cells expressing such high-affinity antibodies.

[0072] In one exemplary embodiment, the disclosed HCAb is produced by immunizing transgenic mice, wherein endogenous mouse antibody expression has been eliminated and a camelid transgene has been introduced. HCAb mice are disclosed in US8,883,150, US8,921,524, US8,921,522, US8,507,748, US8,502,014, US 2014 / 0356908, US2014 / 0033335, US2014 / 0037616, US2014 / 0356908, US2013 / 0344057, US2013 / 0323235, US2011 / 0118444, and US2009 / 0307787, all of which are incorporated herein by reference for all their disclosures regarding heavy-chain-only antibodies and the production of said heavy-chain-only antibodies in transgenic mice. HCAb mice were immunized, and the resulting sensitized spleen cells were fused with mouse myeloma cells to form hybridomas.

[0073] In other embodiments, HCAbs are produced by immunizing llamas with the desired antigen and sequencing the VHH region encoding the resulting antigen-binding antibody. In one embodiment, the VHH is isolated using a phage display library. See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204 (the entire description of the preparation of phage libraries in each of these patents is incorporated herein by reference).

[0074] This document also discloses multispecific or multivalent antibodies in which two or more antigen-binding domains are conjugated to a single fusion protein. Multispecific antibodies can take various forms, including (i) multispecific Fv fragments; (ii) heavy chains having first specificity and having a second VH domain of second specificity associated with (or fused to); (iii) tetrameric monoclonal antibodies having first specificity and having a second VH domain of second specificity associated with it, wherein the second VH domain is associated with the first VH domain; and (iv) Fab fragments (VH-CH1 / VL-CL) having first specificity and having a second VH domain of second specificity associated with it. Exemplary Fab fragments include those in which a second VH sequence of second specificity is associated with the C-terminus or N-terminus of the first VH domain, or with the C-terminus or N-terminus of the first CH1 or first CL domain. In another embodiment, a VH sequence having a second and / or third (or more) specificity may associate (or fuse) with the C-terminus or N-terminus of the first VH domain, or with the C-terminus or N-terminus of the first CH1 or first CL domain. In various embodiments, any of these forms may include at least one of the HCAb variable domains disclosed herein.

[0075] Multispecific or multivalent antibodies may include adapter sequences that link a specific antigen-binding domain (e.g., VH or VHH) to another antigen-binding domain and allow the amino acid sequence to fold correctly to produce the desired three-dimensional conformation and antigen-binding profile. Typically, the adapter sequence will be a short amino acid sequence that provides sufficient space and flexibility between the domains for proper folding. The adapter may also induce steric hindrance to facilitate binding to the target of each domain. Suitable adapters include, but are not limited to, the adapters in Table 15 (SEQ ID No: 100 to SEQ ID No: 119), EPKSCD (SEQ ID NO: 224), and ASTKGP (SEQ ID NO: 225). More adapters will be known to those skilled in the art.

[0076] Amino acid sequence variants of monospecific or multispecific antibodies disclosed herein are also within the scope of this disclosure. These amino acid sequence variants are prepared by introducing appropriate nucleotide alterations into the DNA encoding the antibody, or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibodies exemplified herein. Any combination of deletions, insertions, and substitutions can be made to achieve the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter the post-translational processes of humanized or variant antibodies, such as changing the number or location of glycosylation sites.

[0077] A useful method for identifying specific antibody residues or regions as preferred mutagenic sites is called "alanine scan mutagenesis." A residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and substituted with a neutral amino acid (most preferably alanine or polyalanine) to affect the interaction between the amino acid and the antigen. Those amino acid positions that exhibit functional sensitivity to the substitution are then improved by introducing further or other variations at or targeting the substitution site. Therefore, while the sites for introducing amino acid sequence variations are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scans or random mutagenesis are performed on the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0078] Amino acid sequence insertions include fusion of amino and / or carboxyl ends from one residue to a polypeptide of one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies disclosed herein with an N-terminal methionyl residue, or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include the fusion of an enzyme or polypeptide that extends the serum half-life of the antibody to the N-terminus or C-terminus of said antibody.

[0079] Another type of variant is the amino acid substitution variant. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at its position. The substitution mutagenic sites of most interest include hypervariable regions, but FR alterations are also considered. Conservative substitutions are shown under the “Preferred Substituents” heading in Table 1. If such substitutions result in an alteration of biological activity, more substantial alterations, designated as “Exemplary Substituents” in Table 1 or further described below with reference to the amino acid categories, can be introduced, and products screened.

[0080] Table 1

[0081]

[0082] Substantial modification of the biological properties of antibodies is achieved through substitutions that significantly alter (a) the structure of the polypeptide backbone (e.g., sheet-like or helical conformation) at the substitution region, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volumetric effect of the side chains. Naturally occurring residues are categorized into the following groups based on common side-chain characteristics:

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

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

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

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

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

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

[0089] Non-conservative replacement would require swapping members of one of these categories with members of another category.

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

[0091] Another type of substitution variant involves replacing one or more hypervariable residues of the parent antibody (e.g., humanized or camelid antibody). Typically, the resulting variants selected for further development will have improved biological properties relative to the parent antibody that produced them. A convenient way to generate such substitution variants is through affinity maturation using phage display. In short, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The resulting antibody variants are displayed monovalently from filamentous phage particles as fusions with the M13 gene III product packaged in each particle. The phage-displayed variants are then screened for their biological activities (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively or additionally, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the antigen. According to the techniques detailed herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the group of variants is screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

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

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

[0094] Glycosylation sites can be conveniently added to antibodies (for N-linked glycosylation sites) by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences. Alterations can also be made by adding or substituting one or more serine or threonine residues into the original antibody sequence (for O-linked glycosylation sites).

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

[0096] Other modifications to monospecific or multispecific antibodies are envisioned. For example, modifications to the antibody in terms of effector function may be necessary, such as to enhance the antibody's efficacy in treating diseases. For instance, one or more cysteine ​​residues can be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in that region. The resulting homodimeric antibody may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinking agents. Alternatively, the antibody can be engineered to have a double Fc region, thereby possessing enhanced complement cleavage and ADCC capabilities.

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

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

[0099] The monospecific or multispecific antibodies disclosed herein can be generated through recombinant methods. Therefore, this document discloses nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods for recombinant production are well known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and typically purification to pharmaceutically acceptable purity. To express antibodies in host cells as described above, nucleic acids encoding antibody sequences are inserted into expression vectors using standard methods. Suitable prokaryotic or eukaryotic host cells (such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or Escherichia coli) are used. E. coli Expression is performed in cells, and antibodies are recovered from said cells (lysed supernatant or cells). It should be understood that any recombinantly expressed protein requires an initiator methionine (or formylmethionine) or a signal sequence at its N-terminus, depending on the expression system used and whether the protein is expressed in the cytoplasm or secreted. Therefore, in some embodiments, the protein sequences disclosed herein are modified at their N-terminus with such additional amino acids. In some embodiments, such N-terminal sequences are cut (in whole or in part) from the fully mature sequence, while in other embodiments they are retained.

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

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

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

[0103] As used herein, the term "transformation" refers to the process of transferring a vector / nucleic acid into a host cell. If cells without a strong cell wall barrier are used as host cells, transfection can be performed, for example, by calcium phosphate precipitation. However, other methods can also be used to introduce DNA into the cell, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells with a large cell wall structure are used, one transfection method is, for example, calcium treatment with calcium chloride.

[0104] As used herein, “expression” refers to the process of transcribing nucleic acids into mRNA and / or translating transcribed mRNA (also called transcripts) into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include mRNA splicing.

[0105] A “vector” is a nucleic acid molecule, especially a self-replicating nucleic acid molecule, that transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that primarily function to insert DNA or RNA into cells (e.g., chromosome integration), vectors that primarily function to replicate DNA or RNA, and expression vectors that function to transcribe and / or translate DNA or RNA. It also includes vectors that provide more than one of the functions described above.

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

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

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

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

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

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

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

[0113] Besides prokaryotes, eukaryotic microorganisms, such as filamentous fungi or yeasts, are also suitable cloning or expression hosts for vectors encoding monospecific or multispecific antibodies. Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae *Saccharomyces cerevisiae*, or common baker's yeast, is the most commonly used yeast among microorganisms of lower eukaryotic hosts. However, many other genera, species, and strains are generally available and useful in this paper, such as *Schizosaccharomyces cerevisiae*. Schizosaccharomyces pombe Kluyveromycin ( Kluyveromyces ) hosts, such as Kluyveromyces lactis ( K. lactis Kluyveromyces brittle-walled ( K. fragilis (ATCC 12,424), Kluyveromyces bulgaricus ( K. bulgaricus (ATCC 16,045), Wicklwyn yeast ( K. wickeramii (ATCC 24,178), Walt Kluwer yeast ( K. waltii (ATCC 56,500), Kluyveromyces davidianus ( K. drosophilarum (ATCC 36,906), heat-resistant Kluyveromyces ( K. thermotolerans ), and Max Kluyveromycin ( K. marxianus ); Yersinia genus ( yarrowia (EP 402,226); Pichia pastoris ( Pichia pastoris (EP 183,070); Candida genus ( Candida Trichoderma reesei ( Trichoderma reesia (EP 244,234); Neurospora crassa ( Neurospora crassa ); genus *Schwannium* ( Schwanniomyces), such as Western Schwann yeast ( Schwanniomyces occidentalis ); and filamentous fungi, such as Neurospora ( Neurospora ), Penicillium genus ( Penicillium ), genus *Cyclophorus* ( Tolypocladium ), and Aspergillus ( Aspergillus ) host, such as Aspergillus nidus ( A. nidulans ) and Aspergillus niger ( A. niger ).

[0114] Suitable host cells for expressing glycosylated monospecific or multispecific antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculovirus strains and variants from hosts, along with corresponding permissible insect host cells, have been identified, such as the meadow moth (Eriocheir sinensis). Spodoptera frugiperda (caterpillars), Aedes aegypti ( Aedes aegypti (mosquitoes), Aedes albopictus ( Aedes albopictus (mosquitoes), fruit flies (black-bellied flies) Drosophila melanogaster (fruit fly) and Chinese silkworm ( Bombyx mori The various virus strains used for transfection are publicly available, for example, the alfalfa silver-striped moth (…). Autographa californica The L-1 variant of NPV and the Bm-5 strain of Chinese silkworm NPV are used, and these viruses can be used as the viruses according to the present invention, particularly for transfection of *Hemiberlesia lataniae* cells. Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0115] However, there is the greatest interest in vertebrate cells, and the proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines include: monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or 293 cells subcloned for growth in suspension culture); juvenile 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 (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumors (MMT 060562, ATCC). CCL51); TRI cells; MRC 5 cells; FS4 cells; and the human liver cancer cell line (Hep G2).

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

[0117] Host cells used to produce monospecific or multispecific antibodies can be cultured in a variety of media. Commercially available media, such as Ham's F10 (Sigma), Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and DuPont Modified Eagle Medium (DMEM) (Sigma), are suitable for culturing host cells. Additionally, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or US Re. 30,985 can be used as a culture medium for host cells. Any of these culture media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN™), trace elements (defined as inorganic compounds typically present in micromolar concentrations), and glucose or equivalent energy. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature and pH, are those previously used with the selected host cells for expression and will be readily apparent to those skilled in the art.

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

[0119] Cell-prepared antibody compositions can be purified using techniques such as hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains, although it can also be used to purify antibodies lacking the Fc region. Protein G can be used for all mouse isotypes and human γ3. The matrix to which the affinity ligand is attached is most commonly agarose, but other matrices can also be used. Mechanically stable matrices, such as controlled-pore glass or poly(divinyl)styrene, allow for faster flow rates and shorter processing times than achievable with agarose. Bakerbond ABX™ resin can be used for purification if the antibody contains a CH3 domain. The antibodies and antibody fragments disclosed herein can also be synthesized with histidine tags and purified by metal affinity chromatography.

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

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

[0122] This document also discloses cleavable multispecific single-chain antibodies in the tumor microenvironment. In some embodiments, once the multispecific single-chain antibody reaches the tumor, a tumor-targeting domain (e.g., a tumor antigen-binding domain) or other functional domains (e.g., an anti-HSA domain, which can prolong systemic half-life) are cleaved at the linker to release other domains that provide therapeutic effects. The tumor microenvironment contains a large number of proteases capable of cleaving the linkers disclosed herein. Non-limiting examples of tumor proteases include, but are not limited to, matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAM (de-integrin and metalloproteinases; e.g., ADAM10 and ADAM17), kallikrein-associated peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepsin (HPN), matriptase, podin, or dipeptidyl peptidases (e.g., DDP4).

[0123] antibody composition

[0124] This document also discloses pharmaceutical compositions comprising monospecific or multispecific antibodies, wherein the specific antibodies include CD47, HSA, PD-L1, CD33, CD16, or LAG3. The use of the antibodies described herein in the manufacture of pharmaceutical compositions is also disclosed. Methods for treating various diseases and conditions using the disclosed antibodies and pharmaceutical compositions comprising said antibodies are also disclosed.

[0125] A pharmaceutical composition is a pharmaceutical composition intended for and suitable for use in the treatment of human diseases. That is, it provides an overall beneficial effect and does not contain any amount of ingredients or contaminants that would cause toxicity or other undesirable effects unrelated to the provision of the beneficial effect. The pharmaceutical composition will contain one or more active agents and may further contain solvents, buffers, diluents, carriers, and other excipients to facilitate the administration, solubility, absorption, or bioavailability and / or stability of the active agent or the entire composition.

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

[0127] As used herein, "drug carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, intraocular, intravitreal, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is aqueous.

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

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

[0130] These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. Protection against microorganisms is ensured through the sterilization procedures described above and by including various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.). Isotonic agents, such as sugars and sodium chloride, may also need to be included in the composition. Furthermore, the absorption of injectable drug forms can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

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

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

[0133] The actual dose level of the active ingredient in a pharmaceutical composition can be varied to obtain a quantity of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration method without toxicity to the patient. The selected dose level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the invention used, the route of administration, the time of administration, the excretion rate of the specific compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the specific composition used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical field.

[0134] The disclosed MVSCA and the functions of its constituent antibody domains and linkers

[0135] VHH anti-HSA

[0136] The primary function of the anti-HSA domain within MVSCA is to bind HSA, thereby extending the half-life of MVSCA in vivo. The inclusion of an anti-HSA domain prolongs the half-life, which would otherwise be only a few hours to more than a week. Typically, a single anti-HSA domain is sufficient for this purpose. Therefore, the anti-HSA domain constitutes a means of extending the half-life of MVSCA.

[0137] By 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 only partial depends on the length of the linker between the two domains; the shorter the linker, the more complete the blockade of antigen binding. Partial blockade is often observed as a decrease in the apparent or effective affinity of VHH for its antigen. In some cases, partial blockade has been observed as an increase in VHH specificity, as the domain continues to bind antigens with higher affinity but fails to exhibit significant binding to antigens with lower affinity. Therefore, the anti-HSA domain constitutes a means of inhibiting the binding activity of adjacent binding domains.

[0138] This blockade can also be reversible. By placing the anti-HSA domain at the terminal position in an MVSCA and attaching it to a cleavable linker, the anti-HSA domain can be removed, and full binding activity of adjacent binding domains can be restored. Such antibody constructs are effective prodrugs. For example, if the linker is cleaved by a protease found at the desired site of action, the MVSCA can travel through the body inactive near its binding site, but upon reaching its site of action (e.g., a tumor), the linker is cleaved, the anti-HSA domain is released, and the inhibition of binding activity of adjacent domains is reversed. Therefore, when paired with a cleavable linker, the anti-HSA domain constitutes a means of reversibly inhibiting the binding activity of adjacent binding domains in MVSCAs or multispecific antibodies.

[0139] Anti-CD47

[0140] The function of the anti-CD47 domain is to inhibit the "don't eat me" signaling of CD47 on tumor cells, allowing these tumor cells to be phagocytosed by macrophages. CD47 is widely expressed, and anti-CD47 activity can be problematic if it binds substantially to normal healthy cells. Several ways can be made to avoid this. Apparently, multiple conformations of CD47 exist, and the conformation typically found on tumor cells differs from, for example, the conformation 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. Avoiding binding to RBCs is also important so that the MVSCA is not captured in the bloodstream and blocked from reaching its target.

[0141] Another way to avoid unwanted or harmful binding of MVSCA to CD47 is to place MVSCA near the anti-HSA domain in a manner that reduces or prevents binding to CD47, as described above. Once MVSCA binds to tumor cells through another binding domain within its binding domain, and the anti-HSA domain is cleaved and released by local proteases, the anti-CD47 domain can bind to CD47 and prevent its phagocytic inhibitory interaction with macrophages.

[0142] Therefore, the anti-CD47 domain constitutes a means of reducing the inhibition of phagocytosis.

[0143] Anti-CD16

[0144] The function of the anti-CD16 domain is to upregulate ADCC activity of NK cells. However, CD16B has a wide tissue distribution, while CD16A is specifically expressed in NK cells. Antibodies specific to CD16A are preferred because they will bind only to NK cells (the desired target cells). However, antibodies that bind both CD16A and CD16B, as well as antibodies that bind only CD16A, are agonists that can promote ADCC activity of NK cells.

[0145] CD16 typically interacts with the Fc portion of antibodies. When CD16A on NK cells is bound to the Fc portion of an antibody, the cytolytic activity of the NK cells becomes targeted at the variable domain of the antibody that has been bound to the cell or microorganism. However, there are multiple Fc sequences and multiple types of Fc receptors, resulting in a variety of possible effects mediated by the Fc region. By using the anti-CD16 domain instead of the Fc region, MVSCAs can specifically recruit NK-mediated ADCCs against other specific targets they carry. Therefore, the anti-CD16 domain constitutes a means of recruiting NK-mediated ADCCs.

[0146] Anti-PD-L1

[0147] The anti-PD-L1 domain functions as both an immune checkpoint inhibitor and an anti-tumor antigen antibody. The anti-PD-L1 domain acts as a PD-1 binding antagonist. By blocking the binding of PD-L1 (e.g., on tumor cells) to PD-1 (e.g., on T cells), the anti-PD-L1 domain inhibits the associated immune checkpoint, thereby releasing the development of a T cell-mediated immune response. PD-1 blockade using anti-PD-1 or anti-PD-L1 antibodies is a well-known approach to cancer therapy. Therefore, the anti-PD-L1 domain constitutes a means of PD-1 blockade or a means of releasing PD-1 immune checkpoints.

[0148] Anti-PD-L1 domain antibodies, acting as anti-tumor antigens, can mediate the binding of MVSCAs to tumor cells. If the MVSCA also contains an anti-CD16 domain, it promotes NK-mediated ADCC. In the case of an MVSCA containing an anti-CD47 domain, it promotes macrophage-mediated phagocytosis. Multivalent binding to tumor cells enhances binding affinity and ADCC. This can be achieved through 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 for binding to tumor cells, for binding to tumor antigens, or for binding to PD-L1 tumor antigens.

[0149] Anti-LAG3

[0150] 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 the binding of LAG3 on T cells to class II MHC on tumor cells, the anti-LAG3 domain inhibits the associated immune checkpoint, thereby releasing the development of T cell-mediated immune responses. Therefore, the anti-LAG3 domain constitutes a means for releasing LAG3 immune checkpoints.

[0151] Anti-CD33

[0152] Anti-CD33 domains can be used in two ways. CD33 is expressed on myeloid cells and some lymphoid cells, and is expressed in some hematologic malignancies, such as acute myeloid leukemia (AML), thus serving as a tumor antigen. Anti-CD33 domains, as antibodies against tumor antigens, can mediate the binding of MVSCAs to tumor cells. If the MVSCA also contains an anti-CD16 domain, NK-mediated ADCC is promoted. In the case where the MVSCA also contains an anti-CD47 domain, macrophage-mediated phagocytosis is promoted. Multivalent binding to tumor cells enhances binding affinity and ADCC. This can be achieved by having multiple copies of the anti-CD33 domain and / or one or more binding domains targeting other tumor antigens. Therefore, anti-CD33 domains constitute a means of binding to tumor cells, a means of binding to tumor antigens, or a means of binding to CD33 tumor antigens.

[0153] Furthermore, when CD33 binds to sialic acid residues, such as in β-amyloid or other glycoproteins deposited with glycolipids, an inhibitory signaling cascade leads to inhibition of phagocytic activity. Antibodies containing the anti-CD33 domain can act as antagonists of CD33 stimulation, thereby promoting phagocytic activity and clearance of β-amyloid for the treatment of Alzheimer's disease. Retinal diseases, such as dry age-related macular degeneration (AMD), also involve insoluble deposits that can be cleared by microglia phagocytosis. Therefore, antibodies containing the anti-CD33 domain can also be used to treat 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 the clearance of β-amyloid, or a means of clearing insoluble deposits.

[0154] MVSCAs suitable for treating Alzheimer's disease and retinal diseases are preferably bivalent to CD33 and contain an anti-HSA domain to increase half-life. They may also contain an FC5 nanobody domain (Rissiek et al., Front. Cell. Neurosci. 8:344, 2014) to facilitate migration across the human blood-brain barrier.

[0155] connector

[0156] In many embodiments, the binding domains are not directly connected to each other, but instead have short amino acid sequences interposed between them, i.e., linkers. Examples of linkers are shown in Table 15. The length and sequence of the linkers can significantly affect the expression level and structure of the MVSCA, as well as the binding affinity of the connected domains. Length-adjustable linkers L2 and L4 (see Table 15) can be used to optimize the MVSCA based on these parameters. Linkers L1, L2, and L4 can be referred to as indestructible linker means, flexible linker means, or flexible indestructible linker means.

[0157] When two copies of the same VHH domain are placed adjacent to each other in an MVSCA, they frequently and unfavorably interact. This can be avoided by inserting a relatively short and rigid joint between the two copies. In some embodiments, the short rigid joint has a sequence AAA (L3 in Table 15). Such a joint may be referred to as a short rigid joint means or an indivisible short rigid joint means.

[0158] When anti-HSA domain-HSA complexes are used to generate prodrugs with binding activity associated with adjacent binding domains, a cleavable linker should be inserted between the two domains. L11*3 to L11*18 (see Table 15) are examples of cleavable linkers with various lengths and susceptibility to cleavage by different proteases, which can be used to optimize MVSCA expression levels and structure, binding affinity of the linked domains, and cleavage. Linkers L11*3 to L11*18 may be referred to as cleavable linker means, flexible linker means, or flexible cleavable linker means.

[0159] MVSCA

[0160] The binding domains and connectors described herein can be combined to produce multifunctional MVSCAs suitable for the treatment of specific diseases. The binding domains and connectors can also be further combined with other binding domains. MVSCAs can also be referred to as including means for achieving various functions associated with each component type of the binding domain, and / or including connector means for achieving their respective functions. Exemplary designs will be briefly discussed below.

[0161] HSA / CD47 / PD-L1: This design is suitable for treating PD-L1-expressing tumors, will promote phagocytosis, will release PD-1 immune checkpoints, and will have a prolonged circulating half-life. In various embodiments, the MVSCA may be bivalent for anti-CD47 and / or anti-PD-L1 binding domains. Depending on the adapter used, the anti-HSA domain (once bound to HSA) may or may not inhibit binding to CD47, and inhibition (if present) can be reversed by cleaving the cleavable adapter. In some embodiments, the binding domains are arranged in a different order, but the anti-HSA domain should be at the terminal position if to be cleaved. In addition to describing this design of the MVSCA as a means of including one or more functions for its components, the MVSCA may also be referred to as a means of promoting phagocytosis (and releasing PD-1 immune checkpoints) in PD-L1-expressing tumors. Several embodiments of this design are illustrated in Example 7.

[0162] HSA / LAG3 / PD-L1: This design is suitable for treating PD-L1-expressing tumors, releasing LAG3 and PD-1 immune checkpoints, and will have an extended circulating half-life. In various embodiments, the MVSCA may be bivalent for anti-LAG3 and / or anti-PD-L1 binding domains. In some embodiments, the binding domains are arranged in a different order. Besides describing this type of MVSCA as a means of including one or more functions for its components, the MVSCA may also be referred to as a means of recruiting T effector cells to PD-L1-expressing tumors (and releasing LAG3 and PD-1 immune checkpoints). Several embodiments of this design are illustrated in Example 7.

[0163] CD16A / HSA / CD47 / PD-L1: This design is suitable for treating PD-L1-expressing tumors, will promote phagocytosis, will recruit NK cells to mediate ADCC, will release PD-1 immune checkpoints, and will have an extended circulating half-life. In various embodiments, the MVSCA may be bivalent for anti-CD47 and / or anti-PD-L1 binding domains. In some embodiments, the binding domains are arranged in different orders, but the anti-HSA domain should be placed at the terminal position if it is to be cleaved. Depending on the adapter used, the anti-HSA domain (once bound to HSA) will or will not inhibit binding to CD47, and inhibition (if present) can be reversed by cleaving the cleavable adapter. In addition to describing this design of the MVSCA as a means of including one or more functions for its components, the MVSCA may also be referred to as a means of promoting phagocytosis of PD-L1-expressing tumors and recruiting NK-mediated ADCC to PD-L1-expressing tumors (and releasing PD-1 immune checkpoints). Several embodiments of this design are illustrated in Example 8.

[0164] CD16A / HSA / CD47 / CD33: This design is suitable for treating CD33-expressing tumors, will promote phagocytosis, will recruit NK cells to mediate ADCC, and will have a prolonged circulating half-life. In various embodiments, the MVSCA may be bivalent for anti-CD47 and / or anti-CD33 binding domains. In some embodiments, the binding domains are arranged in different orders, but the anti-HSA domain should be placed at the terminal position if it is to be cleaved. Depending on the adapter used, the anti-HSA domain (once bound to HSA) may or may not inhibit binding to CD47, and inhibition (if present) can be reversed by cleaving the cleavable adapter. In addition to describing this design of the MVSCA as a means of including one or more functions for its components, the MVSCA may also be referred to as a means of promoting phagocytosis of CD33-expressing tumors and recruiting NK-mediated ADCC to CD33-expressing tumors. Several embodiments of this design are illustrated in Example 8.

[0165] Bivalent anti-CD33 MVSCAs: These designs are suitable for treating diseases associated with the deposition of insoluble substances by, for example, blocking the inhibition of phagocytosis by microglia. Such diseases include Alzheimer's disease and dry AMD. HSA / CD33 / CD33 designs have an extended circulating half-life. FC5 / CD33 / CD33 designs will cross the blood-brain barrier. FC5 / CD33 / CD33 / HAS designs will have an extended circulating half-life and cross the blood-brain barrier. Simple CD33 / CD33 designs are suitable for local injection into the eye or brain, in which case the extended circulating half-life or the ability to cross the blood-brain barrier has negligible value. In some embodiments, the binding domains are arranged in different orders. Besides describing such MVSCAs as means of including one or more functions for their components, MVSCAs can also be referred to as means of promoting (microglia) phagocytosis of insoluble deposits. Several embodiments of this design are illustrated in Example 9.

[0166] The disclosed uses of the antibodies

[0167] The disclosed antibodies are medically available. The term "treatment," etc., refers to the medical management of a patient with the intent to cure, improve, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or disorder, and also includes etiological treatment, i.e., treatment aimed at eliminating the cause of the associated disease, pathological condition, or disorder. Furthermore, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or disorder; preventative treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of an associated disease, pathological condition, or disorder; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving an associated disease, pathological condition, or disorder. Various implementations may specifically include or exclude one or more of these treatment modalities.

[0168] The use of the antibodies disclosed in this paper in diagnosis and imaging was also considered.

[0169] Furthermore, the term "treatment" broadly encompasses any type of therapeutic activity, including the diagnosis, relief, or prevention of disease in humans or other animals, or aspects thereof, or any activity that otherwise affects the structure or any function of the body of a human or other animal. Therapeutic activities include, in particular, the administration of the medicines, dosage forms, and pharmaceutical compositions described herein to a patient, whether by a healthcare professional, the patient themselves, or any other person. Therapeutic activities include orders, instructions, and recommendations from healthcare professionals (such as physicians, physician assistants, nursing practitioners, etc.) that are then performed by any other person, including other healthcare professionals or the patient themselves. This includes, for example, instructing a patient to undergo or instructing a clinical laboratory to perform diagnostic procedures, such as those used for cancer diagnosis and staging, so that the patient can ultimately receive appropriate and beneficial treatment. In some implementations, treatment activities may include prescriptions, instructions, and recommendations that encourage, induce, or compel the selection of a particular drug or combination thereof to treat the condition, and the drug is actually used by: approving insurance coverage for the drug, refusing alternative drugs (including the drug being on or excluded from a drug formulary), or providing financial incentives for the use of the drug, such as those provided by insurance companies or pharmacy benefit management companies. In some implementations, treatment activities may also include encouraging, inducing, or compel the selection of a particular drug to treat the disease—and the drug is actually used in accordance with policies or practice standards established by hospitals, clinics, health maintenance organizations, medical practices, or physician groups. All such prescriptions, instructions, and recommendations are considered a benefit of receiving treatment conditional upon compliance with the instructions. In some cases, patients may also receive financial benefits for complying with such prescriptions, instructions, and recommendations. In some cases, healthcare professionals may also receive financial benefits for complying with such prescriptions, instructions, and recommendations.

[0170] The disclosed monospecific HCAbs and multivalent single-chain antibodies specific to CD47, HSA, PD-L1, CD33, CD16, and LAG3 can be used to treat cancer. Each antibody is designed to treat a specific type of cancer based on the antigen-binding specificity contained in the antibody.

[0171] The present invention provides a method for treating cancer, the method comprising administering to a patient in need of such treatment an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising said antibody.

[0172] Examples of cancers that can be treated using the disclosed methods include: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer; bladder cancer; bone cancer; brainstem glioma; brain tumor; breast cancer; bronchial adenoma / carcinoid tumor; carcinoid tumor; islet cell carcinoma; tumors of unknown primary origin; central nervous system lymphoma; cerebellar astrocytoma; brain astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorder; colon cancer; colorectal cancer; transcutaneous T-cell lymphoma; endometrial cancer; ependymoma; ovarian epithelial cancer; esophageal cancer; Ewing's family of tumors. Tumors); Extracranial germ cell tumors; Intraocular melanoma; Retinoblastoma; Gallbladder cancer; Gastric cancer; Germ cell tumors; Gestational trophoblastic tumors; Hairy cell leukemia; Head and neck cancer; Hepatocellular carcinoma; Hodgkin's lymphoma; Hypopharyngeal cancer; Kaposi's sarcoma; Kidney cancer; Laryngeal cancer; Non-small cell lung cancer; Small cell lung cancer; Non-Hodgkin's lymphoma; Warburg's macroglobulinemia; Malignant mesothelioma; Malignant thymoma; Meningoblastoma; Melanoma; Merkel cell carcinoma; Squamous neck cancer; Multiple endocrine tumor syndrome Combination syndromes; multiple myeloma / plasma cell tumors; mycosis fungoides; myelodysplastic syndromes; nasopharyngeal carcinoma; neuroblastoma; oral cancer; oropharyngeal cancer; osteosarcoma; pancreatic cancer; parathyroid carcinoma; penile cancer; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; soft tissue sarcoma; Cezari syndrome; skin cancer; squamous neck cancer; testicular cancer; thymoma; thyroid cancer; trophoblastic tumors; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and Wilms' tumor.

[0173] The effectiveness of cancer treatments is typically measured by "response." Techniques for monitoring response can be similar to tests used to diagnose cancer, such as, but not limited to:

[0174] Lumps or tumors involving some lymph nodes can be felt and measured from the outside through a physical examination.

[0175] Some internal cancerous tumors will be visible on X-rays or CT scans and can be measured with a ruler.

[0176] Blood tests can be performed, including those that measure organ function.

[0177] It can be used to test tumor markers for certain cancers.

[0178] Regardless of the test used—whether it's a blood test, cell count, or tumor marker test—it should be repeated at specific time intervals to allow for comparison of results with earlier, similar tests.

[0179] The response to cancer treatment can be defined in several ways:

[0180] Complete response—all cancers or tumors disappear; no signs of disease. Tumor marker expression levels (if applicable) may fall within the normal range.

[0181] Partial response—the cancer has shrunk by a certain percentage, but the disease is still present. The levels of tumor markers (if applicable) may have decreased (or increased, based on tumor markers as an indicator of reduced tumor burden), but signs of disease are still present.

[0182] Stable disease—cancer neither grows nor shrinks; the amount of disease remains unchanged. Tumor markers (if applicable) show no significant changes.

[0183] Disease progression—the cancer has grown; the disease is now more extensive than before treatment. Tumor marker tests (if applicable) show elevated tumor markers.

[0184] Other measures of cancer treatment efficacy include overall survival time (i.e., the time from diagnosis or the start of the evaluated treatment to death from any cause), cancer-free survival time (i.e., the length of time cancer remains undetectable after a full response), and progression-free survival time (i.e., the length of time tumor growth resumes undetectable after disease stabilization or a partial response).

[0185] There are two standard methods for assessing treatment response to solid tumors based on tumor size (tumor burden): the WHO criteria and the RECIST criteria. These methods measure the solid tumor to compare the current tumor size with past measurements, or to compare changes with future measurements, and to adjust the treatment plan accordingly. In the WHO method, the long and short axes of the solid tumor are measured, and the product of these two measurements is calculated; if there are multiple solid tumors, all products are summed. In the RECIST method, only the long axis is measured. If there are multiple solid tumors, all long axis measurements are summed. However, for lymph nodes, the short axis is measured instead of the long axis.

[0186] This disclosure provides a method for treating an eye disease, the method comprising administering the antibody disclosed herein to a patient requiring such treatment. Exemplary eye diseases include age-related macular degeneration (AMD), such as wet AMD or dry AMD; or macular edema, such as diabetic macular edema. In some embodiments, the eye disease is a retinal disease.

[0187] The present invention also provides a method for treating neurodegenerative diseases, including but not limited to 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.

[0188] The following embodiments, sequence listing, and drawings are provided to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the described processes without departing from the spirit of the invention.

[0189] List of specific implementation methods

[0190] The following list of embodiments is an illustration of various embodiments of the width, combination and sub-combination, invention category, etc., described herein, but is not intended to be an exhaustive list of all embodiments for which support is sought herein.

[0191] Implementation Method 1. A heavy chain variable (VHH) domain with antigen-binding specificity to CD47.

[0192] Implementation Method 2. The VHH domain according to Implementation Method 1, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223.

[0193] Implementation Method 3. A heavy chain variable (VHH) domain with antigen binding specificity to PD-L1.

[0194] Implementation 4. The VHH domain according to Implementation 3, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 31 to SEQ ID NO: 38.

[0195] Implementation Method 5. A heavy chain variable (VHH) domain with antigen-binding specificity to human serum albumin (HSA).

[0196] Implementation 6. The VHH domain according to Implementation 5, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48.

[0197] Implementation Method 7. A heavy chain variable (VHH) domain with antigen-binding specificity to CD33.

[0198] Implementation Method 8. The VHH domain according to Implementation Method 7, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78.

[0199] Implementation Method 9. A heavy chain variable (VHH) domain with antigen-binding specificity to LAG3.

[0200] Implementation 10. The VHH domain according to Implementation 9, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO:80 to SEQ ID NO:93.

[0201] Implementation Method 11. A heavy chain variable (VHH) domain with antigen-binding specificity to CD16.

[0202] Implementation 12. The VHH domain according to Implementation 11, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99.

[0203] Embodiment 13. A heavy chain-only antibody (HCAb) comprising a VHH domain according to any one of Embodiments 1-12.

[0204] Implementation 14. An antibody comprising one or more constant domains and means for binding to CD47, HSA, PD-L1, CD33, CD16 or LAG3.

[0205] Embodiment 15. A multispecific antibody comprising one or more of the VHH domain described in Embodiments 1-12 or means for binding CD47, HSA, PD-L1, CD33, CD16 or LAG3.

[0206] Implementation 16. The multispecific antibody according to Implementation 15, wherein the multispecific antibody further comprises one or more additional antibody-binding domains.

[0207] Implementation 17. The multispecific antibody according to Implementation 16, wherein the additional antibody-binding domain comprises FC5 (SEQ ID NO:222).

[0208] Implementation 18. The multispecific antibody according to Implementation 16, wherein the additional antibody-binding domain comprises Fv or Fab.

[0209] Implementation Method 19. The multispecific antibody according to any one of Implementation Methods 15-17, wherein the multispecific antibody is a multispecific single-chain antibody (MVSCA).

[0210] Implementation 20. The MVSCA according to Implementation 19, wherein the MVSCA comprises 2, 3, 4, 5 or 6 antibody-binding domains.

[0211] Implementation Method 21. The MVSCA according to Implementation Method 20, wherein the MVSCA has binding specificity for one, two, three or four antibodies.

[0212] Embodiment 22. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes HSA, CD47 and PD-L1.

[0213] Embodiment 23. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes HSA, CD47 and CD33.

[0214] Embodiment 24. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes HSA, LAG3 and PD-L1.

[0215] Embodiment 25. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes HSA, LAG3 and CD33.

[0216] Embodiment 26. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes CD16, HSA and PD-L1.

[0217] Embodiment 27. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes CD16, HSA and CD33.

[0218] Embodiment 28. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes CD16, HSA, CD47 and PD-L1.

[0219] Embodiment 29. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain that recognizes CD16, HSA, CD47 and CD33.

[0220] Implementation 30. The MVSCA according to any one of Implementations 14-21 or 26-29, wherein the antibody-binding domain that recognizes CD16 preferentially recognizes CD16A.

[0221] Implementation 31. The MVSCA according to any one of Implementations 19-30, wherein the MVSCA comprises two adjacent antibody-binding domains having the same specificity.

[0222] Implementation 32. The MVSCA according to Implementation 31, wherein the two adjacent antibody-binding domains having the same specificity have a short rigid linker means inserted between them.

[0223] Embodiment 33. The MVSCA according to Embodiment 31, wherein the short rigid connector means consists of the amino acid sequence AAA (SEQ ID NO:102).

[0224] Implementation 34. The MVSCA according to any one of Implementations 31-33, wherein the two adjacent antibody-binding domains bind CD33.

[0225] Implementation 35. The MVSCA according to Implementation 34, wherein the MVSCA further comprises an antibody-binding domain that recognizes HSA.

[0226] Implementation 36. The MVSCA according to implementation 34 or 35 further includes FC5.

[0227] Implementation 37. The MVSCA according to any one of Implementations 31-33, wherein the two adjacent antibody-binding domains bind PD-L1.

[0228] Implementation 38. The MVSCA according to any one of Implementations 31-33, wherein the two adjacent antibody-binding domains bind to LAG3.

[0229] Implementation 39. The MVSCA according to any one of Implementations 31-33, wherein the two adjacent antibody-binding domains bind CD16.

[0230] Implementation 40. The MVSCA according to any one of Implementations 31-33, wherein the two adjacent antibody-binding domains bind CD47.

[0231] Implementation 41. The MVSCA according to any one of Implementations 19-30, wherein the MVSCA includes a linker between adjacent antibody-binding domains.

[0232] Implementation 42. The MVSCA according to Implementation 4', wherein the connector inserted between different antigen-binding domains is L1 (SEQ ID NO: 100), L2 (SEQ ID NO: 101), or L4 (SEQ ID NO: 103).

[0233] Implementation 43. The MVSCA according to any one of Implementations 19-30, wherein the MVSCA includes a flexible, indivisible connector means inserted between different antigen-binding domains.

[0234] Embodiment 44. The MVSCA according to any one of Embodiments 19-30, wherein the MVSCA comprises an antibody-binding domain located at the N-terminus or C-terminus of HSA.

[0235] Implementation 45. The MVSCA according to Implementation 44, wherein when binding to HSA, the antibody-binding domain adjacent to the antibody-binding domain that binds to HSA is inhibited from binding to its antigen.

[0236] Implementation 46. The MVSCA according to Implementation 45, wherein the antibody-binding domain adjacent to the antibody-binding domain that binds the HSA recognizes CD47.

[0237] Embodiment 47. The MVSCA according to Embodiment 45 or 46, wherein a cleavable connector is inserted between the antibody-binding domain that binds to HSA and the adjacent antibody-binding domain, wherein the cleavable connector 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:104), L11*15 (SEQ ID NO:116), L11*16 (SEQ ID NO:107), L11*10 (SEQ ID NO:108), L11*11 (SEQ ID NO:109 ... ID NO:117), L11*17 (SEQ ID NO:118) or L11*18 (SEQ ID NO:119).

[0238] Implementation 48. The MVSCA according to Implementation 45 or 46, wherein a cleavable connector means is inserted between the antibody-binding domain that binds HSA and the adjacent antibody-binding domain.

[0239] Implementation 49. The MVSCA according to any one of Implementations 19-48, wherein all of the antibody-binding domains are VHH domains.

[0240] Embodiment 50. A pharmaceutical composition comprising a VHH domain or an antibody according to any one of Embodiments 1-49.

[0241] Implementation Method 51. A pharmaceutical composition comprising means for binding HSA, means for prolonging the half-life of a multispecific antibody or MVSCA in vivo, and means for reversibly inhibiting the binding activity of adjacent binding domains.

[0242] Implementation 52. A pharmaceutical composition comprising means for binding CD47 or means for reducing inhibition of phagocytosis.

[0243] Implementation 53. A pharmaceutical composition comprising means for binding CD16 or CD16A or means for recruiting NK-mediated ADCC.

[0244] Implementation Method 54. A pharmaceutical composition comprising means for binding PD-L1, means for binding PD-L1 tumor antigen, means for PD-1 blockade, or means for releasing PD-1 immune checkpoints.

[0245] Embodiment 55. A pharmaceutical composition comprising means for binding a tumor antigen, means for binding the PD-L1 tumor antigen, or means for binding the CD33 tumor antigen.

[0246] Embodiment 56. A pharmaceutical composition comprising means for binding LAG3 or means for releasing the LAG3 immune checkpoint.

[0247] Implementation 57. A pharmaceutical composition comprising means for releasing an immune checkpoint, means for releasing the PD-1 immune checkpoint, or means for releasing the LAG3 immune checkpoint.

[0248] Implementation Method 58. A pharmaceutical composition comprising means for binding CD33, means for binding the CD33 tumor antigen, means for promoting the clearance of β-amyloid protein, or means for clearing insoluble deposits.

[0249] Implementation 59. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1.

[0250] Implementation 60. A pharmaceutical composition comprising means for recruiting T effector cells to a tumor expressing PD-L1.

[0251] Implementation 61. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1 and recruiting NK-mediated ADCC to the tumors expressing PD-L1.

[0252] Implementation 61. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1 and recruiting NK-mediated ADCC to the tumors expressing PD-L1.

[0253] Implementation Method 62: A pharmaceutical composition comprising means for promoting phagocytosis of CD33-expressing tumors and recruiting NK-mediated ADCC to the CD33-expressing tumors.

[0254] Embodiment 63. A method for treating cancer, the method comprising administering to a patient in need an antibody according to any one of Embodiments 1-48 or a pharmaceutical composition according to any one of Embodiments 49-61.

[0255] Embodiment 64. A method for treating Alzheimer's disease or retinal disease, the method comprising administering to a patient in need an antibody according to any one of Embodiments 7-8, 13-21, 34-36 or 48-49 or a pharmaceutical composition according to Embodiment 58, wherein the antibody comprises a CD33 binding domain.

[0256] Implementation 65. The method according to Implementation 64, wherein the antibody does not contain an antibody-binding domain that recognizes CD47, PD-L1, LAG3 or CD16.

[0257] Implementation 66. The method according to implementation 64 or 65, wherein the retinal disease is dry AMD.

[0258] For each of embodiments 63-66, there are corresponding embodiments for a composition for treatment, a composition for manufacturing a medicine, the use of the composition in treatment, and the use of the composition in manufacturing a medicine.

[0259] Example

[0260] Example 1. Anti-CD47 HCAb antibody

[0261] Anti-CD47 HCAb antibodies were isolated from immunized llamas.

[0262] Immunization. Two llamas were immunized at Abcore Inc (Ramona, CA) according to their standard protocol. Recombinant human CD47 (extracellular domains 19 to 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). Each llama received six subcutaneous injections at 50 μg / dose at bi-weekly intervals. On day 45, serum was collected from the llamas immunized with recombinant CD47 protein to determine the titer of anti-hCD47 antibodies by ELISA. In the ELISA, 96-well Maxisorp plates (Nunc) were coated with 100 ng / well of hCD47. After blocking and adding diluted serum samples, the presence of anti-CD47 antibodies was confirmed using horseradish peroxidase (HRP)-conjugated goat anti-camel IgG (H+L) antibody (Invitrogen).

[0263] SEQ ID NO:1 Extracellular domain of human CD47 (19-139, Q08722)

[0264] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP

[0265] Construction and selection of phage libraries. Peripheral blood mononuclear cells (PBMCs) were prepared from day 45 blood samples from llamas immunized with recombinant CD47 protein using Ficoll-Paque Plus (GE Healthcare), following the manufacturer's instructions. Total RNA was extracted from PBMCs using the RNeasy Midi kit (Qiagen), following the manufacturer's instructions, and used as starting material for RT-PCR to amplify gene fragments encoding VHH. These fragments were cloned into phage vectors, allowing the generation of recombinant phage particles after infection with helper phages, which displayed VHH as a gene-111 fusion protein on the surface of the phage particles. Phages were prepared according to standard methods and stored at 4°C for later use after filtration sterilization.

[0266] To select phages that bind to CD47, biotinylated CD47 was incubated with a phage library and subsequently captured on streptavidin Dynabeads (Invitrogen). After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The selected outputs were rescued into *E. coli* TG1 cells. Colonies were picked and sequenced at BATJ, Inc. (San Diego, CA).

[0267] At Atum (Newark, CA), cDNA encoding CD47-binding VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. Positive VHH was then purified by IMAC chromatography.

[0268] CD47-binding phage colonies from an immunized llama phage library were sequenced, and their amino acid sequences, listed below (Table 2), were determined for each VHH. cDNA sequences based on these amino acid sequences were fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant anti-CD47 HCAb antibodies. The expressed anti-CD47 HCAb was purified using a HiTrap protein A column.

[0269] A09-10 VHH is a humanization based on the IGHV3-23 human germline sequence.

[0270] Table 2. Alpaca anti-CD47 VHH sequence

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277] *Biv=divalent

[0278] The VHH in Table 2 constitutes a means for binding CD47.

[0279] Octet against CD47 HCAb molecules ® Combined analysis

[0280] Bio-Layer Interferometry (BLI), a label-free technique, was used to measure the binding kinetics of human CD47 (R&D systems) with anti-CD47 VHH. Octet was used with an Octet equipped with an anti-Penta-His capture (HIS1K) biosensor tip (FortéBio®). ® QK e Affinity measurements were performed. The assay was performed at 30°C in 1x PBS buffer (Gibco®, PBS pH 7.2). The sample was stirred at 1000 rpm. The sensor was humidified for 15 min prior to analysis. The binding capacity of purified anti-CD47 VHH was tested using the HIS1K sensor tip. The tip was loaded with 20 g / ml of anti-CD47 VHH. Loading was performed for 300 seconds, resulting in capture levels between 1.8 nm and 2 nm. Human CD47 antigen for binding analysis was prepared by dilution to 100 nM, 150 nM, 250 nM, and 350 nM in 1x PBS. Association was initiated and monitored for 200 seconds, after which the tip was transferred to 1x PBS buffer (Gibco, PBS pH 7.2) to monitor dissociation. Sensor data were collected, processed, and analyzed using Octet throughout the experiment. ® The data was analyzed using FortéBio® data analysis software.

[0281] Table 3 lists the binding affinity of anti-CD47 HCAb. ® Kinetic analysis. HCAb A09-04, A09-06, A09-08, and A09-10 exhibit pM binding affinity.

[0282] Table 3. Binding affinity (K) for anti-CD47 HCAb D Octet ® Kinetic analysis

[0283]

[0284] Flow cytometry analysis of the binding affinity of anti-CD47 HCAb to CHO cell lines overexpressing CD47.

[0285] Add 1×10 to ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3) 6 CHO cells overexpressing CD47 were incubated at concentrations ranging from 100 nM to 0.00128 nM with anti-CD47 HCAb or as a control, along with B6H12 anti-CD47 antibody, on ice for 45 min. Cells were washed with FACS buffer and incubated with goat anti-human IgG Fc, FITC conjugate antibody (ThermoFisher) according to the manufacturer's instructions, followed by incubation at 4°C for 30 min. Data were acquired using a GuavaEasyCyte HT system.

[0286] Using the flow cytometry method described above, the binding affinity of anti-CD47 HCAb was determined to be EC50. 50 ,like Figure 1 What is depicted.

[0287] Competitive ELISA binding assay of multispecific molecules with anti-CD47 domain

[0288] Competitive ELISA binding assays were performed to screen for 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 containing anti-CD47 VHHA09-10, which competitively blocks the binding of the CD47 antigen to its receptor SIRPα. The multispecific antibodies were identified by their binding domain (i.e., CD47) and the linker separating the binding domain (i.e., L1, identified in Table 15). 100 nmol / well of CD47-Fc (R&D systems) was coated into 96-well plates. 10 nM biotinylated human SIRPα was pre-incubated with different concentrations of multispecific molecules 1511 and 3321, followed by the addition of HRP-conjugated streptavidin. Multispecific molecules 1511 and 3321 competitively blocked CD47 from interacting with its receptor SIRPα via EC50. 50 Combination, such as Figure 2 What is depicted.

[0289] Competitive flow cytometry-based analysis of multispecific molecules with anti-CD47 domain

[0290] Competitive flow cytometry binding assays were performed to confirm that multispecific molecules 1511 and 3321 block the binding of CD47 antigen to its receptor SIRPα on the surface of cells naturally expressing CD47. 1 × 10⁻⁶ molecules were added to ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN₃). 6 Jurkat cells / ml (ATCC) were incubated with 1511 or 3321 at concentrations ranging from 100 nM to 0.00128 nM on ice for 45 min, followed by the addition of 25 nM SIRP. -Fc (R&D systems) and incubate for another 45 min. Following the manufacturer's instructions, wash cells with FACS buffer and add goat anti-human VHH FITC conjugate antibody (Jackson Immuno Research), then incubate at 4°C for 30 min. Data were acquired using the Guava EasyCyte HT system. Multispecific molecules 1511 and 3321 competitively block CD47 from its receptor SIRPα on the surface of Jurkat cells via EC50. 50 Combination, such as Figure 3 What is depicted.

[0291] Human RBC agglutination assay with multispecific molecules containing anti-CD47 domain

[0292] Human blood samples were provided from healthy donors. Whole blood was centrifuged at 3000 rpm (1800 rcf) for 5 min to remove plasma and the erythrocyte sedimentation rate (ESR) layer. Red blood cells were resuspended in physiological saline (0.9% NaCl) at approximately twice their volume, and the tubes were inverted to mix. The red blood cells were further centrifuged at 2000 rpm for 20 min, and the RBCs were mixed with physiological saline to obtain a 6% (v / v) cell suspension. The RBCs were then added to 96-well round-bottom plates and mixed with varying amounts of antibody (0 ug / ml to 100 ug / ml). The plates were incubated at 37°C for 2 h. Unlike the Hu5F9 anti-CD47 control antibody, the multispecific molecules 1511 and 3321 did not induce RBC aggregation, as depicted in Figure 4.

[0293] Flow cytometry binding assays were performed to confirm the multispecific molecule, anti-CD47 VHH containing 1511 and 3321, which selectively binds to the surface of tumor cells naturally expressing CD47 but not to CD47 on the surface of RBC cells. 1 × 10⁻⁶ ions were added to ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN₃). 6 HL60 cells / ml (ATCC) 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 min. Cells were washed with FACS buffer and incubated with goat anti-human VHH FITC conjugate antibody (Jackson Immuno Research) according to the manufacturer's instructions, followed by incubation at 4°C for 30 min. Data were acquired using the Guava EasyCyte HT system. The multispecific molecules 1511 and 3321 were analyzed using EC50. 50 It selectively binds to the surface of tumor cells that naturally express CD47, but does not bind to CD47 on the surface of RBC cells, such as... Figure 4B What is depicted.

[0294] Antitumor activity of multispecific molecules with anti-CD47 domain

[0295] 1E6 Raji-Luc cells were intravenously seeded into NSG mice. Mice were treated daily with either 10 mg / kg of multispecific molecule 3321 or a PBS control. Representative bioluminescent images of Raji tumors at the start of treatment (day 0), mid-experiment day 3, and end of the experiment (day 7). Multispecific molecule 3321 protects xenograft mice from developing human leukemia, such as... Figure 5 What is depicted.

[0296] Example 2. Anti-PD-L1 HCAb antibody

[0297] Anti-PD-L1 HCAb antibodies were isolated from immunized camels.

[0298] Two llamas were immunized at Abcore Inc. according to their standard protocol. Recombinant human PD-L1 (extracellular domains 19 to 238, SEQ ID NO: 30) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). Each llama received six subcutaneous injections at 50 μg / dose at bi-weekly intervals. On day 45, serum was collected from the llamas immunized with recombinant PD-L1 protein to determine the anti-PD-L1 titer by ELISA. In the 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 confirmed using HRP-conjugated goat anti-camel IgG (H+L) antibody.

[0299] SEQ ID NO:30 Extracellular domains of human PD-L1 (19-238, Q9NZQ7)

[0300] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0301] Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples taken from llamas immunized with recombinant PD-L1 protein on day 45 using Ficoll-Paque+, following the manufacturer's instructions. Total RNA was extracted from PBMCs using the RNeasy Midi kit, following the manufacturer's instructions, and used as starting material for RT-PCR to amplify gene fragments encoding VHH. These fragments were cloned into phage vectors, allowing the generation of recombinant phage particles after infection with helper phages, which displayed VHH as a gene-111 fusion protein on their surface. Phages were prepared according to standard methods and stored at 4°C for later use after filtration sterilization.

[0302] To select VHHs that bind PD-L1, biotinylated PD-L1 was incubated with a phage library and subsequently captured on streptavidin Dynabeads. After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The selected outputs were rescued into *E. coli* TG1 cells. Colonies were picked and sequenced at BATJ, Inc.

[0303] The cDNA encoding VHH that binds to PD-L1 was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. Positive VHH was then purified by IMAC chromatography.

[0304] Phage colonies binding to PD-L1 from an immunized llama phage library were sequenced. The amino acid sequence of each VHH is listed below (Table 4). cDNA sequences based on the following amino acid sequences were fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant anti-PD-L1 HCAb antibodies. The expressed anti-PD-L1 HCAb was purified using a HiTrap protein A column. Two of the llama VHH, PL14, and PL16 were humanized based on the IGHV3-23 human germline sequence.

[0305] Table 4. Llama anti-PD-L1 VHH sequence

[0306]

[0307]

[0308] The VHH in Table 4 constitutes the means for binding PD-L1.

[0309] Octet ® Dynamics combined analysis

[0310] Perform Octet as in Example 1 ® Kinetic binding analysis. Briefly, the binding capacity of purified anti-PD-L1 VHH was tested using the HIS1K sensor tip. The tip was loaded with 20 g / ml of anti-PD-L1 VHH. Loading was performed for 300 seconds, resulting in capture levels between 1.8 nm and 2 nm. Human PD-L1 antigen for binding analysis was prepared by dilution to 100 nM, 150 nM, 250 nM, and 350 nM in 1x PBS. Association was initiated and monitored for 200 seconds, after which the tip was transferred to 1x PBS buffer without PD-L1 protein to monitor dissociation.

[0311] Table 5 presents the Octet of binding affinity for anti-CD47 HCAb.® Kinetic analysis. The analysis showed that PL14, PL16, and PL17 exhibited pM binding affinity.

[0312] Table 5. Binding kinetics (KD) analysis of anti-PD-L1 HCAbs

[0313]

[0314] Flow cytometry combined analysis of anti-PD-L1 HCAb

[0315] Add 1×10 to ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN3) 6 CHO cells overexpressing PD-L1 were incubated with anti-PD-L1 HCAb at concentrations ranging from 100 nM to 0.00128 nM on ice for 45 min. Cells were washed with FACS buffer and incubated with goat anti-human IgG Fc-FITC conjugate antibody (ThermoFisher) for 30 min at 4°C. Data were acquired using a Guava EasyCyte HT system. The binding affinity of anti-PD-L1 HCAb to PD-L1 on CHO cells overexpressing PD-L1 was determined to be EC50. 50 , Figure 6 What is depicted.

[0316] Cell-based functional assays of multispecific molecules with PD-L1 binding domains

[0317] PD-L1-expressing APC / CHO-K1 cells were seeded at 100 K / well in 96-well plates and incubated at 37°C for 16 hours. Next, starting at 100 nM, multispecific molecule 1511 and the control antibody atezolizumab were serially diluted 1:3 and added to the wells at 25 µl / well. Finally, PD-1 effector cells (PD-1 and luciferase-expressing cells) 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 multilabel microplate reader. Data analysis was performed using GraphPad Prism software.

[0318] Cellular functional data indicated that the multispecific molecule 1511 and the control antibody atezolizumab completely blocked PD-L1 activity and had similar EC50 values, such as... Figure 7 As shown.

[0319] Antitumor activity of multispecific molecules with PD-L1 binding domain

[0320] MC38-hPD-L1 colon cancer cells from mice (Biocytogen Co., Ltd.; 5×10⁶) were used.5 Subcutaneous implantation was performed on homozygous B-hPD-L1 mice (female, 6 weeks old, n=6). When the tumor volume reached approximately 100 mm... 3 At this time, the mice were divided into groups, and they were then treated with the multispecific molecule 1518 (SEQ ID NO: 157; CD16F-L1-HSA-L1-CD47-L3-CD47-L1-PDL1-L3-PDL1) to... Figure 8A Treatment with the indicated dosage and regimen. Weight changes during treatment. Figure 8B As shown in the image. Figure 8A As shown, the multispecific molecule 1518A1 is effective in controlling tumor growth in B-hPD-L1 mice. Values ​​are expressed as mean ± SEM.

[0321] Example 3. Anti-HSA HCAb antibody

[0322] Isolation of anti-HSA VHH antibody

[0323] As described in Example 1, llamas were immunized at 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).

[0324] Human serum albumin (SEQ ID NO:39)

[0325] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0326] To select for anti-HSA VHH, biotinylated HSA was incubated with a phage library and subsequently captured on streptavidin Dynabeads (Invitrogen). After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The selected outputs were rescued into *E. coli* TG1 cells. Colonies were picked and sequenced at BATJ, Inc. (San Diego, California).

[0327] The cDNA encoding HSA-specific VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. Positive VHH was then purified by IMAC chromatography.

[0328] Phage clones binding to HSA from the llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 6). cDNA sequences based on the following amino acid sequences were synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant single-domain antibodies (sdAbs) with a C-terminal his tag. The expressed sdAbs were purified using a HisTrap HP column.

[0329] Two of the llamas, VHH, HS5, and HS10, were humanized based on the IGHV3-23 human germline sequence.

[0330] Table 6. Llama anti-HSA VHH sequence

[0331]

[0332]

[0333] These VHHs constitute the means for binding HSA.

[0334] Octet ® Dynamics combined analysis

[0335] Perform Octet as in Example 1 ® The kinetic analysis was combined with the results, which are presented in Table 7 and... Figure 9 Clones HS5, HS6, HS12, and HS27 exhibited affinity for HSA. Cross-species activity was confirmed and is listed in Table 8.

[0336] Table 7. Binding affinity (K) of anti-HSA VHH antibodies D )

[0337]

[0338] Table 8. Cross-species binding affinity of HS10 and HS5 VHH

[0339]

[0340] Example 4. Anti-CD33 HCAb antibody

[0341] Isolation of anti-CD33 VHH antibody

[0342] Alpacas were immunized at Abcore, Inc. with recombinant human CD33 (SEQ ID NO:49) mixed with either full Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization) and a phage library prepared as described in Example 1.

[0343] Human CD33 (SEQ ID NO:49, P20138|18-259)

[0344] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHV TDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVH

[0345] To select for anti-CD33 VHH, biotinylated CD33 was incubated with a phage library and subsequently captured on streptavidin Dynabeads. After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The selected output was rescued into *E. coli* TG1 cells. Colonies were picked and sequenced at BATJ, Inc.

[0346] The cDNA encoding VHH that binds to CD33 was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. Positive VHH was then purified by IMAC chromatography.

[0347] CD33-binding phage colonies from an immunized llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 9). A cDNA sequence based on the following amino acid sequence was fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant anti-CD33 HCAb antibody. The expressed anti-CD33 HCAb was purified using a HiTrap protein A column.

[0348] One of the llamas, VHH or 33-14, was humanized based on the IGHV3-23 human germline sequence.

[0349] Table 9. Llama anti-CD33 VHH sequence

[0350]

[0351]

[0352]

[0353]

[0354]

[0355] In Table 9, VHH constitutes a means for binding CD33.

[0356] Perform Octet as in Example 1 ® Dynamics combined with analysis, and K D The results are shown in Figure 10 and Table 10.

[0357] Table 10. Binding affinity of anti-CD33 VHH antibodies

[0358]

[0359] Example 5. Anti-LAG3 VHH

[0360] Isolation of anti-LAG3 VHH antibody

[0361] Llamas were immunized at Abcore Inc. according to their standard protocol. Recombinant human LAG3 (extracellular domains 19 to 238, SEQ ID NO: 79) was mixed with either complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). Each llama was administered six subcutaneous injections at 50 μg / dose at bi-weekly intervals. On day 45, serum was collected from llamas immunized with recombinant human LAG3 protein to determine the antibody titer against human LAG3 by ELISA. In the ELISA, 96-well Maxisorp plates were coated with 100 ng / well of LAG3. After blocking and adding diluted serum samples, the presence of anti-LAG3 antibodies was confirmed using the antibody titer of the antiserum as determined by ELISA. 96-well Maxisorp plates were also coated with 100 ng / well of hLAG3. After blocking and adding diluted serum samples, the presence of anti-LAG3 antibodies was confirmed using HRP-conjugated goat anti-camel IgG (H+L) antibody.

[0362] Extracellular domains of human LAG3 (SEQ ID NO:79, P18627, 23-450)

[0363] VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFS LWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPW GCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNA TVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL

[0364] Phage libraries were prepared as described in Examples 1 to 4. cDNA encoding LAG3-binding VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. The LAG3-binding VHH was then purified by IMAC chromatography.

[0365] LAG3-binding phage colonies from an immunized llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 11). A cDNA sequence based on the following amino acid sequence was fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant anti-LAG3 HCAb antibody. The expressed anti-LAG3 HCAb was purified using a HiTrap protein A column.

[0366] One of the llamas, VHH or LG9, was humanized based on the IGHV3-23 human germline sequence.

[0367] Table 11. Llama anti-LAG3 VHH sequence

[0368]

[0369]

[0370] The VHH in Table 11 constitutes the means for binding LAG3.

[0371] Octet for anti-LAG3 VHH as in Examples 1 to 4 ® The combined analysis and results are shown in Table 12.

[0372] Table 12. Binding affinity (K) of anti-LAG3 single-domain antibodies D )

[0373]

[0374] Example 6. Anti-CD16 VHH

[0375] Isolation of anti-CD16 VHH antibody

[0376] Llamas were immunized at Abcore Inc. according to their 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). Each llama was administered six subcutaneous injections at 50 μg / dose at bi-weekly intervals. On day 45, serum was collected from immunized llamas to determine antibody titers by ELISA. In the 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 demonstrated using HRP-conjugated goat anti-camel IgG (H+L) antibody.

[0377] Human CD16A (SEQ ID NO:94)

[0378] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0379] Human CD16B (SEQ ID NO:95)

[0380] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRW VFKEEDPIHLRCHSWKNTALHKVTYLQNGKDRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDTGLYFSVKTNI

[0381] Phage libraries were prepared as described in Example 1. The cDNA encoding CD16A-binding VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. The CD16A-binding VHH was then purified by IMAC chromatography.

[0382] Phage colonies binding to CD16A from an immunized llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 13). A cDNA sequence based on the following amino acid sequence was fused with human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate recombinant anti-CD16 HCAb antibody. The expressed anti-CD16 HCAb was purified using a HiTrap protein A column.

[0383] One of the llamas, VHH or CD16F1, was humanized based on the IGHV3-23 human germline sequence.

[0384] Table 13. Anti-CD16 VHH sequences

[0385]

[0386] The VHH in Table 13 constitutes a means for binding CD16.

[0387] Octet for anti-CD16 VHH, as in Example 1 ® The combined analysis is presented in Table 14 and Figure 11.

[0388] Table 14. Binding affinity (KD) of anti-CD16 VHH antibodies

[0389]

[0390] CD16-F1 is selective for CD16A, while CD16-E11 binds to both CD16A and CD16B.

[0391] CD16F1 and CD16E11 are both agonist anti-CD16 VHH antibodies that activate CD16A in the Jurkat-Lucia NFAT-CD16ADCC reporting assay (Invivogen).

[0392] The functional assays of multispecific molecules 1511 and 3321 (containing anti-CD16A VHH CD16F1) and control anti-CD47 antibodies B6H12 IgG1 and B6H12 IgG4 were performed using the Jurkat-Lucia NFAT-CD16 reporting assay (Invivogen), and the results were expressed as follows: Figure 12 Data indicate that CD16F1 is an effective CD16A agonist.

[0393] Example 7. Trispecific single-chain antibody (HSA / CD47 / PD-L1 or HSA / LAG3 / PD-L1)

[0394] To construct trispecific 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 CD33 VHH sequences, were fused together via adapters in six different ways (Figure 13) using recombinant DNA technology. Figure 13 depicts the structures of exemplary trispecific molecules: 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 adapter sequences are presented in Table 15. These constitute adapter means or means for linking protein domains. These means can be further characterized as cleavable or non-cleavable. The amino acid sequences of exemplary trispecific molecules are presented in Table 16. The adapter sequences in Tables 16 and 17 are underlined.

[0395] Table 15. Non-cuttable and cuttable joint sequences

[0396]

[0397]

[0398] Table 16. Trispecific Molecules

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415]

[0416]

[0417] By binding to HSA, the anti-HSA domain can prolong the in vivo half-life of MVSCA. It may also interfere with the activity of other domains, which is desirable in some cases for MVSCA distributed throughout the body, but undesirable when MVSCA is located at its intended site of action (e.g., a tumor). Therefore, the anti-HSA domain is linked to other antigen-binding domains via a cleavable linker that is preferentially cleaved at the intended site of action. In this way, MVSCA can act as a prodrug. An MVSCA with a linker containing a protease-cleavable sequence is used between HSA·VHH and CD47·VHH or LAG3 VHH, and different linkers are used to link CD47·VHH with PD-L1·VHH or LAG3 VHH with PD-L1, as shown in Figure 13 and Table 9. Figure 14B It shows the Figure 14A SDS-page analysis was performed on the antibodies after protease digestion.

[0418] Protease assay

[0419] (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). rhMMP-9 was then activated by adding APMA (mercuric p-aminophenylacetate, Sigma) to a final concentration of 1 mM and incubating at 37°C for 24 h. The activated rhMMP-9 was titrated with an equal volume of 20 µM antibody in assay buffer and incubated at room temperature for 1 h. The digested substrate was analyzed by SDS-PAGE.

[0420] (2) Assay for u-plasminogen activator (uPA, urokinase). 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 assay buffer. The reaction mixture was incubated at room temperature for 1–2 hours, and the digested substrate was analyzed by SDS-PAGE.

[0421] (3) Assay for matropeptidase activity. The substrate was diluted to 200 µM in assay buffer (50 mM Tris, 50 mM NaCl, 0.01% Tween 20) and titrated with an equal volume of recombinant human matrix enzyme (R&D Systems) in assay buffer. The reaction mixture was incubated at room temperature for 1–2 hours and the digested substrate was analyzed by SDS-PAGE.

[0422] Polyacrylamide gel electrophoresis (SDS-PAGE). Denaturing SDS-PAGE was performed according to the Invitrogen NuPAGE® instructions. Briefly, 7.5 μL of protein sample (3 μg protein) was mixed with 2.5 μL of 4X LDS sample loading buffer (Invitrogen) and heated at 70°C for 10 min. The sample was then loaded into a pre-prepared NuPAGE Novex 4-12% Bis-Tris 1.0 mm microgel (Invitrogen). Then, 5 μL of pre-stained SDS-PAGE standards (Bio-Rad) were loaded for each gel run. Electrophoresis was performed at room temperature in a 1X solution of NuPAGE MOPS SDS run buffer (Invitrogen) using a constant voltage (200V) for approximately 45 min, until the dye front reached the end of the 60 mm gel. The gel was stained with SimplyBlue SafeStain (Invitrogen).

[0423] Figure 15 A real-time kinetic binding assay was described for PD-L1 / pro-CD47 (HSA-CD47-PD-L1 antibody) versus PD-L1 / active CD47 (the same antibody with its HSA-binding domain cleaved) in the presence of 10 mg / ml HSA. PD-L1 / pro-CD47 showed little or no binding to CD47, while PD-L1 / active CD47 exhibited strong binding to CD47. No difference or effect on PD-L1 binding was observed in this assay.

[0424] Example 8. A multispecific molecule containing CD16A / HSA / CD47 / (PD-L1 or CD33)

[0425] To construct multispecific molecules, anti-CD16A, anti-HSA, anti-CD47, and anti-PD-L1 or CD33 VHH sequences were fused together in eight different ways via adapters using recombinant DNA technology. Figure 16 The structures of exemplary multispecific molecules against CD16A, HSA, CD47, and PD-L1 or CD33 VHH are depicted. The amino acid sequences of the exemplary multispecific molecules are listed below (Table 17).

[0426] Figure 17 In a flow cytometry binding assay on HL60 cells, the lengths of the linker G4SG3S(L1, SEQ ID NO:100) versus (G4S)3(L4, SEQ ID NO:103) between VHH2 and VHH3 were compared. HL60 cells express CD47 but not PD-L1; therefore, the binding of 1518-HS5 (SEQ ID 173) and 1518-HS5-GS15 (SEQ ID 184) indicates that these two molecules bind to CD47 on the surface of HL60 cells. Figure 18 Longer linkers, such as (G4S)3 (GS15 represents a 15-amino acid linker), improved CD47 binding compared to G4SG3S (9 amino acids), with an EC50 of 8.4 nM compared to 26 nM.

[0427] Table 17. Multispecific molecules

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460]

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467]

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494] Octet of multispecific molecules, as in Example 8 ® Combined analysis, and the results are expressed in Figure 17 and Figure 18 middle.

[0495] Example 9. MVSCA containing an anti-CD33 domain for the treatment of Alzheimer's disease and retinal diseases.

[0496] Table 18. MVSCAs containing anti-CD33 VHH*

[0497]

[0498]

[0499] *The sequence of connectors inserted between VHH structural domains is underlined.

[0500] This sequence is shown with an optional C-terminal his tag.

[0501] Each of the four MVSCAs in Table 18 contains a pair of anti-CD33 domains that bind via the linker L3 (sequence AAA; SEQ ID NO: 102). The first entry in Table 18, hHS5-L1-H33-14-L3-H33-14, contains an N-terminal anti-HSA domain to increase in vivo half-life. The second entry in Table 18, FC5-L1-H33-14-L3-H33-14, contains an N-terminal FC5 nanobody domain to facilitate crossing the blood-brain barrier. The third entry in Table 18, FC5-L1-H33-14-L3-H33-14-L1-hHS5, contains an N-terminal FC5 nanobody domain to facilitate crossing the blood-brain barrier and a C-terminal anti-HSA domain to increase in vivo half-life. These three forms are generally suitable for systemic administration, such as intravenous or subcutaneous injection or infusion. The fourth entry in Table 18, H33-14-L3-H3314, is a bivalent, monospecific MVSCA that is specific only for CD33. Its small size makes it more suitable for local injection into the brain or eye.

[0502] The amino acid sequence of the FC5 nanobody domain is:

[0503] EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSS (SEQ ID NO: 222).

[0504] Example 10. Multiple sequence alignment

[0505] Figures 21 to 26 Multiple sequence alignments of the disclosed VHH sequences performed via Clustal O (1.2.4) for each specificity are presented, making it easy to see identical, conserved, and highly variable positions. Each position in the alignment is indicated by a symbol: an asterisk for identity, a colon for a higher degree of conservation, a period for a lower degree of conservation, and a space for a general lack of conservation in the aligned sequence.

[0506] Unless otherwise stated, all figures used in the specification and claims to indicate the amount, properties (e.g., molecular weight), reaction conditions, etc., of components should be understood to be modified by the term "about" in all cases. As used herein, the terms "about" and "approximately" mean within 10% to 15%, preferably within 5% to 10%. Therefore, unless otherwise indicated, the numerical parameters listed in the specification and appended claims are approximate values, which may vary according to the desired properties sought to be obtained according to the invention. In any case, without attempting to limit the application of equivalents to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant digits reported and by applying common rounding techniques. Although the numerical ranges and parameters that illustrate the broad scope of the invention are approximate values, the values ​​listed in the specific embodiments are reported as accurately as possible. However, any numerical value inherently contains some error that is necessarily caused by the standard deviation present in its respective test measurement.

[0507] Unless otherwise specified herein or obviously contradicted by the context, the terms “a,” “an,” “the,” and similar designations used in the context of describing the invention (especially in the context of the following claims) should be interpreted as encompassing both the singular and plural. The description of value ranges herein is intended only as a shorthand method of referring to each individual value falling within that range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were individually referenced herein. Unless otherwise stated herein or obviously contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all examples or illustrative language (e.g., “for example”) provided herein is intended only to better illustrate the invention and not to limit the scope of the claimed invention. No language in the specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0508] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements found herein. It is contemplated that one or more members of a group may be included in or removed from the group for convenience and / or patentability reasons. When any such inclusion or removal occurs, this specification is deemed to include the modified group to satisfy the written description of all Markush groups as used in the appended claims.

[0509] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors encourage those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter described in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention covers any combination of all possible variations of the foregoing elements.

[0510] The specific embodiments disclosed herein may be further limited in the claims by using the language of "consisting of" or "substantially consisting of". When used in the claims, whether in the original filing or as amended, the transitional term "consisting of" excludes any element, step, or component not specified in the claims. The transitional term "substantially consisting of" limits the scope of the claims to the specified materials or steps, and those materials or steps that do not substantially affect the essential and novel features. Embodiments of the invention thus claimed are inherently or explicitly described and implemented herein.

[0511] Furthermore, numerous references have been made to patents and print publications throughout the specification. The entire contents of each of the above-cited references and print publications are individually incorporated herein by reference.

[0512] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications may be made within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings herein, exemplified rather than limited. Thus, the invention is not limited to what is precisely shown and described. sequence list <110> Beijing StarMab BioMed Technology Ltd. <120> Monospecific and multispecific antibodies <130> 1959708-00002 <150> US62 / 907,275 <151> 2019-09-27 <150> US62 / 989,327 <151> 2020-03-13 <160> 225 <170> PatentIn version 3.5 <210> 1 <211> 121 <212> PRT <213> Homo sapiens <400> 1 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Phe Ser Pro 115 120 <210> 2 <211> 127 <212> PRT <213> Lama glama <400> 2 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Gly Val Asn Ser Tyr 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn 100 105 110 Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 3 <211> 127 <212> PRT <213> Alpaca (Lama glama) <400> 3 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Gly Val Asn Ser Tyr 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ala Thr Ala Ile Lys 100 105 110 Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 4 <211> 127 <212> PRT <213> Alpaca (Lama glama) <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Gly Val Asn Ser Tyr 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Phe Ala Ser Lys 100 105 110 Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 5 <211> 127 <212> PRT <213> Alpaca (Lama glama) <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Val Met Trp Ser Ser 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn 100 105 110 Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 6 <211> 127 <212> PRT <213> Alpaca (Lama glama) <400> 6 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Arg Phe Phe Gly Ser 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn 100 105 110 Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 129 <212> PRT <213> Alpaca (Lama glama) <400> 7 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 8 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized VHH A09-10 <400> 8 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 9 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-1 <400> 9 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 - 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 10 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-2 <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 11 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-3 <400> 11 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 12 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-04 <400> 12 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 13 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent Humanized hA09-10-45 <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 130 135 140 Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 14 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-5 <400> 14 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 15 <211> 261 <212> PRT <213> Artificial SequenceI <220> <223> Bivalent Humanized hA09-10-55 <400> 15 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 130 135 140 Arg Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 16 <211> 132 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-6 <400> 16 Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 1 5 10 15 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg 20 25 30 Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys 35 40 45 Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Asp 50 55 60 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 65 70 75 80 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr[[ID=三十二]] 85 90 95 Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile 100 105 110 Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val 115 120 125 Thr Val Ser Ser 130 <210> 17 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent Humanized hA09-10-66 <400> 17 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Asp Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trpw Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 18 <211> 129 <212> PRT <213> Artificial Sequence <220> Note: There seems to be a misspelling in the original text where "Trpw" should probably be "Trp". This has been corrected in the translation.<223> Humanized hA09-10-7 <400> 18 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Asp Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 19 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent humanized hA09-10-77 <400> 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Asp Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 20 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-8 <400> 20 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 21 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent Humanized hA09-10-88 <400> 21 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 22 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-9 <400> 22 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 23 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent Humanized hA09-10-99 <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln<00D1974>245 250 255 Val Thr Val Ser Ser 260 <210> 24 <211> 129 <212> PRT <213> Artificial Sequence <220>[[ID=I27]] <223> Humanized hA09-10-10 <400> 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 It should be noted that there seems to be a misspelling in "<00D1974> ", which should probably be " ". This has been translated as is for the purpose of following the instructions.Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 25 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> Bivalent Humanized hA09-10-100 <400> 25 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly [[ID=�4]]1 510 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Ser Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser 260 <210> 26 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-11 <400> 26 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 27 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-12 <400> 27 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 28 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-13 <400> 28 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 29 <211> 129 <212> PRT <213> Artificial Sequence <220> <223> Humanized hA09-10-14 <400> 29< Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 < Phe Val Ser Ala Ile Ser Arg Ser Gly Gly Asn Ile Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 30 <211> 220 <212> PRT <213> Homo sapiens <400> 30 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg 210 215 220 <210> 31 <211> 127 <212> PRT <213> Alpaca (Lama glama) <400> 31 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Thr Leu Ser Arg Tyr 20 25 30 Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Ile Ile Arg Asn Ser Val Ser Thr Phe His Glu Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Met Met Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asn Val Gly Pro Thr Gly Gly Phe Ser Leu Gln Ser Val Gln 100 105 110 Arg Tyr Asp Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 32 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 32 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Thr Tyr 20 25 30 Asp Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asp Trp Tyr Thr Thr Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Ile Arg Ser Thr Ala Thr Ile Thr Gly Gln Ala Asp Tyr 100 105 110 Trp Gly Gln Gly Ala Gln Val Thr Val Ser Ser 115 120 <210> 33 <211> 114 <212> PRT <213> Alpaca (Lama glama) <400> 33 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Gly Ile Tyr 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Val Val 35 40 45 Gly Ile Ile Thr Ser Gly Gly Thr Thr Asn Tyr Ala Asp Phe Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Met Met Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ile Arg Thr Arg Leu Ile Ile Trp Gly Gln Gly Thr Gln Val Thr Val 100 105 110 Ser Ser <210> 34 <211> 124 <212> PRT <213> Alpaca (Lama glama) <400> 34 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Phe Ser Gly Gly Thr Phe Leu Thr Tyr 20 25 30 Ser Leu Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Asn Trp Ser Gly Tyr Met Thr Ser Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Ala Gln Val Thr Val Ser Ser 115 120 <210> 35 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 35 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Thr Tyr 20 25 30 Asp Met Gly Trp Phe Arg Arg Thr Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Met Asp Trp Asn Thr Ile Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Ile Arg Ser Thr Ala Ile Ile Thr Gly Gln Ala Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 36 <211> 127 <212> PRT. <213> Alpaca (Lama glama) <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Ile Tyr 20 25 30 Ser Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Val Arg Ile Asn Trp Asn Arg Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Arg Gly Ser Pro Ser Thr Ile Gly Ala Phe Thr Ser Ala Ser 100 105 110 His Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser [[ID=!46]] <210> 37 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Humanized PL14 <400> 37 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ser Gly Ile Tyr 20 25 30 Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Val Val 35 40 45 Ser Val Ile Thr Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Ile Arg Thr Arg Leu Ile Ile Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 38 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> Humanized PL16 <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 39 <211> 609[[ID=,40]] <212> PRT <213> Homo sapiens <,400> 39 Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala 1 5 10 15 Tyr Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala 20 25 30 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 35 40 45 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 50 55 60 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 65 70 75 80 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 85 90 95 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 100 105 110 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 115 120 125 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 130 135 140 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 145 150 155 160 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 165 170 175 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Phe Thr Glu Cys 180 185 190 Cys Gln Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 195 200 205 Leu Arg Asp Glu Gly Lys Ser Ser Ala Lys Gln Arg Leu Lys Cys 210 215 220 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 225 230 235 240 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 245 250 255 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 260 265 270 Asp Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 275 280 285 Cys Glu Asn Gln Asp Ser Ile Ser Ser Leu Lys Glu Cys Cys Glu 290,295,300 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 305 310 315 320 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 325 330 335 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 340 345 350 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 355 360 365 Thr Thr Thr Thr Thr Glu Lys Cys 370 375 380 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 385 390 395 400 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys 405 410 415 Leu Phe Glu Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 420 425 430 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 435 440 445 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Lys His 450 455 460 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 465 470 475 480 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 485 490 495 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 500 505 510 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 515 520 525 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 530 535 540 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 545 550 555 560​​​​​​​​​​​​​​​​​​​​​​​​​Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ser Ser Tyr 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Arg Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Ser Leu Ser Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Glu Ser Ser Ser Arg Gln Asp Lys Trp Tyr Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 41 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Thr Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 42 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe His Thr Tyr 20 25 30 Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ser Val 35 40 45 Val Ile Ile Asn Trp Ser Ser Asp His Thr Tyr Val Ala Gln Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Arg Ala Lys Asn Thr Phe Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Val Arg Arg Arg Leu Tyr Gly Leu Arg Glu Ser Asp Phe Asp Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 43 <211> 124 <212> PRT <213> Alpaca (Lama glama) <400> 43 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ile Ser Phe 20 25 30 Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ser Ile Thr Asn Ser Gly Ser Gly Ile Leu Tyr Gly Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Thr Gly Ala Gly Arg Tyr Arg Tyr Ala Ser Met Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 44 <211> 128 <212> PRT <213> Alpaca (Lama glama) <400> 44 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Thr Phe Ser Asn Tyr 20 25 30 Phe Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Val Lys Trp Ser Gly Tyr His Thr Tyr Tyr Ser Asp Ser Val 50 55 60 Lys Gly Gln Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Gly Thr Phe Ser Asn Trp Tyr Thr Arg Pro Arg Ser Gly 100 105 110 Asp Ser Tyr Asp His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 124 <212> PRT <213> Alpaca (Lama glama) <400> 45 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Pro Tyr 20 25 30 Thr Met Gly Trp Phe Arg Gln Ala Ser Gly Lys Glu Arg Glu Ser Val 35 40 45 Ala Ala Thr Thr Trp Thr Gly Ser Arg Ser Tyr Tyr Gly Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Thr Lys Asn Thr Met Ser 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Asp Gly Ala Gly Leu Tyr Thr Asn Arg Gly Gln Tyr Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 46 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Humanized hHS5 <400> 46 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 47 <211> 125 <212> PRT <213> Alpaca (Lama glama) <400> 47 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Asp Ser Ala Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Ser Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Pro Asp Ser Arg Leu Tyr Tyr Thr Val Pro Gln Asn Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Humanized hHS10 <400> 48 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Asp Ser Ala Tyr 20 25 30 Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Ser Asp Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Pro Asp Ser Arg Leu Tyr Tyr Thr Val Pro Gln Asn Tyr 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 49 <211> 242 <212> PRT <213> Homo sapiens <400> 49 Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln Glu 1 5 10 15 Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro Tyr 20 25 30 Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly Ala 35 40 45 Ile Ile Ser Arg Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln Glu 50 55 60 Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro Ser 65 70 75 80 Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp Asn 85 90 95 Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser Tyr 100 105 110 Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg Pro 115 120 125 Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn Leu 130 135 140 Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile Phe 145 150 155 160 Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr His 165 170 175 Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr Asn 180 185 190 Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu Arg 195 200 205 Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr Gly 210 215 220 Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly Val 225 230 235 240 Val His <210> 50 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 50 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Leu Lys Gly Ala Ser Trp Tyr Ser Ala Asn Tyr Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 51 <211> 111 <212> PRT <213> Alpaca (Lama glama) <400> 51 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Ala Ser Ala Asn Ile Leu Arg Thr Ala 20 25 30 Pro Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Phe Val 35 40 45 Ala Leu Ile Thr Ala Asp Gly Thr Thr Asp Tyr Gln Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Thr Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Gln Ser Glu Asp Thr Ala Arg Tyr Phe Cys Lys 85 90 95 Val Tyr Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 100 105 110 <210> 52 <211> 118 <212> PRT <213> Alpaca (Lama glama) <400> 52 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Ser Ile Phe Ser Ile Ala 20 25 30 His Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Val Ile Ser Ser Gly Gly Arg Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys Asn 85 90 95 Val Ala Val Val Gly Gly Pro Arg Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 53 <211> 119 <212> PRT <213> Alpaca (Lama glama) <400> 53 Glu Val Gln Leu Met Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Val Ser Thr Tyr 20 25 30 Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Glu Ile Asn Arg Ile Gly Asp Thr Leu Tyr Asn Arg Thr Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Ser Cys 85 90 95 Ala Ala Arg Val Ile Gly Thr Ser Thr Tyr Asn Tyr Trp Gly Gln Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 54 <211> 121 <212> PRT <213> Alpaca (Lama glama) <400> 54 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Ser Ile Asn 20 25 30 Ser Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Lys Val 35 40 45 Ala Gly Ile Asn Ser Ser Gly Asp Thr Asn Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Arg Thr Thr Tyr Leu 65 70 75 80 Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Gly Leu Tyr Tyr Cys Asn 85 90 95 Ala Asp Pro Arg Pro Trp Pro Asn Asp Val Ala Phe Gly Ser Trp Gly 100 105 110 Gln Gly Thr Arg Val Thr Val Ser Ser 115 120 <210> 55 <211> 117 <212> PRT <213> Alpaca (Lama glama) <400> 55 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Thr Lys Asn Met Val Tyr 65 70 75 80 Leu Arg Met Asp Asn Leu Gly Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 56 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Leu Ala Ser Gly Arg Thr Ser Ser Asn Ser 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Gly Ala Ile Thr Trp Asn Gly Asp Thr Thr Leu Tyr Ala Tyr Tyr Val 50 55 60 Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Met Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Thr Asp Lys Phe Ala Ser Ser Gln Ala Asp Ser Tyr Thr Thr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 57 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 57 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Phe Ser Gly Ala Ser Trp Tyr Ser Ala Gln Tyr His Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 58 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 58 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Phe Asn Gly Ala Ser Trp Phe Ala Ala Asn Tyr His Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 59 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 59 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Lys Gly Ala Ser Trp Phe Ala Ser His Tyr Lys Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 60 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 60 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ile Asn Ala Ala Ser Trp Phe Ala Ala Lys Tyr Asp Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 61 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 61 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Arg Gly Ser Ser Trp Phe Ser Ser His Tyr Asp Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 62 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 62 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 >Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Leu Ser Gly Ala Ser Trp Phe Ala Ser Gln Tyr Glu Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 63 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 63 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Lys Gly Ala Ser Trp Phe Ser Ala Asn Tyr Gln Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 64 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 64 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Phe Lys Gly Ala Ser Trp Phe Ala Ser His Tyr Gln Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 65 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 65 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Phe Asn Gly Ala Ser Trp Phe Ser Ser Asn Tyr Glu Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 66 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 66 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ile Arg Gly Ser Ser Trp Phe Ala Thr His Tyr Asp Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 67 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 67 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Leu Arg Gly Ala Ser Trp Phe Ala Ala Asn Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 68 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 68 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Leu Arg Gly Ala Ser Trp Phe Ala Ala Asn Tyr Asp Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 69 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 69 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ile Ser Ala Ala Ser Trp Tyr Ala Ser Gln Tyr Asp Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 70 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 70 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Ser Gly Ala Ser Trp Tyr Ala Ser His Tyr Glu Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 71 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 71 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Ser Glu Ala Ser Trp Phe Ala Ser Lys Tyr Asp Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 72 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 72 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Ile Ser Gly Ala Ser Trp Phe Ala Ala His Tyr Lys Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 73 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 73 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Phe Arg Gly Ala Ser Trp Phe Ser Ser His Tyr Asn Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 74 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 74 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Lys Gly Ala Ser Trp Phe Ser Ser Asn Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 75 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 75 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Ile Lys Ala Ser Ser Trp Tyr Ser Ser His Tyr Asn Tyr 100 – 105 – 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 76 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 76 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asn Val Arg Gly Ala Ser Trp Phe Ala Ala His Tyr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 77 <211> 123 <212> PRT <213> Alpaca (Lama glama) <400> 77 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Thr Asn Tyr 20 25 30 Ala Met Gly Trp Phe Arg Glu Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Gly Ile Asn Asn Asn Gly Asp Thr Leu Tyr Asn Thr Asn Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu His Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Lys Val Arg Ala Ala Ser Trp Phe Ala Ala Gln Tyr Lys Phe 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 78 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Humanized h33-14 <400> 78 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser 115 <210> 79 <211> 422 <212> PRT <213> Homo sapiens <400> 79 Val Pro Val Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys 1 5 10 15 Ser Pro Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 20 25 30 Val Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala Pro 35 40 45 Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser Ser Trp 50 55 60 Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly Pro Gly Gly 65 70 75 80 Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val Gln Leu Asp Glu 85 90 95 Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp Leu Arg Pro Ala Arg 100 105 110 Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala Val His Leu Arg Asp Arg 115 120 125 Ala Leu Ser Cys Arg Leu Arg Leu Arg Leu Gly Gln Ala Ser Met Thr 130 135 140 Ala Ser Pro Pro Gly Ser Leu Arg Ala Ser Asp Trp Val Ile Leu Asn 145 150 155 160 Cys Ser Phe Ser Arg Pro Asp Arg Pro Ala Ser Val His Trp Phe Arg 165 170 175 Asn Arg Gly Gln Gly Arg Val Pro Val Arg Glu Ser Pro His His His 180 185 190 Leu Ala Glu Ser Phe Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser 195 200 205 Gly Pro Trp Gly Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val Ser 210 215 220 Ile Met Tyr Asn Leu Thr Val Leu Gly Leu Glu Pro Pro Thr Pro Leu 225 230 235 240 Thr Val Tyr Ala Gly Ala Gly Ser Arg Val Gly Leu Pro Cys Arg Leu 245 250 255 Pro Ala Gly Val Gly Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro 260 265 270 Pro Gly Gly Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp Phe 275 280 285 Thr Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr Tyr Thr 290 295 300 Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr Val Thr Leu 305 310 315 320 Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser Pro Gly Ser Leu 325 330 335 Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser Gly Gln Glu Arg Phe 340 345 350 Val Trp Ser Ser Leu Asp Thr Pro Ser Gln Arg Ser Phe Ser Gly Pro 355 360 365 Trp Leu Glu Ala Gln Glu Ala Gln Leu Leu Ser Gln Pro Trp Gln Cys 370 375 380 Gln Leu Tyr Gln Gly Glu Arg Leu Leu Gly Ala Ala Val Tyr Phe Thr 385 390 395 400 Glu Leu Ser Ser Pro Gly Ala Gln Arg Ser Gly Arg Ala Pro Gly Ala 405 410 415 Leu Pro Ala Gly His Leu 420 <210> 80 <211> 121 <212> PRT <213> Alpaca (Lama glama) <400> 80 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Phe Ser Ile Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ile Val Thr Phe Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Ala Arg Asn Val Gln Ser Pro Val Gln Tyr His Leu Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 81 <211> 118 <212> PRT <213> Alpaca (Lama glama) <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Ile Phe Ser Ile Ser 20 25 30 Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ser Ile Thr Ala Arg Gly Ser Ala Asp Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Thr Asp Thr Arg Ser Thr Leu Tyr His Tyr Ser Trp Gly Gln Gly Thr 100 105 110 Gln Val Thr Val Ser Ser 115 <210> 82 <211> 128 <212> PRT <213> Alpaca (Lama glama) <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Asp Asp Tyr 20 25 30 Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Asp Asp Ser Asp Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Ile Phe Met Ile Asp Gly Val Asp Val Phe Leu Thr 100 105 110 Tyr Tyr Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 83 <211> 128 <212> PRT <213> Alpaca (Lama glama) <400> 83 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Asp Asp Tyr 20 25 30 Pro Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Asp Ser Asp Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Ile Phe Met Ile Asp Gly Val Asp Val Phe Leu Thr 100 105 110 Tyr Tyr Tyr Asp Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 84 <211> 120 <212> PRT <213> Alpaca (Lama glama) <400> 84 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Arg Arg Gly Ser Cys Arg Gln Tyr Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 85 <211> 121 <212> PRT <213> Alpaca (Lama glama) <400> 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ile Asn Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Arg Tyr Ser Asp Cys Arg Gly Pro Tyr Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 86 <211> 121 <212> PRT <213> Alpaca (Lama glama) <400> 86 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Lys Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Val Pro Asp Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Gly Arg Asp Thr Ser Tyr Ala Asp Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asn Asn Ala Ala Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asp Asn Leu Lys Leu Glu Asp Thr Gly Val Tyr Tyr Cys 85 90 95 Val Lys Cys Leu Glu Val Trp Ala Thr Arg Glu Tyr Asp Gly Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 87 <211> 128 <212> PRT <213> Alpaca (Lama glama) <400> 87 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Val Ala His Gly Phe Ser Leu Asp Ser His 20 25 30 Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Val Val 35 40 45 Ala Cys Ile Lys His Arg Asp Gly Arg Ile Tyr Ile Leu Glu Ala Val 50 55 60 Lys Asp Arg Phe Val Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Glu Met Asn Asn Leu Ser Asp Glu Asp Thr Ala Val Tyr His Cys 85 90 95 Ala Thr Ala Ser Ser Cys Ser Asp Asn Trp Trp Leu Leu Ile Gly Asp 100 105 110 Ala Tyr Ala Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 88 <211> 115 <212> PRT <213> Alpaca (Lama glama) <400> 88 Glu Val Gln Leu Ala Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Ala Phe Arg Ser Tyr 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Asn Ser Asp Ser Arg Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Val Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Lys Gln Ser Pro Gly Thr Ser Gln Arg Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 89 <211> 129 <212> PRT <213> Alpaca (Lama glama) <400> 89 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Ser Ala Thr Tyr 20 25 30 Ser Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Ser Thr Gly Asp Gly Ser Thr Tyr Tyr Ala Pro Ala Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Arg Ala Gly Tyr Gly Ser Ala Trp Phe Cys Pro Leu Asp Pro 100 105 110 Ser Leu Gln Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser <210> 90 <211> 128 <212> PRT <213> Alpaca (Lama glama) <400> 90 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Ile Asp Asp Tyr 20 25 30 Pro Met Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Gly Val 35 40 45 Ala Ala Ile Asp Asp Ser Asp Glu Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Leu Phe Met Ile Asp Gly Val Asp Val Phe Leu Thr 100 105 110 Tyr Tyr Phe Glu Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 91 <211> 126 <212> PRT <213> Alpaca (Lama glama) <400> 91 Gln Val Gln Leu Val Glu Ser Gly Gly Arg Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Gly Leu Ser Cys Ala Ala Ser Gly Leu Ala Phe Arg Glu Tyr 20 25 30 Ser Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Ala Val Asp Trp Thr Gly Ile Gln Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Ala Thr Ile Ser Arg Asp Thr Ala Lys Ser Thr Val Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Phe Tyr Tyr Cys Ala 85 90 95 Ala Gly Lys Lys Leu Thr Gly Ile Val Leu Leu Thr Arg Arg Thr Glu 100 105 110 Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 92 <211> 125 <212> PRT <213> Alpaca (Lama glama) <400> 92 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala His Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Thr Arg Tyr Gly Ser Ser Cys Arg Gly Gly Gln Trp Asp Thr 100 105 110 Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 93 <211> 125 <212> PRT <213> Alpaca (Lama glama) <400> 93 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Leu Asp Tyr Tyr 20 25 30 Thr Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ala Val 35 40 45 Ser Cys Ile Asn Arg Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Thr Thr Tyr Gly Thr Ser Cys Arg Gly Gly Gln Trp Asp Thr 100 105 110 Gly Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 94 <211> 254 <212> PRT <213> Homo sapiens <400> 94 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Phe 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250 <210> 95 <211> 233 <212> PRT <213> Homo sapiens <400> 95 Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 <210> 96 <211> 116 <212> PRT <213> Alpaca (Lama glama) <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser Ala Arg 20 25 30 Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Ala Ile Thr Ser Gly Val Arg Thr Asp Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 97 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Humanized hCD16F1-1 <400> 97 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser Ala Arg 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ser Ala Ile Thr Ser Gly Val Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 98 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Humanized hCD16F1-2 <400> 98 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser Ala Arg 20 25 30 Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ser Ala Ile Thr Ser Gly Val Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn 85 90 95 Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val 100 105 110 Thr Val Ser Ser 115 <210> 99 <211> 119 <212> PRT <213> Alpaca (Lama glama) <400> 99 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Thr Leu Ser Cys Arg Ala Ser Gly Phe Thr Phe Ser Asn His[[ID=Z9]] 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Glu Ile Ser Phe Asn Gly His Ala Thr Arg Tyr Ala Asp Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Arg Lys Gly Trp Asn Ala Thr Pro Gln Ile Gly Glu Arg Gly Arg Gly 100 105 110 Thr Gln Val Thr Val Ser Ser 115 <210> 100 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Connector L1 <400> 100 Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 101 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Connector L2 <220> <221> REPEAT <222> (1)..(5) <223> Repeat = 1-35 <220> <221> REPEAT <222> (5)..(9) <223> Repeat = 0-35 <400> 101 Gly Gly Gly Ser Ser Gly Gly Gly Ser 1 5 <210> 102 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> Connector L3 <400> 102 Ala Ala Ala 1 <210> 103 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Connector L4 <220> <221> REPEAT <222> (1)..(5) <223> Repeat = 1-35 <400> 103 Gly Gly Gly Gly Ser 1 5 <210> 104 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*3 <400> 104 Gly Gly Arg Gly Pro Leu Gly Leu Ala Gly Ser Arg Ser Ala Phe Gly 1 5 10 15 Gly Ser <210> 105 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*4 <400> 105 Gly Ser Pro Leu Gly Leu Ala Gly Ser 1 5 <210> 106 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*5 <400> 106 Gly Gly Ser Gly Pro Leu Gly Leu Ala Gly Ser Arg Ser Ala Phe Gly 1 5 10 15 <210> 107 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*6 <400> 107 Gly Pro Leu Gly Leu Ala Gly Ser Arg Ser Ala Gly Gly Ser Gln Val 1 5 10 15 Gln Leu <210> 108 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*7 <400> 108 Gly Ser Gly Pro Leu Gly Leu Ala Ala Arg Ser Ala Gly Gly Ser 1 5 10 15 <210> 109 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*8 <400> 109 Gly Ser Gly Pro Leu Gly Leu Ala Ala Arg Ser Ala Phe Gly Gly Ser 1 5 10 15 <210> 110 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Connector L11*9 <400> 110 Gly Gly Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala Arg Gln Ala Arg 1 5 10 15 Gln Val Gly Gly Ser 20 <210> 111 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*10 <400> 111 Gly Gly Ser Gly Arg Ser Ala Pro Leu Gly Leu Gly Arg Gln Ala Arg 1 5 10 15 Gly Gly Ser <210> 112 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Connector L11*11 <400> 112 Gly Gly Ser Pro Leu Gly Leu Ala Arg Gln Ala Arg Gly Ser Gly Arg 1 5 10 15 Ser Ala Gly Gly Ser 20 <210> 113 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> Connector L11*12 <400> 113 Gly Gly Ser Gly Arg Ser Ala Pro Leu Gly Leu Ala Arg Gln Ala Arg 1 5 10 15 Val Val Gly Gly Ser 20 <210> 114 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*13 <400> 114 Gly Ser Arg Gln Ala Arg Val Val Gly Ser 1 5 10 <210> 115 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*14 <400> 115 Gly Ser Arg Gln Arg Arg Val Val Gly Ser 1 5 10 <210> 116 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*15 <400> 116 Gly Ser Arg Gln Ala Arg Gly Ser 1 5 <210> 117 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*16 <400> 117 Gly Ser Arg Gln Arg Arg Gly Ser 1 5 <210> 118 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*17 <400> 118 Gly Ser Arg Gln Ala Arg Gly Gly Ser 1 5 <210> 119 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Connector L11*18 <400> 119 Gly Ser Arg Gln Arg Arg Gly Gly Ser 1 5 <210> 120 <211> 401 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hHS5-L11*3-hA09-10-2-L1-hPL16 <400> 120 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met 210 215 220 Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 260 265 270 Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 275 280 285 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly 290 295 300 Thr Phe Leu Thr Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys 305 310 315 320 Glu Arg Glu Phe Val Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr 325 330 335 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser 340 345 350 Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 355 360 365 Ala Val Tyr Tyr Cys Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg 370 375 380 Ser Ser Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 385 390 395 400 Ser <210> 121 <211> 533 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hHS5-L11*3-hA09-10-2-L3-A09-10-L1-hPL16 <400> 121 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met 210 215 220 Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln 260 265 270 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 275 280 285 Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn 290 295 300 Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 305 310 315 320 Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser 325 330 335 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr 340 345 350 Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr 370 375 380 Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 405 410 415 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 420 425 430 Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met Ser Trp Val Arg Gln 435 440 445 Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ser Ile Asn Trp Ser Gly 450 455 460 Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 465 470 475 480 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 485 490 495 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Arg Thr Ala Ile 500 505 510 Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln 515 520 525 Val Thr Val Ser Ser 530 <210> 122 <211> 660 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hHS5-L11*3- hA09-10-2-L3-A09-10-L1-hPL16-L3-hPL16 <400> 122 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met 210 215 220 Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln 260 265 270 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 275 280 285 Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn 290 295 300 Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 305 310 315 320 Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser 325 330 335 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr 340 345 350 Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr 370 375 380 Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 405 410 415 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 420 425 430 Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met Ser Trp Val Arg Gln 435 440 445 Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ser Ile Asn Trp Ser Gly 450 455 460 Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 465 470 475 480 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 485 490 495 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Arg Thr Ala Ile 500 505 510 Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln 515 520 525 Val Thr Val Ser Ser Ala Ala Ala Glu Val Gln Leu Val Glu Ser Gly 530 535 540 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 545 550 555 560 Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met Ser Trp Val Arg Gln Ala 565 570 575 Pro Gly Lys Glu Arg Glu Phe Val Ser Ser Ile Asn Trp Ser Gly Tyr 580 585 590 Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 595 600 605 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala 610 615 620 Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala Arg Thr Ala Ile Ala 625 630 635 640 Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val 645 650 655 Thr Val Ser Ser 660 <210> 123 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hA09-10-1-L1-hHS5-L1-hPL16 <400> 123 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 130 135 140 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg 165 170 175 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly 180 185 190 Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile 195 200 205 Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 210 215 220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr 225 230 235 240 Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met 290 295 300 Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ser 305 310 315 320 Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 355 360 365 Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp 370 375 380 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 <210> 一百二十四 <211> 五百二十六 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hA09-10-***-L3-A09-10-L1-hHS5-L1-hPL16 <400> 124 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 290 295 300 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 305 310 315 320 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 355 360 365 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 370 375 380 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 405 410 415 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu 420 425 430 Thr Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 435 440 445 Phe Val Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp[[ID=**15**]] 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 465 470 475 480 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu 500 505 510 Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 515 520 525 <210> 125 <211> 653 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hA09‑10‑2‑L3‑A09‑10‑L1‑hHS5‑L1‑hPL16‑L3‑hPL16 <400> 125 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 290 295 300 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 305 310 315 320 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 355 360 365 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 370 375 380 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 405 410 415 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu 420 425 430 Thr Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu 435 440 445 Phe Val Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 465 470 475 480 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu 500 505 510 Phe Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala 515 520 525 Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 530 535 540 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr 545 550 555 560 Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 565 570 575 Val Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser 580 585 590 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu 595 600 605 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 610 615 620 Cys Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe 625 630 635 640 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 645 650 <210> 126 <211> 521 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hA09-10-3-L1-hHS5-L1-hPL16-L3-hPL16 <400> 126 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 130 135 140 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg 165 170 175 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly 180 185 190 Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile 195 200 205 Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 210 215 220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr 225 230 235 240 Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met 290 295 300 Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ser 305 310 315 320 Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala 355 360 365 Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp 370 375 380 Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Glu Val Gln 385 390 395 400 Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 405 410 415 Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser Met Ser 420 425 430 Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ser Ile 435 440 445 Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 450 455 460 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 465 470 475 480 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Ala 485 490 495 Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr Trp Gly 500 505 510 Gln Gly Thr Gln Val Thr Val Ser Ser 515 520 <210> 127 <211> 394 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hHS5-L11* -hA09-10-3-L1-H33-14 <400> 127 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Val Tyr Leu Gln Met 210 215 220 Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 260 265 270 Ser Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu 275 280 285 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 290 295 300 Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys 305 310 315 320 Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg 325 330 335 Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 340 345 350 Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr 355 360 365 Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp 370 375 380 Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 <210> 128 <211> 526 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hHS5-L11*3-hA09-10-2-L3-A09-10-L1-H33-14 <400> 128 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met 210 215 220 Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln 260 265 270 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 275 280 285 Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn 290 295 300 Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 305 310 315 320 Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser 325 330 335 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr 340 345 350 Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr 370 375 380 Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser 405 410 415 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 420 425 430 Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln 435 440 445 Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn 450 455 460 Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 465 470 475 480 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 485 490 495 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly 500 505 510 Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 515 520 525 <210> 129 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hHS5‑L1‑hA09‑10‑2‑L3‑A09‑10‑L1‑H33‑14‑L3‑H33‑14 <400> 129 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 [[ID=*25]]Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60<000464>Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95<* Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Note: There seems to be a potential error in the original text where the tag is misspelled as in the provided text. Also, <000464> in the translation is likely a typo and should be . The corrected translation with these considerations is presented above. 115 120 125 Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly 260 265 270 Gly Gly Leu Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 275 280 285 Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg 290 295 300 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser 305 310 315 320 Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 325 330 335 Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu 340 345 350 Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu 355 360 365 Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly 370 375 380 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 405 410 415 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu 420 425 430 Ile Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu 435 440 445 Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 465 470 475 480 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly 500 505 510 Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu 515 520 525 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 530 535 540 Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg 545 550 555 560 Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn 565 570 575 Asn Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile 580 585 590 Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu 595 600 605 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly 610 615 620 Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 625 630 635 <210> 130 <211> 646 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hHS5-L11*3-hA09-10-2-L3-A09-10-L1-H33-14-L3-H3-14 <400> 130 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His 20 25 30 Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Ser Pro Leu 115 120 125 Gly Leu Ala Gly Arg Ser Ala Phe Gly Gly Ser Glu Val Gln Leu Val 130 135 140 Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser 145 150 155 160 Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn Tyr Ala Leu Gly 165 170 175 Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ala Ala Ile 180 185 190 Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser Val Lys Gly Arg 195 200 205 Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr Tyr Leu Gln Met 210 215 220 Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala His 225 230 235 240 Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr Asn Met Tyr Asn 245 250 255 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln 260 265 270 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly Ser 275 280 285 Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser Asn 290 295 300 Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 305 310 315 320 Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp Ser 325 330 335 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr Thr 340 345 350 Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr 355 360 365 Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser Thr 370 375 380 Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 385 390 395 400 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser 405 410 415 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 420 425 430 Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln 435 440 445 Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn 450 455 460 Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 465 470 475 480 Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg 485 490 495 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly 500 505 510 Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala 515 520 525 Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 530 535 540 Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile 545 550 555 560 Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 565 570 575 Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser 580 585 590 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 595 600 605 Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 610 615 620 Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr 625 630 635 640 Gln Val Thr Val Ser Ser 645<00048​​​​​​​​​​​​​​​​​​Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 130 135 140 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg 165 170 175 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly 180 185 190 Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile 195 200 205 Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 210 215 220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr 225 230 235 240 Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 260 265 270 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met 290 295 300 Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg 305 310 315 320 Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly 325 330 335 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 340 345 350 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala 355 360 365 Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr 370 375 380 Val Ser Ser 385 <210> 132 <211> 519 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hA09-10-2-L3-A09-10-L1-hHS5-L1-H33-14 <400> 132 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser [[ID=३७]]20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu [[ID=४१]]35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55, 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 290 295 300 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 305 310 315 320 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 355 360 365 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 370 375 380 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 405 410 415 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu 420 425 430 Ile Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu 435 440 445 Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 465 470 475 480 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly 500 505 510 Thr Gln Val Thr Val Ser Ser 515 <210> 133 <211> 507 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody hA09-10-L1-hHS5-L1-H33-14-L3-H33-14 <400> 133 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 65 70 75 80 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu 130 135 140 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg 165 170 175 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly 180 185 190 Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile 195 200 205 Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 210 215 220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr 225 230 235 240 Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val 260 265 270 Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met 290 295 300 Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg 305 310 315 320 Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly 325 330 335 Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln 340 345 350 Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala 355 360 365 Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr 370 375 380 Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly 385 390 395 400 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 405 410 415 Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly 420 425 430 Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr 435 440 445 Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 450 455 460 Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp 465 470 475 480 Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly 485 490 495 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 500 505 <210> 134 <211> 639 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hA09‑10‑2‑L3‑A09‑10‑L1‑hHS5‑L1‑H33‑14‑L3‑H33‑14 <400> 134 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly Arg Thr Phe Ser 20 25 30 Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile Asn Tyr Ala Asp 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Phe Lys Asn Thr 65 70 75 80 Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr Ile Ser Thr Ser 100 105 110 Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser 115 120 125 Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 130 135 140 Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Gly 145 150 155 160 Arg Thr Phe Ser Asn Tyr Ala Leu Gly Trp Phe Arg Gln Ala Pro Gly 165 170 175 Lys Glu Arg Glu Phe Val Ala Ala Ile Ser Arg Ser Gly Gly Asn Ile 180 185 190 Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200 205 Phe Lys Asn Thr Thr Tyr Leu Gln Met Ser Ser Leu Glu Pro Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Ala His Tyr Leu Leu Leu Pro Ser Tyr 225 230 235 240 Ile Ser Thr Ser Thr Asn Met Tyr Asn Tyr Trp Gly Gln Gly Thr Gln 245 250 255 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 260 265 270 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 275 280 285 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 290 295 300 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 305 310 315 320 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 325 330 335 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 340 345 350 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 355 360 365 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 370 375 380 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 385 390 395 400 Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 405 410 415 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu 420 425 430 Ile Leu Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu 435 440 445 Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp 450 455 460 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr 465 470 475 480 Val Tyr Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 485 490 495 Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly 500 505 510 Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu 515 520 525 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 530 535 540 Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg 545 550 555 560 Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn 565 570 575 Asn Asp Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile 580 585 590 Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu 595 600 605 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly 610 615 620 Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 625 630 635 <210> 135 <211> 374 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody H33-14-L1-hHS5-L1-CD16F1 <400> 135 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 115 120 125 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 145 150 155 160 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 165 170 175 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 210 215 220 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 260 265 270 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser 275 280 285 Ala Arg Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 290 295 300 Leu Val Ala Ala Ile Thr Ser Gly Val Arg Thr Asp Tyr Ala Asp Ser 305 310 315 320 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val 325 330 335 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 340 345 350 Cys Asn Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr 355 360 365 Gln Val Thr Val Ser Ser 370 <210> 136 <211> 493 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody H33-14-L1-hHS5-L1-CD16F1-L3-CD16F1 <400> 136 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val 115 120 125 Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu 130 135 140 Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Thr His Ala Met 145 150 155 160 Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser Ala 165 170 175 Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly 180 185 190 Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala Asn Thr Leu Tyr Leu Gln 195 200 205 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ser 210 215 220 Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly Thr Thr Asn Ser Trp Gly 225 230 235 240 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255 Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 260 265 270 Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser 275 280 285 Ala Arg Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu 290 295 300 Leu Val Ala Ala Ile Thr Ser Gly Val Arg Thr Asp Tyr Ala Asp Ser 305 310 315 320 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val 325 330 335 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 340 345 350 Cys Asn Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr 355 360 365 Gln Val Thr Val Ser Ser Ala Ala Ala Glu Val Gln Leu Val Glu Ser 370 375 380 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 385 390 395 400 Val Ser Gly Ser Leu Phe Ser Ala Arg Val Met Gly Trp Tyr Arg Gln 405 410 415 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Thr Ser Gly Val 420 425 430 Arg Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 435 440 445 Asp Asn Ala Lys Arg Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 450 455 460 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Leu Tyr Asn Thr Gly 465 470 475 480 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 485 490 <210> 137 <211> 494 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody H33-14-L3-H33-14-L1-hHS5-L1-CD16F1 <400> 137 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln Ala 145 150 155 160 Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp 165 170 175 Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser 210 215 220 Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 245 250 255 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 260 265 270 Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 275 280 285 Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr 290 295 300 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr 305 310 315 320 Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 325 330 335 Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg 340 345 350 Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser 355 360 365 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu 370 375 380 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 385 390 395 400 Ala Val Ser Gly Ser Leu Phe Ser Ala Arg Val Met Gly Trp Tyr Arg 405 410 415 Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Thr Ser Gly 420 425 430 Val Arg Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 435 440 445 Arg Asp Asn Ala Lys Arg Ala Val Tyr Leu Gln Met Asn Ser Leu Lys 450 455 460 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Leu Tyr Asn Thr 465 470 475 480 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 485 490 <210> 138 <211> 613 <212> PRT <213> Artificial Sequence <220> <223> Trispecific antibody H33-14-L3-H33-14-L1-hHS5-L1-CD16F1-L3-CD16F1 <400> 138 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Leu Ile Leu 20 25 30 Ala Met Ala Trp Trp Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala 35 40 45 Gly Arg Ile Trp Trp Asn Asn Asp Met Thr Arg Tyr Ser Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Glu Ala Asp Leu Ile Gly Gly Ser Arg Gly Trp Gly Gln Gly Thr Gln 100 105 110 Val Thr Val Ser Ser Ala Ala Ala Gln Val Gln Leu Val Glu Ser Gly 115 120 125 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala 130 135 140 Ser Gly Arg Thr Ser Leu Ile Leu Ala Met Ala Trp Trp Arg Gln Ala 145 150 155 160 Pro Gly Lys Glu Arg Glu Phe Ala Gly Arg Ile Trp Trp Asn Asn Asp 165 170 175 Met Thr Arg Tyr Ser Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 180 185 190 Asp Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Arg Ala 195 200 205 Glu Asp Thr Ala Val Tyr Tyr Cys Glu Ala Asp Leu Ile Gly Gly Ser 210 215 220 Arg Gly Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly 225 230 235 240 Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 245 250 255 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 260 265 270 Arg Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg Gln Ala Pro Gly 275 280 285 Lys Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr 290 295 300 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr 305 310 315 320 Gly Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 325 330 335 Thr Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg 340 345 350 Ala Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser 355 360 365 Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu 370 375 380 Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 385 390 395 400 Ala Val Ser Gly Ser Leu Phe Ser Ala Arg Val Met Gly Trp Tyr Arg 405 410 415 Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Thr Ser Gly 420 425 430 Val Arg Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 435 440 445 Arg Asp Asn Ala Lys Arg Ala Val Tyr Leu Gln Met Asn Ser Leu Lys 450 455 460 Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Leu Tyr Asn Thr 465 470 475 480 Gly Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala 485 490 495 Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 500 505 510 Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ser Leu Phe Ser Ala 515 520 525 Arg Val Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu 530 535 540 Val Ala Ala Ile Thr Ser Gly Val Arg Thr Asp Tyr Ala Asp Ser Val 545 550 555 560 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ala Val Tyr 565 570 575 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 580 585 590 Asn Val Asn Leu Tyr Asn Thr Gly Asn Tyr Trp Gly Gln Gly Thr Gln 595 600 605 Val Thr Val Ser Ser 610 <210> 139 <211> 381 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hPL16-L1-hHS5-L1-CD16F1 <400> 139 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 145 150 155 160 Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 165 170 175 Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly 195 200 205 Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala 225 230 235 240 Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 260 265 270 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 275 280 285 Val Ser Gly Ser Leu Phe Ser Ala Arg Val Met Gly Trp Tyr Arg Gln 290 295 300 Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Thr Ser Gly Val 305 310 315 320 Arg Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg 325 330 335 Asp Asn Ala Lys Arg Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro 340 345 350 Glu Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Leu Tyr Asn Thr Gly 355 360 365 Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 370 375 380 <210> 140 <211> 508 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hPL16-L3-hPL16-L1-hHS5-L1-CD16F1 <400> 140 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr [[ID= Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Glu 115 120 125 Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser 130 135 140 Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr Ser 145 150 155 160 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val Ser 165 170 175 Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val Lys 180 185 190 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 195 200 205 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 210 215 220 Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp Tyr 225 230 235 240 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser 245 250 255 Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val 260 265 270 Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr 275 280 285 Phe Thr Thr His Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu 290 295 300 Arg Glu Phe Val Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr Tyr 305 310 315 320 Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly Ala 325 330 335 Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 340 345 350 Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala Gly 355 360 365 Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 370 375 380 Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly 385 390 395 400 Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Val 405 410 415 Ser Gly Ser Leu Phe Ser Ala Arg Val Met Gly Trp Tyr Arg Gln Ala 420 425 430 Pro Gly Lys Gln Arg Glu Leu Val Ala Ala Ile Thr Ser Gly Val Arg 435 440 445 Thr Asp Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp 450 455 460 Asn Ala Lys Arg Ala Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu 465 470 475 480 Asp Thr Ala Val Tyr Tyr Cys Asn Val Asn Leu Tyr Asn Thr Gly Asn 485 490 495 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 500 505 <210> 141 <211> 500 <212> PRT <213> Artificial Sequence <220> <223> Trispecific Antibody hPL16-L1-hHS5-L1-CD16F1-L3-CD16F1 <400> 141 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Leu Thr Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ser Ser Ile Asn Trp Ser Gly Tyr Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Ala Arg Thr Ala Ile Ala Ala Lys Arg Ser Ser Glu Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu 130 135 140 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg 145 150 155 160 Thr Phe Thr Thr His Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys 165 170 175 Glu Arg Glu Phe Val Ser Ala Ile Asn Trp Gly Gly Arg Thr Thr Tyr 180 185 190 Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Thr Gly 195 200 205 Ala Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Ser Asn Leu Asp Thr Tyr Asn Val Arg Ala 225 230 235 240 Gly Thr Thr Asn Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 245 250 255 Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser 260 265 270 Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala 275 280 285 Val Ser...

Claims

1. A multispecific antibody comprising an antibody binding domain having a first binding specificity and a second antibody binding domain having a second binding specificity different from the first binding specificity, wherein the first binding specificity is specific for CD16; wherein the amino acid sequence of the antibody binding domain of the first binding specificity specific for CD16 is represented by SEQ ID NO: 96, wherein the second antibody binding domain is specific for CD47, PD-L1, HSA, CD33, or LAG3; wherein (a) the amino acid sequence of the second antibody binding domain of the second binding specificity specific for CD47 is represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223; (b) the amino acid sequence of the second antibody binding domain of the second binding specificity specific for PD-L1 is represented by one of SEQ ID NO: 31 to SEQ ID NO: 38; (c) the amino acid sequence of the second antibody binding domain of the second binding specificity specific for HSA is represented by one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the amino acid sequence of the second antibody binding domain of the second binding specificity specific for CD33 is represented by one of SEQ ID NO: 50 to SEQ ID NO: 78; and (e) the amino acid sequence of the second antibody binding domain of the second binding specificity specific for LAG3 is represented by one of SEQ ID NO: 80 to SEQ ID NO:

93.

2. The multispecific antibody of claim 1, further comprising 1 to 5 additional antibody binding domains, wherein each additional antibody binding domain is individually specific for CD47, PD-L1, HSA, CD33, or LAG3; wherein (a) the amino acid sequence of the additional antibody binding domain specific for CD47 is represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223; (b) the amino acid sequence of the additional antibody binding domain specific for PD-L1 is represented by one of SEQ ID NO: 31 to SEQ ID NO: 38; (c) the amino acid sequence of the additional antibody binding domain specific for HSA is represented by one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the amino acid sequence of the additional antibody binding domain specific for CD33 is represented by one of SEQ ID NO: 50 to SEQ ID NO: 78; and (e) the amino acid sequence of the additional antibody binding domain specific for LAG3 is represented by one of SEQ ID NO: 80 to SEQ ID NO:

93.

3. The multispecific antibody of claim 2, wherein the antibody is a multispecific single chain antibody (MVSCA).

4. The multispecific antibody of any one of claims 1 to 3, wherein a linker LI as set forth in SEQ ID NO: 100, a linker L2 as set forth in SEQ ID NO: 101, or a linker L4 as set forth in SEQ ID NO: 103 is interposed between one or more pairs of different antibody binding domains.

5. The multispecific antibody of claim 4, comprising at least one pair of antibody binding domains having the same specificity.

6. The multispecific antibody of claim 5, wherein the at least one pair of antibody binding domains having the same specificity are adjacent to each other and have a linker L3 as set forth in SEQ ID NO: 102 interposed therebetween.

7. The multispecific antibody of claim 6, comprising an N-terminally or C-terminally positioned HSA-specific antibody binding domain with a cleavable linker interposed between the antibody binding domain and the antibody binding domain adjacent thereto.

8. The multispecific antibody of claim 7, wherein the cleavable linker is LI 1*3 as set forth in SEQ ID NO: 104, LI 1*4 as set forth in SEQ ID NO: 105, LI 1*5 as set forth in SEQ ID NO: 106, LI 1*6 as set forth in SEQ ID NO: 107, LI 1*7 as set forth in SEQ ID NO: 108, LI 1*8 as set forth in SEQ ID NO: 109, LI 1*9 as set forth in SEQ ID NO: 110, LI 1*10 as set forth in SEQ ID NO: 111, LI 1*11 as set forth in SEQ ID NO: 112, LI 1*12 as set forth in SEQ ID NO: 113, LI 1*13 as set forth in SEQ ID NO: 114, LI 1*14 as set forth in SEQ ID NO: 115, LI 1*15 as set forth in SEQ ID NO: 116, LI 1*16 as set forth in SEQ ID NO: 117, LI 1*17 as set forth in SEQ ID NO: 118, or LI 1*18 as set forth in SEQ ID NO:

119.

9. The multispecific antibody of claim 8, wherein all of the antibody binding domains are VHH domains.

10. The multispecific antibody of claim 9, comprising antibody binding domains recognizing HSA, CD47, and PD-L1.

11. The multispecific antibody of claim 9, comprising antibody binding domains recognizing HSA, CD47, and CD33.

12. The multispecific antibody of claim 9, comprising antibody binding domains recognizing HSA, LAG3, and PD-L1.

13. The multispecific antibody of claim 9, comprising antibody binding domains that recognize HSA, LAG3, and CD33.

14. The multispecific antibody of claim 9, comprising antibody binding domains that recognize HSA and PD-L1.

15. The multispecific antibody of claim 9, comprising antibody binding domains that recognize HSA and CD33.

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