Monospecific and multi-specific antibodies

Abstract

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

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610213990.X (22) Application Date 2020.09.28 (30) Priority Data 62 / 907,275 2019.09.27 US 62 / 989,327 2020.03.13 US (62) Divisional Application Data 202080082179.3 2020.09.28 (71) Applicant Beijing Shuoxing Biomedical Technology Co., Ltd. Address Room B213, Building 1, No. 29, Life Science Park Road, Changping District, Beijing 102206 (72) Inventor Liang Yanbin (74) Patent Agency Beijing Liu Shen Law Firm 11105 Patent Attorney Jiang Lan (51) Int.Cl. C07K 16 / 28(2006.01) C07K 16 / 18(2006.01) C07K 16 / 46(2006.01) A61K 39 / 395(2006.01) A61P 35 / 00(2006.01) A61P 25 / 28(2006.01) A61P 27 / 02(2006.01) (54) Invention Title: Monospecific and Multispecific Antibodies (57) Abstract: This paper discloses monospecific and multispecific single-chain antibodies that are specific to one or more of CD47, PD-L1, HSA, CD33, LAG3 and CD16. Claims 4 pages, Description 94 pages, Sequence Listing (Electronic Publication), Drawings 31 pages, CN 121975011 A 2026.05.05 CN 1 21 97 50 11 A 1. A heavy chain variable (VHH) domain having antigen-binding specificity for one of: (a) CD16, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) PD-L1, wherein the VHH region has an amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) human serum albumin (HSA), wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) CD33, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: The amino acid sequence shown in one of SEQ ID NO: 78; (e) LAG3, wherein the VHH domain has the amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; or(f) CD47, 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. 2. A heavy chain-only antibody (HCAb) comprising the VHH domain of claim 1. 3. 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 to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) the CD16 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) the PD-L1 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) the HSA binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the CD33 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) the LAG3 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 99. The amino acid sequence represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223 represents the CD47 binding specificity; and the amino acid sequence represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223 represents the CD47 binding specificity. 4. The multispecific antibody according to claim 3, wherein the second antibody-binding domain is specific to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) the CD16 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) the PD-L1 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) the HSA binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the CD33 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; Claims 1 / 4 page 2 CN121975011 A (e) The LAG3 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; and (f) The CD47 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223. 5. The multispecific antibody according to claim 3, further comprising 1 to 5 additional antibody-binding domains, wherein each additional antibody-binding domain is individually specific to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) the CD16 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) the PD-L1 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) the HSA binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the CD33 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) the LAG3 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 99. The amino acid sequence represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223 represents the CD47 binding specificity; and the amino acid sequence represented by one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223 represents the CD47 binding specificity. 6. The multispecific antibody according to claim 4, further comprising 1 to 4 additional antibody-binding domains, wherein each additional antibody-binding domain is specific for CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) the CD16 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) the PD-L1 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) the HSA binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) the CD33 binding specificity is represented by an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 48.The amino acid sequence shown in SEQ ID NO: 78 represents the specificity of LAG3 binding, which is represented by the amino acid sequence shown in SEQ ID NO: 80 to SEQ ID NO: 93; and the amino acid sequence shown in SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO: 223 represents the specificity of CD47 binding. 7. The multispecific antibody according to any one of claims 3-6, wherein the antibody is a multispecific single-chain antibody (MVSCA). 8. The multispecific antibody according to any one of claims 3-7, wherein the linkers L1 (SEQ ID NO: 100), L2 (SEQ ID NO: 101) or L4 (SEQ ID NO: 103) are interposed between one or more pairs of different antibody-binding domains. 9. The multispecific antibody according to any one of claims 3-8, wherein the multispecific antibody comprises at least one pair of antibody-binding domains having the same specificity. 10. The multispecific antibody of claim 9, wherein the at least one pair of antibody-binding domains having the same specificity are adjacent to each other and a linker L3 (SEQ ID NO: 102) is interposed between them. 11. The multispecific antibody of any one of claims 3-10, wherein the multispecific antibody comprises an N-terminal or C-terminal HSA-specific antibody-binding domain, and a cleavable linker is interposed between the antibody-binding domain and an adjacent antibody-binding domain. 12. The multispecific antibody according to claim 11, wherein the cleavable linker is L11*3 (SEQ ID NO: 104), L11*4 (SEQ ID NO: 105), L11*5 (SEQ ID NO: 106), L11*6 (SEQ ID NO: 107), L11*7 (SEQ ID NO: 108), L11*8 (SEQ ID NO: 109), L11*9 (SEQ ID NO: 110), L11*10 (SEQ ID NO: 111), L11*11 (SEQ ID NO: 112), L11*12 (SEQ ID NO: 113), L11*13 (SEQ ID NO: 114), L11*14 (SEQ ID NO: 115), L11*15 (SEQ ID NO: 116), L11*16 (SEQ ID NO: 117), L11*17 (SEQ ID NO: 118) or L11*18 (SEQ ID NO: 104). ID NO:119).13. The multispecific antibody according to any one of claims 3-12, wherein all of the antibody-binding domains are VHH domains. 14. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing HSA, CD47, and PD-L1. 15. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing HSA, CD47, and CD33. 16. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing HSA, LAG3, and PD-L1. 17. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing HSA, LAG3, and CD33. 18. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing CD16, HSA, and PD-L1. 19. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing CD16, HSA, and CD33. 20. A multispecific antibody according to any one of claims 3-13, comprising CD16, HSA, CD47, and PD-L1. 21. A multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain recognizing CD16, HSA, CD47, and CD33. 22. A multispecific antibody comprising a pair of antibody-binding domains having CD33 specificity. 23. A multispecific antibody according to claim 22, further comprising an antibody-binding domain specific to HSA, FC5 nanobody (SEQ ID NO: 222), or both. 24. A pharmaceutical composition comprising the VHH domain according to claim 1, the HCAb according to claim 2, or a multispecific antibody according to any one of claims 3-21. 25. A method of treating cancer, comprising administering the pharmaceutical composition according to claim 24 to a patient in need. 26. A pharmaceutical composition comprising the multispecific antibody according to any one of claims 22-23. Claims 3 / 4 pages 4 CN 121975011 A 27. A method for treating Alzheimer's disease or retinal disease, the method comprising administering the pharmaceutical composition of claim 24 to a patient in need. 28. The method of claim 27, wherein the retinal disease is dry age-related macular degeneration. Claims 4 / 4 pages 5 CN121975011 A Monospecific and Multispecific Antibodies

[0001] This invention is a divisional application of U.S. Patent Application No. 202080082179.3, filed on September 28, 2020, entitled “Monospecific and Multispecific Antibodies”.

[0002] Cross-Reference to Related Applications

[0003] 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.

[0004] This invention discloses monospecific heavy-chain-only antibodies (HCAbs) specific for CD47, human serum albumin (HSA), PD-L1, CD33, CD16, and LAG3; and multivalent single-chain antibodies incorporating two or more variable domains of HCAbs specific for one or more of these antigens.

[0005] Some embodiments are single-domain antibodies that specifically or primarily comprise the VHH domain of camelid antibodies. These embodiments are monospecific and monovalent.

[0006] Some embodiments are HCAbs, or comprise a VHH domain (e.g., the Fc region of a human IgG antibody) fused with one or more constant domains from conventional antibodies. These embodiments are monospecific but typically bivalent. Other titers are also possible depending on, for example, the choice of constant domains. The Fc regions of IgA and IgM can confer higher titers.

[0007] Some embodiments comprise two VHH domains (multivalent single-chain antibodies) that are specific to the same antigen and are bound in a single amino acid chain. These embodiments are also monospecific and bivalent. Additional VHH domains may be bound to achieve higher titers.

[0008] Some embodiments comprise two (or more) VHH domains, wherein each VHH domain is specific to a different antigen bound in a single amino acid chain (multivalent, multispecific single-chain antibodies). These embodiments are multivalent and multispecific. In other embodiments comprising 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 titers that are higher than specificity.

[0009] Each monospecific embodiment in the monospecific embodiments will be specific to CD47, HSA, PD-L1, CD33, CD16, or LAG3. Each of the multispecific embodiments is 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.

[0010] Some embodiments are specific to HSA and one or more other antigens. In one aspect of these embodiments, the HSA-specific domain confers an extended in vivo half-life, while other domains provide a therapeutic effect. In another aspect of these embodiments, the HSA-specific domain may partially or completely inhibit the binding activity of adjacent domains. The HSA-specific domain may bind via a cleavable linker that is cleaved by a protease present at the intended site of action (e.g., in a tumor), such that cleavage relieves the inhibition of adjacent domains. Some multispecific embodiments are trispecific.

[0011] In some embodiments comprising multiple antigen-binding domains, antigen-binding domains derived from conventional VL-VH pairing may be used instead of one or more (but not all) VHH domains in the above embodiments.

[0012] The antigen-binding domains disclosed herein that are specific to a particular antigen may be referred to as means for binding antigens (see specification 1 / 94 page 6 CN 121975011 A).

[0013] Figure 1 depicts flow cytometry analysis of the binding affinity of anti-CD47 HCAbs A09-10 and B6H12 to CD47-overexpressing cell lines.

[0014] Figure 2 depicts competitive ELISA binding analysis of multispecific antibodies 1511 (SEQ ID NO: 156) and 3321 (SEQ ID NO: 157) that have binding specificity to CD47.

[0015] Figure 3 depicts competitive binding analysis of the CD47-binding multispecific molecules 1511 and 3321 using flow cytometry on Jurkat cell lines.

[0016] Figure 4A depicts a hemagglutination assay of human erythrocytes (RBCs) using the CD47-binding multispecific molecules 1511 and 3321. Hu5F9 was used as a control. Figure 4B depicts the binding of 1511 and 3321 to HL-60 cells and human RBCs.

[0017] Figure 5 depicts the antitumor activity of the CD47-binding multispecific molecule 3321 in Raja-Luc xenograft mice.

[0018] Figure 6 depicts flow cytometry binding analysis of anti-PD-L1 HCAb, PL14, and PL16 on CHO cells overexpressing PD-L1. Atezolizumab was used as a control.

[0019] Figure 7. Cell-based functional assays of the PD-L1-binding multispecific molecule 1511 and atezolizumab as a control.

[0020] Figures 8A and 8B depict the inhibition of PD-L1-binding multispecific molecule 1518 (SEQ ID NO: 135) on MC38-hPD-L1 tumor growth in B-hPD-L1 mice.

[0021] Figure 9 depicts the anti-HSA...Octet® binding assay of VHH antibodies.

[0022] Figures 10A and 10B depict the Octet® binding affinity assay of anti-CD33 VHH antibodies.

[0023] Figures 11A and 11B depict the Octet® binding assay of anti-CD16A VHH molecules CD16F1 (Figure 11A) and CD16E11 (Figure 11B).

[0024] Figure 12 depicts the cell-based functional assay of multispecific molecules 1511 and 3321 performed in the Jurkat NFAT CD16 reporter gene assay (ADCC assay) using IgG1 B6H12 and IgG4 B6H12 as controls.

[0025] Figures 13A to 13C depict the trispecific molecular forms of molecules having an HSA-binding domain and a CD47-binding domain (Figure 13A), molecules having an HSA-binding domain and a LAG3-binding domain (Figure 13B), and molecules having an HSA-binding domain and a CD16-binding domain (Figure 13C).

[0026] Figures 14A to 14B depict the trispecific molecular forms (Figure 14A) and SDS-PAGE analysis of pro-CD47 activated by tumor proteases (Figure 14B).

[0027] Figure 15 depicts the real-time kinetic Octet® binding analysis of PD-L1 / pro-CD47 versus PD-L1 / active CD47.

[0028] Figure 16 depicts the multispecific molecular forms. Mon = monovalent binding domain; BiV = bivalent binding domain containing two identical monovalent binding domains.

[0029] Figure 17 depicts the forms of the four-specific antibodies (four specificities, from left to right). The two VHH3s can be the same VHH or different VHHs binding different epitopes to the same antigen. The two VHH4s can be the same VHH or different VHHs binding different epitopes to the same antigen. 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 represent the four-specific antibodies in prodrug form.

[0030] Figure 18 illustrates flow cytometry binding analysis of the multispecific molecules 1518-HS5 (SEQ ID NO: 173) and 1518-HS5-GS15 (SEQ ID NO: 184) binding to CD47 on the HL60 cell line.

[0031] Figure 19 depicts the Octet® binding analysis of multispecific molecule 1511.

[0032] Figure 20 depicts the Octet® binding analysis of multispecific molecule 3321.

[0033] Figure 21 illustrates the amino acid sequence alignment of the anti-CD47 VHH sequence.

[0034] Figure 22 depicts the amino acid sequence alignment of the anti-PD-L1 VHH sequence.

[0035] Figure 23 depicts the amino acid sequence alignment of the anti-HSA VHH sequence.

[0036] Figure 24 depicts the amino acid sequence alignment of the anti-CD33 VHH sequence.

[0037] Figure 25 depicts the amino acid sequence alignment of the anti-LAG3 VHH sequence.

[0038] Figure 26 depicts the amino acid sequence alignment of the anti-CD16A VHH sequence. Detailed Description

[0039] 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 variable domains of two or more HCAbs that are specific to one or more of these antigens.

[0040] 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 monospecific with respect to the antigen. In some of these embodiments, the MVSCA comprises two or more repeated 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 monospecific with respect to the epitope. Such an MVSCA will bind to only a single site on an 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 multispecific with respect to the epitope. Such an MVSCA can bind to multiple sites on an antigen monomer or crosslink multiple copies of that monomer.

[0041] 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 the antigen. In a further embodiment, the MVSCA comprises any combination of multiple HCAb variable domains, wherein the additional variable domains are the same as the first HCAb variable domain, wherein the additional HCAb variable domains are different from the first HCAb variable domains but are specific to different epitopes on the same antigen, or wherein the additional HCAb variable domains are different from the first HCAb variable domains but are specific to different antigens.

[0042] An MVSCA comprising two or more HCAb variable domains may further comprise a constant HCAb domain. 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 the constant region or Fc region of a more conventional antibody (e.g., a human antibody, such as a human IgG antibody). In some embodiments, as will be known to those skilled in the art, the constant domain or the intact Fc region may confer specific functionality. In other embodiments, an MVSCA comprising two or more HCAb variable domains may further comprise an HCAb constant domain, 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 in addition to being located at the C-terminus of the HCAb variable domain.

[0043] Antigen

[0044] CD47 (differentiation 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 A (page 3 / 94, CN 121975011, document number 8). It works with membrane integrins and also binds to ligands thromboretin-1 (TSP-1) and signal regulatory protein α (SIRPα). TSP-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 signaling, 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 “don’t eat me” signal sent to macrophages of the immune system, making CD47 a potential therapeutic target in some cancers.

[0045] 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, and 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 delivered to T cells, which reduces cytokine production and inhibits T cell proliferation.

[0046] 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 can lead to T cell activation, expansion, and enhanced effector function. Thus, 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, US 8,217,149, and WO2014 / 055897.

[0047] Human serum albumin (HSA) is the most abundant protein in human plasma; it constitutes about half of serum proteins. Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains turgor pressure, among other functions. Albumin is synthesized in the liver as prealbuminogen, which has an N-terminal peptide that is removed before the nascent protein 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 about 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 the nascent Fc receptor (FcRn). Fusion with anti-albumin sdAb (single-domain antibody) has been used to increase the half-life of antitumor single-chain antibodies from 1–2 hours to about 10 days.

[0048] 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 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), thus placing CD33 within the immunoglobulin superfamily. The intracellular portion of CD33 contains an immunoreceptor-based tyrosine inhibitory motif.tyrosine-based inhibitory motif (ITIM) is associated with the inhibition of cell 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).

[0049] CD33 is the target of gemtuzumab ozomicin (Mylotarg®; Pfizer / Wyeth-Ayerst Laboratories) specification page 4 / 94 9 CN 121975011 A, gemtuzumab ozomicin is an antibody-drug conjugate (ADC) used to treat patients with acute myeloid leukemia. CD33 is also the target of vasutuximab taglilin (SGN-CD33A), vasutuximab taglilin is a novel antibody-drug conjugate developed by Seattle Genetics using the company's ADC technology.

[0050] Lymphocyte activation gene 3 (LAG-3), a 503-amino acid transmembrane protein, is an immune checkpoint receptor protein that is present on the cell surface of effector T cells and regulatory T cells (Tregs) and acts 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 negative signals to cells expressing LAG3 and downregulation of antigen-dependent CD4 and CD8 T cell responses. LAG3 negatively regulates the ability of T cells to proliferate, produce cytokines, and lyse target cells, a phenomenon known as T cell 'exhaustion'. Because LAG3 plays an important role in tumor immunity and infection immunity, it is an ideal target for immunotherapy. Blocking LAG3 with antagonists, including monoclonal antibodies, has been studied in the treatment of cancer and chronic viral infections.

[0051] CD16, also known as FcγRIII, is a differentiation cluster molecule present on the surface of natural killer cells, neutrophils, monocytes, and macrophages. CD16 has been 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. As the most well-studied membrane receptor associated with NK cell-triggered lysis, CD16 is a member of the immunoglobulin superfamily involved in antibody-dependent cellular cytotoxicity (ADCC).CD16 is a molecule containing IgSF. 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 carried out by human monocytes. In humans, monocytes expressing CD16 possess a variety of 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 this CD16-induced cytokine mRNA 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. Upregulation of specific cytokine genes occurs via CsA sensitivity and calcium-dependent mechanisms.

[0052] CD16 plays an important role in the early activation of natural killer (NK) cells after vaccination. Furthermore, CD16 downregulation represents a possible way to regulate NK cell responses and maintain immune homeostasis in both T cell and antibody-dependent signaling pathways. In normal healthy individuals, antibody-dependent cytotoxicity (ADCC) is induced in NK cells by immune complex crosslinking of CD16 (FcγRIII). However, this pathway can also target cancer cells or diseased cells through immunotherapy. Following influenza vaccination, CD16 downregulation is associated with a significant upregulation of influenza-specific plasma antibodies and is positively correlated with NK cell degranulation.

[0053] CD16 is frequently used as an adjunct marker to reliably identify different subsets of human immune cells. Several other CD molecules, such as CD11b and CD33, have traditionally been used as markers for human myeloid-derived suppressor cells (MDSCs). However, since these markers are also expressed on NK cells and all other cells derived from myeloid cells, other 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. Although these two markers are sufficient to distinguish neutrophils from monocytes...Cells, but eosinophils and neutrophils have similar CD15 expression. Therefore, CD16 is used as a further marker for identifying neutrophils: mature neutrophils are high in CD16, while eosinophils and monocytes are low in CD16. CD16 allows differentiation between the two types of granulocytes. In addition, CD16 expression varies between different stages of neutrophil development: neutrophil progenitors with differentiation capacity are low in CD16, while CD16 expression increases in metamyelocytes, band cells, and mature neutrophils, respectively.

[0054] Due to the expression of CD16 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 the natural killer cell response to B-cell lymphoma. Furthermore, 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.

[0055] Antibodies

[0056] 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 terms "binding site" or "antigen-binding site" as used herein refer 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 a heavy chain-only antibody, the antibody heavy chain variable region.

[0057] Antibody specificity refers to the selective recognition of a specific epitope of an antigen by an antibody. Natural antibodies are, for example, monospecific. The term "monospecific" antibody as used herein refers to 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, monospecific antibodies...This 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 the 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 may be referred to as a means of binding a specific target (e.g., CD47, HSA, PD-L1, CD33, CD16, or LAG3). Therefore, any of the various antibody structures, forms, or constructs disclosed herein that contain a VHH domain or are constructed to contain a VHH domain may be referred to as antibodies comprising means for 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.

[0058] As used herein, “antibody specific to…”, “antibody that recognizes…”, “antibody with affinity for…”, “antibody having a binding site for…”, and similar structures may be used interchangeably.

[0059] “Multispecific antibody” refers to an antibody having binding specificity 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, a multispecific antibody 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 as specified on page 6 / 94 of CN 121975011 A and at least one second antigen. In some embodiments, the multispecific antibody (MVSCA) is specific for at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3. In some embodiments, the multispecific antibodies disclosed herein are single-chain antibodies. Therefore, some multispecific antibodies may be referred to as antibodies comprising means for binding a first target and means for binding a second target, etc.

[0060] A “bispecific antibody” refers to an antibody having binding specificity for 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 sequences encoding the antigen-binding portion of the bispecific antibody can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portion of the bispecific antibody are linked via adapters as disclosed herein.

[0061] A “trispecific antibody” refers to an antibody having three different antigen-binding specificities. In some embodiments, the trispecific antibodies disclosed herein are specific to three of CD47, HSA, PD-L1, CD33, CD16, and LAG3. The amino acid sequences encoding the antigen-binding portion of the trispecific antibody can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portion of the trispecific antibody are linked via adapters as disclosed herein. In some embodiments, two adapters are used, which may be the same or different.

[0062] A “tetraspecific antibody” refers to an antibody having four different antigen-binding specificities. 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 portion of the tetraspecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding portion 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.

[0063] As used herein, the term "valence" indicates the presence of a specified number of binding sites in the antibody molecule. Thus, 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 the 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, are within the scope of this disclosure, wherein multiple antigen-binding sites bind the same antigen. The antigen-binding sites of monospecific bivalent and trivalent (or higher) antibodies may bind the same epitope or different epitopes on the antigen. Similarly, antibodies can be constructed by combining multiple monospecific binding sites with binding sites for one or more other specificities, in which the titer is higher than that of multispecific antibodies, such as trivalent bispecific antibodies.

[0064] “Full-length antibody” herein refers to the structure constituting the natural biological form of the antibody, including variable and constant regions. For example, in most mammals, including humans and mice, full-length antibodies of the IgG class are tetramers and consist of two pairs of phases.Each pair of IgG antibodies consists of two identical immunoglobulin chains, each pair having 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 (HCAb), each heavy chain containing variable domains (CH2 and CH3 domains) attached to the Fc region.

[0065] Tetrameric antibodies typically consist of two pairs of identical polypeptide chains, each pair having one “light chain” (typically with a molecular weight of about 25 kDa) and one “heavy chain” (typically with a molecular weight of about 50–70 kDa). Each of the light and heavy chains consists of two distinct regions called the variable region and the constant region. For IgG immunoglobulins, the heavy chain consists of four immunoglobulin domains linked from the N-terminus to the C-terminus in the order VH-CH1-CH2-CH3. These four immunoglobulin domains refer to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (also referred to as VH-Cγ1-Cγ2-Cγ3, which refer 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 order VL-CL. These two immunoglobulin domains refer to the light chain variable domain and the light chain constant domain, respectively. The constant region exhibits less sequence diversity and is responsible for binding many native proteins to trigger important biochemical events.

[0066] The variable region of an antibody contains the antigen-binding determinants of the molecule and thus determines the specificity of the antibody for its target antigen. The variable region is so named because it is most different in sequence from other antibodies of the same class. In the variable region, three loops are clustered for each V domain of the heavy and light chains to form an antigen-binding site. Each loop in the loop is called a complementarity-determining region (hereinafter referred to as “CDR”), in which the amino acid sequence variation is most significant. There are a total of six CDRs, three for each heavy and light chain, named VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside the CDRs is called the backbone (FR) region. Although not as diverse as the CDRs, there is indeed sequence variability in the FR regions between different antibodies. In general, this characteristic architecture of antibodies provides a stable scaffold (FR region) on which the immune system can explore a large number of antigen-binding diversity (CDRs) to obtain specificity for a variety of antigens.

[0067] The gene encoding the immunoglobulin locus contains multiple V region sequences and shorter nuclei named “D” and “J”.The nucleotide sequence, and it is the combination of V, D, and J nucleotide sequences that produces VH diversity.

[0068] Antibodies are classified into several classes, also known as isotypes, as determined by constant region inheritance. Human constant light chains are classified as κ (Cκ) and λ (Cλ) light chains. Heavy chains are classified as 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 its constant region. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. The disclosed HCAb antibodies, bispecific antibodies, and multispecific antibodies may have constant regions comprising all or part of the aforementioned isotypes.

[0069] The following antibody fragments, including but not limited to: (i) Fab fragments containing VL, CL, VH, and CH1 domains; (ii) Fd fragments containing VH and CH1 domains; (iii) Fv fragments containing VL and VH domains of a single antibody; (iv) dAb fragments containing a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments, 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.

[0070] 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. Multivalent monospecific and multispecific single-chain antibodies are disclosed herein. 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 CD47,At least one of HSA, PD-L1, CD33, CD16, and LAG3 is specific, plus at least one further specific. In some embodiments, the multispecific single-chain antibody is specific for at least two of CD47, HSA, PD-L1, CD33, CD16, and LAG3.

[0071] As used herein, a “humanized” antibody refers to an antibody comprising a human backbone region (FR) and one or more complementarity-determining regions (CDR') derived from a non-human antibody. The non-human antibody providing the CDR' is referred to as the “donor,” and the human immunoglobulin providing the 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, it is necessary to “reverse mutate” selected recipient backbone residues to the corresponding donor residues to regain the affinity lost in the originally transplanted construct. Humanized antibodies will preferably also include at least a portion of an immunoglobulin constant region (typically the constant region of human immunoglobulins) and will typically include 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 methods described in US 6,797,492, all of which discloses CDR transplantation and is incorporated herein by reference. Structure-based methods may be used for humanization and affinity maturation, such as those described in US 7,117,096, all of which discloses humanization and affinity maturation and is incorporated herein by reference.

[0072] In various embodiments herein, the antibody is a heavy-chain-only antibody (HCAb). In addition to antibodies containing both heavy and light chains (one antibody contains two light chains and two heavy chains), camelids (camels, dromedaries, and llamas) also contain double-chain antibodies (containing only a variant heavy chain). Dimeric antibodies are 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 occurring 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 a constant region (CH2, ...When CH3 is applied, antibodies are produced in which the hapten is a single chain rather than a light chain / heavy chain pair (i.e., an antibody with two heavy chains but no light chain interaction). The binding of the antigen differs from that seen when using conventional antibodies, but the high affinity is achieved in the same way, i.e., through hypermutation of the variable region and selection of cells expressing such high-affinity antibodies.

[0073] In one exemplary embodiment, the disclosed HCAb is produced by immunizing transgenic mice in which endogenous mouse antibody expression has been eliminated and camelid transgenes have been introduced. HCAb mice are disclosed in US 8,883,150, US 8,921,524, US 8,921,522, US 8,507,748, US 8,502,014, US 2014 / 0356908, US 2014 / 0033335, US 2014 / 0037616, US 2014 / 0356908, US 2013 / 0344057, US 2013 / 0323235, US 2011 / 0118444, and US 2009 / 0307787, all of which are incorporated herein by reference for all their disclosures relating to 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.

[0074] In other embodiments, HCAbs were produced by immunizing llamas with the desired antigen and sequencing the VHH region encoding the resulting antigen-binding antibody. In one embodiment, the VHH was 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 the phage library in each of these patents is incorporated herein by reference).

[0075] Multispecific or multivalent antibodies are also disclosed herein, in which two or more antigen-binding domains are conjugated in a single fusion protein. Multispecific antibodies can take various forms, including (i) multispecific Fv fragments; (ii) heavy chains with first specificity having a second VH domain with second specificity associated with (or fused to); (iii) tetrameric monoclonal antibodies with first specificity having a second VH domain with 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) with first specificity having a second VH domain with second specificity associated with it. Exemplary Fab fragments include Fab fragments with second specificity.The second VH sequence associates 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.

[0076] Multispecific or multivalent antibodies may include a linker sequence that links a specific antigen-binding domain (e.g., VH or VHH) to another antigen-binding domain and allows the amino acid sequence to fold correctly to produce the desired three-dimensional conformation and antigen-binding profile. Typically, the linker sequence will be a short amino acid sequence that provides sufficient space and flexibility between the domains for the domains to fold correctly. Linkers can also cause steric hindrance to facilitate binding to targets in each domain. Suitable linkers include, but are not limited to, the linkers in Table 15 (SEQ ID No: 100 to SEQ ID No: 119), EPKSCD (SEQ ID NO: 224), and ASTKGP (SEQ ID NO: 225). More linkers will be known to those skilled in the art.

[0077] Amino acid sequence variants of monospecific or multispecific antibodies disclosed herein are also within the scope of this disclosure. Such amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the DNA encoding the antibody, or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues 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 has the desired characteristics. Amino acid changes can also alter the post-translational process of humanized or variant antibodies, for example, by changing the number or location of glycosylation sites.

[0078] A useful method for identifying certain antibody residues or regions as preferred mutagenic sites is called “alanine scanning mutagenesis.” A target residue or set of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral amino acid (most preferably alanine or polyalanine) to affect the interaction between the amino acid and the antigen. The positions of amino acids 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 mutations at a given site, mutations can be performed at the target codon or region.Alanine scanning or random mutagenesis is performed, and the expressed antibody variants are screened for desired activity.

[0079] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to a polypeptide containing 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 with an epitope tag. Other insertion variants of the antibody molecule 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.

[0080] Another type of variant is amino acid substitution variants. These variants have at least one amino acid residue removed from the antibody molecule and a different residue inserted at its position. The substitution mutagenesis sites of most interest include hypervariable regions, but FR alterations are also considered. Conservative substitutions are shown under the “Preferred Substituents” header in Table 1. If such substitutions result in an alteration of biological activity, a more substantial alteration, designated as “Exemplary Substituents” in Table 1 or further described below with reference to the amino acid categories, can be introduced, and the product screened.

[0081] Table 1 Specification 10 / 94 pages 15 CN 121975011 A

[0082]

[0083] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that significantly differ in maintaining (a) the structure of the polypeptide backbone in the substitution region (e.g., sheet or helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the side chains. Naturally occurring residues are categorized into the following groups based on common side-chain properties:

[0084] (1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0085] (2) Neutral hydrophilic: Cys, Ser, Thr;

[0086] (3) Acidic: Asp, Glu;

[0087] (4) Basic: Asn, Gin, His, Lys, Arg;

[0088] (5) Residues affecting chain orientation: Gly, Pro; and

[0089] (6) Aromatic: Trp, Tyr, Phe.

[0090] Non-conservative substitutions will require exchanging members of one of these categories for another.

[0091] Serine may also be used to replace any cysteine ​​residues that do not participate in maintaining the proper conformation of monospecific or multispecific antibodies to improve the oxidative stability of the molecule and prevent aberrant crosslinking. 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).

[0092] Another type of alternative 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 be relative to the parent antibody that produced them (see page 11 / 94 of the specification).16 CN 121975011 A has improved biological properties. A convenient way to generate such substituted variants is to use phage display for affinity maturation. Briefly, several hypervariable sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus generated are displayed in monovalent form from filamentous phage particles as fusions with the gene III product of M13 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 sites 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 exhibiting superior properties in one or more relevant assays can be selected for further development.

[0093] Another amino acid variant of the antibody alters the original glycosylation pattern of the antibody. This alteration refers to 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.

[0094] Antibody glycosylation is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate moieties to a side chain of asparagine residues. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid other than proline, are recognition sequences for the enzymatic attachment of carbohydrate moieties to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates potential glycosylation sites. 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.

[0095] 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 tripeptide sequences described above. Alterations can also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

[0096] 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.

[0097] Other modifications to monospecific or multispecific antibodies are envisioned. For example, it may be necessary to modify the antibody in terms of effector function, such as to enhance the antibody's efficacy in treating diseases. For example, one or more cysteine ​​residues may be introduced into the Fc region, thereby allowing interchain disulfide bonds to form in the 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 may be engineered to have a double Fc region and may thereby have enhanced complement cleavage and ADCC capabilities.

[0098] In another embodiment, the antibody may be conjugated to a "receptor" (e.g., streptavidin) for pre-targeting, wherein the antibody-receptor conjugate is administered to a patient, followed by removal of unbound conjugates from circulation using a scavenger, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide).

[0099] 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. The antibody covalent modification type of exemplary specification 12 / 94 pages 17 CN 121975011 A includes 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.

[0100] The monospecific or multispecific antibodies disclosed herein can be generated by recombinant means. Therefore, this document discloses nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods of recombinant production are well known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and generally purification to pharmaceutically acceptable purity. In order to express the antibody in host cells as described above, nucleic acids encoding the antibody sequence are inserted into the expression vector by 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 (E. coli)).Expression is performed in *C. coli* 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 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 fully mature sequences, while in other embodiments they are retained.

[0101] 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 said antibody molecule from the culture.

[0102] The antibody is appropriately separated from the culture medium by conventional immunoglobulin purification methods, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0103] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Thus, the terms “transformer” and “transformed cell” include primary subject cells and cultures derived therefrom, regardless of the number of transfections. It should also be understood that the DNA content of all progeny may not be exactly the same due to intentional or unintentional mutations. 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 distinction will be clear from the context.

[0104] 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 cells, for example, 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.

[0105] 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 the splicing of mRNA.

[0106] A “vector” is a nucleic acid molecule, especially a self-replicating nucleic acid molecule, that transfers an inserted nucleic acid molecule to a host cell.Within 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.

[0107] 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.

[0108] As used herein, the term “host cell” means 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.

[0109] Control sequences suitable for prokaryotes include, for example, promoters, optionally operon sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0110] When a nucleic acid is positioned to functionally relate to another nucleic acid sequence, the 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, 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, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is localized to facilitate translation, 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 have to be contiguous. Linkage is accomplished by linking at a convenient restriction site. If such a site is not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0111] This document also discloses isolated nucleic acids encoding monospecific or multispecific antibodies, vectors and host cells containing said nucleic acids, and recombinant techniques for producing said antibodies.

[0112] 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 by homologous recombination, as described, for example, in US 5,204,244, all disclosures of which relating to 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, those used in this study.The following are all disclosures relating to protein expression in this patent, including but not limited to: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences, such as those described in US 5,534,615, which are expressly incorporated herein by reference.

[0113] Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (e.g., Escherichia, Escherichia coli), Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (e.g., Salmonella typhimurium), Serratia (e.g., Serratia marcescans), and Shigella, as well as Bacilli (e.g., B. subtilis and B. licheniformis) and Pseudomonas (e.g., Pseudomonas aeruginosa). (aeruginosa), and Streptomyces. An exemplary Escherichia 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.

[0114] In addition to 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, or common baker's yeast, is the most commonly used yeast among lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and useful in this document, such as *Schizosaccharomyces pombe*; hosts of *Kluyveromyces*, such as *Kluyveromyces lactis*, *Kluyveromyces fragilis* (see specification 14 / 94 pages, 19 CN 121975011 A (ATCC 12,424)), and *Kluyveromyces bulgaricus*.*Kluyvesin bulgaricus* (ATCC 16,045), *Kluyvesin wickeramii* (ATCC 24,178), *Kluyvesin waltii* (ATCC 56,500), *Kluyvesin drosophilarum* (ATCC 36,906), *Kluyvesin thermotolerans*, and *Kluyvesin marxianus*; *Yarrowia* (EP 402,226); *Pichia pastoris* (EP 183,070); *Candida*; *Trichoderma reesia* (EP 244,234); *Neurospora crassa*; *Schwanniomyces*, such as *Schwanniomyces yews*. occidentalis); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts, such as Aspergillus nidulans and Aspergillus niger.

[0115] Suitable host cells for expressing glycosylated monospecific or multispecific antibodies are derived from multicellular organisms, including invertebrate cells, such as plant cells and insect cells. Many baculovirus strains and variants from hosts and corresponding permissible insect host cells have been identified, such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various viral strains used for transfection are publicly available, such as the L-1 variant of the alfalfa silver-striped armyworm (Autographa californica) NPV and the Bm-5 strain of the Chinese silkworm NPV, and such viruses can be used as the viruses according to the present invention, particularly for transfection of armyworm cells. Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0116] 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 are SV40 (COS-7, ATCC).Monkey kidney CV1 line transformed with CRL 1651; human embryonic kidney cell line (293 or 293 cells grown in suspension culture after subcloning); 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 (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumors (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and the human hepatocellular carcinoma cell line (Hep G2).

[0117] Host cells were transformed with the above expression vectors for monospecific or multispecific antibody production and cultured in conventional nutrient media that were appropriately modified to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0118] Host cells used for the production of 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 Duchenne Modified Eagle Medium (DMEM) (Sigma), are suitable for culturing host cells. In addition, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or 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, pH, etc., are those previously used with the selected host cells for expression and will be readily apparent to those skilled in the art.

[0119] 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, particulate debris, either host cells or lysed fragments, is removed as a first step, for example, by centrifugation or ultrafiltration.

[0120] Cell-prepared antibody compositions can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains, although protein A can 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)benzene, allow for faster flow rates and shorter processing times than can be achieved using agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin can be used for purification. The antibodies and antibody fragments disclosed herein can also be synthesized with histidine tags and purified by metal affinity chromatography.

[0121] Depending on the antibody to be recovered, other protein purification techniques may also be used, such as fractionation on an ion exchange column, 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.

[0122] After any preliminary purification step, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography at a pH of about 2.5–4.5 using elution buffer, preferably at low salt concentrations (e.g., about 0–0.25 M salt).

[0123] Multispecific single-chain antibodies that can be cleaved in the tumor microenvironment are also disclosed herein. In some embodiments, once the multispecific single-chain antibody reaches the tumor, the tumor-targeting domain (e.g., a tumor antigen-binding domain) or other functional domains (e.g., an anti-HSA domain, which can prolong the 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), ADAMs (de-integrin and metalloproteinases; e.g., ADAM10 and ADAM17), and kallikrein.Related peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepsin (HPN), matriptase, podocyte fibroblasts, or dipeptidyl peptidases (e.g., DDP4).

[0124] Antibody Compositions

[0125] Pharmaceutical compositions comprising monospecific or multispecific antibodies are also disclosed herein, wherein the monospecific or multispecific antibodies specifically include CD47, HSA, PD-L1, CD33, CD16, or LAG3. Use of the antibodies described herein for manufacturing pharmaceutical compositions is also disclosed. Methods of using the disclosed antibodies and pharmaceutical compositions comprising the antibodies to treat various diseases and disorders are also disclosed.

[0126] A pharmaceutical composition is a pharmaceutical composition intended for and suitable for use in treating human diseases. That is, it provides an overall beneficial effect and does not contain any amount of ingredients or contaminants that 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 composition as a whole.

[0127] 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, page 16 / 94, CN 121975011 A. Particularly useful liposomes can be produced by reverse-phase evaporation from 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.

[0128] 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, etc. 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.

[0129] The compositions disclosed herein can be administered by 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. To administer the drug via certain routes of administration...Disclosed antibodies may require association with a substance that prevents their inactivation or co-administration of the antibody with the substance. For example, antibodies may be administered to a subject in a suitable carrier, such as a liposome or a diluent. Pharmaceutically acceptable diluents include saline and buffered aqueous solutions. Drug carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0130] As used herein, the phrases “parenteral administration” and “extragastric administration” refer to modes of administration other than enteral and local administration (usually by injection) and include, but are not limited to, intravenous, intramuscular, intra-articular, intrathecal, intracapsular, intraocular, intraorbital, intracardiac, intravitreal, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0131] These compositions may also contain excipients such as preservatives, wetting agents, emulsifiers, and dispersants. The sterilization procedures described above and the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenolic sorbic acid, etc.) ensure protection against microbial contamination. It may also be necessary to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Furthermore, absorption of injectable drug forms can be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

[0132] In some embodiments, the antibody-containing pharmaceutical composition is a lyophilized cake. This 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.

[0133] Regardless of the chosen route of administration, the disclosed antibodies and / or pharmaceutical compositions containing said antibodies, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0134] The actual dose level of the active ingredient in the pharmaceutical composition can be varied to obtain a certain amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and administration mode 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.

[0135] Function of the disclosed MVSCA and its constituent antibody domains and linkers

[0136] VHH against HSA

[0137] 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 could 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.

[0138] By binding 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 is observed as an increase in the specificity of VHH, 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.

[0139] This blockade can also be reversible. By placing the anti-HSA domain at the terminal position in the MVSCA and attaching it to a cleavable linker, the anti-HSA domain can be removed and the full binding activity of the adjacent binding domain 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 at the adjacent 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 the adjacent domain 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 an MVSCA or multispecific antibody.

[0140] Anti-CD47

[0141] The function of the anti-CD47 domain is to inhibit the "don't eat me" signal of CD47 on tumor cells, allowing the 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. There are several ways to avoid this. Apparently, there are multiple conformations of CD47, 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.

[0142] Another way to avoid unwanted or harmful binding of MVSCA to CD47 can be achieved by placing MVSCA in a manner near the anti-HSA domain, such that binding to CD47 is reduced or blocked, as described above. Once MVSCA binds to tumor cells through another binding domain in 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.

[0143] Thus, the anti-CD47 domain constitutes a means of reducing the inhibition of phagocytosis.

[0144] Anti-CD16

[0145] The function of the anti-CD16 domain is to upregulate the 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.

[0146] CD16 usually interacts with the Fc portion of an antibody. When CD16A on NK cells is bound to the Fc portion of an antibody, the cytolytic activity of NK cells becomes targeted at cells or microorganisms to which the variable domain of the antibody has bound. 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 ADCC against other specific targets they carry. Thus, the anti-CD16 domain constitutes a means of recruiting NK-mediated ADCC. Specification 18 / 94 pages 23 CN 121975011 A

[0147] Anti-PD-L1

[0148] 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 associated immune checkpoints, thereby releasing the development of T cell-mediated immune responses. 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 for PD-1 blockade or for releasing PD-1 immune checkpoints.

[0149] The anti-PD-L1 domain, as an antibody against a tumor antigen, can mediate the binding of MVSCA 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...This promotes macrophage-mediated phagocytosis. Multivalent binding to tumor cells increases binding affinity and ADCC. This can be achieved through multiple copies of an 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 tumor cells, for binding tumor antigens, or for binding PD-L1 tumor antigens.

[0150] Anti-LAG3

[0151] The anti-LAG3 domain acts as an immune checkpoint inhibitor. The anti-LAG3 domain acts as an antagonist for 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.

[0152] Anti-CD33

[0153] The anti-CD33 domain 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), and is therefore 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 increases 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. Thus, the anti-CD33 domain constitutes a means for binding tumor cells, a means for binding tumor antigens, or a means for binding CD33 tumor antigens.

[0154] Furthermore, when CD33 binds to sialic acid residues, such as in β-amyloid or other glycoproteins deposited by glycolipids, an inhibitory signaling cascade leads to inhibition of phagocytic activity. Antibodies containing an anti-CD33 domain can act as antagonists of CD33 stimulation, thereby promoting phagocytic activity and clearance of β-amyloid protein 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 an anti-CD33 domain can also be used to treat dry AMD and other retinal diseases. Thus, the anti-CD33 domain constitutes a means for promoting phagocytic activity (in CD33-expressing cells), a means for promoting clearance of β-amyloid protein, or a means for clearing insoluble deposits.

[0155] MVSCAs suitable for the treatment of Alzheimer's disease and retinal diseases are preferably bivalent for CD33 andThey contain an anti-HSA domain to enhance 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.

[0156] Connectors

[0157] In many embodiments, the binding domains are not directly connected to each other, but have short amino acid sequences interposed between them, i.e., connectors. Examples of connectors are shown in Table 15. The length and sequence of the connector can significantly affect the expression level and structure of the MVSCA and the binding affinity of the connected domains. Length-adjustable connectors L2 and L4 (see Table 15) can be used to optimize the MVSCA according to these parameters. Connectors L1, L2, and L4 may be referred to as indestructible connector means, flexible connector means, or flexible indestructible connector means.

[0158] When two copies of the same VHH domain are placed adjacent to each other in an MVSCA, they frequently and adversely interact. This can be avoided by inserting a relatively short and rigid linker between the two copies. In some embodiments, the short rigid linker has the sequence AAA (L3 in Table 15). Such a linker may be referred to as a short rigid linker means or an uncleavable short rigid linker means.

[0159] When the anti-HSA domain-HSA complex is used to generate a prodrug associated with the binding activity of 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 the MVSCA in terms of expression level 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.

[0160] MVSCA

[0161] The binding domains and linkers described herein can be combined to produce a multifunctional MVSCA suitable for the treatment of a specific disease. The binding domains and linkers can also be further combined with other binding domains. MVSCAs may also be referred to as including means for achieving various functions associated with each component type of the binding domain, and / or including linker means for achieving their respective functions. Exemplary designs will be briefly discussed below.

[0162] HSA / CD47 / PD-L1: This design is suitable for treating tumors expressing PD-L1, will promote phagocytosis, will release the PD-1 immune checkpoint, and will have a prolonged circulating half-life. In various embodiments, the MVSCA is effective against CD47 and / or...Alternatively, the anti-PD-L1 binding domain may be bivalent. Depending on the linker used, the anti-HSA domain (once bound to HSA) may or may not inhibit binding to CD47, and the inhibition (if present) can be reversed by cleaving the cleavable linker. In some embodiments, the binding domains are arranged in a different order, but the anti-HSA domain should be at the terminal position if to be cleaved. In addition to 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 promoting phagocytosis (and release of PD-1 immune checkpoints) of PD-L1-expressing tumors. Several embodiments of this design are illustrated in Example 7.

[0163] HSA / LAG3 / PD-L1: This design is suitable for treating PD-L1-expressing tumors, will release LAG3 and PD-1 immune checkpoints, and will have an extended circulating half-life. In various embodiments, the MVSCA may be bivalent for the anti-LAG3 and / or anti-PD-L1 binding domains. In some embodiments, the binding domains are arranged in a different order. In addition to describing this type of MVSCA as a means of including one or more functions for its components, the MVSCA can 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.

[0164] 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 inhibit or will not inhibit binding to CD47, and inhibition (if present) can be reversed by cleaving the cleavable adapter. In addition to describing this type of MVSCA as a means of including one or more functions used for its components, the MVSCA can 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 release specification 20 / 94 pages 25 CN 121975011 A PD-1 immune checkpoint). Several embodiments of this design are illustrated in Example 8.

[0165] 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) 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 CD33-expressing tumors and recruiting NK-mediated ADCC to CD33-expressing tumors. Several embodiments of this design are illustrated in Example 8.

[0166] 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. In addition to 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.

[0167] Use of the Disclosed Antibodies

[0168] 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 aggressive 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; and preventative treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the associated disease, pathological condition, or disorder.Treatment of the development of a condition or disease; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving an associated disease, pathological condition, or disease. Various implementations may specifically include or exclude one or more of these treatment modalities.

[0169] The use of antibodies disclosed herein in diagnostics and imaging is also considered.

[0170] Furthermore, the term “treating / treatment” broadly includes any type of therapeutic activity, including the diagnosis, relief, or prevention of a disease in a person or other animal, or any aspect thereof, or any activity that otherwise affects the structure or any function of the body of a person or other animal. Therapeutic activities include, in particular, the administration of the medicines, dosage forms, and pharmaceutical compositions described herein to a patient by a healthcare professional, the patient himself, or any other person. Therapeutic activities include orders, instructions, and recommendations from healthcare professionals (such as physicians, physician assistants, nursing practitioners, etc.) which are then performed by any other person, including other healthcare professionals or the patient himself. This includes, for example, instructing patients to undergo or instructing clinical laboratories to perform diagnostic procedures, such as for cancer diagnosis and staging, so that patients can ultimately receive appropriate and beneficial treatment. In some embodiments, the order, instruction, and recommendation of treatment activities may also include encouraging, inducing, or compelling the selection of a specific drug or combination thereof to treat the condition, and the drug is actually used in a manner that includes: approving the drug's insurance coverage, refusing alternative drugs (including the drug being on or 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 embodiments, treatment activities may also include encouraging, inducing, or compelling the selection of a specific 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 orders, 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 orders, instructions, and recommendations. In some cases, healthcare professionals may also gain financial benefits from following such medical advice, instructions, and recommendations.

[0171] 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 class of cancer based on the antigen-binding specificity contained in the antibody.

[0172] The present invention provides a method of treating cancer, the method comprising administering to a patient in need of such treatment a...Effective doses of the antibodies disclosed herein or pharmaceutical compositions containing said antibodies.

[0173] Examples of cancers that can be treated by 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; tumor 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; renal cell carcinoma; laryngeal carcinoma; non-small cell lung cancer; small cell lung cancer; non-Hodgkin's lymphoma; Warburg's macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell carcinoma; squamous neck carcinoma; multiple endocrine neoplasia syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome; nasopharyngeal carcinoma; neuroblastoma; oral cancer; oropharyngeal carcinoma; osteosarcoma; pancreatic cancer; parathyroid carcinoma; penile cancer; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland carcinoma; soft tissue sarcoma; Cezari syndrome; skin cancer; squamous neck carcinoma; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and Wilms' tumor.

[0174] The effectiveness of cancer therapy is usually measured by "response". Techniques for monitoring response can be similar to tests used to diagnose cancer, such as, but not limited to:

[0175] A mass or tumor involving some lymph nodes can be felt and measured externally through a physical examination.

[0176] Some internal cancerous tumors will be visible on an X-ray or CT scan and can be measured with a ruler.

[0177] Blood tests can be performed, including those that measure organ function.

[0178] Tumor marker tests can be performed for certain cancers.

[0179] Regardless of the test used, whether it is a blood test, cell count, or tumor marker test, it should be repeated at specific time intervals to compare the results with earlier similar tests.

[0180] Response to cancer treatment can be defined in several ways:

[0181] Complete response—all cancer or tumors have disappeared; there are no signs of disease. Tumor markers (if applicable)The expression level of tumor markers may fall within the normal range.

[0182] Partial response—the cancer has shrunk by a certain percentage, but the disease still exists. The level of tumor markers (if applicable, see page 22 / 94 of the specification, 27 CN 121975011 A) may have decreased (or increased, based on tumor markers as an indicator of reduced tumor burden), but signs of disease still exist.

[0183] Stable disease—the cancer has neither grown nor shrunk; the amount of disease has not changed. Tumor markers (if applicable) have not changed significantly.

[0184] Disease progression—the cancer has grown; the disease is now more than before treatment. Tumor marker tests (if applicable) show that tumor markers have increased.

[0185] Other measures of cancer treatment efficacy include the overall survival time interval (i.e., the time from diagnosis or the start of the evaluated treatment to death from any cause), the cancer-free survival time interval (i.e., the length of time the cancer remains undetectable after a complete response), and the progression-free survival time interval (i.e., the length of time the tumor growth that has resumed after disease stabilization or partial response is undetectable).

[0186] There are two standard methods for assessing the response to treatment of solid tumors based on tumor size (tumor burden): the WHO standard and the RECIST standard. These methods measure solid tumors to compare the current tumor with past measurements, or to compare changes with future measurements, and to adjust the treatment regimen. In the WHO method, the long axis and short axis of the solid tumor are measured, and then 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.

[0187] This disclosure provides a method for treating an eye disease, the method comprising administering an antibody disclosed herein to a patient in need of 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.

[0188] 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.

[0189] The following embodiments, sequence listing, and drawings are provided to aid in understanding the invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures without departing from the spirit of the invention.

[0190] List of Specific Embodiments

[0191] The following list of embodiments is an illustration of various embodiments with respect to the width, combination and sub-combination, invention category, etc., described herein, but is not intended to be an exhaustive list of all embodiments sought to be supported herein.

[0192] Embodiment 1. A heavy chain variable (VHH) domain having antigen-binding specificity to CD47.

[0193] Embodiment 2. The VHH domain according to Embodiment 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.

[0194] Embodiment 3. A heavy chain variable (VHH) domain having antigen-binding specificity to PD-L1.

[0195] Embodiment 4. The VHH domain according to Embodiment 3, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 31 to SEQ ID NO: 38.

[0196] Embodiment 5. A heavy chain variable (VHH) domain having antigen-binding specificity to human serum albumin (HSA).

[0197] Embodiment 6. The VHH domain according to Embodiment 5, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48. Specification 23 / 94 pages 28 CN 121975011 A

[0198] Embodiment 7. A heavy chain variable (VHH) domain having antigen-binding specificity to CD33.

[0199] Embodiment 8. The VHH domain according to Embodiment 7, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78.

[0200] Embodiment 9. A heavy chain variable (VHH) domain having antigen-binding specificity to LAG3.

[0201] Embodiment 10. The VHH domain according to Embodiment 9, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93.

[0202] Embodiment 11. A heavy chain variable (VHH) domain having antigen-binding specificity to CD16.

[0203] Embodiment 12. The VHH domain according to Embodiment 11, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99.

[0204] Embodiment 13. A heavy chain-only antibody (HCAb) comprising the VHH domain according to any one of Embodiments 1-12.

[0205] Embodiment 14.An antibody comprising one or more constant domains and means for binding CD47, HSA, PD-L1, CD33, CD16, or LAG3.

[0206] Embodiment 15. A multispecific antibody comprising one or more of the VHH domains described in Embodiments 1-12 or means for binding CD47, HSA, PD-L1, CD33, CD16, or LAG3.

[0207] Embodiment 16. The multispecific antibody according to Embodiment 15, further comprising one or more additional antibody-binding domains.

[0208] Embodiment 17. The multispecific antibody according to Embodiment 16, wherein the additional antibody-binding domain comprises FC5 (SEQ ID NO: 222).

[0209] Embodiment 18. The multispecific antibody according to Embodiment 16, wherein the additional antibody-binding domain comprises Fv or Fab.

[0210] Embodiment 19. The multispecific antibody according to any one of Embodiments 15-17, wherein the multispecific antibody is a multispecific single-chain antibody (MVSCA).

[0211] Embodiment 20. The MVSCA according to Embodiment 19, wherein the MVSCA comprises 2, 3, 4, 5, or 6 antibody-binding domains.

[0212] Embodiment 21. The MVSCA according to Embodiment 20, wherein the MVSCA has 1, 2, 3, or 4 antibody-binding specificities.

[0213] Embodiment 22. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA comprises antibody-binding domains recognizing HSA, CD47, and PD-L1.

[0214] Embodiment 23. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA comprises antibody-binding domains recognizing HSA, CD47, and CD33.

[0215] Embodiment 24. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain recognizing HSA, LAG3, and PD-L1.

[0216] Embodiment 25. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain recognizing HSA, LAG3, and CD33.

[0217] Embodiment 26. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain recognizing CD16, HSA, and PD-L1.

[0218] Embodiment 27. The MVSCA according to any one of Embodiments 19-21, wherein the MVSCA includes an antibody-binding domain recognizing CD16, HSA, and CD33. DescriptionPage 24 / 94, 29 CN 121975011 A

[0219] 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.

[0220] 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.

[0221] Embodiment 30. The MVSCA according to any one of Embodiments 14-21 or 26-29, wherein the antibody-binding domain that recognizes CD16 preferentially recognizes CD16A.

[0222] Embodiment 31. The MVSCA according to any one of Embodiments 19-30, wherein the MVSCA includes two adjacent antibody-binding domains having the same specificity.

[0223] Embodiment 32. The MVSCA according to Embodiment 31, wherein the two adjacent antibody-binding domains having the same specificity have a short rigid linker means inserted between them.

[0224] Embodiment 33. The MVSCA according to Embodiment 31, wherein the short rigid linker means consists of the amino acid sequence AAA (SEQ ID NO: 102).

[0225] Embodiment 34. The MVSCA according to any one of Embodiments 31-33, wherein the two adjacent antibody-binding domains bind CD33.

[0226] Embodiment 35. The MVSCA according to Embodiment 34, wherein the MVSCA further comprises an antibody-binding domain that recognizes HSA.

[0227] Embodiment 36. The MVSCA according to Embodiment 34 or 35, wherein the MVSCA further comprises FC5.

[0228] Embodiment 37. The MVSCA according to any one of Embodiments 31-33, wherein the two adjacent antibody-binding domains bind PD-L1.

[0229] Embodiment 38. The MVSCA according to any one of Embodiments 31-33, wherein the two adjacent antibody-binding domains bind LAG3.

[0230] Embodiment 39. The MVSCA according to any one of Embodiments 31-33, wherein the two adjacent antibody-binding domains bind CD16.

[0231] Embodiment 40. The MVSCA according to any one of Embodiments 31-33, wherein the two adjacent antibody-binding domains bind CD47.

[0232] Embodiment 41. The MVSCA according to any one of Embodiments 19-30, wherein the MVSCA includes a linker between adjacent antibody-binding domains.

[0233] Embodiment 42.According to Embodiment 4', the MVSCA 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).

[0234] Embodiment 43. According to any one of Embodiments 19-30, the MVSCA includes a flexible, non-cuttable connector means inserted between different antigen-binding domains.

[0235] Embodiment 44. According to any one of Embodiments 19-30, the MVSCA includes an antibody-binding domain located at the N-terminus or C-terminus of HSA.

[0236] Embodiment 45. According to Embodiment 44, when binding HSA, the antibody-binding domain adjacent to the antibody-binding domain of HSA is inhibited from binding to its antigen.

[0237] Embodiment 46. According to Embodiment 45, the antibody-binding domain adjacent to the antibody-binding domain of HSA recognizes CD47.

[0238] Embodiment 47. The MVSCA according to Embodiment 45 or 46, wherein a cleavable connector is inserted between the antibody-binding domain that binds 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:112), L11*12 (SEQ ID NO:113), L11*13 (SEQ ID NO:114), L11*14 (SEQ ID NO:115 ... NO:115), L11*15 (SEQ ID NO:116), L11*16 (SEQ ID NO:117), L11*17 (SEQ ID NO:118), or L11*18 (SEQ ID NO:119).

[0239] Embodiment 48. The MVSCA according to Embodiment 45 or 46, wherein a cleavable connector means is inserted between the antibody-binding domain that binds the HSA and the adjacent antibody-binding domain.

[0240] Embodiment 49.According to any one of Embodiments 19-48, the MVSCA is wherein all of the antibody-binding domains are VHH domains.

[0241] Embodiment 50. A pharmaceutical composition comprising a VHH domain or antibody according to any one of Embodiments 1-49.

[0242] Embodiment 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.

[0243] Embodiment 52. A pharmaceutical composition comprising means for binding CD47 or means for reducing inhibition of phagocytosis.

[0244] Embodiment 53. A pharmaceutical composition comprising means for binding CD16 or CD16A or means for recruiting NK-mediated ADCC.

[0245] Embodiment 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.

[0246] Embodiment 55. A pharmaceutical composition comprising means for binding tumor antigen, means for binding the PD-L1 tumor antigen, or means for binding the CD33 tumor antigen.

[0247] Embodiment 56. A pharmaceutical composition comprising means for binding LAG3 or means for releasing the LAG3 immune checkpoint.

[0248] Embodiment 57. A pharmaceutical composition comprising means for releasing immune checkpoints, means for releasing the PD-1 immune checkpoint, or means for releasing the LAG3 immune checkpoint.

[0249] Embodiment 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.

[0250] Embodiment 59. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1.

[0251] Embodiment 60. A pharmaceutical composition comprising means for recruiting T effector cells to tumors expressing PD-L1.

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

[0253] Embodiment 61.A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing PD-L1 and recruiting NK-mediated ADCC to the PD-L1-expressing tumors.

[0254] Embodiment 62. A pharmaceutical composition comprising means for promoting phagocytosis of tumors expressing CD33 and recruiting NK-mediated ADCC to the CD33-expressing tumors. Specification 26 / 94 pages 31 CN 121975011 A

[0255] Embodiment 63. A method of 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.

[0256] Embodiment 64. A method of 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.

[0257] Embodiment 65. The method according to Embodiment 64, wherein the antibody does not contain an antibody-binding domain that recognizes CD47, PD-L1, LAG3, or CD16.

[0258] Embodiment 66. The method according to Embodiment 64 or 65, wherein the retinal disease is dry AMD.

[0259] For each of Embodiments 63-66, there are corresponding embodiments for a composition for treatment, a composition for manufacturing a medicament, use of the composition in treatment, and use of the composition in manufacturing a medicament.

[0260] Examples

[0261] Example 1. Anti-CD47 HCAb antibody

[0262] Anti-CD47 HCAb antibody was isolated from immunized llamas.

[0263] Immunization. Two llamas were immunized at Abcore Inc (Ramona, CA) according to its 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). Six subcutaneous injections of 50 μg / dose were administered to each llama at bi-weekly intervals. On day 45, serum was collected from llamas immunized with recombinant CD47 protein to determine the antibody titer against hCD47 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, horseradish peroxidase (HRP)-conjugated goat anti-camel antibodies were used.IgG (H+L) antibody (Invitrogen) confirmed the presence of anti-CD47 antibody.

[0264] SEQ ID NO:1 Extracellular domain of human CD47 (19-139, Q08722)

[0265] QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIE VSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSP

[0266] Construction and selection of phage library. Peripheral blood mononuclear cells were prepared from blood samples from llamas immunized with recombinant CD47 protein on day 45 using Ficoll-Paque Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi kit (Qiagen) according to the manufacturer's instructions and used as starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were cloned into a phage vector, thereby allowing the generation of recombinant phage particles after infection with helper phages, the recombinant phage particles displaying 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.

[0267] To select phages that bind CD47, biotinylated CD47 was incubated with a phage library and then 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).

[0268] Attum (Newark, CA) synthesized cDNA encoding CD47-binding VHH with a C-terminal His tag and transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0269] CD47-binding phage colonies from an immunized llama phage library were sequenced, and their amino acid sequences were determined for each VHH as listed below (Table 2). 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-CD47 HCAb antibodies. The expressed anti-CD47 HCAb was purified using a HiTrap protein A column.

[0270] A09-10VHH is a humanized sequence based on the IGHV3-23 human germline sequence.

[0271] Table 2. Llama anti-CD47 VHH sequence specification 28 / 94 pages 33 CN 121975011 A

[0272] Specification 29 / 94 pages 34 CN 121975011 A

[0273] Specification 30 / 94 pages 35 CN 121975011 A

[0274] Specification 31 / 94 pages 36 CN 121975011 A

[0275]

[0276] *Biv=bivalent

[0277] The VHH in Table 2 constitutes a means for binding CD47.

[0278] Octet® Binding Analysis of Anti-CD47 HCAb Molecules

[0279] 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. Affinity measurements were performed using an Octet® QKe equipped with an anti-Penta-His Capture (HIS1K) biosensor tip (FortéBio®). 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 the 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® data analysis software 7 (FortéBio®) throughout the experiment.

[0280] Table 3 lists the anti-CD47 Octet® kinetic analysis of binding affinity of HCAbs. HCAbs A09-04, A09-06, A09-08, and A09-10 exhibited pM binding affinity.

[0281] Table 3. Octet® kinetic analysis of binding affinity (KD) of anti-CD47 HCAbs

[0282]

[0283] Flow cytometry analysis of binding affinity of anti-CD47 HCAbs to CHO cell lines overexpressing CD47.

[0284] Ice-cold FACS buffer (PBS, 1%) was used to bind the HCAbs to CHO cell lines overexpressing CD47.CHO cells overexpressing CD47 at a concentration of 1×10⁶ cells / ml in BSA (0.1% NaN₃) were incubated with anti-CD47 HCAb at concentrations ranging from 100 nM to 0.00128 nM or as a control, with B6H12 anti-CD47 antibody, and incubated 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 Guava EasyCyte HT system.

[0285] The binding affinity of anti-CD47 HCAb was determined to be EC50 using the flow cytometry method described above, as depicted in Figure 1.

[0286] Competitive ELISA binding assay of multispecific molecules with anti-CD47 domain

[0287] Competitive ELISA binding assays were performed to screen for multispecific molecules 1511 (SEQ ID NO 32 / 94, page 37, CN 121975011 A 156; CD16F-L1-HSA-L1-CD47-L3-CD47-L1-PDL1-L3-PDL1) and 3321 (SEQ ID NO 159; CD16F-L1-HSA-L1-CD47-L1-CD33-L3-CD33), both of which contain anti-CD47 VHH A09-10, which competitively blocks the binding of the CD47 antigen to its receptor SIRPα. 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 ng / well of CD47-Fc (R&D systems) was coated in 96-well plates, and 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 binding to its receptor SIRPα at EC50, as depicted in Figure 2.

[0288] Competitive Flow Cytometry Binding Assay of Multispecific Molecules with Anti-CD47 Domain

[0289] Competitive flow cytometry binding assays were performed to confirm that multispecific molecules 1511 and 3321 blocked the binding of the CD47 antigen to its receptor SIRPα on the surface of cells naturally expressing CD47. Jurkat cells (ATCC) at 1×10⁶ cells / ml in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN₃) were incubated with 1511 or 3321 at concentrations ranging from 100 nM to 0.00128 nM and incubated on ice for 45 minutes.min, then add 25 nM SIRPα-Fc (R&D systems) and incubate for another 45 min. Wash cells with FACS buffer and add goat anti-human VHH FITC conjugate antibody (Jackson Immuno Research) according to the manufacturer's instructions, 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 binding to its receptor SIRPα on the surface of Jurkat cells at EC50, as depicted in Figure 3.

[0290] Human RBC agglutination assay with multispecific molecules containing anti-CD47 domains

[0291] Human blood samples were provided from healthy donors. Whole blood was centrifuged at 3000 rpm (1800 rcf) for 5 min and plasma and erythrocyte sedimentation rate (ESR) layer were removed. Red blood cells were resuspended in physiological saline (0.9% NaCl) at approximately twice the volume of red blood cells and the tubes were inverted to mix. Red blood cells were further centrifuged at 2000 rpm for 20 min, and 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 different amounts of antibody (0 ug / ml to 100 ug / ml). The plates were incubated at 37°C for 2 hours. Unlike the Hu5F9 anti-CD47 control antibody, the multispecific molecules 1511 and 3321 did not induce RBC aggregation, as depicted in Figure 4.

[0292] Flow cytometry binding assays were performed to confirm that the multispecific molecules, the anti-CD47 VHH containing 1511 and 3321, selectively bind to the surface of tumor cells that naturally express CD47, but do not bind to CD47 on the surface of RBC cells. HL60 cells (ATCC) at 1×10⁶ cells / ml or 10% washed human RBC cells (Rockland Immunochemicals, Inc.) in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN₃) 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 goat anti-human VHH FITC conjugate antibody (Jackson Immuno Research) was added according to the manufacturer's instructions, followed by incubation at 4°C for 30 min. Data were acquired using the Guava EasyCyte HT system. Multispecific molecules 1511 and 3321 selectively bound to the surface of tumor cells naturally expressing CD47 at EC50, but not to CD47 on the surface of RBC cells, as depicted in Figure 4B.

[0293] Antitumor activity of multispecific molecules with anti-CD47 domains

[0294] 1E6 Raji-Luc cells were intravenously inoculated into NSG mice. Mice were treated daily with intravenous injection of 10 mg / kg of multispecific molecule 3321 or PBS control. Representative bioluminescent images of Raji tumors at the start of treatment (day 0), mid-experiment day 3, and end of experiment (day 7). Multispecific molecule 3321 protects xenograft mice from developing human leukemia, as depicted in Figure 5.

[0295] Example 2. Anti-PD-L1 HCAb antibody

[0296] Anti-PD-L1 HCAb antibody isolated from immunized camels

[0297] Two llamas were immunized at Abcore Inc. according to their standard protocol. Recombinant human PD-L1 (extracellular datasheet, page 33 / 94, CN 121975011 A, domains 19 to 238, SEQ ID NO:30) was mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (post-immunization). Each llama was administered six subcutaneous injections at a time interval of 50 μg / dose, every two weeks. On day 45, serum was collected from llamas immunized with recombinant PD-L1 protein to determine the anti-PD-L1 antibody 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.

[0298] SEQ ID NO:30 Extracellular domains of human PD-L1 (19-238, Q9NZQ7)

[0299] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARL LKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAE VIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0300] Peripheral blood mononuclear cells were prepared from blood samples from llamas immunized with recombinant PD-L1 protein on day 45 using Ficoll-Paque+, according to the manufacturer's instructions for use. Total RNA was extracted from peripheral blood mononuclear cells using the RNeasy Midi kit, following the manufacturer's instructions, and used as starting material for RT-PCR to amplify the gene fragment encoding VHH. These fragments were then cloned into a phage vector, allowing for the generation of recombinant phage particles after infection with helper phages.The recombinant phage particles 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.

[0301] To select VHHs that bind PD-L1, biotinylated PD-L1 was incubated with a phage library and then 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.

[0302] The cDNA encoding VHHs that bind PD-L1 was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells, and positive VHHs were purified by IMAC chromatography.

[0303] PD-L1-bound phage colonies from an immunized llama phage library were sequenced. The amino acid sequence of each VHH is listed below (Table 4). The 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 produce recombinant anti-PD-L1 HCAb antibody. The expressed anti-PD-L1 HCAb was purified by HiTrap protein A column. Two of the llama VHH, PL14 and PL16 were humanized based on the IGHV3-23 human germline sequence.

[0304] Table 4. Llama anti-PD-L1 VHH sequence specification 34 / 94 pages 39 CN 121975011 A

[0305]

[0306] The VHHs in Table 4 constitute the means for binding PD-L1.

[0307] Octet® kinetic binding analysis

[0308] Octet® kinetic binding analysis was performed as in Example 1. In short, the binding affinity 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.

[0309] Table 5 presents the Octet® kinetic analysis of binding affinity for anti-CD47 HCAb. The analysis showed that PL14, PL16, and PL17 exhibited pM binding affinity.

[0310] Table 5. Binding kinetics (KD) of anti-PD-L1 HCAb Specification 35 / 94 pages 40 CN 121975011 A

[0311]

[0312] Flow Cytometry Binding Analysis of Anti-PD-L1 HCAb

[0313] PD-L1-overexpressing CHO cells at 1×10⁶ cells / ml in ice-cold FACS buffer (PBS, 1% BSA, 0.1% NaN₃) were incubated with anti-PD-L1 HCAb at concentrations ranging from 100 nM to 0.00128 nM for 45 min on ice. Cells were washed with FACS buffer and then incubated with goat anti-human IgG Fc-FITC conjugate antibody (ThermoFisher) at 4°C for 30 min. Data were acquired using a Guava EasyCyte HT system. The binding affinity of anti-PD-L1 HCAb to PD-L1 on PD-L1-overexpressing CHO cells was determined as EC50, as depicted in Figure 6.

[0314] Cell-based functional assay of a multispecific molecule with a PD-L1 binding domain

[0315] PD-L1-expressing APC / CHO-K1 cells were seeded at 100 K / well in 96-well plates and incubated at 37°C for 16 h. 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 h. After 6 h, 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.

[0316] Cell-based functional data showed that multispecific molecule 1511 and the control antibody atezolizumab completely blocked PD-L1 activity with similar EC50 values, as shown in Figure 7.

[0317] Antitumor activity of a multispecific molecule with a PD-L1 binding domain

[0318] Rat colon cancer MC38-hPD-L1 cells (Biocytogen Co., Ltd; 5×10⁵) were subcutaneously implanted into homozygous B-hPD-L1 mice (female, 6 weeks old, n=6). When the tumor volume reached approximately 100 mm³, the mice were grouped and treated with multispecific molecule 1518 (SEQ ID NO: 157; CD16F-L1-HSA-L1-CD47-L3-CD47-L1-PDL1-L3-PDL1) at the doses and regimens indicated in Figure 8A. Changes in body weight during treatment are shown in Figure 8B. As shown in Figure 8A, multispecific molecule 1518A1 is effective in controlling tumor growth in B-hPD-L1 mice. Values ​​are expressed as mean ± SEM.

[0319] Example 3. Anti-HSA HCAb antibody

[0320] Anti-HSAIsolation of VHH antibodies

[0321] As described in Example 1, llamas were immunized at Abcore, Inc. with recombinant human HSA (SEQ ID NO:39) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization). Human Serum Albumin (SEQ ID NO:39) MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNE VTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDV MCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLK CASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLK ECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHPDYSVVLLLRLAKTY ETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNL Instructions for Use 36 / 94 pages 41 CN 121975011 A GKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETF TFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALG L

[0324] To select for anti-HSA VHH, biotinylated HSA was incubated with a phage library and then captured on streptavidin Dynabeads (Invitrogen). 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. (San Diego, California).

[0325] The cDNA encoding HSA-specific VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. Positive VHHs were purified by IMAC chromatography.

[0326] HSA-binding phage clones from a llama phage library were sequenced, and the amino acid sequence of each VHH is listed below (Table 6). The cDNA sequence based on the following amino acid sequence was synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate a recombinant single-domain antibody (sdAb) with a C-terminal his tag. The expressed sdAb was purified by HisTrap HP column.

[0327] Two of the llama VHH, HS5, and HS10 were humanized based on the IGHV3-23 human germline sequence.

[0328] Table 6. Llama anti-HSA VHH sequence specification 37 / 94 pages 42 CN 121975011 A

[0329]

[0330] These VHHs constitute a means for binding HSA.

[0331] Octet® kinetic binding analysis

[0332] Octet® kinetic binding analysis was performed as in Example 1, and the results are presented in Table 7 and Figure 9. Clones HS5, HS6, HS12, and HS27 showed affinity for HSA. Cross-species activity was confirmed and is listed in Table 8.

[0333] Table 7. Binding affinity (KD) of anti-HSA VHH antibodies Specification 38 / 94 pages 43 CN 121975011 A

[0334]

[0335] Table 8. Cross-species binding affinity of HS10 and HS5 VHH

[0336]

[0337] Example 4. Anti-CD33 HCAb antibody

[0338] Isolation of anti-CD33 VHH antibody

[0339] At Abcore, Inc., llamas were immunized with recombinant human CD33 (SEQ ID NO:49) mixed with complete Freund's adjuvant (day 0) or incomplete Freund's adjuvant (after immunization) and a phage library prepared as in Example 1.

[0340] Human CD33 (SEQ ID NO:49, P20138|18-259)

[0341] DPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISRDSPVATNKLDQEVQEET QGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTC

[0342] 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 outputs were rescued into E. coli TG1 cells. Colonies were picked and sequenced at BATJ, Inc.

[0343] The cDNA encoding CD33-bound VHH was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells, and positive VHH was purified by IMAC chromatography.

[0344] 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 a human Fc and synthesized in the pJ607 expression vector. The expression plasmid was transfected into the HEK293 cell line to generate a recombinant anti-CD33 HCAb antibody. The expressed anti-CD33 HCAb was purified using a HiTrap protein A column.

[0345] One of the llama VHHs, 33-14, was humanized based on the IGHV3-23 human germline sequence.

[0346] Table 9. Llama anti-CD33 VHH sequence specification 39 / 94 pages 44 CN 121975011 A

[0347] Specification 40 / 94 pages 45 CN 121975011 A

[0348] Specification 41 / 94 pages 46 CN 121975011 A

[0349]

[0350] VHH in Table 9 constitutes the means for binding CD33.

[0351] Octet® kinetic binding analysis was performed as in Example 1, and the KD results are presented in Figure 10 and Table 10.

[0352] Table 10. Binding affinity of anti-CD33 VHH antibody

[0353]

[0354] Example 5. Anti-LAG3 VHH

[0355] Isolation of anti-LAG3 VHH antibody

[0356] 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 treated with 50 [units of] CN 121975011 A (page 42 / 94 of the instruction manual).μg / dose was administered subcutaneously six times at two-week 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. Alternatively, 96-well Maxisorp plates were 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. Extracellular domain of human LAG3 (SEQ ID NO:79, P18627, 23-450) RYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTAS PPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFNV SIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQA GTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQL LSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHL

[0359] Phage libraries were prepared as described in Examples 1 to 4. cDNA encoding LAG3-binding VHHs was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells. The LAG3-binding VHHs were then purified by IMAC chromatography.

[0360] 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). 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-LAG3 HCAb antibody. The expressed anti-LAG3 HCAb was purified using a HiTrap protein A column.

[0361] One of the llama VHH and LG9 was humanized based on the IGHV3-23 human germline sequence.

[0362] Table 11. Llama anti-LAG3 VHH sequence specification 43 / 94 pages 48 CN 121975011 A

[0363] Specification 44 / 94 pages 49 CN 121975011 A

[0364]

[0365] The VHH in Table 11 constitutes a means for binding LAG3.

[0366] Octet® binding analysis of anti-LAG3 VHH was performed as in Examples 1 to 4, and the results are shown in Table 12.

[0367] Table 12. Binding affinity (KD) of anti-LAG3 single-domain antibodies

[0368]

[0369] Example 6. Anti-CD16 VHH

[0370] Isolation of anti-CD16 VHH antibodies

[0371] 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 given 50 μg / dose, administered subcutaneously six times at intervals of two weeks. 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. Human CD16A (SEQ ID NO:94) MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISS QASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGK GRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDT GLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0374] Human CD16B (SEQ ID NO:95)

[0375] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYSVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVNDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTALHKVTYLQNGK DRKYFHHNSDFHIPKATLKDSGSYFCRGLVGSKNVSSETVNITITQGLAVSTISSFSPPGYQVSFCLVMVLLFAVDT Instruction Manual 45 / 94 pages 50 CN 121975011 A GLYFSVKTNI

[0376] Phage libraries were prepared as described in Example 1. The cDNA encoding CD16A-binding VHHs was synthesized with a C-terminal His tag and transiently transfected into HEK293 cells, and the CD16A-binding VHHs were purified by IMAC chromatography.

[0377] CD16A-binding phage colonies from immunized llama phage libraries were sequenced, and the amino acid sequence of each VHH is listed below (Table 13). The 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 by HiTrap protein A column.

[0378] One of the llama VHH and CD16F1 was humanized based on the IGHV3-23 human germline sequence.

[0379] Table 13. Anti-CD16 VHH sequence

[0380]

[0381] The VHH in Table 13 constitutes the means for binding CD16.

[0382] Octet® binding analysis of anti-CD16 VHH was performed as in Example 1, and the results are shown in Table 14 and Figure 11.

[0383] Table 14. Binding Affinity (KD) of Anti-CD16 VHH Antibodies

[0384]

[0385] CD16-F1 is selective for CD16A, while CD16-E11 binds to both CD16A and CD16B.

[0386] Both CD16F1 and CD16E11 are agonist anti-CD16 VHH antibodies that activate CD16A in the Jurkat-Lucia NFAT-CD16 ADCC Reporting Assay (Invivogen).

[0387] 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 are shown in Figure 12. The data indicate that CD16F1 is an effective CD16A agonist.

[0388] Example 7. Trispecific Single-Chain Antibody (HSA / CD47 / PD-L1 or HSA / LAG3 / PD-L1)

[0389] To construct a trispecific single-chain antibody, anti-HSA, anti-CD47, and anti-PD-L1 or anti-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 (46 / 94 pages, 51 CN 121975011 A), 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 may be further characterized as cleavable or non-cleavable. The amino acid sequences of exemplary trispecific molecules are presented in Table 16. The linker sequences in Tables 16 and 17 are underlined.

[0390] Table 15. Indivisible and divisible connector sequences

[0391]

[0392] Table 16. Trispecific molecules Specification 47 / 94 pages 52 CN 121975011 A

[0393] Specification 48 / 94 pages 53 CN 121975011 A

[0394] Specification 49 / 94 pages 54 CN 121975011 A

[0395] Specification 50 / 94 pages 55 CN 121975011 A

[0396] Specification 51 / 94 pages 56 CN 121975011 A

[0397] Specification 52 / 94 pages 57 CN 121975011 A

[0398] Specification 53 / 94 pages 58 CN 121975011 A

[0399] Specification 54 / 94 pages 59 CN 121975011 A

[0400] Specification 55 / 94 pages 60 CN 121975011 A

[0401] Specification 56 / 94 pages 61 CN 121975011 A

[0402] Specification 57 / 94 pages 62 CN 121975011 A

[0403]

[0404] 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 when MVSCA is located in its intended action...The target site (e.g., tumor) is undesirable. 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, the MVSCA can act as a prodrug. One such 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 to link LAG3 VHH with PD-L1, as shown in Figure 13 and Table 9. Figure 14B shows the SDS-page analysis of the antibody of Figure 14A after protease digestion.

[0405] Protease assay

[0406] (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 hours. The activated rhMMP-9 was titrated with an equal volume of 20 µM antibody in assay buffer and incubated at room temperature for 1 hour. The digested substrate was analyzed by SDS-PAGE.

[0407] (2) u-plasminogen activator (uPA, urokinase) activity assay. The substrate was diluted to 200 µM in assay buffer (50 mM Tris, 0.01% Tween 20, pH 8.5) and titrated with an equal volume of recombinant human u-plasminogen activator (rhuPA, R&D Systems) in assay buffer. The reaction mixture was incubated at room temperature for 1–2 hours and the digested substrate was analyzed by SDS-PAGE.

[0408] (3) Assay for mastropeptidase 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.

[0409] Polyacrylamide gel electrophoresis (SDS-PAGE). Denaturing SDS-PAGE was performed according to the Invitrogen NuPAGE® instructions. In short, mix 7.5 μL of protein sample (3 μg protein) with 2.5 μL of 4X LDS sample loading buffer (Invitrogen) and heat at 70°C for 10 min. Then load the sample onto a pre-prepared NuPAGE Novex container.4–12% Bis-Tris in 1.0 mm microgels (Invitrogen). Then, 5 μL of pre-stained SDS-PAGE standards (Bio-Rad) were loaded in each gel run. Electrophoresis was performed at room temperature in 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).

[0410] Figure 15 illustrates the real-time kinetic binding analysis of PD-L1 / pro-CD47 (HSA-CD47-PD-L1 antibody) versus PD-L1 / active CD47 (the same antibody with the 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 showed strong binding to CD47. In this assay, there was no difference in PD-L1 binding or no effect on PD-L1 binding.

[0411] Example 8. Multispecific molecules containing CD16A / HSA / CD47 / (PD-L1 or CD33)

[0412] 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 depicts the structure of exemplary multispecific molecules containing anti-CD16A, anti-HSA, anti-CD47, and anti-PD-L1 or CD33 VHH. The amino acid sequences of exemplary multispecific molecules are listed below (Table 17)

[0413] Figure 17 compares the length of the linker G4SG3S (L1, SEQ ID NO:100) between VHH2 and VHH3 in a flow cytometry binding assay on HL60 cells. 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) compared to G4SG3S (9 amino acids) improved CD47 binding, with an EC50 of 8.4 nM compared to 26 nM.

[0414] Table 17. Multispecific molecule specification 59 / 94 pages 64 CN 121975011 A

[0415] Specification60 / 94 pages, 65 CN 121975011 A

[0416] Specification 61 / 94 pages, 66 CN 121975011 A

[0417] Specification 62 / 94 pages, 67 CN 121975011 A

[0418] Specification 63 / 94 pages, 68 CN 121975011 A

[0419] Specification 64 / 94 pages, 69 CN 121975011 A

[0420] Specification 65 / 94 pages, 70 CN 121975011 A

[0421] Specification 66 / 94 pages, 71 CN 121975011 A

[0422] Specification 67 / 94 pages, 72 CN 121975011 A

[0423] Specification 68 / 94 pages, 73 CN 121975011 A

[0424] Specification page 69 / 94, page 74, CN 121975011 A

[0425] Specification page 70 / 94, page 75, CN 121975011 A

[0426] Specification page 71 / 94, page 76, CN 121975011 A

[0427] Specification page 72 / 94, page 77, CN 121975011 A

[0428] Specification page 73 / 94, page 78, CN 121975011 A

[0429] Specification page 74 / 94, page 79, CN 121975011 A

[0430] Specification page 75 / 94, page 80, CN 121975011 A

[0431] Specification page 76 / 94, page 81, CN 121975011 A

[0432] Specification page 77 / 94, page 82, CN 121975011 A

[0433] Specification page 78 / 94, page 83 CN 121975011 A

[0434] Specification page 79 / 94, page 84 CN 121975011 A

[0435] Specification page 80 / 94, page 85 CN 121975011 A

[0436] Specification page 81 / 94, page 86 CN 121975011 A

[0437] Specification page 82 / 94, page 87 CN 121975011 A

[0438] Specification page 83 / 94, page 88 CN 121975011 A

[0439] Specification page 84 / 94, page 89 CN 121975011 A

[0440] Specification page 85 / 94, page 90 CN 121975011 A

[0441] Specification 86 / 94 pages 91 CN 121975011 A

[0442] Specification 87 / 94 pages 92 CN 121975011 A

[0443] Specification 88 / 94 pages 93 CN 121975011 A

[0444] Specification 89 / 94 pages 94 CN 121975011 A

[0445] Specification 90 / 94 pages 95 CN 121975011 A

[0446]

[0447] Octet® binding analysis of multispecific molecules was performed as in Example 8, and the results are shown in Figures 17 and 18.

[0448] Example 9. MVSCA containing anti-CD33 domains for the treatment of Alzheimer's disease and retinal diseases

[0449] Table 18. MVSCA containing anti-CD33 VHH* Specification 91 / 94 pages 96 CN 121975011 A

[0450]

[0451] *The connector sequence interposed between the VHH domains is underlined

[0452] **The sequence is shown with an optional C-terminal his tag

[0453] Each of the four MVSCAs in Table 18 contains a pair of anti-CD33 domains that are joined by the connector 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 the in vivo half-life Specification 92 / 94 pages 97 CN 121975011 A 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 its half-life in vivo. 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 smaller size makes it more suitable for local injection into the brain or eye.

[0454] The amino acid sequence of the FC5 nanobody domain is:

[0455] EVQLQASGGGLVQAGGSLRLSCAASGFKITHYTMGWFRQAPGKEREFVSRITWGGDNTFYSNSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAGSTSTATPLRVDYWGKGTQVTVSS (SEQ ID NO:222).

[0456] Example 10. Multiple sequence alignment

[0457] Figures 21 to 26 present multiple sequence alignments of the VHH sequences disclosed herein performed by Clustal O (1.2.4) for each specificity, making it easy to see the same, conserved and highly variable positions. Each position in the alignment is indicated by a symbol: an asterisk indicates identity, a colon indicates a higher degree of conservation, a period indicates a lower degree of conservation, and a space indicates a general lack of conservation in the aligned sequence.

[0458] 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 are to 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 approximations, which may vary according to the desired properties sought to be obtained according to the invention. In any case, not in an attempt to limit the application of the principle 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 ordinary rounding techniques. Although the numerical ranges and parameters that set forth the broad scope of the invention are approximations, the numerical 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 their respective test measurements.

[0459] Unless otherwise indicated herein or obviously contradicted by the context, the terms “a,” “an,” “the,” and similar indicators used in the context of describing the invention (especially in the context of the following claims) should be interpreted as covering both the singular and the plural. The description of the range of values ​​herein is intended only as a shorthand method of referring to each individual value falling within the range. Unless otherwise stated herein, each individual value is incorporated into the specification as if it were separately referenced herein. Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all 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.

[0460] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each group member may...It is mentioned and claimed individually or in any combination with other members of the group or other elements found herein. It is contemplated that one or more members of the 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 contain the modified group, thereby satisfying the written description of all Markush groups as used in the appended claims.

[0461] Certain embodiments of the invention have been described herein, 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 intend that those skilled in the art appropriately employ such variations, and the inventors intend that the invention be practiced in a manner different from the specific description 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.

[0462] The specific embodiments disclosed herein may be further limited by the use of the language "consisting of" or "consisting substantially of". When used in the claims, whether in the original filing or as amended, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transitional term "consisting substantially of" limits the scope of the claims to the specified materials or steps, and those materials or steps that do not substantially affect the basic and novel features. Embodiments of the invention thus claimed are inherently or explicitly described and practiced herein.

[0463] 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.

[0464] 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 employed within the scope of the invention. Therefore, alternative configurations of the invention may be used in accordance with the teachings herein, by way of example rather than limitation. Therefore, the invention is not limited to what is precisely shown and described. Instruction manual 94 / 94 pages 99 CN 121975011 A Figure 1 Instruction manual Figure 1 / 31 pages 100 CN 121975011 A Figure 2 Instruction manual Figure 2 / 31 pages 101 CN 121975011 A Figure 3 Instruction manual Figure 3 / 31 pages 102 CN 121975011 A Figure 4A Instruction manual Figure 4 / 31 pages 103 CN121975011 A Figure 4B Instruction Manual Appendix 5 / 31 Page 104 CN 121975011 A Figure 5 Instruction Manual Appendix 6 / 31 Page 105 CN 121975011 A Figure 6 Instruction Manual Appendix 7 / 31 Page 106 CN 121975011 A Figure 7 Instruction Manual Appendix 8 / 31 Page 107 CN 121975011 A Figure 8A Instruction Manual Appendix 9 / 31 Page 108 CN 121975011 A Figure 8B Instruction Manual Appendix 10 / 31 Page 109 CN 121975011 A Figure 9 Instruction Manual Appendix 11 / 31 Page 110 CN 121975011 A Figure 10A Instruction Manual Appendix 12 / 31 Page 111 CN 121975011 A Figure 10B Instruction Manual Appendix 13 / 31 Page 112 CN 121975011 Figure 11A, Instruction Manual Appendix, Page 14 / 31, CN 121975011; Figure 11B, Instruction Manual Appendix, Page 15 / 31, CN 121975011; Figure 12, Instruction Manual Appendix, Page 16 / 31, CN 121975011; Figure 13A, Instruction Manual Appendix, Page 17 / 31, CN 121975011; Figure 13B, Instruction Manual Appendix, Page 18 / 31, CN 121975011; Figure 13C, Instruction Manual Appendix, Page 19 / 31, CN 121975011; Figure 14A, Figure 14B, Instruction Manual Appendix, Page 20 / 31, CN 121975011; Figure 15, Instruction Manual Appendix, Page 21 / 31, CN 120; Figure 16, Instruction Manual Appendix, Page 22 / 31, CN 121 121975011 A Figure 17 Instruction Manual Appendix 23 / 31 Page 122 CN 121975011 A Figure 18 Instruction Manual Appendix 24 / 31 Page 123 CN 121975011 A Figure 19 Instruction Manual Appendix 25 / 31 Page 124 CN 121975011 A Figure 20 Instruction Manual Appendix 26 / 31 Page 125 CN 121975011 A Figure 21 Figure 22 Instruction Manual Appendix 27 / 31 Page 126 CN 121975011 A Figure 23 Instruction Manual Appendix 28 / 31 Page 127 CN121975011 A Figure 24 Appendix to the Specification Page 29 / 31 128 CN 121975011 A Figure 24 (continued) Appendix to the Specification Page 30 / 31 129 CN 121975011 A Figure 25 Figure 26 Appendix to the Specification Page 31 / 31 130 CN 121975011 A Abstract Disclosed herein are monospecific and multi-specific single chain antibodies with specificities for one or more of CD47, PD-L1, HSA, CD33, LAG3, and CD16.

Claims

1. A heavy chain variable (VHH) domain having antigen-binding specificity for one of the following: (a) CD16, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) PD-L1, wherein the VHH region has the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) Human serum albumin (HSA), wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d)CD33, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) LAG3, wherein the VHH domain has an amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; or (f)CD47, 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.

2. A heavy chain-only antibody (HCAb), said heavy chain-only antibody comprising the VHH domain of claim 1.

3. A multispecific antibody, said 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 to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) The HSA binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; and (f) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO:

223.

4. The multispecific antibody according to claim 3, wherein the second antibody-binding domain is specific to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) The HSA binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; and (f) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO:

223.

5. The multispecific antibody according to claim 3, wherein the multispecific antibody further comprises 1 to 5 additional antibody-binding domains, wherein each additional antibody-binding domain is individually specific to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) The HSA binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; and (f) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO:

223.

6. The multispecific antibody according to claim 4, wherein the multispecific antibody further comprises 1 to 4 additional antibody-binding domains, wherein each additional antibody-binding domain is specific to CD47, PD-L1, HSA, CD33, LAG3, or CD16; wherein (a) The CD16 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 96 to SEQ ID NO: 99; (b) The PD-L1 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 38, SEQ ID NO: 37, SEQ ID NO: 31 to SEQ ID NO: 36; (c) The HSA binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 40 to SEQ ID NO: 48; (d) The CD33 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 50 to SEQ ID NO: 78; (e) The LAG3 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 80 to SEQ ID NO: 93; and (f) The CD47 binding specificity is represented by the amino acid sequence shown in one of SEQ ID NO: 2 to SEQ ID NO: 29 or SEQ ID NO:

223.

7. The multispecific antibody according to any one of claims 3-6, wherein the antibody is a multispecific single-chain antibody (MVSCA).

8. The multispecific antibody according to any one of claims 3-7, wherein the linkers L1 (SEQ ID NO: 100), L2 (SEQ ID NO: 101) or L4 (SEQ ID NO: 103) are interposed between one or more pairs of different antibody-binding domains.

9. The multispecific antibody according to any one of claims 3-8, wherein the multispecific antibody comprises at least one pair of antibody-binding domains having the same specificity.

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

11. The multispecific antibody according to any one of claims 3-10, wherein the multispecific antibody comprises an HSA-specific antibody-binding domain located at the N-terminus or C-terminus, and a cleavable linker is inserted between the antibody-binding domain and an adjacent antibody-binding domain.

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

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

14. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes HSA, CD47 and PD-L1.

15. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes HSA, CD47 and CD33.

16. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes HSA, LAG3 and PD-L1.

17. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes HSA, LAG3 and CD33.

18. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes CD16, HSA and PD-L1.

19. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes CD16, HSA and CD33.

20. The multispecific antibody according to any one of claims 3-13, comprising CD16, HSA, CD47, and PD-L1.

21. The multispecific antibody according to any one of claims 3-13, wherein the multispecific antibody comprises an antibody-binding domain that recognizes CD16, HSA, CD47 and CD33.

22. A multispecific antibody comprising a pair of antibody-binding domains having CD33 specificity.

23. The multispecific antibody according to claim 22, wherein the multispecific antibody further comprises an antibody-binding domain specific to HSA, FC5 nanobody (SEQ ID NO: 222) or both.

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

25. A method of treating cancer, the method comprising administering the pharmaceutical composition according to claim 24 to a patient in need.

26. A pharmaceutical composition comprising a multispecific antibody according to any one of claims 22-23.

27. A method of treating Alzheimer's disease or retinal disease, the method comprising administering the pharmaceutical composition of claim 24 to a patient in need.

28. The method of claim 27, wherein the retinal disease is dry age-related macular degeneration.

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