Multispecific cancer targeting antibodies

Abstract

Disclosed herein are multivalent, multispecific single-chain antibodies having specificity for four antigens selected from the group consisting of EGFR, PD-L1, HSA, CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 596,555, filed November 6, 2023, and U.S. Provisional Patent Application No. 63 / 601,931, filed November 22, 2023, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list named 1959708-00018_SequenceListing.xml, which is 185KB in size and was created on November 5, 2024. The entire contents of this sequence list are incorporated herein by reference.

[0005] Summarize

[0006] This article discloses multivalent single-chain antibodies (MVSCAs) containing four or more VHH domains, each VHH domain being specific for one or more antigens. MVSCAs contain at least four binding specificities and have the following structures:

[0007] A—B—C—D

[0008] A, B, C, and D each contain a VHH domain specifically targeting EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E, respectively, and the amino acid sequences of the VHH domains of A, B, C, and D are linked by amino acid linker sequences.

[0009] In some implementations, A, B, and D each contain a VHH domain specifically targeting EGFR, PD-L1, or HSA, and C contains a VHH domain specifically targeting 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E.

[0010] In some embodiments, the VHH domain specifically targeting EGFR comprises an amino acid sequence selected from SEQ ID NO: 134-161. In some embodiments, the VHH domain specifically targeting PD-L1 comprises an amino acid sequence selected from SEQ ID NO: 66-74. In some embodiments, the VHH domain specifically targeting HSA comprises an amino acid sequence selected from SEQ ID NO: 76-92 and 188. In some embodiments, the VHH domain specifically targeting 4-1BB comprises an amino acid sequence selected from SEQ ID NO: 120-132. In some embodiments, the VHH domain specifically targeting OX40 comprises an amino acid sequence selected from SEQ ID NO: 101 or 102. In some embodiments, the VHH domain specifically targeting CD40 comprises an amino acid sequence selected from SEQ ID NO: 104-118. In some embodiments, the VHH domain specifically targeting CD16A comprises an amino acid sequence selected from SEQ ID NO: 94-99. In some embodiments, the VHH domain specifically targeting CD3E comprises an amino acid sequence selected from SEQ ID NO: 47-64. In some embodiments, the VHH domain specifically targeting CD28 comprises an amino acid sequence selected from SEQ ID NO: 25-45. In some embodiments, the VHH domain specifically targeting LAG3 comprises an amino acid sequence selected from SEQ ID NO: 174-187.

[0011] In some embodiments, the order of the A, B, C, and D domains is ABCD. In some embodiments, the linker comprises an amino acid sequence of one of SEQ ID NO: 1-23.

[0012] In some embodiments, a domain comprises two VHH sequences specifically targeting the same target. In some embodiments, a domain is a C domain. In some embodiments, the two VHH sequences specifically target the same epitope on the target. In some embodiments, the two VHH sequences have the same amino acid sequence. In some embodiments, the two VHH sequences have different amino acid sequences. In some embodiments, the two VHH sequences are linked by a linker of one of SEQ ID NO: 1-23.

[0013] In some embodiments, the MVSCA contains specificity in the following order: EGFR—PD-L1—4-1BB—4-1BB—HSA. In some embodiments, the MVSCA contains the amino acid sequence of SEQ ID NO: 165.

[0014] In some embodiments, the MVSCA contains specificity in the following order: EGFR—PD-L1—CD28—HSA. In some embodiments, the MVSCA contains an amino acid sequence of one of SEQ ID NO: 167-172.

[0015] In some embodiments, the MVSCA contains specificity in the following order: EGFR—PD-L1—CD40—HSA. In some embodiments, the MVSCA contains the amino acid sequence of SEQ ID NO: 162.

[0016] In some embodiments, the MVSCA contains specificity in the following order: EGFR—PD-L1—HSA—CD40. In some embodiments, the MVSCA contains the amino acid sequence of SEQ ID NO: 163.

[0017] In some embodiments, the MSCA contains specificity in the following order: CD16A—HSA—CD47—CD33. In some embodiments, the MSCA contains the amino acid sequence of SEQ ID NO: 164.

[0018] This article discloses pharmaceutical compositions comprising the MVSCA disclosed herein.

[0019] This article also discloses methods for treating cancer, which include administering the disclosed MVSCA or pharmaceutical composition to subjects in need of such treatment.

[0020] This article also discloses the use of the MVSCA or pharmaceutical composition disclosed herein for the treatment of cancer in subjects with such need. Attached Figure Description

[0021] Figure 1 The structures of the SM2272 and SM2272A multispecific single-chain antibodies were depicted. The SM2272A sequence has a mutation in the anti-EGFRVHH complementarity-determining region (CDR3) binding domain, resulting in a reduced binding affinity to human cancer cells compared to SM2272.

[0022] Figure 2 The mechanism of action of the SM2272 multispecific single-chain antibody was described.

[0023] Figure 3 The binding of SM2272 to normal cells was compared with that to cancer cells.

[0024] Figure 4A -D describes SM2272 and EGFR ( Figure 4A ), PD-L1 ( Figure 4B ), 4-1BB ( Figure 4C) and HSA Figure 4D ) combined with ELISA.

[0025] Figure 5A -C describes the blocking effect of SM2272 on EGF and EGFR ( Figure 5A ), PD-1 and PD-L1 ( Figure 5B ) and 4-1BB and 4-1BBL ( Figure 5C ELISA blocking assay for binding.

[0026] Figure 6A -C describes the SM2272 and 2272A with A431 ( Figure 6A ), MB231 Figure 6B ) and HCC827 ( Figure 6C Flow cytometry analysis of cell binding. SM2272 binds to EGFR+PD-L1+ dual-expressing cells with enhanced affinity and specificity.

[0027] Figure 7A -C describes SM2272 binding with enhanced affinity to cancer cells that simultaneously express EGFR and PD-L1. Figure 7A -A431 cells; Figure 7B -MB231 cells; Figure 7C -HCC827 cells.

[0028] Figure 8A -D describes SM2272 and A431 ( Figure 8A HCC827 Figure 8B ) and MB231 ( Figure 9C Assay for blocking PD-1 binding on cells. Figure 8D The SM2272, SM2272A, and Keytruda are depicted. ® The luciferase assay showed that antibody binding with enhanced affinity enabled SM2272 to selectively block the interaction between PD-L1 and PD-1 on the surface of EGFR+PD-L1+ double-positive cancer cells, thereby allowing local (re)activation of tumor-resident cytotoxic T cells without systemic immune cell activation.

[0029] Figure 9A -C describes the SM2272 and SM2272A with A431 ( Figure 9A ), MB231 Figure 9B ) and HCC827 ( Figure 9C ) Reporter gene assay for cell binding. Effective activation of 4-1BB via SM2272 requires binding to two tumor antigens.

[0030] Figure 10This study describes the inhibition of CT26-hEGFR / hPDL1 tumor growth by SM2272 in the Balb / c-hPD-1-hPD-L1-h4-1BB mouse model.

[0031] Figure 11 The pharmacokinetic analysis of SM2272 in mice following a single 3 mg / kg dose was described.

[0032] Figure 12 The structures of the SM2275-700 and SM2275-649 multispecific antibodies were depicted.

[0033] Figure 13 Flow cytometry analysis of Jurkat cells by SM2275-649 and SM2275-700 was described.

[0034] Figure 14A -G depicts SM2275 cells and cells expressing CHO-hEGFR-hPD-L1 ( Figure 14A ), MB231 Figure 14B A431 Figure 14C H292 Figure 14D ) and cells expressing CHO-hCD28 ( Figure 14E Jurkat cells () Figure 14F ) and human T cells ( Figure 14G Combined with flow cytometry analysis.

[0035] Figure 15 The blocking assays of SM2275 and SM2275A on PD-1 and PD-L1 binding on A431 cells were depicted. Enhanced antibody binding enabled SM2275 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells compared to single-target binding. The mutant SM2275 (SM2275A), lacking EGFR binding ability, showed reduced PD1 / PDL1 blocking ability on A431 cancer cells compared to SM2275.

[0036] Figure 16 This study describes the enhanced inhibition of PD-L1 activity in EGFR+PD-L1+ double-positive cells compared to single-target binding. The increased affinity resulted in enhanced PD-L1 signaling blocking efficacy in CHO-Dual-OKT3 cells compared to CHO-hPD-L1-OKT3 cells.

[0037] Figure 17 Cell-based functional assays of SM2275 cells using Jurkat-PD1-NFAT luciferase reporter cells were depicted.

[0038] Figure 18A-D depicts the cytokine release from isolated human PBMCs induced by SM2275 and TGN1412. Figure 18A -TNF-α; Figure 18B -IL-6; Figure 18C -IL-2; Figure 18D -INF-γ.

[0039] Figure 19A -D depicts the release of cytokines in humanized hCD28 / hPD-L1 mouse model cells induced by SM2275 and TGN1412. Figure 19A -IL-2; Figure 19B -IL-6; Figure 19C -TNF-α; Figure 19D -INF-γ.

[0040] Figure 20 This study describes the inhibition of SM2275 on the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted. When the tumor size reached approximately 80 mm³, animals were randomly assigned to groups and treated with either the medium or SM2275 every 2 days for 22 days (N=6).

[0041] Figure 21 This study describes the inhibition of SM2275 on the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 / hPD-L1 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted. When the tumor size reached approximately 90 mm³, animals were randomly assigned to groups and treated with either the medium or SM2275 every 2 days for 18 days (N=5).

[0042] Figure 22 illustrates the pharmacokinetics of SM2275 in cynomolgus monkeys following multiple doses administered via IV. Invention Details

[0044] This article discloses multivalent and multispecific single-chain antibodies (MVSCAs) containing four or more variable domains of VHH, wherein the antibody has the following structure:

[0045] A—B—C—D

[0046] In this context, A, B, C, and D are VHH domains specifically targeting EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E, respectively; and the amino acid sequences of the VHH domains A, B, C, and D are linked by amino acid linker sequences. VHH domains A, B, C, and D, as well as multiple copies of any one of these domains, are individually linked by cleavable or non-cleavable linkers. Exemplary, non-limiting linkers are disclosed in Table 1.

[0047] As used herein, the term VHH refers to the variable domain of a heavy chain antibody and is the antigen-binding fragment of a heavy chain antibody only.

[0048] As used herein, the term "immune cell connector" refers to a molecularly specific VHH domain expressed on cells of the immune system that functions to attach immune cells to PD-L1 and EGFR cancer cells (see [link to article]). Figure 2 Exemplary, non-restrictive immune cell adaptor target molecules include 4-1BB, OX-40, CD40, 4-1BB, CD16A, CD3E, LAG3, and CD28.

[0049] In some embodiments, the MVSCA is tetravalent and tetraspecific, meaning it has four VHH domains that are specific to four different antigens. In some embodiments, the MVSCA is pentavalent and tetraspecific, meaning it has five different VHH domains that are specific to four different antigens; two VHH domains are specifically targeted at the same antigen. In some embodiments, the MVSCA includes more than one VHH domain that is specific to the same antigen, and two VHH domains may be adjacent to each other or separated by domains with different specificities. In some embodiments, the VHH domains are specific to the same antigen, and this domain may be specific to different epitopes on the same antigen. In some embodiments, the VHH domains specific to the same antigen may have different amino acid sequences.

[0050] antigen

[0051] Programmed cell death 1 (PD-1), also known as CD279, is a type I membrane protein encoded by the PDCD1 gene in humans. It has two ligands, PD-L1 and PD-L2. PD-L1, also known as CD274 or B7 homolog 1 (B7-H1), is a 40 kDa type I transmembrane protein encoded by the CD274 gene in humans. PD-1 is expressed on the surface of activated T cells, and PD-L1 is also expressed on the surface of antigen-presenting cells (APCs) such as dendritic cells and macrophages. PD-L1 is also overexpressed in several tumors, including breast cancer, lung cancer, bladder cancer, head and neck cancer, and other cancers. When PD-L1 or PD-L2 binds to PD-1, an inhibitory signal is transmitted to T cells, which reduces cytokine production and inhibits T cell proliferation.

[0052] The PD-1 pathway is a key immunosuppressive mediator of T cell exhaustion. During infection-induced inflammatory responses, PD-1 acts to limit the activity of peripherally activated T cells in order to restrict autoimmune responses. Blocking this pathway leads to T cell activation, expansion, and enhanced effector function. Therefore, PD-1 negatively regulates T cell responses. PD-1 has been identified as a marker of exhausted T cells in chronic disease states, and blockade of 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.

[0053] Human serum albumin (HSA) is the most abundant protein in human plasma; it accounts for approximately half of all serum proteins. Albumin transports hormones, fatty acids, and other compounds, buffers pH, maintains colloid osmotic pressure, and performs other functions. Albumin is synthesized in the liver as prealbumin, which has an N-terminal peptide that is removed before nascent albumin is released from the rough endoplasmic reticulum. The prealbumin product is then cleaved in Golgi vesicles to produce secreted albumin. It has a serum half-life of approximately 20 days. The long serum half-life of albumin is partly due to its size, which inhibits clearance through the kidneys, and partly due to its interaction with the neonatal Fc receptor (FcRn). Fusion with anti-albumin sdAbs (single-domain antibodies) has been used to increase the half-life of antitumor single-chain antibodies from 1–2 hours to approximately 10 days.

[0054] CD16, also known as FcγRIII, is a differentiation cluster molecule found on the surface of natural killer cells, neutrophils, monocytes, and macrophages. CD16, identified as an Fc receptor, exists in two forms, encoded by separate genes: FcγRIIIa (CD16A), a transmembrane protein; and FcγRIIIb (CD16B), a GPI-anchored protein; both involved in signal transduction. The most well-studied membrane receptor involved in triggering NK cell lysis, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cytotoxicity (ADCC). It can be used to separate specific immune cell populations using antibodies against CD16 via fluorescence-activated cell sorting (FACS) or magnetically activated cell sorting. These receptors bind to the Fc portion of IgG antibodies, thereby activating antibody-dependent cell-mediated cytotoxicity (ADCC) in human NK cells. CD16 is essential for the ADCC process performed by human monocytes. In humans, CD16-expressing monocytes possess various ADCC capabilities in the presence of specific antibodies and can kill primary leukemia cells, cancer cell lines, and cells infected with hepatitis B virus. Furthermore, CD16 can mediate the direct killing of some viral infections and cancer cells in the absence of antibodies. Upon binding to the conserved portion of ligands such as IgG antibodies, CD16 on human NK cells induces the transcription of surface activating molecules such as IL-2-R (CD25) and inflammatory cytokines such as IFN-γ and TNF. The expression of these CD16-induced cytokine mRNAs in NK cells is mediated by activating T cell nuclear factor (NFATp), a cyclosporine A (CsA)-sensitive factor that regulates the transcription of various cytokines. The upregulation of specific cytokine genes occurs via CsA-sensitivity and calcium-dependent mechanisms.

[0055] CD16 plays a crucial role in the early activation of natural killer (NK) cells following vaccination. Furthermore, CD16 downregulation represents a possible mechanism for regulating NK cell responses and maintaining immune homeostasis in both T cell and antibody-dependent signaling pathways. In healthy individuals, antibody-dependent cytotoxicity (ADCC) in NK cells is induced by cross-linking of CD16 (FcγRIII) in immune complexes. However, this pathway can also be targeted at cancer cells or diseased cells via immunotherapy. Following influenza vaccination, CD16 downregulation was associated with a significant upregulation of influenza-specific plasma antibodies and was positively correlated with NK cell degranulation.

[0056] CD16 is commonly used as an additional marker to reliably identify different subsets of human immune cells. Several other CD molecules, such as CD11b and CD33, have traditionally been used as markers for human myeloid-derived suppressor cells (MDSCs). However, because 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 have high on CD14 and low on CD15. While these two markers are sufficient to distinguish between neutrophils and monocytes, eosinophils have similar CD15 expression to neutrophils. Therefore, CD16 has been used as a further marker to identify neutrophils: mature neutrophils are high on CD16, while eosinophils and monocytes are low on CD16. CD16 can differentiate between these two types of granulocytes. In addition, CD16 expression varies at different stages of neutrophil development: CD16 is low in neutrophil progenitor cells with differentiation capacity, while CD16 expression increases sequentially in postmyeloid cells, band cells, and mature neutrophils.

[0057] The expression of CD16 on neutrophils makes it a potential target for cancer immunotherapy. Margetuximab, an Fc-optimized monoclonal antibody, recognizes human epidermal growth factor receptor 2 (HER2) expressed on tumor cells in breast cancer, bladder cancer, and other solid tumor cancers, targeting CD16A instead of CD16B. Additionally, 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. The anti-CD19 / CD16 bivalent has been shown to enhance natural killer cell responses against B-cell lymphoma. Furthermore, targeting exogenous factors such as FasL or TRAIL to the surface of tumor cells can trigger death receptors, inducing apoptosis through autocrine and paracrine processes.

[0058] OX40 (CD134; TNFRSF4) is a T-cell costimulatory molecule of the tumor necrosis factor (TNF) receptor superfamily, which coordinates with other costimulatory factors (CD28, CD40, CD30, CD27, and 4-1BB) to manage the activation of the immune response. Under co-stimulation of CD40-CD40 ligands and CD28-B7, OX40 activates antigen-activated CD40 receptors. + and CD8 +OX40 is upregulated on T cells. The interaction of OX40 with its ligand on antigen-presenting cells enhances T cell survival, proliferation, and cytokine production. It also inhibits the conversion of effector T cells into regulatory T cells (Tregs) and promotes the maintenance and recall of memory T cells. OX40 is constitutively expressed on Tregs, where it promotes Treg proliferation and immunosuppressive activity. OX40-OX40 ligand signaling is involved in allergic airway inflammation, graft-versus-host disease, and autoimmune diseases.

[0059] CD40, also known as TNFRSF5, is a 45-50 kDa type I transmembrane glycoprotein member of the TNF receptor superfamily. Mature human CD40 consists of a 173-amino acid (aa) extracellular domain, a transmembrane domain, and a 62-aa cytoplasmic domain. The extracellular domain of human CD40 shares 58% and 56% aa sequence identity with mouse and rat CD40, respectively. Antagonistic soluble human CD40 splice variants contain alternative sequences in the extracellular and transmembrane domains and lack the cytoplasmic domain. CD40 is expressed on the surface of B cells, dendritic cells, macrophages, monocytes, platelets, and endothelial and epithelial cells. The interaction between CD40 and its ligand, CD40 ligand, leads to the aggregation of CD40 molecules, resulting in the initiation of bidirectional intracellular signaling in both CD40- and CD40 ligand-expressing cells. CD40 ligand-CD40 linkage promotes B cell activation and T cell-dependent humoral responses. CD40 has multiple functions in both hematopoiesis and epithelial cancer, and is a target for tumor immunotherapy.

[0060] Epidermal growth factor receptor (EGFR) is a transmembrane protein that acts as a receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. EGFR is a member of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Mutations affecting EGFR expression or activity can lead to cancer in many cancer types. EGFR is a transmembrane protein that is activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Defects in EGFR and other receptor tyrosine kinase signaling in humans are associated with diseases such as tumors, while overexpression is associated with the development of various tumors. By blocking the EGFR binding site on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, EGFR signaling can be inhibited, thus preventing the growth of EGFR-expressing tumors and improving patient outcomes.

[0061] 4-1BB, also known as CD137 and TNFRSF9, is a transmembrane glycoprotein of approximately 30 kDa in the TNF receptor superfamily. 4-1BB plays a role in the development and activation of various immune cells. Mature human 4-1BB consists of a 163aa extracellular domain (ECD) with four TNFR cysteine-rich repeat sequences (SEQ ID NO: 54), a 27aa transmembrane fragment, and a 42aa cytoplasmic domain. Within the ECD, human 4-1BB shares 60% aa sequence identity with mouse and rat 4-1BB. 4-1BB is expressed as a disulfide-linked homodimer on various activated T cell populations, including CD4+, CD8+, memory CD8+, NKT, and regulatory T cells, as well as in bone marrow and mast cell progenitors, dendritic cells, mast cells, and bacterially infected osteoblasts. It binds with high affinity to the transmembrane 4-1BB ligand / TNFSF9, which is expressed on antigen-presenting cells and bone marrow progenitor cells. This interaction co-stimulates the proliferation, activation, and / or survival of 4-1BB-expressing cells. It can also enhance cell death induced by repeated stimulation of T cell activation. Mice lacking 4-1BB exhibit enhanced T cell activation, likely due to its absence on regulatory T cells. 4-1BB can bind to OX40 on activated T cells, forming a complex that responds to either ligand and inhibits the proliferation of Treg and CD8+ T cells. Reverse signaling via the 4-1BB ligand inhibits the development of dendritic cells, B cells, and osteoclasts, but supports the survival of mature dendritic cells and co-stimulates mast cell proliferation and activation. 4-1BB activation enhances CD8+ T cell and NK cell-mediated antitumor immunity. It also contributes to the development of inflammation in high-fat diet-induced metabolic syndrome. Soluble forms of 4-1BB and 4-1BB ligands circulate at elevated levels in the serum of patients with rheumatoid arthritis and hematologic malignancies, respectively.

[0062] CD28 is a transmembrane glycoprotein expressed by T cells and some other hematopoietic cells. It is one of the proteins expressed on T cells, providing the co-stimulatory signals required for T cell activation and survival. In addition to the T cell receptor (TCR), stimulation of T cells via CD28 can provide effective signals for the production of various interleukins, particularly IL-6. CD28 is part of the CD28 family of receptors, a group of regulatory cell surface receptors expressed on immune cells. The CD28 family of receptors is a subgroup of the immunoglobulin superfamily. Two family members, CD28 and ICOS, act as positive regulators of T cell function, while three others, BTLA, CTLA-4, and PD-1, act as repressors. The CD28 protein contains 220 amino acids and is encoded by a gene consisting of four exons. It is a glycosylated disulfide-linked homodimer with a molecular weight of 44 kDa. The structure of CD28 contains a paired domain of the V-set immunoglobulin superfamily (IgSF). CD28 bispecific antibodies are designed to specifically co-stimulate T cells within the tumor microenvironment. By bridging T cells to malignant cells expressing selected tumor-associated antigens (TAAs), CD28 bispecific antibodies deliver a so-called signaling agent II to the T cells, releasing their full cytotoxic potential².

[0063] CD3E is a protein complex and T cell co-receptor involved in activating both cytotoxic T cells (CD8+ naive T cells) and helper T cells (CD4+ naive T cells). It consists of four distinct chains: the CD3γ chain, the CD3δ chain, and two CD3ε chains. CD3E is also known as CD3 or T3 and is primarily expressed on T cells, NK-T cells, and at varying levels on thymocytes during T cell differentiation. The CD3ε subunit of the T cell receptor complex is encoded by the CD3E gene. CD3 bispecific antibodies are a type of immunotherapy that has gained considerable attention in cancer treatment. They are designed to redirect T cells to recognize and kill tumor cells. These antibodies bind to both the CD3 protein complex on T cells and tumor-associated antigens (TAAs) on cancer cells. By doing so, they form an artificial immune synapse between T cells and cancer cells, leading to T cell activation and tumor cell killing. Multispecific antibodies are another class of immunotherapies that can bind to multiple targets simultaneously. They can be engineered to target different antigens on cancer cells or to connect immune cells while targeting cancer cells. Both CD3 bispecific and multispecific antibodies have shown promise in preclinical and clinical studies for the treatment of various cancers. They are being investigated as monotherapy or in combination with other immunomodulators. The goal is to enhance anti-tumor immune responses and improve patient clinical outcomes.

[0064] Lymphocyte activation gene 3 (LAG-3) is a 503-amino acid transmembrane protein, an immune checkpoint receptor protein found on the surface of effector T cells and regulatory T cells (Tregs), whose function is 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 leads to the transmission of negative signals to LAG3-expressing cells 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." Due to its important role in tumor and infection immunity, LAG3 is an ideal target for immunotherapy. Blocking LAG3 with antagonists, including monoclonal antibodies, has been investigated in the treatment of cancer and chronic viral infections.

[0065] Patent applications PCT / US2020 / 053064, PCT / US2023 / 069262, and PCT / US2024 / XXXXX (filed on the same day as this application and claiming priority to U.S. Provisional Application 63 / 596,552, filed November 6, 2023, and U.S. Provisional Application 63 / 604,398, filed November 30, 2023) are incorporated herein by reference for all information disclosed therein concerning antibodies specific to a variety of antigens.

[0066] Antibody

[0067] Antibodies and their uses in treating diseases are well known in the art. As used herein, the term "antibody" refers to a polymeric, multispecific protein comprising one or more polypeptide chains containing antigen-binding sites. Antibodies specifically bind to antigens and can modulate the biological activity of antigens. As used herein, the term "antibody" may include "full-length antibody" and "antibody fragment." As used herein, the term "binding site" or "antigen-binding site" refers to the region of an antibody molecule to which the ligand actually binds. The term "antigen-binding site" includes the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL), or, in the case of heavy chain-only antibodies, the antibody heavy chain variable region.

[0068] Antibody specificity refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, natural antibodies are monospecific. As used herein, the term "monospecific" antibody means an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen. A VHH domain may be referred to as a component for binding a specific target antigen (e.g., CD28, CD3E, OX-40, CD40, 4-1BB, HSA, PD-L1, CD16A, LAG3, and EFGR). Therefore, any of the various antibody structures, forms, or constructs disclosed herein containing a VHH domain, or constructed to contain a VHH domain, may be referred to as an antibody containing a component for binding an indicator 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.

[0069] As used in this article, phrases such as "antibody that is specific to...", "antibody that recognizes...", "antibody that has affinity for...", "antibody that has a binding site for...", and similar sentence structures can be used interchangeably.

[0070] As used herein, the term "multispecific antibody" or "MVSCA" refers to an antibody that has specificity for four antigens. The multispecific antibodies disclosed herein are specific for EGFR, PD-L1, and HSA, and one of CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A. Furthermore, an MVSCA may comprise at least two copies of the same antigen-binding sequence, or two antigen-binding sequences specific for different epitopes (double complementary sites) on the same antigen, provided that the multispecific antibody is specific for at least EGFR, PD-L1, and HSA, and at least one additional antigen. In some embodiments, the MVSCAs disclosed herein are single-chain antibodies.

[0071] This article discloses a multivalent and multispecific MVSCA containing four or more variable domains of VHH, wherein the antibody has the following structure:

[0072] A—B—C—D

[0073] in

[0074] (i) A, B, C, and D are respectively specific to the EGFR, PD-L1, HSA, CD28, CD3E, 4-1BB, OX-40, CD40, 4-1BB, LAG3, and CD16AVHH domains; or

[0075] (ii) A, B and D are each a VHH domain specifically targeting EGFR, PD-L1 or HSA, and C contains one or more VHH domains specific to CD28, CD3E, 4-1BB, OX-40, CD40, 4-1BB, LAG3 and CD16A.

[0076] In some implementations, the domains A, B, C, and D are arranged in the order ABCD; however, in other implementations, the order of the domains may be different. Alternative domain orders include, but are not limited to, A—B—D—C.

[0077] VHH domains A, B, C, and D, and multiple copies of each domain, are individually connected by cuttable or non-cuttable joints. Exemplary, non-limiting joints are disclosed in Table 1.

[0078] In some embodiments, A comprises a VHH domain specifically targeting EGFR. In some embodiments, B comprises a VHH domain specifically targeting PD-L1. In some embodiments, D comprises a VHH domain specifically targeting HSA. In some embodiments, A comprises a VHH domain specifically targeting EGFR, B comprises a VHH domain specifically targeting PD-L1, and D comprises a VHH domain specifically targeting HSA. In some embodiments, C comprises a single specificity selected from CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

[0079] In some embodiments, a domain comprises two VHH sequences, each VHH sequence being specific to the same antigen and having the same amino acid sequence. In some embodiments, a domain comprises two VHH domains, each VHH domain being specific to the same antigen and having a different amino acid sequence. If two VHHs have the same antigen specificity but different amino acid sequences, then the VHH is specific to the same epitope on the antigen or to different epitopes on the same antigen. In some embodiments, the domain comprising two VHHs is a C domain.

[0080] The term "tetraspecific antibody" refers to an antibody that has four different antigen-binding specificities. In some embodiments, the tetraspecific antibodies disclosed herein are specific to four antigens selected from EGFR, PD-L1, and HSA, and CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A. The amino acid sequence encoding the antigen-binding moiety of the tetraspecific antibody can be linked in various conformations. In some embodiments, the amino acid sequence encoding the antibody-binding moiety of the tetraspecific antibody is linked via a linker as disclosed herein. In some embodiments, two linkers are used, which can be the same or different.

[0081] As used herein, the term "valence" indicates the presence of a specified number of binding sites in an antibody molecule. Therefore, the terms "tetravalent," "pentavalent," "hexavalent," "heptavalent," and "octavalent" indicate that the antibody molecule contains four, five, six, seven, and eight binding sites, respectively. The tetraspecific antibodies disclosed herein are "tetravalent." However, within the scope of this disclosure are MVSCAs in which multiple antigen-binding sites bind to the same antigen. The antigen-binding sites of MVSCAs can bind to the same epitope or different epitopes on the antigen. Similarly, by combining multiple monospecific binding sites with one or more other specific binding sites, antibodies with a higher valence than multispecific antibodies can be constructed, such as pentavalent tetraspecific antibodies.

[0082] In this article, "full-length antibody" refers to the structure of the naturally occurring biological form of the antibody, including variable and constant regions. For example, in most mammals (including humans and mice), full-length IgG antibodies are tetramers composed of two pairs of identical immunoglobulin chains, each pair containing one light chain and one heavy chain. Each light chain contains immunoglobulin domains VL and CL, and each heavy chain contains immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, such as camels and llamas, IgG antibodies may also consist of only two variable heavy chains, each containing a variable domain (VHH) attached to the Fc region (CH2 and CH3 domains).

[0083] Tetrameric antibodies typically consist of two pairs of identical polypeptide chains, each pair having a "light" chain (typically with a molecular weight of about 25 kDa) and a "heavy" chain (typically with a molecular weight of about 50-70 kDa). Each of the light and heavy chains contains two distinct regions, called the variable region and the constant region. For IgG immunoglobulins, the heavy chain contains four immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VH-CH1-CH2-CH3, namely the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 (also referred to as VH-Cγ1-Cγ2-Cγ3, namely the heavy chain variable domain, constant γ1 domain, constant γ2 domain, and constant γ3 domain, respectively). The IgG light chain contains two immunoglobulin domains linked from the N-terminus to the C-terminus in the sequence VL-CL, namely the light chain variable domain and the light chain constant domain. The constant region exhibits less sequence diversity and is responsible for binding many native proteins to trigger important biochemical events.

[0084] The variable region of an antibody contains the molecule's antigen-binding determinants, thus determining the antibody's specificity for its target antigen. The variable region is so named because it differs most significantly in sequence from other antibodies in its class. Within the variable region, three loops are clustered in each of the V domains of the heavy and light chains to form an antigen-binding site. Each of these loops is called a complementarity-determining region (hereinafter referred to as "CDR"), where the variation in amino acid sequence is most significant. There are a total of six CDRs, three for each heavy and light chain, named VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside the CDRs is called the frame (FR) region. Although not as diverse as the CDRs, sequence variability does exist in the FR regions between different antibodies. Overall, this characteristic architecture of an antibody provides a stable scaffold (FR region) upon which the immune system can explore rich antigen-binding diversity (CDRs) to achieve specificity against a broad array of antigens.

[0085] The gene encoding the immunoglobulin locus contains multiple V region sequences as well as shorter nucleotide sequences named “D” and “J”, and it is the combination of V, D and J nucleotide sequences that produces VH diversity.

[0086] Antibodies are classified into different classes, also known as isotypes, as determined genetically by constant regions. Human constant light chains are classified as kappa (Cκ) and lambda (Cλ) light chains. Heavy chains are classified as mu (μ), delta (δ), gamma (γ), alpha (α), or epsilon (ε), and antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is most commonly used for therapeutic purposes. In humans, this class includes subclasses IgG1, IgG2, IgG3, and IgG4. In mice, this class includes subclasses IgG1, IgG2a, IgG2b, and IgG3. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Therefore, as used herein, “isotype” refers to any one of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of its constant regions. Known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. Disclosed VHH antibodies, bispecific and multispecific antibodies may have constant regions comprising all or part of the above isotypes.

[0087] Also within the scope of this disclosure are antibody fragments, including but not limited to (i) Fab fragments containing VL, CL, VH, and CH1 domains; (ii) Fd fragments containing VH and CH1 domains; (iii) Fv fragments containing the VL and VH domains of a single antibody; (iv) dAb fragments containing a single variable region; (v) isolated CDR regions; (vi) F(ab')2 fragments; bivalent fragments containing two linked Fab fragments; and (vii) single-chain Fv molecules (scFv) wherein the VH and VL domains are linked by a peptide linker that allows the two domains to bind to form an antigen-binding site. Trivalent or tetravalent antibody fragments containing three variable domains having three different specificities linked by cleavable or non-cleavable linkers are also disclosed. In some embodiments, the antibody is generated using recombinant DNA technology. In other embodiments, the antibody is generated by enzymatic or chemical cleavage of a naturally occurring antibody.

[0088] As used herein, a “single-chain antibody” refers to a fusion protein of an antibody’s antigen-binding portion (i.e., variable region) typically linked by a linker peptide. This article discloses a multivalent, multispecific single-chain antibody. MVSCA exhibits specificity against EGFR, PD-L1, and HSA, as well as one of CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

[0089] As used herein, a “humanized” antibody refers to an antibody comprising a human frame region (FR) and one or more complementarity-determining regions (CDRs) derived from a non-human antibody. The non-human antibody providing the CDR is referred to as the “donor,” and the human immunoglobulin providing the frame is referred to as the “recipient.” In some embodiments, humanization primarily relies on transplanting the donor CDR onto the recipient (human) VL or VH frame. This strategy is referred to as “CDR transplantation.” Typically, selected receptor frame residues need to be “reverted” to the corresponding donor residues to regain affinity lost in the initial transplanted construct. Humanized antibodies may also preferably contain at least a portion of an immunoglobulin constant region, typically the constant region of a human immunoglobulin, and may typically contain a human Fc region. Humanization or other methods that reduce the immunogenicity of the variable region of a non-human antibody may include surface remodeling methods. In one embodiment, a selection-based approach may be used to humanize and / or affinity-mature the antibody variable region, i.e., to increase the affinity of the variable region for its target antigen. Other humanization methods may involve transplanting only portions of the CDR, including but not limited to the methods described in US 6,797,492, all of which discloses information regarding CDR transplantation and is incorporated herein by reference. Structure-based methods may be employed for humanization and affinity maturation, such as those described in US 7,117,096, all of which discloses information regarding humanization and affinity maturation and is incorporated herein by reference.

[0090] In the various embodiments described herein, the antibody is VHH. In addition to conventional heavy and light chain antibodies (two light chains and two heavy chains in one antibody), camelids (camels, dromedaries, and llamas) also possess double-chain antibodies (containing only the variable heavy chain). The dimer antibody is encoded by a set of distinct VH fragments called the VHH gene. VH and VHH are scattered throughout the genome (i.e., they appear mixed together). Identification of the same D fragment in the VH and VHH cDNA indicates that VH and VHH share the D fragment. Antibodies containing native VHH lack the entire CH1 domain of the heavy chain constant region. The exon encoding the CH1 domain is present in the genome but is spliced ​​out due to the loss of the functional splice acceptor sequence at the 5' end of the CH1 exon. As a result, the VDJ region is spliced ​​onto the CH2 exon. When VHH recombines into such constant regions (CH2, CH3), antibodies are produced, where the half-antibody is a single chain rather than a light / heavy chain pair (i.e., an antibody with two heavy chains but no light chain interaction). The binding of the antigen differs from that seen with conventional antibodies, but high affinity is achieved in the same way, i.e., through hypermutation of the variable region and screening of cells expressing such high-affinity antibodies.

[0091] In an exemplary embodiment, the disclosed VHH is generated by immunizing transgenic mice, wherein endogenous mouse antibody expression has been eliminated and a camelidoid transgene has been introduced. Transgenic VHH mice are disclosed in US8,883,150, US8,921,524, US8,921,522, US8,507,748, US8,502,014, US 2014 / 0356908, US2014 / 0033335, US2014 / 0037616, US2014 / 0356908, US2013 / 0344057, US2013 / 0323235, US2011 / 0118444, and US2009 / 0307787, all of which are incorporated herein by reference for their entire disclosure regarding heavy chain antibodies and their generation in transgenic mice. In these implementations, transgenic VHH mice are immunized, and the resulting sensitized spleen cells are fused with mouse myeloma cells to form hybridomas.

[0092] In other embodiments, VHHs are generated by immunizing the llama with the desired antigen and isolating the sequence encoding the VHH region of the resulting antigen-binding antibody. In one embodiment, VHHs are isolated using a phage display library. See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204 (each document is incorporated by reference in its entirety for describing the preparation of phage libraries).

[0093] This document discloses multispecific or multivalent antibodies in which two or more antigen-binding domains are linked in a single fusion protein. Multispecific antibodies can take various forms, including (i) multispecific Fv fragments; (ii) heavy chains with first specificity to which a second VH domain with second specificity is bound (or fused to); (iii) tetrameric monoclonal antibodies with first specificity to which a second VH domain with second specificity is bound, wherein the second VH domain binds to the first VH domain; and (iv) Fab fragments (VH-CH1 / VL-CL) with first specificity to which a second VH domain with second specificity is bound. Exemplary Fab fragments include those in which the second VH sequence with second specificity binds to the C-terminus or N-terminus of the first VH domain or the C-terminus or N-terminus of the first CH1 or first CL domain. In another embodiment, the VH sequence having second and / or third (or more) specificity may bind (or fuse) to the C-terminus or N-terminus of the first VH domain or the C-terminus or N-terminus of the first CH1 or first CL domain. In various embodiments, any of these forms may include at least one of the VHH domains disclosed herein. Examples of multispecific antibody configurations can be found in WO2021 / 062361, the entire contents of which are disclosed regarding multispecific antibody configurations and are incorporated herein by reference.

[0094] Multispecific multivalent antibodies may include adapter sequences that link a specific antigen-binding domain (such as VH or VHH) to another antigen-binding domain, and these sequences allow for proper folding of the amino acid sequences to produce the desired three-dimensional conformation and antigen-binding profile. Typically, the adapter sequence can be a short amino acid sequence that provides sufficient space and flexibility between the domains for proper folding. The length and sequence of the adapter can have a substantial impact on the expression level and structure of the MVSCA, as well as the binding affinity of the linking domain. The adapter can also induce steric hindrance to facilitate binding to targets in each domain. For example, length-adjustable adapters L2 and L4 (see Table 1) can be used to optimize the MVSCA according to these parameters. For example, adapters L1, L2, and L4 may be referred to as indestructible adapter members, flexible adapter members, or flexible indestructible adapter members. Suitable adapters include, but are not limited to, the adapters in Table 1 (SEQ ID NO: 38-28, 55, and 259). Other adapters are known to those skilled in the art.

[0095] When two copies of the same VHH structural domain are placed adjacent to each other in an MVSCA, they often interact harmfully with each other. This can be avoided by inserting a relatively short and rigid joint between the two copies. In some embodiments, the short rigid joint has a sequence AAA (e.g., L3 in Table 1). Such a joint may be referred to as a short rigid joint member or an incuttable short rigid joint member.

[0096] When anti-HSA domain-HSA complexes are used to generate prodrugs with respect to the binding activity of adjacent binding domains, a cleavable linker should be inserted between the two domains. L11*3 to L11*18 (see Table 1) are examples of cleavable linkers with varying lengths and cleavage sensitivities to different proteases, which can be used to optimize MVSCA expression levels and structure, binding affinity of the linker domains, and cleavage. Linkers L11*3 to L11*18 may be referred to as cleavable linker members, flexible linker members, or flexible cleavable linker members.

[0097] Table 1. Sequences of non-cuttable and cuttable joints

[0098]

[0099] The binding domains and connectors described herein can be combined to create multifunctional MVSCAs suitable for treating specific diseases. They can also be further combined with other binding domains. MVSCAs may also be referred to as containing components for realizing various functions associated with the binding domains of each component type and / or containing connector components for realizing their associated functions.

[0100] Amino acid sequence variants of the multispecific antibodies disclosed herein are also within the scope of this disclosure. Amino acid sequence variants are prepared by introducing appropriate nucleotide changes into the antibody-encoding DNA or by peptide synthesis. Such variants include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibodies described herein. Any combination of deletions, insertions, and substitutions is performed to obtain a final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter the post-translational processes of humanized or variant antibodies, such as changing the number or location of glycosylation sites.

[0101] A useful method for identifying specific residues or regions of an antibody as preferred sites for mutagenesis is called "alanine scan mutagenesis." Residues or target residue groups (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) are identified and substituted with neutral amino acids (preferably alanine or polyalanine) to affect the interaction of the amino acids with the antigen. Those amino acid positions that demonstrate functional sensitivity to the substitution are then refined by introducing further or other variants at or against the substitution site. Therefore, while the sites used to introduce amino acid sequence variations are predetermined, the nature of the mutation itself does not need to be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scans or random mutagenesis are performed at the target codon or region, and antibody variants expressed are screened for the desired activity.

[0102] Amino acid sequence insertions include fusions of amino and / or carboxyl terms to peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionine residue as disclosed herein, or antibodies fused to an epitope tag. Other insertion variants of antibody molecules include fusions to the N-terminus or C-terminus of an antibody with an enzyme or peptide that increases the antibody's serum half-life.

[0103] Another type of variant is the amino acid substitution variant. These variants have at least one removed amino acid residue in the antibody molecule and a different residue inserted at its position. The sites of most interest for substitution mutagenesis include hypervariable regions, but FR alterations are also considered. Conservative substitutions are shown under the heading “Preferred Substitutions” in Table 2. If such substitutions result in an alteration of biological activity, more substantial changes can be introduced, named “Exemplary Substitutions” in Table 1, or as further described below regarding amino acid categories, and the products can be screened.

[0104] Table 2.

[0105]

[0106] Substantial modification of antibody biological properties is achieved by selecting substitutions that significantly differ in their role in maintaining (a) the structure of the polypeptide backbone in the substitution region, for example, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chains. Naturally occurring residues are grouped into several groups based on shared side-chain characteristics:

[0107] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0108] (2) Neutral hydrophilicity: Cys, Ser, Thr;

[0109] (3) Acidity: Asp, Glu;

[0110] (4) Alkaline: Asn, Gin, His, Lys, Arg;

[0111] (5) Residues affecting chain orientation: Gly, Pro; and

[0112] (6) Aromatics: Trp, Tyr, Phe.

[0113] Non-conservative substitution may involve exchanging members of one of these categories for another category.

[0114] Any cysteine ​​residues that do not participate in maintaining the proper conformation of a multispecific antibody can be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent undesirable cross-linking. Conversely, cysteine ​​bonds can be added to antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0115] Another type of substitution variant involves replacing one or more hypervariable residues of the parent antibody (e.g., humanized or camelid antibody). Typically, the resulting variants selected for further development possess improved biological properties relative to the parent antibody from which they originate. A convenient method for generating such substitution variants is affinity maturation using phage display. Briefly, several hypervariable sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The resulting antibody variant is displayed monovalently from filamentous phage particles as a fusion with the M13 gene III product packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened as disclosed herein. To identify candidate hypervariable sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable residues that significantly contribute to antigen binding. Alternatively, or additionally, analysis of the crystal structure of the antigen-antibody complex to identify contact sites between the antibody and antigen may be beneficial. Such contact residues and adjacent residues are candidates for substitution according to the techniques described herein. Once such variants are generated, the variant group is screened as described herein, and antibodies that exhibit superior properties in one or more relevant assays can be selected for further development.

[0116] Another type of amino acid variant of an antibody alters its original glycosylation pattern. This alteration involves the deletion of one or more carbohydrate moieties found in the antibody, and / or the addition of one or more glycosylation sites not present in the antibody.

[0117] Antibody glycosylation is typically N-linked or O-linked. N-linking refers to the attachment of the carbohydrate moiety to the asparagine residue side chain. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for the enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Therefore, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation involves attaching one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyl amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0118] Adding glycosylation sites to antibodies can be conveniently accomplished by altering the amino acid sequence to include one or more of the aforementioned tripeptide sequences (for N-linked glycosylation sites). Alternatively, alterations can be made by adding one or more serine or threonine residues to the original antibody sequence, or by substituting one or more serine or threonine residues (for O-linked glycosylation sites).

[0119] Nucleic acid molecules encoding amino acid sequence variants of multispecific antibodies are prepared using a variety of methods known in the art. These methods include, but are not limited to, the preparation of variant or non-variant forms of the antibodies disclosed herein from early preparations, either isolated from natural sources (in the case of naturally occurring amino acid sequence variants) or by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis.

[0120] Other modifications to multispecific antibodies have been considered. For example, antibodies may need to be modified in terms of effector function to enhance their effectiveness, such as in treating diseases. For instance, cysteine ​​residues can be introduced into the Fc region, allowing interchain disulfide bonds to form in that region. The resulting homodimeric antibodies may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cytotoxicity (ADCC). Homodimeric antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinking agents. Alternatively, antibodies with dual Fc regions can be engineered to possess enhanced complement cleavage and ADCC capabilities.

[0121] In another implementation, the antibody can be conjugated to a "receptor" (such as streptavidin) for pre-targeting, wherein the antibody-receptor conjugate is administered to the patient, followed by the removal of unbound conjugates from circulation using a scavenger, and then the administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionuclide).

[0122] Covalent modifications of multispecific antibodies are also included within the scope of this disclosure. Where applicable, they can be prepared by chemical synthesis or by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of antibodies are introduced into the molecule by reacting the target amino acid residues of the antibody with an organic 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, all of which discloses covalent modifications of peptides and are specifically incorporated herein by reference. Exemplary types of covalent modifications of antibodies include linking the antibody to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylene, in a manner as described in US4,640,835, US4,496,689, US4,301,144, US4,670,417, US4,791,192, or US4,179,337.

[0123] The multispecific antibodies disclosed herein can be generated recombinantly. Therefore, this document discloses nucleic acids encoding antibodies, expression vectors containing nucleic acids encoding antibodies, and cells containing nucleic acids encoding antibodies. Methods for recombinant production are widely known in the art and involve protein expression in prokaryotic and eukaryotic cells, followed by antibody isolation and typically purification to pharmaceutically acceptable purity. To express the antibodies as described above in host cells, nucleic acids encoding the antibody sequence are inserted into an expression vector using standard methods. Expression is performed in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2 / 0 cells, HEK293 cells, COS cells, PER.C 6 cells, yeast, or E. coli cells, and the antibody is recovered from the cells (supernatant or lysed cells). It should be understood that any recombinantly expressed protein requires an initiating methionine (or formyl-methionine) or signal sequence at its N-terminus, depending on the expression system used and whether the protein is expressed in the cytoplasm or secreted. Therefore, in some embodiments, the protein sequences disclosed herein are modified at their N-terminus with such additional amino acids. In some implementations, such N-terminal sequences are cut (in whole or in part) from fully mature sequences, while in other implementations they are preserved.

[0124] Therefore, some embodiments disclosed herein include a method for preparing multispecific antibodies, comprising the steps of: a) transforming host cells with at least one expression vector containing a nucleic acid molecule encoding an antibody; b) culturing the host cells under conditions that allow for the synthesis of antibody molecules; and c) recovering the antibody molecules from the culture.

[0125] Antibodies are appropriately separated from the culture medium using conventional immunoglobulin purification methods, such as protein A-agarose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0126] As used herein, the terms “cell,” “cell line,” and “cell culture” are used interchangeably, and all such names include progeny. Therefore, the terms “transformation” and “transformed cell” include primary subject cells and cultures derived from them, regardless of the number of passages. It should also be understood that the DNA content of all progeny may not be entirely identical due to intentional or unintentional mutations. This includes variant progeny with the same function or biological activity as those screened in the initially transformed cells. Where the intention is to distinguish the names, it will be clear from the context.

[0127] As used herein, the term "transformation" refers to the process of transferring a vector / nucleic acid into a host cell. If cells without a robust cell wall barrier are used as host cells, transfection can be performed, for example, by calcium phosphate precipitation. However, other methods of introducing DNA into the cell can also be used, such as nuclear injection or protoplast fusion. If prokaryotic cells or cells with abundant cell wall structures are used, one transfection method is calcium treatment with calcium chloride.

[0128] As used herein, “expression” refers to the process by which nucleic acids are transcribed into mRNA and / or the transcribed mRNA (also called transcript) is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in eukaryotic cells may include the splicing of mRNA.

[0129] A “vector” is a nucleic acid molecule, particularly a self-replicating one, that transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into a cell (e.g., chromosome integration), replication vectors whose primary function is to replicate DNA or RNA, and expression vectors whose primary function is to transcribe and / or translate DNA or RNA. Vectors that provide more than one of these functions are also included.

[0130] An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell containing an expression vector that can function to produce the desired expression product.

[0131] As used herein, the term "host cell" refers to any type of cell system that can be engineered to generate the antibodies disclosed herein. In one embodiment, HEK293 cells and CHO cells are used as host cells.

[0132] Control sequences suitable for prokaryotes include, for example, promoters, optional operator gene sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

[0133] When a nucleic acid is in a functional relationship with another nucleic acid sequence, that nucleic acid is "operably linked." For example, if the DNA of a pre-sequence or secretory leader sequence is expressed as a pre-protein involved in polypeptide secretion, then the DNA of the pre-sequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a sequence, then the promoter or enhancer is operably linked to the coding sequence; or if a ribosome binding site is located to facilitate translation, then the ribosome binding site is operably linked to the coding sequence. Generally, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, contiguous and within the reading frame. However, enhancers do not need to be contiguous. Ligation is accomplished by ligating at a convenient restriction site. If such a site is not available, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

[0134] This article also discloses isolated nucleic acids encoding multispecific antibodies, vectors and host cells containing nucleic acids, and recombinant technologies for antibody production.

[0135] To generate antibodies through recombination, the nucleic acid encoding it can be isolated and inserted into a reproducible vector for further cloning (DNA amplification) or for expression. In some embodiments, antibodies can be generated through homologous recombination, as described, for example, in US 5,204,244, all of which discloses information regarding antibody generation and is specifically incorporated herein by reference. The DNA encoding the antibody can be readily isolated and sequenced using conventional methods, such as by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody. Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence, as described, for example, in US 5,534,615, all of which discloses information regarding protein expression and is specifically incorporated herein by reference.

[0136] Suitable host cells for cloning or expressing the DNA in the vectors described herein are the aforementioned prokaryotes, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia coli, Enterobacter spp., Erwinia spp., Klebsiella spp., Proteus spp., Salmonella, for example, Salmonella typhimurium, Serratia spp., for example, Serratia marcescens, and Shigella spp., as well as bacilli such as Bacillus subtilis and Bacillus licheniformis, Pseudomonas spp. and Pseudomonas aeruginosa, and Streptomyces. An exemplary Escherichia coli cloning host is Escherichia coli 294 (ATCC 31,446), although other strains such as Escherichia coli B, Escherichia coli X1776 (ATCC 31,537), and Escherichia coli W3110 (ATCC 27,325) are suitable. These examples are illustrative and not limiting.

[0137] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable cloning or expression hosts for multispecific antibody encoding vectors. Saccharomyces cerevisiae or Bacillus baker's yeast are the most commonly used lower eukaryotic host microorganisms. However, many other genera, species, and strains are generally available and are used herein, such as *Schizosaccharomyces cerevisiae*; hosts of the *Kluyveromyces* genus such as *Kluyveromyces lactis*, *Kluyveromyces brittle* (ATCC 12, 424), *Kluyveromyces bulgaricus* (ATCC 16, 045), *Kluyveromyces wickham* (ATCC 24, 178), *Kluyveromyces walter* (ATCC 56, 500), *Kluyveromyces fruitfly* (ATCC 36, 906), *Kluyveromyces thermosus*, and *Kluyveromyces maculae*; *Yarlostomium lipolytica* (EP402, 226); *Pichia pastoris* (EP 183, 070); *Candida* genus; *Trichoderma reesei* (EP 244, 234); *Neurospora crassa*; *Schwanyophytes* genus such as *Schwanyophyces serrata*; and filamentous fungi such as, for example, *Neurospora*, *Penicillium*, *Cyclophorus*, and *Aspergillus* hosts such as *Aspergillus nidus* and *Aspergillus niger*.

[0138] Suitable host cells for expressing glycosylated multispecific antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells from hosts such as the fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm, have been identified. Several viral strains for transfection are publicly available, such as the L-1 variant of the silver-striped armyworm NPV and the Bm-5 strain of the silkworm NPV, and such viruses can be used as the viruses described herein according to this disclosure, particularly for transfecting fall armyworm cells. Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

[0139] However, people are most interested in vertebrate cells, and propagating vertebrate cells in cultures (tissue cultures) has become a routine procedure. Examples of available mammalian host cell lines include: monkey kidney CV1 line (COS-7, ATCC CRL 1651) transformed with SV40; human embryonic kidney line (for 293 or 293 cell subclones grown in suspension culture); juvenile hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse Support cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL 34); Buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumors (MMT). 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatocellular carcinoma cell line (Hep G2).

[0140] The above expression vector was used to transform host cells for the production of multispecific antibodies, and the cells were cultured in conventional nutrient media that were appropriately modified to induce promoters, screen transformants, or amplify genes encoding desired sequences.

[0141] Host cells used to produce multispecific antibodies can be cultured in a variety of media. Commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing host cells. Additionally, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or US Re. 30,985 can be used as a culture medium for host cells. Any of these culture media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™), trace elements (defined as inorganic compounds typically present in micromolar final concentrations), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0142] When using recombinant technology, antibodies can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If antibodies are produced intracellularly, as a first step, particulate debris, host cell fragments, or lysed fragments are removed, for example, by centrifugation or ultrafiltration.

[0143] Cell-prepared antibody compositions can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains, although it can also be used to purify antibodies lacking an Fc region. Protein G can be used for all mouse isotypes and human γ3. The most common matrix for linking affinity ligands is agarose, but other matrices can also be used. Mechanically stable matrices such as controlled-pore glass or poly(divinyl styrene)benzene allow for faster flow rates and shorter processing times compared to what is achievable with agarose. When the antibody contains a CH3 domain, Bakerbond ABX™ resin can be used for purification. The antibodies and antibody fragments disclosed herein can also be synthesized using histidine tags and purified by metal affinity chromatography.

[0144] Depending on the antibody to be recovered, other techniques for protein purification can also be used, such as fractionation based on ion exchange columns, ethanol precipitation, reversed-phase HPLC, silica-based chromatography, heparin SEPHAROSE™-based chromatography, anion or cation exchange resin-based chromatography (e.g., polyaspartic acid columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0145] Following any initial purification step, the mixture containing the antibody of interest and contaminants can be subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH between approximately 2.5 and 4.5, preferably at a low salt concentration (e.g., approximately 0–0.25 M salt).

[0146] This document also discloses cleavable multispecific single-chain antibodies in the tumor microenvironment. In some embodiments, once the multispecific single-chain antibody reaches the tumor, a tumor-targeting domain (such as a tumor antigen-binding domain) or other functional domain is cleaved at the linker to release other domains that produce therapeutic effects. The tumor microenvironment contains a variety of proteases capable of cleaving the linkers disclosed herein. Non-limiting examples of tumor proteases include, but are not limited to, matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAMs (de-integrin and metalloproteinases; e.g., ADAM10 and ADAM17), kallikrein-associated peptidases (e.g., KLK1, KLK2, KLK3, and KLK6), cathepsins (e.g., CTS-B, CTS-L, and CTS-S), urokinase plasminogen activator (uPA), hepatic serine protease (HPN), matriptase, asparagine endopeptidase, or dipeptidyl peptidase (e.g., DDP4).

[0147] antibody composition

[0148] This document also discloses pharmaceutical compositions comprising the multispecific antibodies disclosed herein, wherein specificity includes one or more of EGFR, PD-L1, HSA, and CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A. Use of the antibodies described herein for preparing pharmaceutical compositions is also disclosed. Methods for treating various diseases and conditions using the disclosed antibodies and antibody-containing pharmaceutical compositions are also disclosed.

[0149] A pharmaceutical composition is a composition intended and suited for treating a disease in a human body. That is, it provides an overall beneficial effect and does not contain any amount of any ingredient or contaminant that causes toxicity or other undesirable effects unrelated to providing the beneficial effect. A pharmaceutical composition may contain one or more active agents and may further contain solvents, buffers, diluents, carriers, and other excipients to aid in the administration, solubility, absorption, or bioavailability and / or stability of the active agent or the entire composition.

[0150] The multispecific antibodies disclosed herein can be formulated into liposomes. Liposomes containing antibodies are prepared using methods known in the art, such as those described in US4,485,045, US4,544,545, and US5,013,556. Particularly useful liposomes can be generated by reverse-phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derived phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter with defined pore sizes to produce liposomes with the desired diameter. The Fab' fragment of the antibody can be conjugated to the liposome via a disulfide exchange reaction.

[0151] As used herein, "drug carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents, and absorption delay agents. Preferably, the carrier is suitable for intravenous, intramuscular, intraocular, intravitreal, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). In some embodiments, the carrier is aqueous.

[0152] The compositions disclosed herein can be administered by a variety of methods known in the art. As those skilled in the art will understand, the route and / or manner of administration may vary depending on the desired outcome. To administer the disclosed antibodies via certain routes of administration, it may be necessary to bind the antibody to a material, or co-administer the antibody with the material, to prevent its inactivation. For example, the antibody may be administered to the subject in a suitable carrier such as liposomes or diluents. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Drug carriers include sterile aqueous solutions or dispersions and sterile powders for the provisional preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

[0153] As used herein, the phrases “extragastric administration” and “post-extragastric administration” refer to administration methods other than intravenous and local administration, usually by injection, and including but not limited to intravenous, intramuscular, intra-arterial, intrasheath, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0154] These compositions may also contain excipients such as preservatives, humectants, emulsifiers, and dispersants. The presence of microorganisms can be prevented by the sterilization procedures described above and by including various antimicrobial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It is also desirable to include isotonic agents, such as sugars, sodium chloride, etc., in the composition. Furthermore, prolonged absorption of injectable drug forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0155] In some embodiments, the antibody-containing pharmaceutical composition is a lyophilized cake. The lyophilized cake may further contain fillers, buffers, and / or salts or other excipients, as described herein. The lyophilized composition may be reconstituted by adding sterile water or an aqueous buffer for administration to a patient.

[0156] Regardless of the chosen route of administration, the disclosed antibodies and / or antibody-containing pharmaceutical compositions that can be used in a suitable hydrated form can be formulated into pharmaceutically acceptable dosage forms using conventional methods known to those skilled in the art.

[0157] The actual dose level of the active ingredient in a pharmaceutical composition can be varied to obtain an amount of active ingredient that is non-toxic to the patient and effectively achieves the desired therapeutic response for a particular patient, composition, and route of administration. The selected dose level may depend on a variety of pharmacokinetic factors, including the activity of the particular composition used in this disclosure, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health status, and prior medical history of the patient being treated, and similar factors well known in the medical field.

[0158] Publicly disclosed uses of antibodies

[0159] The disclosed antibodies are for medical use. The terms "treatment," "treating," etc., refer to the medical management of a patient aimed at curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or symptom, and also includes etiological treatment, i.e., treatment aimed at eliminating the associated disease, pathological condition, or cause. Additionally, this term includes palliative treatment, i.e., treatment aimed at relieving symptoms rather than curing a disease, pathological condition, or symptom; preventative treatment, i.e., treatment aimed at minimizing or partially or completely suppressing the development of an associated disease, pathological condition, or symptom; and supportive treatment, i.e., treatment used to complement another specific therapy aimed at improving an associated disease, pathological condition, or symptom. Various implementation schemes may specifically include or exclude one or more of these treatment modalities.

[0160] The use of the antibodies disclosed in this paper in diagnosis and imaging was also considered.

[0161] Furthermore, the term "treating" or "treatment" broadly encompasses any type of therapeutic activity, including any activity that diagnoses, alleviates, or prevents a disease or aspect thereof in a person or other animal, or otherwise affects the structure or any function of the body of a person or other animal. Therapeutic activities include administering the medicines, dosage forms, and pharmaceutical compositions described herein to a patient, particularly according to the various treatment methods disclosed herein, whether administered by a medical professional, the patient themselves, or any other person. Therapeutic activities include orders, instructions, and recommendations from medical professionals such as physicians, physician assistants, nurse practitioners, etc., which are then carried out by any other person, including other medical professionals or the patient themselves. This includes, for example, instructing a patient to undergo or instructing a clinical laboratory to perform diagnostic procedures, such as those used for cancer diagnosis and staging, so that the patient can ultimately receive appropriate and beneficial treatment. In some implementations, the commands, instructions, and recommendations of treatment activities may also include encouraging, guiding, or compelling the use of a specific drug (or combination thereof) to treat a condition—and that drug is actually being used—through approving insurance coverage for the drug, refusing coverage for alternative drugs, including (or excluding) the drug in a drug list, or providing financial incentives for its use; as insurance companies or pharmacy benefit management companies may do. In some implementations, treatment activities may also include encouraging, inducing, or compelling the selection of a specific drug to treat a condition—and that drug is actually being used—through policies or standards of practice that may be established by hospitals, clinics, health maintenance organizations, medical institutions, or physician groups. All such commands, instructions, and recommendations should be considered as conditions of receiving treatment benefits on the basis of compliance with the instructions. In some cases, patients also receive financial benefits for complying with such commands, instructions, and recommendations. In some cases, healthcare professionals also receive financial benefits for complying with such commands, instructions, and recommendations.

[0162] The disclosed multivalent single-chain antibodies specific to EGFR, PD-L1, HSA, CD47, CD33, and LAG3 can be used to treat cancer. Based on the antigen-binding specificity included in the antibody, each MVSCA can be designed to treat a specific class of cancer, or a single MVSCA can be used for multiple different types of cancer.

[0163] This disclosure provides methods for treating cancer, including administering an effective amount of the disclosed antibody or a pharmaceutical composition containing the antibody to a patient in need of such treatment.

[0164] Examples of cancers treatable through the disclosed methods include: acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer; bladder cancer; bone cancer; brainstem glioma; brain tumors; breast cancer; bronchial adenoma / carcinoid; carcinoid tumor; islet cell carcinoma; cancer of unknown primary site; central nervous system lymphoma; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; endometrial cancer; ependymoma; ovarian epithelial cancer; esophageal cancer; Ewing's family tumors; extracranial germ cell tumors; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; germ cell tumors; gestational trophoblastic tumors; pilonidal tumors. Leukemia; head and neck cancer; hepatocellular carcinoma; Hodgkin's lymphoma; hypopharyngeal cancer; Kaposi's sarcoma; kidney cancer; laryngeal cancer; non-small cell lung cancer; small cell lung cancer; non-Hodgkin's lymphoma; Waldenström macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell carcinoma; squamous cell carcinoma of the neck; multiple endocrine adenoma syndrome; multiple myeloma / plasma cell tumor; mycosis fungoides; myelodysplastic syndrome; nasopharyngeal carcinoma; neuroblastoma; oral cancer; oropharyngeal cancer; osteosarcoma; pancreatic cancer; parathyroid carcinoma; penile cancer; pheochromocytoma; pituitary adenoma; pleural pulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; soft tissue sarcoma; Cezari syndrome; skin cancer; squamous cell carcinoma of the neck; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and nephroblastoma.

[0165] The effectiveness of cancer treatments is typically measured by "response." Techniques for monitoring this response can be similar to tests used to diagnose cancer, such as, but not limited to:

[0166] A lump or tumor involving certain lymph nodes can be felt and measured externally through physical examination.

[0167] Some internal cancerous tumors can appear on X-rays or CT scans and can be measured with a ruler.

[0168] Blood tests can be performed, including tests that measure organ function.

[0169] It can be used to test tumor markers for certain cancers.

[0170] Regardless of the type of test used—whether it's a blood test, cell count, or tumor marker test—it is repeated at specific intervals so that the results can be compared with earlier tests of the same type.

[0171] There are several definitions of response to cancer treatment:

[0172] Complete response – all cancers or tumors disappear; no evidence of disease. Tumor marker expression levels (if applicable) may be within the normal range.

[0173] Partial remission – the cancer has shrunk by a certain percentage, but the disease is still present. Tumor marker levels (if applicable) may have decreased (or increased, based on tumor markers as an indicator of reduced tumor burden), but evidence of disease remains.

[0174] Disease stable - the cancer neither grows nor shrinks; the disease load remains unchanged. Tumor markers (if applicable) show no significant changes.

[0175] Disease progression - Cancer has grown; the disease is now more extensive than before treatment. Tumor marker tests (if applicable) show elevated tumor markers.

[0176] Other measures of cancer treatment efficacy include overall survival (i.e., the time to death from any cause, measured from the time of diagnosis or from the start of the evaluated treatment), cancer-free survival (i.e., the length of time after a full response when cancer remains undetectable), and progression-free survival (i.e., the length of time after disease stabilization or partial remission when tumor growth is undetectable).

[0177] There are two standard methods for assessing treatment response to solid tumors (regarding tumor size (tumor burden)): the WHO and RECIST criteria. These methods measure the solid tumor to compare the current tumor size with past measurements or to compare changes with future measurements and adjust treatment accordingly. In the WHO method, the long and short axes of the solid tumor are measured, and then the product of these two measurements is calculated; if multiple solid tumors are present, all products are summed. In the RECIST method, only the long axis is measured. If multiple solid tumors are present, all long axis measurements are summed. However, for lymph nodes, the short axis is measured instead of the long axis.

[0178] The following embodiments, sequence listing, and drawings are provided to aid in understanding the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the described procedures without departing from the spirit of the invention. Example

[0179] Example 1: Generation of VHH

[0180] Immunization. Rillas were immunized at Pacific Immunology, Inc (Ramona, CA) according to their standard protocol. The antigen of interest was mixed with Freund's complete adjuvant (day 0) or Freund's incomplete adjuvant (after immunization) (Difco, BD Biosciences). Each llama was administered six subcutaneous injections at two-week intervals at a dose of 50 μg. On day 45, serum was collected from the immunized llamas to determine antibody titers against the immunogenic antigens by ELISA.

[0181] Phage library construction and selection. Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples taken from immunized llamas on day 45 using Ficoll-Paque™ Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from PBMCs using the RNeasy™ Midi Kit (Qiagen) according to the manufacturer's instructions and used as starting material for RT-PCR to amplify VHH-encoding gene fragments. These fragments were cloned into a self-made phage particle vector, allowing the generation of recombinant phage particles after infection with helper phages. These phage particles displayed VHH as a gene III fusion protein on their surface. Phages were prepared according to standard methods and stored at 4°C for future use after filtration sterilization. The phage library obtained from the llamas was used for screening. In screening, biotinylated recombinant antigens were incubated with the phage library and then captured on streptavidin Dynabeads™ (Invitrogen). After thorough washing, the bound phages were eluted with 1 mg / ml trypsin. The products obtained from screening were rescued in E. coli TG1 cells. Clones were selected and sequenced.

[0182] The cDNA encoding positive VHH was fused with a C-terminal His-tag and cloned into the expression vector SVT003, which was then transiently transfected into HEK293 cells. Positive VHH was purified by IMAC chromatography for in vitro functional assays.

[0183] VHH specific to human CD28

[0184] An antibody targeting the extracellular domain of human CD28 was generated (Asn19-Pro152; accession number P10747; SEQ ID NO:24):

[0185] NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0186] Table 3 lists the VHH amino acid sequences specific to human CD28. Table 3 also outlines the methods by which VHH structures bind to human CD28.

[0187] Table 3. Anti-CD28 VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0188]

[0189]

[0190] The humanization of D28-12E10 is based on the IGHV-74 germline sequence, and the humanization of D28-A1 is based on the IGHV3-23 germline sequence.

[0191] VHH specific to human CD3E

[0192] Generate antibodies against the extracellular domain of human CD3E (amino acids 23-126; accession number P07766; SEQ ID NO:46):

[0193] DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0194] Table 4 lists the VHH amino acid sequences specific to CD3E. Table 4 also outlines the means by which VHH binds to human CD3E.

[0195] Table 4. Anti-CD3E VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0196]

[0197]

[0198] *These CD3E-specific VHHs cross-react with cynomolgus monkey CD3E

[0199] VHH specific to human PD-L1

[0200] Generate an antibody targeting the extracellular domain of human PD-L1 (amino acids 19-238; accession number Q9NZQ7; SEQ ID NO: 65):

[0201] FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITV KVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNER

[0202] Table 5 lists the VHH amino acid sequences specific to human PD-L1. The VHH structure in Table 5 represents the means by which human PD-L1 is bound.

[0203] Table 5. Anti-PD-L1 VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0204]

[0205] VHH specific to human HSA

[0206] Generate antibodies against HSA (SEQ ID NO: 75):

[0207] MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFH DNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCE KPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHHDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGK VGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0208] Table 6 lists the VHH amino acid sequences specific to human HSA. The VHH structures in Table 6 are used to bind to human HSA.

[0209] Table 6. Anti-HSA VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0210]

[0211]

[0212] VHH specific to human CD16A

[0213] An antibody against human CD16A was generated (SEQ ID NO: 93):

[0214] MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLISSQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLR CHSWKNTALHKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFPPGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0215] Table 7 lists the VHH amino acid sequences specific to human CD16A. The VHH structure in Table 7 represents the means of binding to human CD16A.

[0216] Table 7. Anti-CD16A VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0217]

[0218] VHH specific to human OX40

[0219] Generate antibodies against the extracellular domains of human OX40 (amino acids 29-216; accession number P43489; SEQ ID NO:100):

[0220] LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA

[0221] Table 8 lists the VHH amino acid sequences specific to human OX40. Table 8 also outlines the methods by which VHH structures bind to human OX40.

[0222] Table 8. Anti-OX40 VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0223]

[0224] VHH specific to human CD40

[0225] An antibody targeting the extracellular domain of human CD40 (amino acids 21-193; accession number P25942-1; SEQ ID NO: 103) was generated.

[0226] EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLGLRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSSAFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLR

[0227] Table 9 lists the VHH amino acid sequences specific to human CD40. The VHH structure in Table 9 represents the means by which human CD40 is bound.

[0228] Table 9. Anti-CD40 VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0229]

[0230]

[0231] VHH specific to human 4-1BB

[0232] Antibodies were generated targeting the extracellular domain of human 4-1BB (amino acids 24-183; accession number Q07011; SEQ ID NO: 119):

[0233] LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGH

[0234] Table 10 lists the VHH amino acid sequences specific to human 4-1BB. The VHH structure in Table 10 represents the means by which human 4-1BB is bound.

[0235] Table 10. Anti-4-1BB VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0236]

[0237] VHH specific to human EGFR

[0238] Antibodies against the extracellular domains of human EGFR (amino acids 1-645; accession number CAA25240; SEQ ID NO: 133) were generated.

[0239] MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEITYVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELPMRNLQEILHGAVRFSNNPALCNVES IQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGEENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPTTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDG VRKCKKCEGPCRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGT SGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCKLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0240] Table 11 lists the VHH amino acid sequences specific to human EGFR. The VHH structure in Table 11 is used for binding to human EGFR.

[0241] Table 11. Anti-EGFR VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0242]

[0243]

[0244] VHH specific to human LAG3

[0245] Generate antibodies against the extracellular domains of human LAG3 (amino acids 19-238; P18627; SEQ ID NO: 173):

[0246] VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVS PMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCH IHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQ

[0247] Table 12 lists the VHH amino acid sequences specific to human LAG3. The VHH structure in Table 12 is used for binding to human LAG3.

[0248] Table 12. Anti-LAG3 VHH sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0249]

[0250] Example 2: Multispecific Antibody

[0251] Table 13 describes multispecific single-chain antibodies specific to EGFR, PD-L1, CD40, and HSA.

[0252] Table 13. Multispecific antibodies (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0253]

[0254] Example 2. SM2272, with enhanced cancer cell selectivity, four specificities, dual tumor antigen targeting, and conditional... Sex 4-1BB agonists

[0255] While 4-1BB agonist monoclonal antibodies have demonstrated promising antitumor efficacy in preclinical animal models, their clinical development has been hampered by lethal "extratumor-targeted" hepatotoxicity. To improve the safety profile and therapeutic window of 4-1BB agonists, we developed SM2272, a quadruple-specific VHH antibody targeting EGFR, PD-L1, 4-1BB, and HSA, for the treatment of EGFR+PD-L1+ solid tumors. Preclinical studies have shown that SM2272 conditionally activates 4-1BB-expressing immune cells upon co-binding to EGFR+PD-L1+ cancer cells. It is well-tolerated in vivo and exhibits potent antitumor activity in syngeneic tumor transplantation models.

[0256] SM2272 possesses two monovalent, low-affinity binding domains targeting EGFR and PD-L1, and a bivalent, high-affinity 4-1BB binding domain arranged in tandem, followed by a half-life-extended HSA binding domain. The binding affinity and specificity of SM2272 for recombinant human EGFR, PD-L1, 4-1BB, and HSA were confirmed by biolayer interferometry and ELISA. Flow cytometry was used to analyze the differential binding affinity of SM2272 for EGFR / PD-L1 monoexpressing versus dual-expressing cell lines. Competitive ligand binding assays were used to analyze the PD-1 binding affinity for EGFR compared to PD-L1+ monopositive cells. + PD-L1 + Affinity-enhancing blockade with double positivity was achieved. EGFR and PD-L1 dual-target-dependent 4-1BB activation was confirmed using 4-1BB-NFκB-Luc reporter gene assays and primary human CD8+ T cell IFNγ release assays. EGFR-targeting therapy was evaluated using a CT26-hEGFR / hPD-L1 cell line-derived tumor transplantation model into Balb / c-h4-1BB humanized mice. + / PD-L1 + The in vivo efficacy of double-positive human tumors.

[0257] The aim is to develop VHH-based dual tumor antigen-specific conditional 4-1BB agonists to enhance antibody tumor-targeting specificity. This strategy involves conditional 4-1BB activation based on dual tumor antigen (EGFR and PD-L1) binding to minimize systemic immune activation. This strategy also enhances the efficacy of PD-L1 signaling blockade against tumor cells expressing both EGFR and PD-L1. Figure 2 and 3SM2272 is a humanized pentavalent tetraspecific VHH antibody targeting EGFR, PD-L1, 4-1BB, and HSA, with optimized pharmacokinetic properties. Structural diagrams of SM2272 and SM2272A are shown in [the diagram / image / etc.]. Figure 1 The sequences of these two antibodies are shown in Table 14.

[0258] Table 14. SM2272 and SM2272A sequences (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0259]

[0260] * indicates the stop codon

[0261] SM2272 binds to EGFR+PD-L1+ dual-expressing cells with increased affinity and specificity. Compared to single-target expression cells, SM2272 binds to EGFR+ and PD-L1+ dual-expressing cells with increased affinity. The monovalent EGFR, PDL1, and HSA module binds to recombinant proteins with moderate affinity. The bivalent 4-1BB module binds to its target with high affinity.

[0262] SM2272 and EGFR + PD-L1 + The affinity of double-positive cancer cells to EGFR + or PD-L1 + The affinity for binding to single-positive normal cells was significantly increased (>500-fold), improving antibody specificity. SM2272 in EGFR + PD-L1 + The IC50 of PD-L1 blockade on double-positive cancer cells was significantly lower (>50-fold) than that on PD-L1. + IC50 on single-positive normal cells allows for the reactivation of TME-restricted T cells. SM2272 conditionally stimulates 4-1BB cells upon cancer cell conjugation. + Tumor-infiltrating T / NK cells, in synergy with PD-L1 inhibition, further enhance anti-tumor immunity while avoiding systemic toxicity.

[0263] SM2272 cross-reacts with cynomolgus monkey targets but not with rodent targets. SM2272 binds to recombinant human EGFR and PD-L1 with intermediate affinity and to 4-1BB with high affinity (Table 15 and Figure 4).

[0264] Table 15. Biolayer Interferometry (BLI) Binding Affinity Analysis of SM2272

[0265]

[0266] SM2272 blocks the binding of ligands to recombinant human EGFR and PD-L1 at nanomolar concentrations. Figure 5A and 5B SM2272 does not block the interaction between 4-1BB and 4-1BBL. Figure 5C SM2272 does not block the binding of HSA to FcRN.

[0267] Compared to SM2272, the mutant SM2272 (SM2272A) lacking EGFR binding ability showed reduced binding affinity to human cancer cell lines, indicating that effective binding of SM2272 to target cells requires dual-target binding, which improves antibody selectivity against EGFR+PD-L1+ cancer cells. Figure 6A -C).

[0268] As determined by flow cytometry, the monovalent EGFR and PDL1 module binds to cell surface targets with intermediate affinity. The bivalent 4-1BB module also binds to cell surface targets with high affinity. Compared to normal tissues expressing a single target, SM2272 selectively binds to EGFR+PD-L1+ double-positive cancer cells with enhanced affinity.

[0269] Enhanced antibody binding allows SM2272 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells. Figure 7A -C). Compared with the single-target binding SM2272A, the dual-target binding SM2272 showed a significantly enhanced ability to block the interaction between PD-1 and PD-L1.

[0270] Enhanced affinity PD-L1 blockade allows for local T cell reactivation. Enhanced antibody binding allows SM2272 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells. Figure 8A -D). Compared with the single-target binding SM2272A, the dual-target binding SM2272 showed a significantly enhanced ability to block the interaction between PD-1 and PD-L1.

[0271] Enhanced antibody binding enables SM2272 to selectively block the interaction between PD-L1 and PD-1 on the surface of EGFR+PD-L1+ double-positive cancer cells, thereby allowing local (re)activation of tumor-resident cytotoxic T cells without systemic immune cell activation.

[0272] SM2272 conditionally activates the HEK293-4-1BB-NFκB-Luc reporter cell line upon conjugation to cells expressing both EGFR and PD-L1. Figure 9A-C). In the presence of a broadly EGFR and PD-L1 dual-expressing human cancer cell line, SM2272 showed a significantly enhanced ability to activate the HEK293-4-1BB-NFκB-Luc reporter cell line compared to SM2272A. In the absence of target cells, SM2272 did not activate 4-1BB.

[0273] SM2272 conditionally activates 4-1BB-expressing immune cells only upon dual TAA binding, thereby avoiding hepatotoxicity associated with overall 4-1BB activation.

[0274] SM2272 significantly inhibited the growth of CT26-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in the humanized BALB / c-hPD1 / hPD-L1 / h4-1BB mouse model. Figure 10 Cancer cells were implanted subcutaneously. When the tumor size reached ~80 mm³, the animals were randomly divided into groups and treated with a medium or SM2272 every 3 days.

[0275] The pharmacokinetics of SM2272 were evaluated in mice after a single 3 mg / kg dose. Figure 11 ).

[0276] SM2272 exhibits moderate binding affinity for CHO-hEGFR and CHO-hPD-L1 cells and high affinity for CHO-4-1BB cells. Notably, SM2272 shows a significantly increased affinity for cancer cells co-expressing EGFR and PD-L1 compared to mono-expressing cells. This leads to PD-L1 blockade with enhanced affinity specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, allowing for local reactivation of tumor-infiltrating T cells without systemic immunotoxicity. In the presence of SM2272, EGFR and PD-L1 double-positive cancer cells induce significantly enhanced 4-1BB activation and cytokine secretion compared to cells expressing a single antigen. In an endogenetic tumor transplantation model, SM2272 effectively inhibits the growth of CT26-hEGFR / hPD-L1 tumors in humanized Balb / c-h4-1BB mice without significant hepatotoxicity or immunotoxicity.

[0277] SM2272 is a novel bispecific antibody targeting conditional 4-1BB agonists. Its unique mechanism of action has demonstrated promising efficacy and good safety profile in preclinical models, distinguishing it from existing monoclonal and bispecific antibodies.

[0278] Example 3. SM2275 is a four-specificity, dual tumor antigen-targeting, conditional CD28 agonist with enhanced cancer cell selectivity.

[0279] While CD28 agonist monoclonal antibodies have demonstrated promising antitumor efficacy in preclinical animal models, their clinical development has been hampered by a series of acute and serious adverse events in Phase I trials. To improve the safety profile and therapeutic window of CD28 agonists, we developed SM2275, a quadruple-specific VHH antibody targeting EGFR, PD-L1, CD28, and HSA, for the treatment of EGFR+PD-L1+ solid tumors. Preclinical studies have shown that SM2275 conditionally activates CD28-expressing immune cells upon co-binding with EGFR+PDL1+ cancer cells. It is well-tolerated in vivo and exhibits potent antitumor activity in syngeneic tumor transplantation models.

[0280] SM2275 uses Starmab's proprietary Quadbody. TM Developed using a multifunctional VHH antibody platform, SM2275 possesses two monovalent low-affinity binding domains targeting EGFR and PD-L1, along with a sub-nanomolar affinity CD28 binding domain arranged in tandem, followed by a half-life-extended HSA binding domain. The binding affinity and specificity of SM2275 for recombinant human EGFR, PD-L1, CD28, and HSA were confirmed by biolayer interference and ELISA. Flow cytometry analysis was used to analyze the differential binding affinity of SM2275 to EGFR / PD-L1 monoexpressing versus dual-expressing cell lines. Competitive ligand binding assays showed that PD-1 exhibited enhanced affinity blockade against EGFR+PD-L1+ dual-positive cells compared to PD-L1+ monopositive cells. EGFR and PD-L1 dual-target-dependent CD28 activation was confirmed using Jurkat IL2 promoter Luc reporter gene assays and primary human T cell IL2 release assays. The in vivo efficacy against EGFR+PD-L1+ double-positive human tumors was evaluated using a homologous tumor transplantation model of the MC38-hEGFR / hPDL1 cell line into C57-hPD-l1 and hCD28 humanized mice.

[0281] The multispecific antibody against CD28 VHH, comprising Example 1, is presented in Figure 12 And in Table 16.

[0282] Table 16. Multispecific antibodies SM2275 with different linkers (CDRs for each sequence are indicated by underline [CDR1], bold [CDR2], or double underline [CDR3])

[0283]

[0284]

[0285] Following the flow cytometry method described above, the binding affinity of the multispecific antibodies SM2275-679 (low hCD28 binding affinity) and SM2275-700 (high hCD28 binding affinity) was determined as follows: Figure 13 and 14 and EC in Table 16 50 .

[0286] Cell-based functional assays showed that SM2275-649 and SM2275-700 are fully functional, multispecific VHH antibodies (Table 5). SM2275 cross-reacted with cynomolgus monkey targets at the same KD, but did not bind to rodent targets.

[0287] Table 17. BLI binding affinity analysis of SM2275-700 and SM2275-649

[0288]

[0289] As determined by flow cytometry, the monovalent EGFR and PDL1 modules bind to cell surface targets with moderate affinity. Compared to normal tissues expressing a single target, SM2275 selectively binds to EGFR+PD-L1+ double-positive cancer cells with enhanced affinity. This enhanced affinity antibody binding allows SM2275 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells. Figure 15 The dual-target binding SM2272 showed a significantly enhanced ability to block PD-1 and PD-L1 interactions on CHO-bis-OKT3 compared to hPD-L1-OKT3. Figure 16 Tumor site-specific blockade of PD-L1 / PD-1 synergistically with CD28 agonist activity to allow SM2275 to locally maximize T cell activation.

[0290] SM2275 exhibits dual-target-dependent CD28 activation in synergy with PD1 / PD-L1 signaling. Figure 17 The Jurkat NFAT-PD-1 reporter cell line was used in this assay. The CHO-hEGFR-hPD-L1-OKT3 cell line was used as the target cell line. The target-free control was the CHO-OKT3 cell line. SM2275 showed no activity in the target-free control, indicating target-dependent CD28 activation. Compared with SM2275, SM2275(CD28-), SM2275 with a mutated and inactivated CD28 module, showed a reduced ability to inhibit PD-L1 signaling, indicating that CD28 activation significantly enhances the inhibition of PDL1 signaling.

[0291] Compared with TGN1412, SM2275 does not induce the release of cytokines from human PBMCs. Figure 16Compared with TGN1412, SM2275 did not show systemic IL-2 release in vivo (Figure 18). Humanized hPD-L1 / hCD28 mice received a single dose of PBS, SM2275 (5 mg / kg, 15 mg / kg), or TGN1412 (2.5 mg / kg). Blood was collected 4 hours after administration and serum cytokines were measured. SM2275 was well tolerated and did not induce cytokine release 4 hours after treatment. In contrast, TGN1412 induced significant systemic cytokine release, consistent with its in vivo hyperagonist activity.

[0292] SM2275 significantly inhibited the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted, and animals were randomly assigned to groups when the tumor size reached ~80 mm³. Treatment with either the drug or SM2275 was administered every 2 days for 10 days. Body weight and tumor volume were measured on days 0, 2, 4, 6, 8, 10, 12, 14, 18, and 22 after grouping (N=6). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized CD28 mice in a dose-dependent manner. Figure 20 ).

[0293] SM2275 significantly inhibited the growth of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenografts in a humanized hCD28 / hPD-L1 mouse model. MC38-hEGFR-hPD-L1 cells were subcutaneously implanted, and animals were randomly assigned to groups when the tumor size reached approximately 90 mm³. Treatment with either the drug or SM2275 was administered every 2 days for 12 days, and body weight and tumor volume were measured on days 0, 2, 4, 6, 8, 10, 12, 15, and 18 (N=5). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized hCD28 / hPD-L1 mice. Figure 21 )

[0294] The pharmacokinetics of SM2275 were evaluated in cynomolgus monkeys. Eight healthy adult cynomolgus monkeys were randomly assigned to four groups based on body weight: a solvent control group and low, medium, and high dose groups of SM2275 (administered at doses of 5, 15, and 50 mg / kg, respectively). Each group consisted of two monkeys, with an equal number of males and females. The SM2275 administration groups received the corresponding concentration of SM2275 via intravenous infusion at a volume of 10 mL / kg, while the solvent control group received the solvent in the same manner. No dose-limiting toxicities were observed at the maximum dose of 50 mg / kg. SM2275 exhibited favorable pharmacokinetic properties with a terminal half-life of >60 hours, consistent with the albumin-mediated FcRn cycle in cynomolgus monkeys (Figure 22).

[0295] Unless otherwise stated, all figures describing the amount and characteristics of the expressed components, such as molecular weight, reaction conditions, etc., as used in the specification and claims should be understood to be modified 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 stated, the numerical parameters set forth in the specification and appended claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained according to the invention. At least, and not intended to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying conventional rounding techniques. Although the numerical ranges and parameters that illustrate the broad scope of the invention are approximate values, the values ​​set forth in the specific embodiments are reported as precisely as possible. However, any numerical value inherently contains a certain degree of error, which is necessarily caused by the standard deviation found in their respective test measurements.

[0296] The terms “a,” “an,” “the,” and similar designations used in the context of describing the invention (particularly in the context of the appended claims) should be interpreted to cover both the singular and plural forms, unless otherwise indicated herein or clearly contradicted by the context. The description of ranges of values ​​herein is intended only as a shorthand method for individually referring to each individual value falling within that range. Each individual value is incorporated into the specification as if it were individually described herein, unless otherwise stated herein or clearly contradicted by the context. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the otherwise claimed invention. No language in the specification should be construed as indicating any unclaimed element essential to the practice of the invention.

[0297] The grouping of alternative elements or embodiments of the invention disclosed herein should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of that group or other elements found herein. It is contemplated that one or more members of a group may be included in or removed from the group for convenience and / or patentability reasons. When any such inclusion or removal occurs, the specification is deemed to contain the modified group, thereby satisfying the written description of all Markush groups as used in the appended claims.

[0298] This document describes certain embodiments of the invention, including the best mode known to the inventors for carrying out the invention. Of course, variations of these described embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to appropriately employ such variations, and the inventors intend to practice the invention in ways other than those specifically described herein. Therefore, the invention includes all modifications and equivalents of the subject matter set forth in the appended claims as permitted by applicable law. Furthermore, unless otherwise stated herein or clearly contradicted by the context, the invention covers any combination of the foregoing elements in all possible variations.

[0299] The specific embodiments disclosed herein may be further limited in the claims using the language “consisting of…” or “substantially consisting of…”. When used in the claims, whether as submitted or added according to amendments, the transitional term “consisting of…” excludes any element, step, or ingredient not specified in the claims. The transitional term “substantially consisting of…” limits the scope of the claims to the specified materials or steps and those that do not substantially affect the essential and novel features. Embodiments of the claimed invention are inherently or explicitly described and implemented herein.

[0300] Furthermore, numerous references have been made to patents and print publications throughout this specification. Each of the above-cited references and print publications is incorporated herein by reference in its entirety.

[0301] Finally, it should be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the invention. Other modifications that may be made are also within the scope of the invention. Therefore, alternative configurations of the invention can be utilized in accordance with the teachings herein by way of example rather than limitation. Thus, the invention is not limited to what is precisely shown and described.

Claims

1. Multivalent and multispecific single-chain antibodies (MVSCAs) containing at least four binding specificities and having the following structure: A—B—C—D The A, B, C, and D regions each contain VHH domains specifically targeting EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E, respectively. The amino acid sequences of the A, B, C, and D VHH domains are linked by amino acid linker sequences.

2. The MVSCA of claim 1, wherein A, B and D each comprise a VHH domain specifically targeting EGFR, PD-L1 or HSA, and C comprises a VHH domain specific to 4-1BB, OX40, CD40, CD16A, CD28, LAG3 and CD3E.

3. The MVSCA of claim 1, wherein the VHH domain specifically targeting EGFR comprises an amino acid sequence selected from SEQ ID NO:134-161.

4. The MVSCA of claim 1, wherein the VHH domain specifically targeting PD-L1 comprises an amino acid sequence selected from SEQ ID NO:66-74.

5. The MVSCA of claim 1, wherein the VHH domain specifically targeting the HSA comprises an amino acid sequence selected from SEQ ID NO: 76-92 and 188.

6. The MVSCA of claim 1, wherein the VHH domain specifically targeting 4-1BB comprises an amino acid sequence selected from SEQ ID NO:120-132.

7. The MVSCA of claim 1, wherein the VHH domain specifically targeting OX40 comprises an amino acid sequence selected from SEQ ID NO: 101 or 102.

8. The MVSCA of claim 1, wherein the VHH domain specifically targeting CD40 comprises an amino acid sequence selected from SEQ ID NO:104-118.

9. The MVSCA of claim 1, wherein the VHH domain specifically targeting CD16A comprises an amino acid sequence selected from SEQ ID NO:94-99.

10. The MVSCA of claim 1, wherein the VHH domain specifically targeting CD3E comprises an amino acid sequence selected from SEQ ID NO:47-64.

11. The MVSCA of claim 1, wherein the VHH domain specifically targeting CD28 comprises an amino acid sequence selected from SEQ ID NO:25-45.

12. The MVSCA of claim 1, wherein the VHH domain specifically targeting LAG3 comprises an amino acid sequence selected from SEQ ID NO:174-187.

13. The MVSCA of any one of claims 1-12, wherein the order of the A, B, C and D structural domains is A—B—C—D.

14. The MVSCA of any one of claims 1-13, wherein the adapter comprises an amino acid sequence of one of SEQ ID NO: 1-23.

15. The MVSCA of claim 1, wherein one of its domains contains two VHH sequences specifically targeting the same target.

16. The MVSCA of claim 15, wherein one of the domains is a C domain.

17. The MVSCA of claim 15, wherein the two VHH sequences specifically target the same epitope on the target.

18. The MVSCA of claim 15, wherein the two VHH sequences have the same amino acid sequence.

19. The MVSCA of claim 15, wherein the two VHH sequences have different amino acid sequences.

20. The MVSCA of any one of claims 15-19, wherein the two VHH sequences are connected by a connector of one of SEQ ID NO: 1-23.

21. The MVSCA of any one of claims 2-20, wherein the MVSCA comprises specificity in the following order: EGFR—PD-L1—4-1BB—4-1BB—HAS.

22. The MVSCA of claim 21, comprising the amino acid sequence of SEQ ID NO:

165.

23. The MVSCA of any one of claims 2-20, wherein the MVSCA comprises specificities in the following order: EGFR—PD-L1—CD28—HSA.

24. The MVSCA of claim 23, comprising an amino acid sequence of one of SEQ ID NO: 167-172.

25. The MVSCA of any one of claims 2-20, wherein the MVSCA comprises specificities in the following order: EGFR—PD-L1—CD40—HSA.

26. The MVSCA of claim 25, comprising the amino acid sequence of SEQ ID NO:

162.

27. The MVSCA of any one of claims 2-20, wherein the MVSCA comprises specificities in the following order: EGFR—PD-L1—HSA—CD40.

28. The MVSCA of claim 27, comprising the amino acid sequence of SEQ ID NO:

163.

29. The MVSCA of any one of claims 1 or 3-20, wherein the MVSCA comprises specificities in the following order: CD16A—HSA—CD47—CD33.

30. The MVSCA of claim 29, wherein the MVSCA comprises the amino acid sequence of SEQ ID NO:

164.

31. A pharmaceutical composition comprising the MVSCA according to any one of claims 1-30.

32. A method of treating cancer, the method comprising administering to a subject in need the MVSCA of any one of claims 1-30 or the pharmaceutical composition of claim 31.

33. Use of the MVSCA of any one of claims 1-30 or the pharmaceutical composition of claim 31 for the treatment of cancer in a subject of need.

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