Multi-specific cancer-targeting antibodies

Abstract

Disclosed herein are multi-valent, multi-specific, single-chain antibodies with specificities for four antigens selected from EGFR, PD-L1, HSA, CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

Description

PCT Patent Application 1959708-00018 MULTI-SPECIFIC CANCER-TARGETING ANTIBODIES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional patent applications 63 / 596,555 filed November 6, 2023 and 63 / 601,931 filed November 22, 2023, the entire contents of both of which are incorporated by reference herein. SEQUENCE LISTING

[0002] This application contains a sequence listing having the filename 1959708- 00018WO_Sequence_Listing.xml, which is 185KB in size, and was created on November 5, 2024. The entire content of this sequence listing is incorporated herein by reference. SUMMARY

[0003] Disclosed herein are multivalent single chain antibodies (MVSCA), incorporating four or more VHH domains, each having specificity for one or more antigens. The MVSCA comprise at least four binding specificities and have the structure: A—B—C—D wherein each of the A, B, C, and D comprise VHH domains individually specific for EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E, and the amino acid sequences of A, B, C, and D VHH domains are connected by an amino acid linker sequence.

[0004] In some embodiments, each of the A, B, and D comprise VHH domains individually specific for EGFR, PD-L1, or HSA, and C comprises a VHH domain specific for 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E.

[0005] In some embodiments, the VHH domain specific for EGFR comprises an amino acid sequence selected from SEQ ID NOs:134-161. In some embodiments, the VHH domain specific for PD-L1 comprises an amino acid sequence selected from SEQ ID NOs:66-74. In some embodiments, the VHH domain specific for HSA comprises an amino acid sequence selected from SEQ ID NOs:76-92 and 188. In some embodiments, the VHH domain specific for 4-1BB comprises an amino acid sequence selected from SEQ ID NOs:120-132. In some embodiments, the VHH domain specific for OX40 comprises an amino acid sequence selected from SEQ ID NOs:101 or 102. In some embodiments, the VHH domain specific for CD40 comprises an amino acid sequence selected from SEQ ID NOs:104-118. In some embodiments, the VHH domain specific for CD16A comprises an amino acid sequence selected from SEQ ID NOs:94-99. In some embodiments, the VHH domain specific for CD3E comprises an amino acid sequence selected from SEQ ID NOs:47-64. In some embodiments,PCT Patent Application 1959708-00018 the VHH domain specific for CD28 comprises an amino acid sequence selected from SEQ ID NOs:25-45. In some embodiments, the VHH domain specific for LAG3 comprises an amino acid sequence selected from SEQ ID NOs:174-187.

[0006] In some embodiments, the order of the A, B, C, and D domains is A—B—C—D. In some embodiments, the linker comprises an amino acid sequence of one of SEQ ID NOs:1- 23.

[0007] In some embodiments, one domain comprises two VHH sequences specific for the same target. In some embodiments, the one domain is the C domain. In some embodiments, the two VHH sequences are specific for 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 NOs:1-23.

[0008] In some embodiments, the MVSCA comprises the following specificities in the following order: EGFR—PD-L1—4-1BB—4-1BB—HSA. In some embodiments, the MVSCA comprises an amino acid sequence of SEQ ID NO:165.

[0009] In some embodiments, the MVSCA comprises the following specificities in the following order: EGFR—PD-L1—CD28—HSA. In some embodiments, the MVSCA comprises an amino acid sequence of one of SEQ ID NOs:167-172.

[0010] In some embodiments, the MVSCA comprises the following specificities in the following order: EGFR—PD-L1—CD40—HSA. In some embodiments, the MVSCA comprises an amino acid sequence of SEQ ID NO:162.

[0011] In some embodiments, the MVSCA comprises the following specificities in the following order: EGFR—PD-L1—HSA—CD40. In some embodiments, the MVSCA comprises an amino acid sequence of SEQ ID NO:163.

[0012] In some embodiments, the MVSCA comprises the following specificities in the following order: CD16A—HSA—CD47—CD33. In some embodiments, the MVSCA comprises an amino acid sequence of SEQ ID NO:164.

[0013] Also disclosed herein are pharmaceutical compositions comprising an MVSCA disclosed herein.

[0014] Also disclosed herein are methods of treating cancer comprising administering an MVSCA or the pharmaceutical composition disclosed herein to a subject in need thereof.

[0015] Also disclosed herein are use of a MVSCA or a pharmaceutical composition disclosed herein for the treatment of cancer in a subject in need thereof.PCT Patent Application 1959708-00018 BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG.1 depicts the structure of SM2272 and SM2272A multi-specific single chain antibodies. The SM2272A sequence has a mutation in the anti-EGFR VHH complementary determining region (CDR3) binding domain which causes reduced binding affinity to human cancer cells relative to SM2272.

[0017] FIG. 2 depicts a mechanism of action of SM2272 multi-specific single chain antibody.

[0018] FIG.3 depicts comparison of SM2272 binding to normal versus cancer cells.

[0019] FIG.4A-D depicts ELISA binding of SM2272 to EGFR (FIG.4A), PD-L1 (FIG.4B), 4-1BB (FIG.4C), and HSA (FIG.4D).

[0020] FIG. 5A-C depict an ELISA blocking assay of SM2272 binding to EGF to EGFR (FIG.5A), PD-1 to PD-L1 (FIG.5B), and 4-1BB to 4-1BBL (FIG.5C).

[0021] FIG. 6A-C depicts flow cytometry analysis of binding of SM2272 and 2272A to A431 (FIG.6A), MB231 (FIG.6B), and HCC827 (FIG.6C) cells. SM2272 binds EGFR+PD- L1+ dual-expressing cells with increased affinity and specificity.

[0022] FIG.7A-C depicts that SM2272 binds cancer cells expressing both EGFR and PD- L1 with enhanced affinity. FIG.7A – A431 cells; FIG.7B – MB231 cells; FIG.7C – HCC827 cells.

[0023] FIG.8A-D depicts a blocking assay of SM2272 binding to PD-1 on A431 (FIG.8A), HCC827 (FIG.8B), and MB231 (FIG.9C) cells. FIG.8D depicts a luciferase assay of SM2272, SM2272A, and Keytruda®. Avidity-enhanced antibody binding enables SM2272 to selectively block PD-L1 and PD-1 interaction on the surface of EGFR+PD-L1+ double-positive cancer cells, thus allowing local (re-)activation of tumor-resident cytotoxic T cells without systemic immune cell activation.

[0024] FIG.9A-C depict reporter assay of binding of SM2272 and SM2272A to A431 (FIG. 9A), MB231 (FIG.9B), and HCC827 (FIG.9C) cells. Dual tumor antigen binding is required for efficient 4-1BB activation by SM2272.

[0025] FIG. 10 depicts inhibition of CT26-hEGFR / hPDL1 tumor growth by SM2272 in Balb / c-hPD-1-hPD-L1-h4-1BB mouse model.

[0026] FIG.11 depicts pharmacokinetic analysis of SM2272 in mice after a single 3 mg / kg dose.

[0027] FIG. 12 depicts the structure of SM2275-700 and SM2275-649 multispecific antibodies.PCT Patent Application 1959708-00018

[0028] FIG.13 depicts flow cytometry analysis of SM2275-649 and SM2275-700 to Jurkat cells.

[0029] FIG.14A-G depicts flow cytometry analysis of SM2275 binding to CHO-hEGFR- hPD-L1 expressing cells (FIG. 14A) MB231 (FIG. 14B), A431 (FIG. 14C), H292 (FIG.14D) and CHO-hCD28 expressing cells (FIG.14E), Jurkat cells (FIG.14F), and human T cells (FIG. 14G).

[0030] FIG. 15 depicts a blockade assay of PD-1 binding to PD-L1 on A 431 cells by SM2275 and SM2275A. Avidity-enhanced antibody-binding allows SM2275 to selectively inhibit PD-L1 activity on EGFR+PD-L1+ double-positive cells than single-target-binding. Mutated SM2275 (SM2275A) lacking EGFR-binding ability shows reduced PD1 / PDL1 blocking compared to SM2275 on cancer cell A431.

[0031] FIG 16 depicts enhanced inhibition of PD-L1 activity on EGFR+PD-L1+ double- positive cells in comparison to single-target-binding. Avidity enhanced the efficacy of PD-L1 signaling blockage on CHO-Dual-OKT3 than CHO-hPD-L1-OKT3 cells.

[0032] FIG.17 depicts a cell-based functional assay of SM2275 using Jurkat-PD1-NFAT luciferase reporter cells.

[0033] FIG.18A-D depicts cytokine release from isolated human PBMC cells induced by SM2275 and TGN1412. FIG.18A – TNF-α; FIG.18B – IL-6; FIG.18C – IL-2; FIG.18D – INF- γ.

[0034] FIG.19A-D depicts cytokine release in humanized hCD28 / hPD-L1 mouse model cells induced by SM2275 and TGN1412. FIG.19A – IL-2; FIG.19B – IL-6; FIG.19C – TNF- α; FIG.19D – INF-γ.

[0035] FIG.20 depicts inhibition of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenograft growth in the humanized hCD28 mouse model by SM2275. MC38-hEGFR-hPD-L1 cells were implanted subcutaneously. When the tumor size reached ~80 mm³, animals were randomly grouped and treated with vehicle or SM2275 every 2 days for 22 days, N=6.

[0036] FIG.21 depicts inhibition of MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenograft growth in the humanized hCD28 / hPD-L1 mouse model by SM2275. MC38-hEGFR-hPD-L1 cells were implanted subcutaneously. When the tumor size reached ~90 mm³, animals were randomly grouped and treated with vehicle or SM2275 every 2 days for 18 days, N=5.

[0037] FIG.22 depicts the pharmacokinetics of SM2275 in cynomolgus monkey following multiple doses by IV injection.PCT Patent Application 1959708-00018 DETAILED DESCRIPTION

[0038] Disclosed herein are multivalent, multi-specific, single chain antibodies (MVSCA), incorporating the variable domains of four or more VHH, wherein the antibodies have the structure: A—B—C—D wherein each of A, B, C, and D are VHH domains individually specific for EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E; and the amino acid sequences of A, B, C, and D VHH domains are connected by an amino acid linker sequence. VHH domains A, B, C, and D, and multiples of any domain, are individually linked by a cleavable or non-cleavable linker. Exemplary, non-limiting, linkers are disclosed in Table 1.

[0039] As used herein, the term VHH refers to the variable domain of heavy-chain antibodies and is the antigen binding fragment of heavy chain only antibodies.

[0040] As used herein, the term “immune cell engager” refers to a VHH domain specific for a molecule expressed on cells of the immune system and functions to attach an immune cell to PD- L1 and EGFR cancer cells (see FIG. 2). Exemplary, non-limiting, immune cell engager target molecules include 4-1BB, OX-40, CD40, 4-1BB, CD16A, CD3E, LAG3,and CD28.

[0041] In some embodiments the MVSCA are tetravalent and tetra-specific, that is having four VHH domains specific for four different antigens. In some embodiments, the MVSCA are pentavalent and tetra-specific, that is having five different VHH domains specific for four different antigens; two VHH domains are specific for the same antigen. In the embodiments wherein the MVSCA includes more than one VHH domain specific for the same antigen, the two VHH domains can be adjacent to each other or separated by a domain of a different specificity. In some embodiments, the VHH domains with specificity for the same antigen, the domains can be specific to different epitopes on the same antigen. In some embodiments, the VHH domains with specificity for the same antigen can have different amino acid sequences. Antigens

[0042] Programmed cell death 1 (PD-1), also called CD279, is a type I membrane protein encoded in humans by the PDCD1 gene. It has two ligands, PD-L1 and PD-L2. PD-L1, also called CD274 or B7 homolog 1 (B7-H1) is a 40 kDa type I transmembrane protein encoded in humans by the CD274 gene. PD-1 is expressed on the surface of activated T cells, and PD- L1 is expressed on the surface of antigen presenting cells (APCs), such as dendritic cells and macrophages. PD-L1 is also overexpressed in several tumors, including breast, lung, bladder, head and neck, and other cancers. When PD-L1 or PD-L2 bind to PD-1, an inhibitory signal isPCT Patent Application 1959708-00018 transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation.

[0043] The PD-1 pathway is a key immune-inhibitory mediator of T-cell exhaustion. PD-1 functions to limit the activity of already activated T cells in the periphery during the inflammatory response to infection in order to limit autoimmunity. Blockade of this pathway can lead to T- cell activation, expansion, and enhanced effector functions. As such, 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 PD-1:PD-L1 interactions has been shown to partially restore T cell function. (Sakuishi et al., JEM, 207:2187-2194, 2010). Methods and compositions for the treatment of persistent infections and cancer by inhibiting the PD-1 pathway are disclosed in WO 2006 / 133396. Human monoclonal antibodies to PD-L1 are described in WO 2007 / 005874, US2011 / 209230, US 8,217,149 and WO2014 / 055897.

[0044] Human serum albumin (HSA) is the most abundant protein in human blood plasma; it constitutes about half of serum protein. Albumin transports hormones, fatty acids, and other compounds, buffers pH, and maintains oncotic pressure, among other functions. Albumin is synthesized in the liver as preproalbumin, which has an N-terminal peptide that is removed before the nascent protein is released from the rough endoplasmic reticulum. The product, proalbumin, is in turn cleaved in the Golgi vesicles to produce the secreted albumin. It has a serum half-life of approximately 20 days. The long serum half-life of albumin is achieved in part by its size which prevents clearance through the kidney, and by its interaction with the neonatal Fc receptor (FcRn). Fusion to an anti-albumin sdAb (single domain antibody) has been used to increase the half-life of an antitumor single chain antibody from 1-2 hr to approximate 10 days.

[0045] CD16, also known as FcγRIII, is a cluster of differentiation 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; and participates in signal transduction. The most well-researched membrane receptor implicated in triggering lysis by NK cells, CD16 is a molecule of the immunoglobulin superfamily (IgSF) involved in antibody-dependent cellular cytotoxicity (ADCC). It can be used to isolate populations of specific immune cells through fluorescent-activated cell sorting (FACS) or magnetic-activated cell sorting, using antibodies directed towards CD16. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) in human NK cells. CD16 is required for ADCC processes carried out by human monocytes. In humans, monocytes expressing CD16 have a variety of ADCC capabilities in the presence of specific antibodies, and can kill primary leukemic cells, cancer cell lines, and cells infectedPCT Patent Application 1959708-00018 with hepatitis B virus. In addition, CD16 is able to mediate the direct killing of some virally infected and cancer cells without antibodies. After binding to ligands such as the conserved section of IgG antibodies, CD16 on human NK cells induce gene transcription of surface activation molecules such as IL-2-R (CD25) and inflammatory cytokines such as IFN-gamma and TNF. This CD16-induced expression of cytokine mRNA in NK cells is mediated by the nuclear factor of activated T cells (NFATp), a cyclosporin A (CsA)-sensitive factor that regulates the transcription of various cytokines. The upregulated expression of specific cytokine genes occurs via a CsA-sensitive and calcium-dependent mechanism.

[0046] CD16 plays a significant role in early activation of natural killer (NK) cells following vaccination. In addition, CD16 downregulation represents a possible way to moderate NK cell responses and maintain immune homeostasis in both T cell and antibody-dependent signaling pathways. In a normal, healthy individual, cross-linking of CD16 (FcγRIII) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) in NK cells. However, this pathway can also be targeted in cancerous or diseased cells by immunotherapy. After influenza vaccination, CD16 downregulation was associated with significant upregulation of influenza-specific plasma antibodies, and positively correlated with degranulation of NK cells.

[0047] CD16 is often used as an additional marker to reliably identify different subsets of human immune cells. Several other CD molecules, such as CD11b and CD33, are traditionally used as markers for human myeloid-derived suppressor cells (MDSCs). However, since these markers are also expressed on NK cells and all other cells derived from myelocytes, other markers are required, such as CD14 and CD15. Neutrophils are found to be CD14 low and CD15 high, whereas monocytes are CD14 high and CD15 low. While these two markers are sufficient to differentiate between neutrophils and monocytes, eosinophils have a similar CD15 expression to neutrophils. Therefore, CD16 is used as a further marker to identify neutrophils: mature neutrophils are CD16 high, while eosinophils and monocytes are both CD16 low. CD16 allows for distinction between these two types of granulocytes. Additionally, CD16 expression varies between the different stages of neutrophil development: neutrophil progenitors that have differentiation capacity are CD16 low, with increasing expression of CD16 in metamyelocytes, banded, and mature neutrophils, respectively.

[0048] With its expression on neutrophils, CD16 represents a possible target in cancer immunotherapy. Margetuximab, an Fc-optimized monoclonal antibody that recognizes the human epidermal growth factor receptor 2 (HER2) expressed on tumor cells in breast, bladder, and other solid tumor cancers, targets CD16A in preference to CD16B. In addition, CD16 could play a role in antibody-targeting cancer therapies. Bispecific antibody fragments, such as anti-CD19 / CD16, allow the targeting of immunotherapeutic drugs to the cancer cell. Anti-CD19 / CD16 diabodies have been shown to enhance the natural killer cell response to B-PCT Patent Application 1959708-00018 cell lymphomas. Furthermore, targeting extrinsic factors such as FasL or TRAIL to the tumor cell surface triggers death receptors, inducing apoptosis by both autocrine and paracrine processes.

[0049] OX40 (CD134; TNFRSF4) is a T cell costimulatory molecule of the tumor necrosis factor (TNF) receptor superfamily that coordinates with other co-stimulators (CD28, CD40, CD30, CD27, and 4-1BB) to manage the activation of the immune response. OX40 is upregulated on antigen activated CD4+and CD8+T cells with co-stimulation by CD40-CD40 ligand and CD28-B7. OX40 interactions with OX40 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 can promote the maintenance of, and recall, response in 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 disease.

[0050] 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. An antagonistic soluble human CD40 splice variant contains an alternate sequence within the extracellular and transmembrane domains and lacks a cytoplasmic domain. CD40 is expressed on the surface of B cells, dendritic cells, macrophages, monocytes and platelets, as well as endothelial and epithelial cells. Interaction of CD40 with 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 ligation by CD40 ligand promotes B cell activation and T cell-dependent humoral responses. CD40 serves multiple functions in both hematopoietic and epithelial cancers and is a target for tumor immunotherapy.

[0051] The epidermal growth factor receptor (EGFR) is a transmembrane protein that is a receptor for members of the epidermal growth factor (EGF) family of extracellular protein ligands. The EGFR is a member of the ErbB family of receptors, 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). In many cancer types, mutations affecting EGFR expression or activity could result in cancer. Epidermal growth factor receptor is a transmembrane protein that is activated by binding of its specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Deficient signaling of the EGFR and other receptor tyrosine kinases in humans is associated with diseases such as tumors, while over-expression is associated with the development of a wide variety of tumors. Interruption of EGFR signaling, either by blockingPCT Patent Application 1959708-00018 EGFR binding sites on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity, can prevent the growth of EGFR-expressing tumors and improve the patient's condition.

[0052] 4-1BB, also known as CD137 and TNFRSF9, is an approximately 30 kDa transmembrane glycoprotein in the TNF receptor superfamily. 4-1BB functions in the development and activation of multiple immune cells. Mature human 4-1BB consists of a 163 aa extracellular domain (ECD) with four TNFR cysteine-rich repeats (SEQ ID NO:54), a 27 aa transmembrane segment, and a 42 aa 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 populations of activated T cell including CD4+, CD8+, memory CD8+, NKT, and regulatory T cells as well as on myeloid 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 myeloid progenitor cells. This interaction co-stimulates the proliferation, activation, and / or survival of the 4-1BB expressing cell. It can also enhance the activation-induced cell death of repetitively stimulated T cells. Mice lacking 4-1BB show augmented T cell activation, perhaps due to its absence on regulatory T cells.4-1BB can associate with OX40 on activated T cells, forming a complex that responds to either ligand and inhibits Treg and CD8+ T cell proliferation. Reverse signaling through 4-1BB ligand inhibits the development of dendritic cells, B cells, and osteoclasts but supports mature dendritic cell survival and co-stimulates the proliferation and activation of mast cells.4-1BB activation enhances CD8+ T cell and NK cell mediated anti-tumor immunity. It also contributes to the development of inflammation in high fat diet-induced metabolic syndrome. Soluble forms of 4-1BB and 4-1BB ligand circulate at elevated levels in the serum of rheumatoid arthritis and hematologic cancer patients, respectively.

[0053] CD28 is a transmembrane glycoprotein expressed by T cells and some additional hematopoietic cells. It is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation and survival. T cell stimulation through CD28, in addition to the T-cell receptor (TCR), can provide a potent signal for the production of various interleukins, particularly IL-6. CD28 is part of the CD28 family receptors, which are a group of regulatory cell surface receptors expressed on immune cells. The CD28 family receptors are a subgroup of the immunoglobulin superfamily. Two family members, CD28 and ICOS, act as positive regulators of T cell function, while another three, BTLA, CTLA-4, and PD-1, act as inhibitors. 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 kDa1. The structure of CD28 comprises paired domains of the V-set immunoglobulinPCT Patent Application 1959708-00018 superfamilies (IgSF). CD28 bispecific antibodies are designed to co-stimulate T cells specifically within the tumor microenvironment. By bridging T cells to malignant cells expressing a selected tumor-associated antigen (TAA), CD28 bispecific antibodies deliver the so-called signal two to T cells, unleashing their full cytotoxic potential ².

[0054] CD3E is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains: a CD3γ chain, a 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 different 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 the field of cancer treatment. They are designed to redirect T cells to recognize and kill tumor cells. These antibodies bind to both CD3, a protein complex on T cells, and a tumor-associated antigen (TAA) 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. Multi-specific antibodies are another class of immunotherapies that can simultaneously bind to multiple targets. They can be engineered to target different antigens on cancer cells or to engage immune cells in addition to cancer cells. Both CD3 bispecific and multi-specific antibodies have shown promise in preclinical and clinical studies for the treatment of various cancers. They are being investigated as monotherapies or in combination with other immunomodulatory agents. The goal is to enhance anti-tumor immune responses and improve clinical outcomes for patients.

[0055] Lymphocyte-activation gene 3 (LAG-3), a 503 amino acid transmembrane protein, is an immune checkpoint receptor protein found on the cell surface of effector T cells and regulatory T cells (Tregs) and functions to control T cell response, activation and growth. LAG3 is a member of the immunoglobulin (Ig) superfamily. LAG3 binding to MHC class II molecules results in delivery of a negative signal to LAG3-expressing cells and down-regulates antigen- dependent CD4 and CD8 T cell responses. LAG3 negatively regulates the ability of T cells to proliferate, produce cytokines and lyse target cells, termed as 'exhaustion' of T cells. Since LAG3 plays an important role in tumor immunity and infectious immunity, it is an ideal target for immunotherapy. Blocking LAG3 with antagonists, including monoclonal antibodies, has been studied in treatments of cancer and chronic viral infections.

[0056] Patent applications PCT / US2020 / 053064, PCT / US2023 / 069262, PCT / US2024 / XXXXX (having Attorney Docket No.1959708.00017, filed on the same day as the present application and claiming priority to U.S. provisional applications 63 / 596,552 filed 11 / 06 / 2023 and 63 / 604,398 filed 11 / 30 / 2023) are incorporated by reference herein for all they disclose regarding antibodies specific for multiple antigens.PCT Patent Application 1959708-00018 Antibodies

[0057] Antibodies, and their use for treatment of diseases, are well known in the art. As used herein, the term "antibody" refers to a multimeric, multi-specific protein comprising one or more polypeptide chains that comprise antigen-binding sites. An antibody binds specifically to an antigen and may be able to modulate the biological activity of the antigen. As used herein, the term "antibody" can include "full length antibody" and "antibody fragments." The terms "binding site" or "antigen-binding site" as used herein denotes the region(s) of an antibody molecule to which a ligand actually binds. The term "antigen-binding site" comprises an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL), or in the case of heavy chain only antibodies, an antibody heavy chain variable region.

[0058] Antibody specificity refers to selective recognition of the antibody for a particular epitope of an antigen. Natural antibodies, for example, are monospecific. The term "monospecific" antibody as used herein denotes an antibody that has one or more binding sites each of which bind to the same epitope of the same antigen. A VHH domain may be referred to as means for binding a particular target antigen (such as, CD28, CD3E, OX-40, CD40, 4-1BB, HSA, PD-L1, CD16A, LAG3, and EFGR). Any of the various antibody structures, formats, or constructs disclosed herein that contains a VHH domain or is constructed to contain a VHH domain can thus be referred to an antibody comprising means for binding the indicated target. Some embodiments may specifically include one or more particular antibody structures, formats, or constructs. Other embodiments may specifically exclude one or more particular antibody structures, formats, or constructs.

[0059] As used herein “an antibody having specificity for”, “an antibody recognizing”, “an antibody having affinity for”, “an antibody with a binding site for”, and similar constructions may be used interchangeably.

[0060] As used herein, the terms “multi-specific antibodies” or “MVSCA” refers to antibodies that have four antigen-binding specificities. Multi-specific 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, MVSCA can include at least two copies of the same antigen-binding sequence, or two antigen-binding sequences which are specific for different epitopes on the same antigen (biparatopic) as long as the multi-specific antibody has specificity for at least EGFR, PD-L1, and HSA and at least one further antigen. In some embodiments, the MVSCA disclosed herein are single chain antibodies.

[0061] Disclosed herein are multivalent, multi-specific, MVSCA, incorporating the variable domains of four or more VHH, wherein the antibodies have the structure: A—B—C—DPCT Patent Application 1959708-00018 wherein (i) each of A, B, C and D are VHH domains individually specific for EGFR, PD-L1, HSA, CD28, CD3E, 4-1BB, OX-40, CD40, 4-1BB, LAG3, and CD16A; or (ii) each of A, B, and D are VHH domains individually specific for EGFR, PD-L1, or HSA and C comprises one or more VHH domains, both specific for CD28, CD3E, 4-1BB, OX-40, CD40, 4-1BB, LAG3, and CD16A.

[0062] In some embodiments, the A, B, C, and D domains are in the order A—B—C—D, however in some embodiments, the order of the domains can be different. Alternative domain orders include, but are not limited to, A—B—D—C.

[0063] VHH domains A, B, C, and D, and multiples of each domain, are individually linked by a cleavable or non-cleavable linker. Exemplary, non-limiting, linkers are disclosed in Table 1.

[0064] In some embodiments, A comprises a VHH domain specific for EGFR. In some embodiments, B comprises a VHH domain specific for PD-L1. In some embodiments, D comprises a VHH domain specific for HSA. In some embodiments, A comprises a VHH domain specific for EGFR, B comprises a VHH domain specific for PD-L1, and D comprises a VHH domain specific for HSA. In some embodiments, C comprises a single specificity selected from CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

[0065] In some embodiments, a domain comprises two VHH sequences, each specific for the same antigen and having the same amino acid sequence. In some embodiments, a domain comprises two VHH domains, each specific for the same antigen and having different amino acid sequences. If the two VHH having the same antigen specificity but having different amino acid sequences, the VHH have specificity for the same epitope on the antigen or specificity for different epitopes on the same antigen. In some embodiments, the domain comprising two VHH is the C domain.

[0066] The term “quadbodies” refers to antibodies which have four different antigen- binding specificities. In some embodiments, the quadbodies disclosed herein are specific for four specificities selected from EGFR, PD-L1, and HSA, and one of CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A. Amino acid sequences encoding antigen-binding portions of the quadbodies can be linked in various configurations. In some embodiments, the amino acid sequences encoding the antibody-binding portions of the quadbodies are connected by a linker as disclosed herein. In some embodiments two linkers are used, which can be the same of different.

[0067] The term "valent" as used herein denotes the presence of a specified number of binding sites in an antibody molecule. As such, the terms "tetravalent", “pentavalent”,PCT Patent Application 1959708-00018 "hexavalent", “heptavalent”, and “octavalent” denote the presence of four binding sites, five binding sites, six binding sites, seven binding sites, and eight binding sites, respectively, in an antibody molecule. The quadbodies disclosed herein are “tetravalent.” However, within the scope of the present disclosure are MVSCAs in which multiple antigen-binding sites bind the same antigen. The antigen-binding sites of MVSCAs can bind either the same epitope or different epitopes on the antigen. Similarly, by combining multiple monospecific binding sites with binding sites for one or more other specificities antibodies can be constructed in which the valency is of a higher order than the multi-specificity, for example, a pentavalent, tetra- specific antibody.

[0068] By "full length antibody" herein is meant the structure that constitutes the natural biological form of an antibody, including variable and constant regions. For example, in most mammals, including humans and mice, the full-length antibody of the IgG class is a tetramer and consists of two identical pairs of two immunoglobulin chains, each pair having one light and one heavy chain, each light chain comprising immunoglobulin domains VL and CL, and each heavy chain comprising immunoglobulin domains VH, CH1, CH2, and CH3. In some mammals, for example in camels and llamas, IgG antibodies can also consist of only two variable heavy chains, each heavy chain comprising a variable domain (VHH) attached to the Fc region (CH2 and CH3 domains).

[0069] Tetrameric antibodies are typically composed of two identical pairs of polypeptide chains, each pair having one "light" (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50-70 kDa). Each of the light and heavy chains are made up of two distinct regions, referred to as the variable and constant regions. For the IgG class of immunoglobulins, the heavy chain is composed of four immunoglobulin domains linked from N- to C-terminus in the order VH-CH1-CH2-CH3, referring to the heavy chain variable domain, heavy chain constant domain 1, heavy chain constant domain 2, and heavy chain constant domain 3 respectively (also referred to as VH- Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constant gamma 1 domain, constant gamma 2 domain, and constant gamma 3 domain respectively). The IgG light chain is composed of two immunoglobulin domains linked from N- to C-terminus in the order VL-CL, referring to the light chain variable domain and the light chain constant domain respectively. The constant regions show less sequence diversity and are responsible for binding a number of natural proteins to elicit important biochemical events.

[0070] The variable region of an antibody contains the antigen-binding determinants of the molecule, and thus determines the specificity of an antibody for its target antigen. The variable region is so named because it is the most distinct in sequence from other antibodies within the same class. In the variable region, three loops are gathered for each of the VPCT Patent Application 1959708-00018 domains of the heavy chain and light chain to form an antigen-binding site. Each of the loops is referred to as a complementarity-determining region (hereinafter referred to as a "CDR"), in which the variation in the amino acid sequence is most significant. There are six CDRs total, three each per heavy and light chain, designated VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3. The variable region outside of the CDRs is referred to as the framework (FR) region. Although not as diverse as the CDRs, sequence variability does occur in the FR region between different antibodies. Overall, this characteristic architecture of antibodies provides a stable scaffold (the FR region) upon which substantial antigen-binding diversity (the CDRs) can be explored by the immune system to obtain specificity for a broad array of antigens.

[0071] The genes encoding the immunoglobulin locus comprise multiple V region sequences along with shorter nucleotide sequences named “D” and “J” and it is the combination of the V, D, and J nucleotide sequence that give rise to the VH diversity.

[0072] Antibodies are grouped into classes, also referred to as isotypes, as determined genetically by the constant region. 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 define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG class is the most commonly used for therapeutic purposes. In humans this class comprises subclasses IgG1, IgG2, IgG3, and IgG4. In mice this class comprises subclasses IgG1, IgG2a, IgG2b, IgG3. IgM has subclasses, including, but not limited to, IgM1 and IgM2. IgA has several subclasses, including but not limited to IgA1 and IgA2. Thus, "isotype" as used herein is meant any of the classes or subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions. The known human immunoglobulin isotypes are IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE. The disclosed VHH antibodies, bispecific, and multi-specific antibodies can have constant regions comprising all, or part, of the above-described isotypes.

[0073] Also within the scope of the present disclosure are antibody fragments including, but are not limited to, (i) a Fab fragment comprising VL, CL, VH, and CH1 domains, (ii) a Fd fragment comprising VH and CH1 domains, (iii) a Fv fragment comprising VL and VH domains of a single antibody; (iv) a dAb fragment comprising a single variable region, (v) isolated CDR regions, (vi) F(ab')2fragment, a bivalent fragment comprising two linked Fab fragments, and (vii) a single chain Fv molecule (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen-binding site. Trivalent or tetravalent antibody fragments comprising variable domains of having three different specificities and linked by cleavable or uncleavable linkers are also disclosed. In certain embodiments, antibodies are produced by recombinant DNA techniques. In additionalPCT Patent Application 1959708-00018 embodiments, antibodies are produced by enzymatic or chemical cleavage of naturally occurring antibodies.

[0074] “Single-chain antibody” as used herein, refers to a fusion protein of the antigen- binding portions of antibodies (i.e., variable regions) generally connected by a linker peptide. Disclosed herein are multivalent multi-specific single chain antibodies. The MVSCA have specificity for EGFR, PD-L1, and HSA, and one of CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A.

[0075] By "humanized" antibody as used herein is meant an antibody comprising a human framework region (FR) and one or more complementarity determining regions (CDR's) from a non-human antibody. The non-human antibody providing the CDR's is called the "donor" and the human immunoglobulin providing the framework is called the "acceptor". In certain embodiments, humanization relies principally on the grafting of donor CDRs onto acceptor (human) VL or VH frameworks. This strategy is referred to as "CDR grafting". "Backmutation" of selected acceptor framework residues to the corresponding donor residues is often required to regain affinity that is lost in the initial grafted construct. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin, and often will typically comprise a human Fc region. Humanization or other methods of reducing the immunogenicity of nonhuman antibody variable regions may include resurfacing methods. In one embodiment, selection-based methods may be employed to humanize and / or affinity mature antibody variable regions, that is, to increase the affinity of the variable region for its target antigen. Other humanization methods may involve the grafting of only parts of the CDRs, including but not limited to methods described in US 6,797,492, incorporated by reference herein for all it discloses regarding CDR grafting. Structure-based methods may be employed for humanization and affinity maturation, for example as described in US 7,117,096, incorporated by reference herein for all it discloses regarding humanization and affinity maturation.

[0076] In various embodiments herein, the antibodies are VHH. Camelids (camels, dromedary, and llamas) contain, in addition to conventional heavy and light chain antibodies (2 light chains and 2 heavy chains in one antibody), two-chain antibodies (containing only variant heavy chains). The dimeric antibodies are coded for by a distinct set of VH segments referred to as VHH genes. The VH and VHH are interspersed in the genome (i.e., they appear mixed in between each other). The identification of an identical D segment in a VH and VHH cDNA suggests the common use of the D segment for VH and VHH. Natural VHH-containing antibodies are missing the entire CH1 domain of the constant region of the heavy chain. The exon coding for the CH1 domain is present in the genome but is spliced out due to the loss of a functional splice acceptor sequence at the 5' side of the CH1 exon. As a result, the VDJPCT Patent Application 1959708-00018 region is spliced onto the CH2 exon. When a VHH is recombined onto such constant regions (CH2, CH3), an antibody is produced in which the half-antibody is a single chain instead of a light chain / heavy chain pair (i.e., an antibody of two heavy chains without a light chain interaction). Binding of an antigen is different from that seen with a conventional antibody, but high affinity is achieved the same way, i.e., through hypermutation of the variable region and selection of the cells expressing such high affinity antibodies.

[0077] In an exemplary embodiment, the disclosed VHH are produced by immunizing a transgenic mouse in which endogenous murine antibody expression has been eliminated and camelid transgenes have 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 all they disclose regarding heavy chain only antibodies and their production in transgenic mice. In these embodiments, the transgenic VHH mice are immunized and the resulting primed spleen cells fused with murine myeloma cells to form hybridomas.

[0078] In other embodiments, VHH are produced by immunizing llamas with a desired antigen, and isolating sequencing encoding the VHH regions of resulting antigen-binding antibodies. In one embodiment, the VHH are isolated using a phage display library. See, for example, WO 91 / 17271; WO 92 / 01047; and WO 92 / 06204 (each of which is incorporated by reference in its entirety for description of making phage libraries).

[0079] Disclosed herein are multi-specific or multivalent antibodies in which two or more antigen-binding domains are joined in a single fusion protein. Multi-specific antibodies can take many forms including (i) multi-specific Fv fragments; (ii) a heavy chain of a first specificity having associated therewith (or fused thereto) a second VH domain having a second specificity; (iii) tetrameric monoclonal antibodies with a first specificity having associated therewith with a second VH domain having a second specificity, wherein the second VH domain is associated with a first VH domain); (iv) Fab fragments (VH-CH1 / VL-CL) of a first specificity having associated therewith a second VH domain with a second specificity. Exemplary Fab fragments include those in which the second VH sequence having the second specificity is associated with the C-terminus or the N-terminus of the first VH domain, or the C-terminus or the N-terminus of the first CH1 or first CL domains. In additional embodiments, VH sequences having a second and / or a third specificity (or more) can be associated with (or fused to) the C-terminus or the N-terminus of the first VH domain, or the C-terminus or the N- terminus of the first CH1 or first CL domains. In various embodiments any of these formats can include at least one of the herein disclosed VHH domains. Examples of configurations ofPCT Patent Application 1959708-00018 multi-specific antibodies can be found in WO2021 / 062361, which is incorporated by reference herein for all it discloses regarding configuration of multi-specific antibodies.

[0080] Multi-specific multivalent antibodies may include linker sequences linking a particular antigen-binding domain (such as a VH or VHH) to another antigen-binding domain and which allows for proper folding of the amino acid sequences to generate the desired three- dimensional conformation and antigen-binding profiles. Generally, a linker sequence will be a short amino acid sequence that provides sufficient space and flexibility between the domains for them to fold properly. The length and sequence of the linker can have substantial effects on the expression level, and structure of the MVSCA, and the binding affinity of the linked domains. The linker may also cause steric hindrance so as to facilitate binding to the target of each domain. The adjustable length linkers L2 and L4, for example, (see Table 1) can be used to optimize the MVSCA in terms of these parameters. Linkers L1, L2, and L4, for example, may be termed non-cleavable linker means, flexible linker means, or flexible, non-cleavable linker means. Suitable linkers include, but are not limited to, the linkers of Table 1 (SEQ ID Nos:38-28, 55, and 259). Further linkers will be known to the person of skill in the art.

[0081] When two copies of the same VHH domain are placed adjacent to each other in a MVSCA, they frequently interact detrimentally with each other. This can be avoided by interposing a relatively short and rigid linker between the two copies. In some embodiments, the short, rigid linker has the sequence AAA (L3, for example, in Table 1). Such linkers may be termed short, rigid linker means or non-cleavable short, rigid linker means.

[0082] When an anti-HSA domain-HSA complex is being used to generate a prodrug with respect to the binding activity of an adjacent binding domain, a cleavable linker should be interposed between the two domains. L11*3 through L11*18 (see Table 1) are examples of cleavable linkers of various lengths and susceptibility to cleavage by different proteases that can be used to optimize the MVSCA in terms of expression level, and structure of the MVSCA, the binding affinity of the linked domains, and cleavage. Linkers L11*3 through L11*18 may be termed cleavable linker means, flexible linker means, or flexible, cleavable linker means. Table 1. Non-cleavable and cleavable linker sequences Linker Reference Sequence Cleavable?PCT Patent Application 1959708-00018 Linker Reference Sequence Cleavable? L11*5 (SEQ ID NO:7)GGSGPLGLAGSRSAFGYes*

[0083] The binding domains and linkers described herein can be combined to create multifunctional MVSCA, adapted for the treatment of particular diseases. They can also be further combined with other binding domains. The MVSCA can also be referred to as comprising means for accomplishing the various functions associated with each component type of binding domain and / or comprising linker means for accomplishing their associated functions.

[0084] Also within the scope of the present disclosure are amino acid sequence variants of the multi-specific antibodies disclosed herein. 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 from, and / or insertions into and / or substitutions of, residues within the amino acid sequences of the antibodies of the examples herein. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the humanized or variant antibodies, such as changing the number or position of glycosylation sites.

[0085] A useful method for identification of certain residues or regions of the antibodies that are preferred locations for mutagenesis is called "alanine scanning mutagenesis". A residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral amino acid (most preferably alanine orPCT Patent Application 1959708-00018 polyalanine) to affect the interaction of the amino acids with antigen. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, alanine scanning or random mutagenesis is conducted at the target codon or region and the expressed antibody variants are screened for the desired activity.

[0086] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody disclosed herein with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecules include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0087] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 2 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened. Table 2 Original Exemplary Preferred Residue Substitutions SubstitutionsPCT Patent Application 1959708-00018 Original Exemplary Preferred Residue Substitutions Substitutions Ph F L V l Il Al T T

[0088] body are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; (3) Acidic: Asp, Glu; (4) Basic: Asn, Gin, His, Lys, Arg; (5) Residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0089] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0090] Any cysteine residue not involved in maintaining the proper conformation of the multi-specific antibodies also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0091] Another type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or camelid antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants is affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product ofPCT Patent Application 1959708-00018 M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identified hypervariable region residues contributing significantly to antigen-binding. Alternatively, or in addition, it may be beneficial to analyze a crystal structure of the antigen- antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0092] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody.

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

[0094] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0095] Nucleic acid molecules encoding amino acid sequence variants of the multi- specific antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of an antibody disclosed herein.PCT Patent Application 1959708-00018

[0096] Other modifications of the multi-specific antibodies are contemplated. For example, it may be desirable to modify the antibodies with respect to effector function, so as to enhance the effectiveness of the antibody in treating disease, for example. For example, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody- dependent cellular cytotoxicity (ADCC). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers. Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities.

[0097] In another embodiment, an antibody may be conjugated to a "receptor" (such streptavidin) for utilization in pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand" (e.g., avidin) which is conjugated to a cytotoxic agent (e.g., a radionuclide).

[0098] Covalent modifications of the multi-specific antibodies are also included within the scope of this disclosure. They may be made by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of the antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C-terminal residues. Exemplary covalent modifications of polypeptides are described in US5,534,615, specifically incorporated herein by reference for all it discloses regarding covalent modifications of polypeptides. An exemplary type of covalent modification of the antibody comprises linking the antibody to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in US4,640,835, US4,496,689, US4,301,144, US4,670,417, US4,791,192, or US4,179,337.

[0099] The multi-specific antibodies disclosed herein may be produced by recombinant means. Thus, disclosed herein are nucleic acids encoding the antibodies, expression vectors containing nucleic acids encoding the antibodies, and cells comprising the nucleic acid encoding the antibodies. Methods for recombinant production are widely known in the state of the art and comprise protein expression in prokaryotic and eukaryotic cells with subsequent isolation of the antibody and usually purification to a pharmaceutically acceptable purity. For the expression of the antibodies as aforementioned in a host cell, nucleic acids encoding the antibody sequences are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells like CHO cells, NS0 cells, SP2 / 0PCT Patent Application 1959708-00018 cells, HEK293 cells, COS cells, PER.C6 cells, yeast, or E. coli cells, and the antibody is recovered from the cells (supernatant or cells after lysis). It is to be understood that any recombinantly-expressed protein requires an initiator 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. Thus in some embodiments, the herein disclosed protein sequences are modified with such additional amino acids at their N-terminus. In some embodiments such N-terminal sequences are cleaved (in whole or in part) from the fully mature sequence, while in other embodiments they are retained.

[0100] Accordingly certain embodiments disclosed herein include a method for the preparation of a multi-specific antibody, comprising the steps of a) transforming a host cell with at least one expression vector comprising nucleic acid molecules encoding the antibody; b) culturing the host cell under conditions that allow synthesis of the antibody molecule; and c) recovering said antibody molecule from the culture.

[0101] The antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny which have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

[0103] The term "transformation" as used herein refers to process of transfer of a vectors / nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection can be carried out e.g. by the calcium phosphate precipitation method. However, other methods for introducing DNA into cells such as by nuclear injection or by protoplast fusion may also be used. If prokaryotic cells or cells which contain substantial cell wall constructions are used, e.g. one method of transfection is calcium treatment using calcium chloride.

[0104] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and / or to the process by which the transcribed mRNA (also referred to as transcript) is subsequently being translated into peptides, polypeptides, or proteins. The transcripts and the encoded polypeptides are collectively referred to as gene product. If thePCT Patent Application 1959708-00018 polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

[0105] A "vector" is a nucleic acid molecule, in particular self-replicating, which transfers an inserted nucleic acid molecule into and / or between host cells. The term includes vectors that function primarily for insertion of DNA or RNA into a cell (e.g., chromosomal integration), replication of vectors that function primarily for the replication of DNA or RNA, and expression vectors that function for transcription and / or translation of the DNA or RNA. Also included are vectors that provide more than one of the functions as described.

[0106] An "expression vector" is a polynucleotide which, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to yield a desired expression product.

[0107] The term "host cell" as used herein denotes any kind of cellular system which can be engineered to generate the antibodies disclosed herein. In one embodiment HEK293 cells and CHO cells are used as host cells.

[0108] The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

[0109] A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0110] Also disclosed herein are isolated nucleic acid encoding the multi-specific antibodies, vectors and host cells comprising the nucleic acids, and recombinant techniques for the production of the antibodies.

[0111] For recombinant production of the antibodies, the nucleic acid encoding it may be isolated and inserted into a replicable vector for further cloning (amplification of the DNA) orPCT Patent Application 1959708-00018 for expression. In some embodiments, the antibody may be produced by homologous recombination, e.g. as described in US 5,204,244, specifically incorporated herein by reference for all it discloses regarding antibody production. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The vector components generally 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, e.g., as described in US 5,534,615, specifically incorporated herein by reference for all it discloses regarding protein expression.

[0112] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa, and Streptomyces. One exemplary E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

[0113] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for multi-specific antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0114] Suitable host cells for the expression of glycosylated multi-specific antibodies are derived from multicellular organisms, including invertebrate cells such as plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-1 variantPCT Patent Application 1959708-00018 of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

[0115] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO); mouse sertoli cells (TM4); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[0116] Host cells are transformed with the above-described expression vectors for multi- specific antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0117] The host cells used to produce the multi-specific antibodies may 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 the host cells. In addition, US4,767,704; US4,657,866; US4,927,762; US4,560,655; or US5,122,469; WO 90 / 03430; WO 87 / 00195; or US Re.30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary 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 usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression and will be apparent to the ordinarily skilled artisan.PCT Patent Application 1959708-00018

[0118] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.

[0119] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite 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 species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2, or γ4 heavy chains, although Protein A can be used to purify antibody that do not have Fc regions. Protein G is useful for all mouse isotypes and for human γ3. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™ resin is useful for purification. Antibodies and antibody fragments disclosed herein can also be synthesized with histidine tags and affinity purified by metal affinity chromatography.

[0120] Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[0121] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25M salt).

[0122] Also disclosed herein are multi-specific single chain antibodies that are cleavable in a tumor microenvironment. In some embodiments, a tumor targeting domain (such as a tumor antigen-binding domain) or other functional domain is cleaved at the linker once the multi-specific single chain antibody reaches the tumor, in order to release the other domain(s) which bring about the therapeutic effect. The tumor microenvironment contains a multitude of proteases capable of cleaving the linkers disclosed herein. Non-limiting examples of tumor proteases include, but are not limited to, matrix metalloproteinases (e.g., MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP12, and MMP14), ADAM (a disintegrin and metalloproteinase; e.g., ADAM10 and ADAM17), a kallikrein-related peptidase (e.g., KLK1,PCT Patent Application 1959708-00018 KLK2, KLK3, and KLK6), a cathepsin (e.g., CTS-B, CTS-L, and CTS-S), a urokinase plasminogen activator (uPA), a hepsin (HPN), a matriptase, a legumain, or a dipeptidyl peptidase (e.g., DDP4). Antibody compositions

[0123] Also disclosed herein are pharmaceutical compositions comprising a multi-specific antibody disclosed herein in which the specificities include EGFR, PD-L1, HSA, and one or more of CD28, CD3E, OX-40, CD40, 4-1BB, LAG3, and CD16A. Also disclosed is the use of the antibodies described herein for the manufacture of a pharmaceutical composition. Also disclosed are methods of using the disclosed antibodies and pharmaceutical compositions comprising the antibodies for the treatment of various diseases and disorders.

[0124] A pharmaceutical composition is one intended and suitable for the treatment of disease in humans. That is, it provides overall beneficial effect and does not contain amounts of ingredients or contaminants that cause toxic or other undesirable effects unrelated to the provision of the beneficial effect. A pharmaceutical composition will contain one or more active agents and may further contain solvents, buffers, diluents, carriers, and other excipients to aid the administration, solubility, absorption or bioavailability, and or stability, etc. of the active agent(s) or overall composition.

[0125] The multi-specific antibodies disclosed herein may also be formulated in liposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in US4,485,045, US4,544,545, and US5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibodies can be conjugated to the liposomes via a disulfide interchange reaction.

[0126] As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. 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.

[0127] A composition disclosed herein can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. To administer the disclosed antibodies by certain routes of administration, it may be necessary to associate the antibodies with, or co-administerPCT Patent Application 1959708-00018 the antibodies with, a material to prevent its inactivation. For example, the antibodies may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

[0128] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intraocular, intravitreal, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0129] These compositions may also contain excipients such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0130] In some embodiments, the pharmaceutical composition comprising the antibody is a lyophilization cake. The lyophilization cake may further comprise bulking agents, buffers and / or salts, or other excipients, such as described herein. The lyophilized composition can be reconstituted by addition of sterile water or aqueous buffer, for administration to the patient.

[0131] Regardless of the route of administration selected, the disclosed antibodies, which may be used in a suitable hydrated form, and / or the pharmaceutical compositions containing the antibodies, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0132] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, otherPCT Patent Application 1959708-00018 drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Use of the Disclosed Antibodies

[0133] The disclosed antibodies are useful in medicine. The terms “treatment” “treating”, etc., refer to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder. Various embodiments may specifically include or exclude one or more of these modes of treatment.

[0134] Use of the herein disclosed antibodies in diagnostics and imaging is also contemplated.

[0135] Further, the term “treating” or “treatment” broadly includes any kind of treatment activity, including the diagnosis, mitigation, or prevention of disease, or aspect thereof, in man or other animals, or any activity that otherwise affects the structure or any function of the body of man or other animals. Treatment activity includes the administration of the medicaments, dosage forms, and pharmaceutical compositions described herein to a patient, especially according to the various methods of treatment disclosed herein, whether by a healthcare professional, the patient his / herself, or any other person. Treatment activities include the orders, instructions, and advice of healthcare professionals such as physicians, physician’s assistants, nurse practitioners, and the like, that are then acted upon by any other person including other healthcare professionals or the patient him / herself. This includes, for example, direction to the patient to undergo, or to a clinical laboratory to perform, a diagnostic procedure, such as for cancer diagnosis and staging, so that ultimately the patient may receive the benefit appropriate treatment. In some embodiments, the orders, instructions, and advice aspect of treatment activity can also include encouraging, inducing, or mandating that a particular medicament, or combination thereof, be chosen for treatment of a condition - and the medicament is actually used - by approving insurance coverage for the medicament,PCT Patent Application 1959708-00018 denying coverage for an alternative medicament, including the medicament on, or excluding an alternative medicament, from a drug formulary, or offering a financial incentive to use the medicament, as might be done by an insurance company or a pharmacy benefits management company, and the like. In some embodiments, treatment activity can also include encouraging, inducing, or mandating that a particular medicament be chosen for treatment of a condition - and the medicament is actually used - by a policy or practice standard as might be established by a hospital, clinic, health maintenance organization, medical practice or physicians group, and the like. All such orders, instructions, and advice are to be seen as conditioning receipt of the benefit of the treatment on compliance with the instruction. In some instances, a financial benefit is also received by the patient for compliance with such orders, instructions, and advice. In some instances, a financial benefit is also received by the healthcare professional for compliance with such orders, instructions, and advice.

[0136] The disclosed multivalent single chain antibodies having specificity for EGFR, PD- L1, HSA, CD47, CD33, and LAG3 are useful for treating cancer. Each MVSCA may be designed for treatment for a specific class of cancers based on the antigen-binding specificities included in the antibody or a single MVSCA may be useful for a variety of different types of cancer.

[0137] The present disclosure provides a method of treating cancer comprising administering to a patient in need of such treatment an effective amount of an antibody disclosed herein or a pharmaceutical composition comprising the antibody.

[0138] Examples of cancers which can be treated by the disclosed methods include acute lymphoblastic leukemia; acute myeloid leukemia; adrenocortical carcinoma; AIDS-related lymphoma; AIDS-related malignancies; anal cancer; astrocytoma; bile duct cancer, bladder cancer; bone cancer; brain stem glioma; brain tumor; breast cancer; bronchial adenomas / carcinoids; carcinoid tumor; islet cell carcinoma; carcinoma of unknown primary; central nervous system lymphoma; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; cervical cancer; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; cutaneous T-cell lymphoma; endometrial cancer, ependymoma; ovarian epithelial cancer; esophageal cancer; Ewing's family of tumors; extracranial germ cell tumor; intraocular melanoma; retinoblastoma; gallbladder cancer; gastric cancer; germ cell tumor; gestational trophoblastic tumor; hairy cell leukemia; head and neck cancer; hepatocellular cancer; 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; Waldenstrom's macroglobulinemia; malignant mesothelioma; malignant thymoma; medulloblastoma; melanoma; Merkel cell carcinoma; squamous neck cancer; multiple endocrine neoplasiaPCT Patent Application 1959708-00018 syndrome; multiple myeloma / plasma cell neoplasm; mycosis fungoides; myelodysplastic syndromes; nasopharyngeal cancer; neuroblastoma; oral cancer; oropharyngeal cancer; osteosarcoma; pancreatic cancer; parathyroid cancer; penile cancer; pheochromocytoma; pituitary tumor; pleuropulmonary blastoma; prostate cancer; rectal cancer; rhabdomyosarcoma; salivary gland cancer; soft tissue sarcoma; Sezary syndrome; skin cancer; squamous neck cancer; testicular cancer; thymoma; thyroid cancer; trophoblastic tumor; urethral cancer; uterine cancer; vaginal cancer; vulvar cancer; and Wilms' tumor.

[0139] The effectiveness of cancer therapy is typically measured in terms of "response." The techniques to monitor responses can be similar to the tests used to diagnose cancer such as, but not limited to: • A lump or tumor involving some lymph nodes can be felt and measured externally by physical examination. • Some internal cancer tumors will show up on an x-ray or CT scan and can be measured with a ruler. • Blood tests, including those that measure organ function can be performed. • A tumor marker test can be done for certain cancers.

[0140] Regardless of the test used, whether blood test, cell count, or tumor marker test, it is repeated at specific intervals so that the results can be compared to earlier tests of the same type.

[0141] Response to cancer treatment is defined several ways: • Complete response - all of the cancer or tumor disappears; there is no evidence of disease. Expression level of tumor marker (if applicable) may fall within the normal range. • Partial response - the cancer has shrunk by a percentage but disease remains. Levels of a tumor marker (if applicable) may have fallen (or increased, based on the tumor marker, as an indication of decreased tumor burden) but evidence of disease remains. • Stable disease - the cancer has neither grown nor shrunk; the amount of disease has not changed. A tumor marker (if applicable) has not changed significantly. • Disease progression - the cancer has grown; there is more disease now than before treatment. A tumor marker test (if applicable) shows that a tumor marker has risen.

[0142] Other measures of the efficacy of cancer treatment include intervals of overall survival (that is time to death from any cause, measured from diagnosis or from initiation of the treatment being evaluated)), cancer-free survival (that is, the length of time after aPCT Patent Application 1959708-00018 complete response cancer remains undetectable), and progression-free survival (that is, the length of time after disease stabilization or partial response that resumed tumor growth is not detectable).

[0143] There are two standard methods for the evaluation of solid cancer treatment response with regard to tumor size (tumor burden), the WHO and RECIST standards. These methods measure a solid tumor to compare a current tumor with past measurements or to compare changes with future measurements and to make changes in a treatment regimen. In the WHO method, the solid tumor’s long and short axes are measured with the product of these two measurements is then calculated; if there are multiple solid tumors, the sum of all the products is calculated. In the RECIST method, only the long axis is measured. If there are multiple solid tumors, the sum of all the long axes measurements is calculated. However, with lymph nodes, the short axis is measured instead of the long axis.

[0144] The following examples, sequence listing, and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention. EXAMPLES Example 1. Generation of VHH

[0145] Immunizations. Llamas were immunized at Pacific Immunology, Inc (Ramona, CA) following their standard protocols. The antigen of interest was mixed with Complete Freund's Adjuvant (day 0) or Incomplete Freund's Adjuvant (following immunizations) (Difco, BD Biosciences). Six subcutaneous injections per llama was performed at 50 μg / dose at biweekly intervals. At day 45, serum was collected from immunized llamas to determine antibody titers against the immunizing antigen by ELISA.

[0146] Phage library construction and selection. Peripheral blood mononuclear cells were prepared from blood samples at das 45 from immunized llamas using Ficoll-Paque™ Plus (GE Healthcare) according to the manufacturer's instructions. Total RNA was extracted from the peripheral blood mononuclear cells using RNeasy™ Midi Kit (Qiagen) following manufacturer instructions and used as a starting material for RT-PCR to amplify VHH encoding gene fragments. These fragments were cloned into a house-made phagemid vector, allowing production of recombinant phage particles, after infection with helper phage, which display the VHH as gene-lll fusion proteins on the surface of the phage particles. Phage was prepared according to standard methods and stored after filter sterilization at 4°C for further use. Phage libraries obtained from llamas were used for selection. In the selection, biotinylated recombinant antigen was incubated with the phage libraries and subsequently captured onPCT Patent Application 1959708-00018 streptavidin Dynabeads™ (Invitrogen). Following extensive washing, bound phages were eluted with 1 mg / ml trypsin. The output from the selections was rescued in E. coli TG1 cells. Colonies were picked and sequenced.

[0147] cDNAs encoding the positive VHH were fused with a C-terminal His-tag and cloned into expression vector SVT003 and transiently transfected in HEK293 cells. The positive VHH were purified by IMAC chromatography for in vitro functional assays.

[0148] VHH Specific for Human CD28

[0149] Antibodies were generated against the extracellular domain of human CD28 (Asn19-Pro152; Accession # P10747; SEQ ID NO:24): NKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNC DGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0150] VHH amino acid sequences specific for human CD28 are listed in Table 3. The VHH of Table 3 constitute means for binding human CD28. Table 3. Anti-CD28 VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) D28-12E10 QVQLVESGGGLVQPGGSLRLSCAASGFTFSGYWMFWVRQAPGKGLEWVSGI (SMB0649) NADGDFTGYADSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYFCVKGTSG I A I W I G I T G R I Y I W I TPCT Patent Application 1959708-00018 D28-F2 QVQLVESGGRLVQPGGSLRLSCAAYGFSFSTYAMNWVRQAPGKGLEWVSLI (SMB0705） NPSGSSTNYAHSVEGRFTISRDNAKNMLYLEMNSLKPEDTAVYYCAKYLFS SEQ ID NO:34 TNWGISPNDYWGQGTQVTVSS I C I L G A T A I G I G I W I W I W I W I W

[0151] The humanization of D28-12E10 was based on IGHV-74 human germline sequence and humanized D28-A1 was based on IGHV3-23 human germline sequence.

[0152] VHH Specific for Human CD3E

[0153] Antibodies were generated against the extracellular domain of human CD3E (amino acids 23-126; Accession # P07766; SEQ ID NO:46): DGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSEL EQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD

[0154] VHH amino acid sequences specific for CD3E are listed in Table 4. The VHH of Table 4 constitute means for binding human CD3E.PCT Patent Application 1959708-00018 Table 4. Anti-CD3E VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) G5-1B5 QVQLVESGGGLVEPGGSLRLSCVVSSRRFSAYAMAWYRQVPGKQHELVASMA (SMB0675N) STLATQYADDVNGRFTISRDNTRNTLYLQMNSLQSHDTGVYYCNRRDVGDVG SEQ ID NO:47 WPWGQGTQVTVSS T G S L D S N R S E S L A R Y Y D S S S A D S L T D T K I R S GPCT Patent Application 1959708-00018 54D11 QVQLVESGGGLVQPGGSLRLSCVVSGGTFSSYPMGWFRQAPGKEREFVAAIS (SMB0681N) WSGGRTYYADSVKGRFTIARDNAKNTVYLQMNSLKPEDTAVYYCNAAAQVDR SEQ ID NO:64 PEYDYWGQGTQVTVSS

[0155] VHH Specific for Human PD-L1

[0156] Antibodies were generated against the extracellular domain of human PD-L1 (amino acids 19-238; Accession # Q9NZQ7; SEQ ID NO:65): FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRAR LLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELT CQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTA ELVIPELPLAHPPNER

[0157] VHH amino acid sequences specific for human PD-L1 are listed in Table 5. The VHH of Table 5 constitute means for binding human PD-L1. Table 5. Anti-PD-L1 VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) PL2 QVQLVESGGGLVQAGGSLRLSCAVSGLTLSRYTMAWFRQAPGKEREFVAAIIRN SEQ ID NO:66 SVSTFHEESVKGRFTISRDNVKNMMYLQMNTLKPEDTAVYYCATNVGPTGGFSLPCT Patent Application 1959708-00018

[0158] VHH Specific for Human HSA

[0159] Antibodies were generated against HSA (SEQ ID NO:75): MKWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNL PRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHG DLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVC KNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEP QNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDY LSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKE RQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0160] VHH amino acid sequences specific for human HSA are listed in Table 6. The VHH of Table 6 constitute means for binding human HSA. Table 6. Anti-HSA VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) HS2 EVQLVESGGGLVQAGGSLRLSCAASGRAFSSYAMGWFRQAPRKEREFVATISL SEQ ID NO:76 SGGYTYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAAGESSSRQDPCT Patent Application 1959708-00018 MSA4 EVQLVEFGGGLVQAGGSLRLSCAASGRTDSAYRMGWFRQAPGKEREFVSAINW SEQ ID NO:87 SDGRTVYLDSVKGRFTISRDNAKNMVYLQMNSLKPEDTAVYYCAADPDSRLYY TVPQNYDYWGQGTQVTVSS

[0161] VHH Specific for Human CD16A

[0162] Antibodies were generated against human CD16A (SEQ ID NO:93): MWQLLLPTALLLLVSAGMRTEDLPKAVVFLEPQWYRVLEKDSVTLKCQGAYSPEDNSTQWFHNESLIS SQASSYFIDAATVDDSGEYRCQTNLSTLSDPVQLEVHIGWLLLQAPRWVFKEEDPIHLRCHSWKNTAL HKVTYLQNGKGRKYFHHNSDFYIPKATLKDSGSYFCRGLFGSKNVSSETVNITITQGLAVSTISSFFP PGYQVSFCLVMVLLFAVDTGLYFSVKTNIRSSTRDWKDHKFKWRKDPQDK

[0163] VHH amino acid sequences specific for human CD16A are listed in Table 7. The VHH of Table 7 constitute means for binding human CD16A. Table 7. Anti-CD16A VHH sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) CD16F1 EVQLVESGGGLVQPGGSLRLSCAVSGSLFSARVMGWYRQAPGKQRELVAAIT SEQ ID NO:94 SGVRTDYADSVKGRFTISRDNAKRAVYLQMNSLKPEDTAVYYCNVNLYNTGNPCT Patent Application 1959708-00018 hCD16A-E11 QVQLVESGGGLVQPGGSLRLSCRASGFTFSNHAMSWVRQAPGKGLEWVSEIS SEQ ID NO:99 FNGHATRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCRKGWNATP QIGERGRGTQVTVSS

[0165] Antibodies were generated against the extracellular domain of human OX40 (amino acids 29-216; Accession # P43489; SEQ ID NO:100): LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERK QLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDA ICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVA

[0166] VHH amino acid sequences specific for human OX40 are listed in Table 8. The VHH of Table 8 constitute means for binding human OX40. Table 8. Anti-OX40 VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) pgXX88-101 QVQLVESGGGLMQPGGSLRLSCAASGGPLSSYAMAWFRQAPGKEREFVTFIS SEQ ID NO:101 YNGGRIFYADSVKGRFTISRDNAKNTVSLQMNSLKPEDTAVYYCAGGLRSSN S N

[0167] VHH Specific for Human CD40

[0168] Antibodies were generated against the extracellular domain of human CD40 (amino acids 21-193; Accession # P25942-1; SEQ ID NO:103): EPPTACREKQYLINSQCCSLCQPGQKLVSDCTEFTETECLPCGESEFLDTWNRETHCHQHKYCDPNLG LRVQQKGTSETDTICTCEEGWHCTSEACESCVLHRSCSPGFGVKQIATGVSDTICEPCPVGFFSNVSS AFEKCHPWTSCETKDLVVQQAGTNKTDVVCGPQDRLR

[0169] VHH amino acid sequences specific for human CD40 are listed in Table 9. The VHH of Table 9 constitute means for binding human CD40. Table 9. Anti-CD40 VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) pgDD40-SZ-1 QVQLVESGGALAQPGGSLRLSCEASTNVRSVNAMAWYRQGPGKPRELVANIT SEQ ID NO:104 SDDATYYADSVKGRFTLSREGARNTVYLQMNSLKPEDTAVYQCNVHYTSWGG T G S VPCT Patent Application 1959708-00018 pgDD40-SZ-5 QVQLVESGGALAQPGGSLRLSCEASENVRSVNAMAWYRQGPGKPRELVANIT SEQ ID NO:107 SGDATYYADSVKGRFTLSREGARNTVYLQMNSLKPEDTAVYQCNVHYTSWGG IRRDYWGRGTQVTVSS T G D S S V S C E S T S G P N P S V S V E S

[0170] VHH Specific for Human 4-1BB

[0171] Antibodies were generated against the extracellular domain of human 4-1BB (amino acids 24-183; Accession # Q07011; SEQ ID NO:119): LQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPG FHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCG PSPADLSPGASSVTPPAPAREPGH

[0172] VHH amino acid sequences specific for human 4-1BB are listed in Table 10. The VHH of Table 10 constitute means for binding human 4-1BB.PCT Patent Application 1959708-00018 Table 10. Anti-4-1BB VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) 4-1BB-1 QVQLVESGGGLVQAGGSLRLSCAASENIFSFNAMAWYRQAPGKQRELVALI SEQ ID NO:120 TGDGTTKYADSVKGRFTIARLNAKNTVNLEMNSLKPEDTAVYTCAALRNSD RTSWGQGTQVTVSS I A V G I S I G I S I T I D I I I D I D I I I I

[0173] VHH Specific for Human EGFR

[0174] Antibodies were generated against the extracellular domain of human EGFR (amino acids 1-645; Accession # CAA25240; SEQ ID NO:133): MRPSGTAGAALLALLAALCPASRALEEKKVCQGTSNKLTQLGTFEDHFLSLQRMFNNCEVVLGNLEIT YVQRNYDLSFLKTIQEVAGYVLIALNTVERIPLENLQIIRGNMYYENSYALAVLSNYDANKTGLKELP MRNLQEILHGAVRFSNNPALCNVESIQWRDIVSSDFLSNMSMDFQNHLGSCQKCDPSCPNGSCWGAGE ENCQKLTKIICAQQCSGRCRGKSPSDCCHNQCAAGCTGPRESDCLVCRKFRDEATCKDTCPPLMLYNPPCT Patent Application 1959708-00018 TTYQMDVNPEGKYSFGATCVKKCPRNYVVTDHGSCVRACGADSYEMEEDGVRKCKKCEGPCRKVCNGI GIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQ AWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTIN WKKLFGTSGQKTKIISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCKLLEG EPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYAD AGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPS

[0175] VHH amino acid sequences specific for human EGFR are listed in Table 11. The VHH of Table 11 constitute means for binding human EGFR. Table 11. Anti-EGFR VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) pgEG1 QVQLVESGGGLVQPGGSLRLSCAASGIIFEAEAMGWVRQAPGKQRESVAFI SEQ ID NO:134 GSGGNTNVGWSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCNTHPLRS I G I S I D V Y I P I G F G L P I Y I Y I T V RPCT Patent Application 1959708-00018 pgEG-SX56 QVQLVESGGGFVQAGGSLRLSCAASGRFLSIADMDWYRRVPGKQRELVASI SEQ ID NO:147 TRAGDTALEDSVKGRFTISRDNAKNTVHLQMNSVKPEDTAVYYCRADVTRS GTPYFEVWGQGTLVTVSS F G I G I Y I Y I Y I Y I Y F G F G F G F G F G I Y I Y

[0176] VHH Specific for Human LAG3

[0177] Antibodies were generated against the extracellular domain of human LAG3 (amino acids 19-238; P18627; SEQ ID NO:173): VPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRP RRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRL RLGQASMTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPCT Patent Application 1959708-00018 PMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTP PGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGKLL CEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQ

[0178] VHH amino acid sequences specific for human LAG3 are listed in Table 12. The VHH of Table 12 constitute means for binding human LAG3. Table 12. Anti-LAG3 VHH Sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) LG2 QVQLVESGGGLVQPGGSLRLSCAASGTIFSINAMGWYRQAPGKQRELVAIVT SEQ ID NO:174 FGGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCSARNVQSPV YHLAVWG GTLVTVSS T Y D I D I S C I C S A K D N Q S S D I D I S S N SPCT Patent Application 1959708-00018 Example 2. Multi-Specific Antibodies

[0179] Multi-specific single chain antibodies specific for EGFR, PD-L1, CD40, and HSA are depicted in Table 13. Table 13. Multi-specific antibodies (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) 2270, pgEG-SX-40-403-F3Y QVQLVESGGGLVQPGGSLRLSCTASGIRFSFYTLGWYRQAPGKG – hhPL14-- pgDD40-SZ-3- RELVAEIYSSDNAKYADSVKGRFTISRDNAKNSLYLQMNSLRAE 4F1GS15 MSA41 DTAVYYCNVRATDYWGPGTQVTVSSGGGGSGGGSEVQLVESGGG G R R S R V G P G E G G R R S T S A P Q E S I Y E K R Q K R G Q M S I YPCT Patent Application 1959708-00018 Example 2. SM2272, A Tetra-Specific, Dual Tumor-Antigen-Targeting, Conditional 4- 1BB Agonist with Enhanced Cancer Cell Selectivity

[0180] While 4-1BB agonistic monoclonal antibodies demonstrated promising anti-tumor efficacy in preclinical animal models, their clinical development has been hindered by fatal "on-target, off-tumor" hepatotoxicity. To improve the safety profile and therapeutic window of 4-1BB agonists, we developed SM2272, a tetra-specific VHH antibody against EGFR, PD-L1, 4-1BB and HSA, for the treatment of EGFR+PD-L1+ solid tumors. Preclinical study showed that SM2272 conditionally activated 4-1BB-expressing immune cells upon co-engagement of EGFR+PDL1+ cancer cells. It was well-tolerated in vivo and exhibited robust anti-tumor activity in syngeneic tumor transplantation models.

[0181] SM2272 features two monovalent, low-affinity binding domains for EGFR and PD- L1, along with bivalent high-affinity 4-1BB binding domains arranged in a tandem format, followed by a half-life extending HSA binding domain. Binding affinity and specificity of SM2272 to recombinant human EGFR, PD-L1, 4-1BB and HSA were confirmed by bio-layer interferometry and ELISA. Differential binding affinities of SM2272 to EGFR / PD-L1 single- expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity- enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single- positive cells was analyzed via competitive ligand binding assay. EGFR and PD-L1 dual- target-dependent 4-1BB activation was confirmed using a 4-1BB-NFκB-Luc reporter assay and primary human CD8+ T cell IFNγ release assay. In vivo efficacy against EGFR+PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of CT26-hEGFR / hPD-L1 cell line into Balb / c-h4-1BB humanized mice.

[0182] The objective was to develop a VHH-based dual-tumor-antigen-specific, conditional 4-1BB agonist to improve antibody tumor-targeting specificity. This strategy involves conditional 4-1BB activation upon dual tumor antigen (EGFR and PD-L1) binding to minimize systemic immune activation. The strategy also enhances the efficacy of PD-L1 signaling blockage on tumor cells expressing both EGFR and PD-L1 (FIGs.2 and 3). SM2272 is a humanized pentavalent tetra-specific VHH antibody against EGFR, PD-L1, 4-1BB, and HSA with optimized pharmacokinetic properties. A graphic of the structure of SM2272 and SM2272A are presented in FIG.1 and the sequences of these two antibodies is presented in Table 14.PCT Patent Application 1959708-00018 Table 14. SM2272 and SM2272A sequences (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) SM2272 QVQLVESGGGLVQPGGSLRLSCTASGIRFSFYTLGWYRQAPGKG RELVAEIYSSDNAKYADSVKGRFTISRDNAKNSLYLQMNSLRAE EGFR-PD-L1-41BBbiv-HSA DTAVYYCNVRATDYWGPGTQVTVSSGGGGSGGGSEVQLVESGGG G R A T T F Y S M T T G E G G R A T T F Y S M T T

[0183] SM2272 binds EGFR+PD-L1+ dual-expressing cells with increased affinity and specificity. SM2272 binds EGFR+ and PD-L1+ double-expressing cells with increased affinity compared to single-target expressing cells. Monovalent EGFR, PDL1, and HSA modules bind recombinant proteins with intermediate affinity. Bivalent 4-1BB module binds target with high affinity.

[0184] SM2272 binds to EGFR+PD-L1+double positive cancer cells with significantly increase affinity (>500 fold) than to EGFR+or PD-L1+single positive normal cells, improving antibody specificity. SM2272 blocks PD-L1 with significantly lower IC50 (> 50-fold) on EGFR+PD-L1+double positive cancer cells than on PD-L1+single positive normal cells, allowing TME-restricted T cell reactivation. SM2272 conditionally stimulates 4-1BB+tumor infiltrating T / NK cells upon cancer cell engagement, synergizing with PD-L1 inhibition to further boost anti-tumor immunity while avoiding systemic toxicity.PCT Patent Application 1959708-00018

[0185] SM2272 cross reacts with cynomolgus monkey targets but not rodent targets. SM2272 binds recombinant human EGFR and PD-L1 with modest affinity and 4-1BB with high affinity (Table 15 and FIG.4). Table 15. Bio-Layer Interferometry (BLI) binding affinity analysis of SM2272 Molecule Target koff(1 / s) kon(1 / Ms) KD(M) SM2272 rhEGFR-hFc 6.97E-03 2.36E+05 2.95E-08oc s ga g o eco a u a a -L1 at nanomolar concentration (FIG. 5A and 5B). SM2272 does not block 4-1BB and 4-1BBL interaction (FIG.5C). SM2272 does not block HSA binding to FcRN.

[0187] Mutated SM2272 (SM2272A) lacking EGFR-binding ability shows reduced binding affinity towards human cancer cell lines compared to SM2272, suggesting dual-target-binding is required for effective engagement of SM2272 with target cells, which improves antibody selectivity against EGFR+PD-L1+ cancer cells (FIG.6A-C).

[0188] Monovalent EGFR and PDL1 modules bind cell surface targets with intermediate affinity as assayed by flow cytometry. Bivalent 4-1BB module also binds cell surface target with high affinity. SM2272 selectively binds EGFR+PD-L1+ double-positive cancer cells with enhanced affinity compared to single-target-expressing normal tissues.

[0189] Avidity-enhanced antibody-binding allows SM2272 to selectively inhibits PD-L1 activity on EGFR+PD-L1+ double-positive cells (FIG. 7A-C). Dual-target-binding SM2272 shows significantly enhanced ability to block PD-1 and PD-L1 interaction than single-target- binding SM2272A.

[0190] Avidity-enhanced PD-L1 blockade allows local T cell reactivation. Avidity- enhanced antibody-binding allows SM2272 to selectively inhibits PD-L1 activity on EGFR+PD- L1+ double-positive cells (FIG. 8A-D). Dual-target-binding SM2272 shows significantly enhanced ability to block PD-1 and PD-L1 interaction than single-target-binding SM2272A.

[0191] Avidity-enhanced antibody binding enables SM2272 to selectively block PD-L1 and PD-1 interaction on the surface of EGFR+PD-L1+ double-positive cancer cells, thus allowing local (re-)activation of tumor-resident cytotoxic T cells without systemic immune cell activation.

[0192] SM2272 conditionally activates HEK293-4-1BB-NFκB-Luc reporter cell line upon engaging EGFR and PD-L1 dual-expressing cells (FIG.9A-C). SM2272 shows significantlyPCT Patent Application 1959708-00018 enhanced ability to activates HEK293-4-1BB-NFκB-Luc Reporter cell line compared to SM2272A in the presence of a wide-range of EGFR and PD-L1 dual-expressing human cancer cell lines. In the absence of target cell, SM2272 does not activate 4-1BB.

[0193] SM2272 conditionally activates 4-1BB-expressing immune cells only upon dual- TAA engagement, thus avoiding hepatotoxicity associated with global 4-1BB activation.

[0194] SM2272 significantly inhibits CT26-hEGFR-hPD-L1 (mPD-L1null) tumor xenograft growth in humanized BALB / c-hPD1 / hPD-L1 / h4-1BB mouse model (FIG. 10). Cancer cells were implanted subcutaneously, when tumor size reaches ~80 mm³, animals were randomly grouped and treated with vehicle or SM2272 every 3 days.

[0195] The pharmacokinetics of SM2272 was evaluated in mice after a single 3 mg / kg dose (FIG.11).

[0196] SM2272 exhibits modest binding affinity to CHO-hEGFR and CHO-hPD-L1 cells and high affinity to CHO-4-1BB cells. Notably, SM2272 displays significantly increased affinity for cancer cells co-expressing EGFR and PD-L1 compared to single-expressing cells. This leads to an avidity-enhanced PD-L1 blockade specifically on the surface of EGFR+PD-L1+ double-positive cancer cells, enabling localized reactivation of tumor-infiltrating T cells without systemic immune toxicity. In the presence of SM2272, EGFR and PD-L1 double-positive cancer cells induce significantly enhanced 4-1BB activation and cytokine secretion compared to single-antigen-expressing cells. In a syngeneic tumor transplantation model, SM2272 effectively inhibits the growth of CT26-hEGFR / hPD-L1 tumors in humanized Balb / c-h4-1BB mice, with no apparent hepatic or immune toxicity.

[0197] SM2272 is a novel dual-TAA-targeting, conditional 4-1BB agonistic antibody. It demonstrated promising efficacy and favorable safety profile in preclinical models through a unique mechanism of action, differentiating it from existing monoclonal and bispecific antibodies. Example 3. SM2275 Tetra-Specific, Dual Tumor-Antigen-Targeting, Conditional CD28 Agonist with Enhanced Cancer Cell Selectivity

[0198] While CD28 agonistic monoclonal antibodies demonstrated promising anti-tumor efficacy in preclinical animal models, their clinical development has been hindered by a series of acute and serious adverse events in phase I trial. To improve the safety profile and therapeutic window of CD28 agonists, we developed SM2275, a tetra-specific VHH antibody against EGFR, PD-L1, CD28 and HSA, for the treatment of EGFR+PD-L1+ solid tumors. Preclinical study showed that SM2275 conditionally activated CD28-expressing immune cellsPCT Patent Application 1959708-00018 upon co-engagement of EGFR+PDL1+ cancer cells. It was well-tolerated in vivo and exhibited robust anti-tumor activity in syngeneic tumor transplantation models.

[0199] SM2275 was developed using Starmab’s proprietary QuadbodyTMmultifunctional VHH antibody platform. It features two monovalent, low-affinity binding domains for EGFR and PD-L1, along with a sub nanomolar-affinity CD28 binding domain arranged in a tandem format, followed by a half-life extending HSA binding domain. Binding affinity and specificity of SM2275 to recombinant human EGFR, PD-L1, CD28, and HSA were confirmed by bio-layer interferometry and ELISA. Differential binding affinities of SM2275 to EGFR / PD-L1 single- expressing versus double-expressing cell lines were analyzed by flow cytometry. Avidity- enhanced blockade of PD-1 on EGFR+PD-L1+ double-positive compared to PD-L1+ single- positive cells was analyzed via competitive ligand binding assay. EGFR and PD-L1 dual- target-dependent CD28 activation was confirmed using a Jurkat IL2 promoter-Luc reporter assay and primary human T cell IL2 release assay. In vivo efficacy against EGFR+PD-L1+ double-positive human tumors were evaluated using a syngeneic tumor transplantation model of MC38-hEGFR / hPDL1 cell line into C57-hPD-l1 and hCD28 humanized mice.

[0200] Multi-specific antibodies incorporating the anti-CD28 VHH of Example 1 are presented in Figure 12 and Table 16. Table 16. Multi-specific antibodies SM2275 with different linkers (CDRs are underlined [CD1], bolded [CDR2], or double underlined [CDR3] for each sequence) hEG403-F3Y- QVQLVESGGGLVQPGGSLRLSCTASGIRFSFYTLGWYRQAPGKGRELVAEI hhPL14-KPG4- YSSDNAKYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCNVRATDY Y E S R G R E I Y W A L S G F APCT Patent Application 1959708-00018 E(G4SG3S)P- QVQLVESGGGLVQPGGSLRLSCTASGIRFSFYTLGWYRQAPGKGRELVAEI KPgggg-649- YSSDNAKYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCNVRATDY MSA4-1 WGPGTQVTVGGGGSGGSEVQLVESGGGLVQPGGSLRLSCAASGSTSGIYDM L L T S G L I Y Y E S R V Y M I Y W A L S Q A Q I Y M L L T G D Y

[0201] Following the flow cytometric methods described above, the binding affinity for multi-specific antibodies SM2275-679 (Low hCD28 binding affinity) and SM2275-700 (high hCD28 binding affinity) was determined as EC50in FIGs.13 and 14 and Table 17.

[0202] The cell-based functional assay indicated that SM2275-649 and SM2275-700 are fully functional multi-specific VHH antibodies (Table 5). SM2275 cross reacts with cynomolgus monkey targets at the same KD but does not bind to rodent targets.PCT Patent Application 1959708-00018 Table 17. BLI binding affinity analysis of SM2275-700 and SM2275-649 Target Koff (1 / s) Kon (1 / Ms) KD (M) rEGFR 4.47x10-32.36x1051.89x10-83 5 8ate affinity as assayed by flow cytometry. SM2275 selectively binds EGFR+PD-L1+ double- positive cancer cells with enhanced affinity compared to single-target-expressing normal tissues. Avidity-enhanced antibody-binding allows SM2275 to selectively inhibits PD-L1 activity on EGFR+PD-L1+ double-positive cells (FIG.15). Dual-target-binding SM2272 shows significantly enhanced ability to block PD-1 and PD-L1 interaction on CHO-Dual-OKT3 than CHO- hPD-L1-OKT3 (Fig 16). Tumor site-specific blockage of PD-L1 / PD-1 synergizes with CD28 agonist activity to allow SM2275 to maximize T cell activity locally.

[0204] SM2275 showed the dual target dependent CD28 activation synergizing with PD1 / PD-L1 signaling (FIG. 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 cells. The no target control was the CHO-OKT3 cell line. SM2275 showed no activity in no target control indicating target dependent CD28 activation. SM2275 (CD28-), a SM2275 with a mutated and inactivated CD28 module, reduced ability to inhibit PD-L1 signaling compared to SM2275, indicating CD28 activation significantly enhanced the inhibition of PDL1 signaling.

[0205] SM2275 does not induce cytokines release from human PBMC cells compared to TGN1412 (FIG.16). SM2275 demonstrates no systemic IL-2 release compared to TGN1412 in vivo (FIG.18). humanized hPD-L1 / hCD28 mice received a single dose of PBS, SM2275 (5mg / kg, 15mg / kg), or TGN1412 (2.5 mg / kg). Blood was collected 4 hr after dosing and serum cytokines were measured. SM2275 was well tolerated and did not induce cytokine release after 4 h of treatment. In contrast, TGN1412 induced significant systemic cytokines release, consistent with its super-agonistic activity in vivo.

[0206] SM2275 significantly inhibited MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenograft growth in the humanized hCD28 mouse model. MC38-hEGFR-hPD-L1 cells were implanted subcutaneously and when the tumor size reached ~80 mm³, animals were randomly grouped and treated with vehicle or SM2275 every 2 days for 10 days, and body weight and tumor volume were measured on days 0, 2, 4, 6, 8, 10, 12, 14, 18, and 22 post-grouping (N=6). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized CD28 mice in a dose-dependent manner (FIG.20).PCT Patent Application 1959708-00018

[0207] SM2275 significantly inhibited MC38-hEGFR-hPD-L1 (mPD-L1null) tumor xenograft growth in the humanized hCD28 / hPD-L1 mouse model. MC38-hEGFR-hPD-L1 cells were implanted subcutaneously and when the tumor size reached ~90 mm³, animals were randomly grouped and treated with vehicle or SM2275 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 days (N=5). SM2275 effectively inhibited the growth of MC38-hEGFR / hPD-L1 tumors in humanized hCD28 / hPD-L1 mice (FIG.21)

[0208] The pharmacokinetics of SM2275 was evaluated in cynomolgus monkeys. Eight healthy adult cynomolgus monkeys were randomly divided into four groups based on body weight: a solvent control group and low, medium, and high-dose SM2275 groups (administered doses of 5, 15, and 50 mg / kg, respectively). Each group consisted of two monkeys, with equal numbers of males and females. The SM2275 dosage groups received intravenous infusions of the corresponding concentrations of SM2275 dosage formulation at 10 mL / kg volume, while the solvent control group received the solvent in the same manner. No dose limited toxicity observed up to 50mg / kg. SM2275 exhibits favorable pharmacokinetic properties with a terminal half-life of > 60 hours, consistent with the albumin-mediated FcRn cycle process in cynomolgus monkeys (FIG.22).

[0209] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” As used herein the terms "about" and “approximately” means within 10 to 15%, preferably within 5 to 10%. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0210] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthandPCT Patent Application 1959708-00018 method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0211] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0212] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0213] Specific embodiments disclosed herein may be further limited in the claims using consisting of or consisting essentially of language. When used in the claims, whether as filed or added per amendment, the transition term “consisting of” excludes any element, step, or ingredient not specified in the claims. The transition term “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0214] Furthermore, numerous references have been made to patents and printed publications throughout this specification. Each of the above-cited references and printed publications are individually incorporated herein by reference in their entirety.PCT Patent Application 1959708-00018

[0215] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

PCT Patent Application 1959708-00018 What is claimed is:

1. A multi-valent, multi-specific, single-chain antibody (MVSCA) comprising at least four binding specificities and having the structure: A—B—C—D wherein each of the A, B, C, and D comprise VHH domains individually specific for EGFR, PD-L1, HSA, 4-1BB, OX40, CD40, CD16A, CD28, LAG3, and CD3E, and the amino acid sequences of A, B, C, and D VHH domains are connected by an amino acid linker sequence.

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

3. The MVSCA of claim 1, wherein the VHH domain specific for EGFR comprises an amino acid sequence selected from SEQ ID NOs:134-161.

4. The MVSCA of claim 1, wherein the VHH domain specific for PD-L1 comprises an amino acid sequence selected from SEQ ID NOs:66-74.

5. The MVSCA of claim 1, wherein the VHH domain specific for HSA comprises an amino acid sequence selected from SEQ ID NOs:76-92 and 188.

6. The MVSCA of claim 1, wherein the VHH domain specific for 4-1BB comprises an amino acid sequence selected from SEQ ID NOs:120-132.

7. The MVSCA of claim 1, wherein the VHH domain specific for OX40 comprises an amino acid sequence selected from SEQ ID NOs:101 or 102.

8. The MVSCA of claim 1, wherein the VHH domain specific for CD40 comprises an amino acid sequence selected from SEQ ID NOs:104-118.

9. The MVSCA of claim 1, wherein the VHH domain specific for CD16A comprises an amino acid sequence selected from SEQ ID NOs:94-99.

10. The MVSCA of claim 1, wherein the VHH domain specific for CD3E comprises an amino acid sequence selected from SEQ ID NOs:47-64.

11. The MVSCA of claim 1, wherein the VHH domain specific for CD28 comprises an amino acid sequence selected from SEQ ID NOs:25-45.

12. The MVSCA of claim 1, wherein the VHH domain specific for LAG3 comprises an amino acid sequence selected from SEQ ID NOs:174-187.PCT Patent Application 1959708-00018 13. The MVSCA of any one of claims 1-12, wherein the order of the A, B, C, and D domains is A—B—C—D.

14. The MVSCA of any one of claims 1-13, wherein the linker comprises an amino acid sequence of one of SEQ ID NOs:1-23.

15. The MVSCA of claim 1, wherein one domain comprises two VHH sequences specific for the same target.

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

17. The MVSCA of claim 15, wherein the two VHH sequences are specific for 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 linked by a linker of one of SEQ ID NOs:1-23.

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

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

165.

23. The MVSCA of any one of claims 2-20, wherein the MVSCA comprises the following 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 NOs:167-172.

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

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

162.

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

28. The MVSCA of claim 27, comprising an amino acid sequence of SEQ ID NO:163.PCT Patent Application 1959708-00018 29. The MVSCA of any one of claims 1 or 3-20, wherein the MVSCA comprises the following specificities in the following order: CD16A—HSA—CD47—CD33.

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

164.

31. A pharmaceutical composition comprising the MVSCA of any one of clams 1- 30.

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

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 in need thereof.

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